CN101189345A - Reagents, Methods, and Libraries for Bead-Based Sequencing - Google Patents

Abstract

The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. In certain embodiments the methods make use of extension probes containing an abasic residue or a damaged base and employ agents appropriate to cleave linkages between a nucleoside and an abasic residue and/or agents appropriate to remove a damaged base from a nucleic acid. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to beads, which are immobilized in or on a semi-solid support. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages or trigger residues that are suitable for use in the method. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides. The invention further provides efficient methods for preparing templates, particularly for performing sequencing multiple different templates in parallel. The invention also provides methods for performing ligation and cleavage. The invention also provides new libraries of nucleic acid fragments containing paired tags, and methods of preparing microparticles having multiple different templates (e.g., containing paired tags) attached thereto and of sequencing the templates individually.

Description

Reagents, Methods, and Libraries for Bead-Based Sequencing

Cross References to Related Applications

This application claims provisional applications USSN 60/649,294, filed February 1, 2005; USSN 60/656,599, filed February 25, 2005; USSN 60/673,749, filed April 21, 2005, July 15, 2005 Priority and benefit of USSN 60/699,541, filed September 30, 2005, and USSN 60/722,526, filed September 30, 2005, all of which are incorporated herein by reference.

Background of the invention

Nucleic acid sequencing technology is important in a variety of fields from basic research to clinical diagnosis. Results obtained from such techniques can include varying degrees of specific information. For example, useful information may include: determining whether the sequence of a specific polynucleotide differs from a reference polynucleotide, confirming the presence or absence of a specific polynucleotide sequence in a sample, determining partial sequence information such as identifying one or more nuclei within a polynucleotide Nucleotides, determine the type and sequence of nucleotides in polynucleotides, etc.

DNA strands are generally polymers composed of four types of subunits, namely deoxynucleosides containing the bases adenine (A), cytosine (C), guanine (G) and thymine (T) acid. These subunits are interconnected by a covalent phosphodiester bond that connects the 5' carbon of one deoxyribose group to the 3' carbon of the next. Most naturally occurring DNA consists of two such strands, arranged in an antiparallel orientation, held together by hydrogen bonds formed between complementary bases, A and T and G and C.

With chain termination or dideoxynucleotide method (Sanger et al., Proc.Natl.Acad.Sci./4:5463-5467, 1977) and chemical degradation method (Maxam and Gilbert, Proc.Natl.Acad.Sci.74 : 560-564, 1977), the development of large-scale DNA sequencing is possible, where the former has been widely used, improved and automated. Specifically, the use of fluorescently labeled chain terminators is important in the development of automated DNA sequencers. Common to both of the above methods is the generation of one or more aggregates of labeled DNA fragments of different sizes, which must then be separated according to length to identify the nucleotides at the 3' ends of the fragments (chain termination method) or Nucleotides recently cleaved from the fragment (chemical degradation).

While currently available sequencing technologies have achieved major advances, such as the sequencing of many complete genomes, these technologies have a number of shortcomings and in many ways are in great need of improvement. Typically, labeled DNA fragments are separated by polyacrylamide gel electrophoresis. However, this step has proven to be the major bottleneck limiting the speed and accuracy of sequencing in many cases. Although capillary electrophoresis (CAE) has been proven to be a breakthrough capable of completing the Human Genome Project (Venter et al., Science, 291:1304-1351, 2001; Lander et al., Nature, 409:860-921, 2001), there are still significant shortcomings. For example, CAE still requires time-consuming separation steps and still involves differentiation based on size, which can be inaccurate.

Various alternatives to the chain termination method have been proposed. In a method commonly referred to as "sequencing by synthesis," oligonucleotide primers are first hybridized to a target template. The primer is then extended by successive cycles of polymerase-catalyzed addition of differently labeled nucleotides whose incorporation into the growing strand is detected. Identification of the label serves as identification of the complementary nucleotide in the template. Alternatively, multiple reactions can be performed in parallel for each nucleotide, and incorporation of the labeled nucleotide in a reaction using a particular nucleotide identifies the complementary nucleotide in the template. (See, e.g., Melamede, U.S. Patent 4,863,849; Cheeseman, U.S. Patent 5,302,509, Tsien et al., International Application WO 91/06678; Rosenthal et al., International Application WO 93/21340; Canard et al., Gene, 148:1-6 (1994); Metzker et al., Nucleic Acids Research, 22: 4259-4267 (1994)).

Efficient sequencing of polynucleotides of any significant length requires the polymerase to incorporate exactly one nucleotide per cycle. Therefore, it is often desirable to employ nucleotides that act as chain terminators, ie their incorporation prevents further extension by the polymerase. The incorporated nucleotide must then be modified enzymatically or chemically to allow the polymerase to incorporate the next nucleotide. Various nucleotide analogues have been proposed which can be used as chain terminators, but which can be modified after their incorporation so that their extension continues in subsequent steps. Such "reversible terminators" have been described, for example, in US Patent Nos. 5,302,509; 6,255,475; 6,309,836; 6,613,513. However, it has proven difficult to identify reversible terminators that can be efficiently incorporated by polymerases, possibly due to the fact that, given the small size of nucleotides, modifications that affect the use of nucleotides as terminators also affect their incorporation into growing polynucleotide chains .

Other sequencing methods include pyrosequencing, which is based on the detection of pyrophosphate (PPi) released during DNA polymerization (see eg, US Patents 6,210,891 and 6,258,568). Although electrophoretic separation is not required, pyrosequencing has a number of disadvantages that still limit its widespread application (Franca et al., Quarterly Reviews of Biophysics, 35(2):169-200, 2002). Sequencing by hybridization has also been proposed as an alternative (US Patent 5,202,231; WO 99/60170; WO 00/56937; Drmanac et al., Advances in Biochemical Engineering/Biotechnology, 11:16-101, 2002), but has many disadvantages, including in distinguishing Errors may occur with highly similar sequences. In theory, single-molecule sequencing by exonucleases is a very efficient method for rapidly determining the sequence of long DNA molecules, which involves labeling each base on one strand and then detecting sequentially excised 3' ends in the sample stream Nucleotides (Stephan et al., J BiotechnoL, 86:255-267, 2001). However, there are many technical hurdles to overcome before this possible approach can be realized (Stephan et al., 2001).

Diagnostic tests based on specific sequence changes are already available for a variety of different diseases. It is widely believed that the sequencing of the human genome has ushered in an era of personalized medicine, in which treatments, including prophylactic treatments, are tailored to a patient's specific genetic makeup or selected based on the identification of specific alleles or mutations. There is an increasing need for rapid and accurate determination of sequence variants of pathogens such as HIV. Therefore, there will certainly be a greater need for accurate and rapid sequence determination in the near future. Therefore, improved methods for all types of sequence determination are needed.

Summary of the invention

The present invention provides new and improved sequencing methods that do not require fragment isolation and, in certain embodiments, do not require the use of polymerases. US Patents 5,740,341 and 6,306,597 to Macevicz describe alternatives to the methods discussed in the Background of the Invention. The method is based on repeated cycles of duplex extension along a single-stranded template. In preferred embodiments of these methods, one nucleotide is identified in each cycle. The present invention improves upon these methods. These improvements enable efficient implementation of the method and are particularly suitable for high-throughput sequencing. In addition, the present invention provides methods for sequence determination that include repeated cycles of duplex extension along a single-stranded template but do not include identifying any single nucleotide in each cycle.

In one aspect, the invention provides improved methods for sequencing based on sequential cycles of duplex extension along a single-stranded template, ligation of a labeled extension probe, and detection of the label. Typically, extension is initiated from the duplex formed by the starting oligonucleotide and the template. The starting oligonucleotide is extended by ligation of the oligonucleotide to the end of the starting oligonucleotide to form an extending duplex, and the extending duplex is then repeatedly extended by successive ligation cycles. During each cycle, one or more nucleotides in the template are identified by identifying a label that is successfully attached to or associated with the oligonucleotide probe. The labeling of newly added probes can also be detected before ligation, or, additionally, after ligation. It is generally preferred to detect the label after ligation.

In a preferred embodiment, the probe has a non-extendable portion in its terminal position (the opposite end of the probe to the nucleotides attached to the growing duplex nucleic acid strand) so that only extension of the duplex occurs in a single cycle. single extension. "Non-extensible" means that the moiety cannot serve as a ligase substrate without modification. For example, the moiety can be a nucleotide residue lacking a 5' phosphate or a 3' hydroxyl. The moiety may be a nucleotide attached with a capping group that prevents ligation. In a preferred embodiment of the invention, the non-extendable portion is removed after ligation to regenerate the extendable end so that the duplex can be further extended in subsequent cycles.

To enable removal of non-extendable moieties, in certain embodiments of the invention, probes contain at least one internucleoside linkage that is cleavable without substantially cleaving phosphodiester bonds. Such linkages are referred to herein as "cleavable internucleoside linkages" or "sharp linkages". Cleavage of the scissile internucleoside linkage removes the non-extendable portion and either regenerates the extendable probe terminus or leaves the terminal residues modified to form the extensible probe terminus. A scissile internucleoside linkage can be located between any two nucleosides in the probe. Preferably, the easy linkage is at least a few nucleotides away from (ie distal to) the newly formed bond. The nucleotides between the terminal nucleotides attached to the extendable termini and the easy junction in the extension probe need not fully hybridize to the template. These nucleotides can be used as "spacers" and used to identify nucleotides located at intervals in the template without performing a cycle for each nucleotide within the interval.

Preferably, the scissile internucleoside linkage and label are positioned such that cleavage of the scissile internucleoside linkage separates the extension probe into a labeled moiety and a moiety that remains part of the growing nucleic acid strand, thereby allowing the labeled moiety to diffuse on (e.g. by raising the temperature). For example, the label can be attached to the terminal nucleotide of the extension probe at the opposite end from the linking nucleotide. Alternatively, the marker can be removed by any other method.

The inventors have found that a phosphorothioate linkage in which one of the bridging oxygen atoms in the phosphodiester linkage is replaced by a sulfur atom is a particularly advantageous scissile internucleoside linkage. The sulfur atom in the phosphorothioate linkage can be attached to the 3' carbon of one nucleoside or the 5' carbon of an adjacent nucleoside.

In certain embodiments of the methods described above, a number of sequencing reactions are performed. These reactions use starting oligonucleotides that hybridize to different sequences of the template so that the ends where the initial ligation occurs are at different positions on the template. For example, the position where the initial ligation occurs can be shifted, or "phased" from each other, by adding 1 nucleotide. Thus, after each cycle of extension with oligonucleotide probes of the same length, the same relative phase exists between the ends of the starting oligonucleotides on different templates. Reactions can be run in parallel in separate vessels each containing a copy of the same template, or they can be run sequentially, with the initial starting oligonucleotides used to obtain sequence information, the extended duplexes on the template removed, and then the extended duplexes hybridized to that template Starting oligonucleotides of different sequences were subjected to other reactions.

In another aspect, the present invention provides solutions useful for various nucleic acid manipulations. In one embodiment, the invention provides a solution comprising or consisting essentially of an aqueous solution of 1.0-3.0% SDS, 100-300 mM NaCl, and 5-15 mM sodium bisulfate ( _NaHSO4 ). The solution may contain or consist essentially of an aqueous solution of about 2% SDS, about 200 mM NaCl, and about 10 mM sodium bisulfate ( _NaHSO4 ). For example, in one embodiment, the solution contains 2% SDS, 200 mM NaCl, and 10 mM sodium bisulfate ( _NaHSO4 ) in water. In another embodiment, the solution consists essentially of an aqueous solution of 2% SDS, 200 mM NaCl, and 10 mM sodium bisulfate ( _NaHSO4 ). In certain embodiments, the pH of the solution is 2.0-3.0, such as 2.5. This solution can be used to separate double-stranded nucleic acids, such as double-stranded DNA, into single strands, even if the double-stranded nucleic acids are denatured (melted). In certain embodiments, both strands are DNA. In other embodiments, both strands are RNA. In other embodiments, one strand is DNA and the other strand is RNA. In other embodiments, one or both strands contain both RNA and DNA. In other embodiments, one or both strands contain at least one nucleotide other than A, G, C, or T. In some embodiments, one or both strands contain non-naturally occurring nucleotides. In other embodiments, one or both residues are priming residues, such as abasic residues or damaged bases. In some embodiments, one or more residues contain a universal base. In some embodiments, one or both strands contain easy cleavage linkages.

Double-stranded nucleic acids can be fully or partially double-stranded. They may be free molecules in solution, or one or both chains may be physically associated (eg, covalently or non-covalently attached) to a solid or semisolid support or substrate. Of particular note is that double-stranded nucleic acids incubated in these solutions efficiently dissociate into single strands in the absence of heat or the presence of strong denaturing agents that would cause gel stratification (eg, when nucleic acids are located on or attached to semi-solid supports such as polyacrylamide gels) or disruptable non-covalent linkages such as streptavidin (SA)-biotin linkages (such as when nucleic acids are attached to supports or substrates via SA-biotin linkages) . In one embodiment, the solution is used to isolate double-stranded nucleic acids in which one nucleic acid is attached to a bead via an SA-biotin linkage.

The present invention also provides a method of separating strands of a double-stranded nucleic acid, said method comprising the step of: contacting the double-stranded nucleic acid with any of the above solutions, such as containing about 1.0-3.0% SDS, about 100-300 mM NaCl and about 5-15 mM sulfuric acid Sodium hydrogen sulfate (NaHSO ₄ ), such as an aqueous solution containing 1.0-3.0% SDS, 100-300 mM NaCl, and 5-15 mM sodium hydrogen sulfate (NaHSO ₄ ). In one embodiment, the solution contains about 2% SDS, 200 mM NaCl, and 10 mM sodium bisulfate (NaHSO ₄ ), such as 2% SDS, 200 mM NaCl, and 10 mM sodium bisulfate (NaHSO ₄ ). In another embodiment, the solution consists essentially of an aqueous solution of 2% SDS, 200 mM NaCl, and 10 mM sodium bisulfate ( _NaHSO4 ). In certain embodiments, the pH of the solution is 2.0-3.0, such as 2.5. In some embodiments, double-stranded nucleic acids are incubated in the solution. In other embodiments, the solution is used to wash double-stranded nucleic acids (preferably nucleic acids attached to a support or substrate). In some embodiments, the double-stranded nucleic acid is contacted with the solution for a time sufficient to separate at least 10% of the double-stranded nucleic acid molecules into single strands. In some embodiments, the double-stranded nucleic acid is contacted with the solution for a time sufficient to remove at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% % or more of the double-stranded nucleic acid separates into single strands. In an exemplary embodiment, the double-stranded nucleic acid is contacted with the solution for 15 seconds to 3 hours. In another embodiment, the double stranded nucleic acid is contacted with the solution for 1 minute to 1 hour. In certain embodiments, the double-stranded nucleic acid is contacted with the solution for about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. The method may also include the step of removing the solution or removing some or all of the nucleic acid from the solution after a period of incubation.

This solution can be used in one or more steps of many of the sequencing methods described herein, and can be used in any of these methods. For example, the solution can be used to separate extended duplexes from templates. This solution can be used after cleavage of the easy junction to remove the portion of the extension probe that is no longer attached to the extension duplex. The solution can also be used to separate strands of triple-stranded nucleic acids or to separate double-stranded regions of single-stranded nucleic acids containing self-complementary portions that hybridize to each other.

In another aspect, the invention provides a method for obtaining sequence information using a collection of at least two distinctly labeled oligonucleotide probe families. Probes in a probe family contain an undefined portion and a defined portion. Extension is initiated from the duplex formed by the starting oligonucleotide and template as described in the above method. The initial oligonucleotide is extended by ligation of the oligonucleotide probe to its terminus to form an extension duplex, and the extension is then repeated through successive ligation cycles. The probe contains a non-extendable portion in its terminal position (the opposite end of the probe from the nucleotides attached to the growing nucleic acid strand of the duplex) so that only one extension occurs in a single cycle of extending the duplex. During each cycle, labels on or attached to successfully ligated probes are detected and non-extendable portions are removed or modified to generate extendable ends. The label corresponds to the probe family to which the probe belongs.

Successive cycles of extension, ligation, and detection generate an ordered list of probe families to which successive successfully ligated probes belong. Sequence information was obtained using the ordered list of probe families. However, knowing which probe family a newly ligated probe belongs to is not, by itself, sufficient to determine the nucleotide species in the template. Conversely, knowing which probe family a newly ligated probe belongs to excludes certain sequences from being part of the probe's defined sequence, but leaves at least two possible nucleotide species at each position. Thus, there are at least two possibilities for the nucleotide species in the template relative to the nucleotides of the defining portion of the newly ligated probe (ie, nucleotides complementary to the nucleotides of the defining portion of the probe).

In certain embodiments, after performing the desired number of cycles, an ordered list of probe family species is used to generate a set of candidate sequences. This set of candidate sequences can provide enough information to achieve the goal. In a preferred embodiment of the invention, one or more additional steps are performed to select the correct sequence from the candidate sequences. For example, the sequence can be compared to a database of known sequences, and the candidate sequence that is closest to one of the sequences in the database is selected as the correct sequence. In other embodiments, the template is subjected to another round of sequencing by successive cycles of extension, ligation, detection and cleavage using the differentially encoded set of probe families, and the information obtained in the second round is used to select the correct sequence. In other embodiments, at least one item of information is combined with information obtained from an ordered list of probe families to determine the sequence.

The present invention also provides methods for error checking when sequencing with probe families. Certain methods can distinguish between single nucleotide polymorphisms (SNPs) and sequencing errors.

The present invention also provides nucleic acid fragments (such as DNA fragments) comprising at least two segments of interest (such as at least two tags) and at least three primer binding regions (PBRs), so that at least two different Templates, each corresponding to a segment of interest. A "primer binding region" is a nucleic acid portion of an oligonucleotide that is hybridizable, thereby rendering the oligonucleotide useful as an amplification primer, sequencing primer, starting oligonucleotide, and the like. Therefore, the primer binding region should have a known sequence to select an appropriate complementary oligonucleotide. As used herein and in the Figures, a portion of a nucleic acid strand used in the methods of the invention may be referred to as a primer binding region, whether a primer actually binds to this region or to the corresponding portion of the complementary strand of the nucleic acid strand in practice of the method of the invention. Thus, when used in the methods described herein, a portion of a nucleic acid may be referred to as a primer binding region, whether the primer actually binds to that region (in which case the sequence of the primer is complementary or substantially complementary to that of the region) or is the complementary region that binds to the region (in which case the sequence of the primer is identical or substantially identical to the sequence of the region). A segment of interest is any nucleic acid segment for which sequence information is desired. For example, a sequence of interest may be a tag, and for the purposes of this disclosure, a segment of interest may be assumed to be a tag (also referred to herein and elsewhere as an "end tag"). It should be understood, however, that the invention is not limited to segments of interest as labels. In certain embodiments, at least two tags are paired tags. A nucleic acid fragment may contain one or more pairs of tags, such as one or more pairs of tag pairs, such as 2, 3, 4, 5 or more pairs of tag pairs. The invention also provides libraries containing such nucleic acid fragments, and methods for preparing templates and libraries.

The invention also provides microparticles, such as beads, to which at least two different populations of nucleic acids are attached, wherein each of the at least two populations of nucleic acids consists of a plurality of substantially identical nucleic acids, and wherein the populations of nucleic acids are amplified by amplification such as PCR. Increased) single nucleic acid fragment generation. In some embodiments, the single nucleic acid fragment contains a 5' tag and a 3' tag, wherein the 5' and 3' tags are paired tags. In some embodiments wherein the single nucleic acid fragment contains a pair of 5' and 3' tags, one of the populations of nucleic acids attached to the microparticle includes at least a portion of a 5' tag, and the population of nucleic acids attached to the microparticle One includes at least a portion of a 3' tag. In a preferred embodiment, one of the populations of nucleic acids includes a complete 5' tag and one of the populations of nucleic acids includes a complete 3' tag.

The nucleic acid fragment contains a plurality of PBRs, at least one of which is located between the tags, and at least two of which flank the portion of the nucleic acid fragment containing the tags, thereby enabling amplification of a region containing at least a portion of the 5' tag and enabling A region containing at least a portion of the 3' tag is amplified to generate two distinct nucleic acid populations. In a preferred embodiment, the entire 5' tag and the entire 3' tag can be amplified. For example, the nucleic acid fragment may contain first and second primer binding sites flanking the 5' tag, and third and fourth primer binding sites flanking the 3' tag. The 5' tag is amplified by PCR amplification using primers that bind to the first and second primer binding sites. The 3' tag is amplified by PCR amplification using primers that bind to the third and fourth primer binding sites. It will be appreciated that the primers should be selected so as to extend from each primer to the region of the DNA fragment containing the tag to be amplified. Alternatively, a first primer binding site may be located upstream of one of the tags, a second primer binding site may be located downstream of the other tag, and a third primer binding site may be located between the two tags. The third primer binding site serves as the binding site for the forward primer for PCR amplification to amplify one tag and as the binding site for the reverse primer for PCR amplification to amplify the other tag. Accordingly, in one embodiment of the invention there is provided a microparticle, such as a bead, to which at least two different populations of nucleic acids are attached, wherein each of the at least two populations of nucleic acids consists of a plurality of substantially identical nucleic acids, and wherein the first A different population of nucleic acids includes a 5' tag and a second different population of nucleic acids includes a 3' tag.

The invention also provides populations of microparticles, such as beads wherein each microparticle has attached at least two different populations of nucleic acids, wherein each of the at least two populations of nucleic acids consists of a plurality of substantially identical nucleic acids, wherein the populations of nucleic acids are amplified by (eg, PCR amplification) the production of individual nucleic acid fragments. A population of substantially identical nucleic acids can be, for example, a 5' tag and a 3' tag. The invention also provides arrays of such microparticles and methods of sequencing comprising sequencing substantially identical populations of nucleic acids. For example, in one embodiment, the two substantially identical populations of nucleic acids attached to a single microparticle each include a different primer binding region (PBR), so that by using different sequencing primers, the sequence can be targeted without interference from the other population. A population is sequenced. If two or more substantially identical populations of substantially identical nucleic acids are attached to a microparticle, each population may have a unique PBR such that a primer that binds a particular PBR does not bind to any other substantially identical populations of substantially identical nucleic acids that are attached to the microparticle. PBR. Thus, the methods of the invention are capable of producing microparticles (e.g., multiple copies of a template containing a 5' tag and multiple copies of a template containing a 3' tag) to which at least two different populations of substantially identical nucleic acids are attached, wherein the tags are pairwise labels. According to the method of the present invention, the template contains different PBRs that provide binding sites for sequencing primers. Therefore, by selecting sequencing primers that are complementary to the PBR in the template containing the 5' tag, sequence information can be obtained from the 5' tag without interference from the template containing the 3' tag, even if there is also a PBR containing the 3' tag on the same particle. template. By selecting sequencing primers that are complementary to the PBR in the template containing the 3' tag, sequence information can be obtained from the 3' tag without interference from the template containing the 5' tag, even if the template containing the 5' tag is also present on the same particle. The presence of both paired tags on the same particle means that the sequences of the 5' and 3' paired tags can be linked to each other, as is known in the art when they are present on a single template.

The invention also provides automated sequencing systems that can be used, for example, to sequence templates arrayed in or on a substantially planar support. The present invention also provides an image processing method, which can be stored in computer-readable media such as hard disk, CD, zip disk, flash memory and the like. In certain preferred embodiments, the system achieves identification of 40,000 or more nucleotides per second. In certain preferred embodiments, the system generates 8.6 gigabytes (Gb) of sequence data or more per day (24 hours). In certain preferred embodiments, the system generates 48 Gb of sequence information (nucleotide identification) or more per day.

Additionally, the invention provides computer readable media storing information generated using the sequencing methods of the invention. The information can be stored in a database.

This application refers to various patents, patent applications, journal articles, and other publications, all of which are hereby incorporated by reference. In addition, the following standard references are incorporated herein by reference: Current Protocols in Molecular Biology, John Wiley & Sons, New York, ed. July 2002; Sambrook, Russell, and Sambrook, Molecular Cloning : Laboratory Manual "(Molecular Cloning: A Laboratory Manual), third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001. In the event of a conflict between this specification and any document incorporated by reference, this specification shall control, with the understanding that the inventors can determine at any time whether a contradiction or inconsistency exists.

Brief description of the drawings

Figure 1A is a schematic diagram of priming followed by two cycles of extension, ligation and identification.

Figure IB is a schematic illustration of initiation followed by two cycles of extension, ligation and identification in an embodiment of inward extension from the free end of the template to the support.

Figure 2 shows the color assignment scheme for oligonucleotide probes, where the 3' base species of the probe is determined by identifying the color of the fluorophore.

Figure 3A shows a schematic diagram of hybridization of initial oligonucleotides to different positions in the template binding region followed by ligation of extension probes to form extension duplexes.

Figure 3B shows a schematic diagram of assembly of contiguous sequences by extension, ligation and cleavage with extension probes designed to read every 6 bases on the template molecule.

Figure 4A shows a 5'-S-phosphorothioate linkage (3'-O-P-S-5').

Figure 4B shows a 3'-S-phosphorothioate linkage (3'-S-P-O-5').

Figure 5A shows a schematic of one extension, ligation and cleavage cycle for sequencing in the 5'→3' direction with an extension probe containing a 3'-O-P-S-5' phosphorothioate linkage.

Figure 5B shows a schematic of one extension, ligation and cleavage cycle for sequencing in the 3'→5' direction with an extension probe containing a 3'-O-P-S-5' phosphorothioate linkage.

6A-6F are more detailed schematics of several sequencing reactions performed on a single template. These reactions utilize starting oligonucleotides that bind to different parts of the template.

Figure 7 is a schematic diagram showing the synthetic scheme of 3'-phosphoramidites of dA and dG.

Figures 8A-8E are the results of two cycles of gel shift assays showing smooth ligation and cleavage of extension probes containing phosphorothioate linkages.

Figure 8F shows a schematic diagram of the ligation mechanism of DNA ligase.

Figure 9 is the results of a gel shift assay showing the ligation efficiency of inosine-containing degenerate oligonucleotide probes.

Figure 10 is the results of a gel shift assay showing the ligation efficiency of inosine-containing degenerate oligonucleotide probes on various substrates.

Figure 11 shows the results of analyzes evaluating the conservation of each of the two DNA ligases (T4 DNA ligase and Tag DNA ligase) on the 3'→5' extension.

Figure 12 is a gel shift test result (A) and ligation of the ligation efficiency of the inosine-containing degenerate oligonucleotide probes used to evaluate the conservation of T4 DNA ligase in ligated oligonucleotide probes Results of direct sequencing analysis of the reaction (B). The results are tabulated into panels C-F.

Figures 13A-13C show the results of experiments with ligation in gels when bead-based templates were embedded in polyacrylamide gels on slides. Figure 13A shows the ligation reaction scheme. Ligation reactions performed in gels in the presence (B) and absence (C) of T4 DNA ligase.

Figure 14A shows an image of an emulsion PCR reaction performed on a bead attached with a first amplification primer using fluorescently labeled second amplification primers and excess template.

Figure 14B (top) shows a fluorescent image of a portion of a slide immobilized in a polyacrylamide gel with beads attached to templates hybridized to Cy3-labeled oligonucleotides. (This slide was used in a different experiment, but the slide used here is representative). Figure 14B (bottom) shows a schematic of a slide fitted with a Teflon mask to block polyacrylamide solution.

Figure 15 shows three sets of labeled oligonucleotide probes designed to address probe specificity and selectivity issues, and also shows excitation and emission values for a set of four spectrally resolved labels.

Figure 16 shows the results of experiments confirming the 4-color spectral properties of oligonucleotide probes. Hybridization and ligation reactions were performed on slides containing four unique single-stranded template populations (A) using an oligonucleotide probe mix containing four unique fluorophore probes, imaged under bright light before and after ligation ( B), and imaged with fluorescence excitation using four bandpass filters. Individual populations appear in false color (C). Spectral features showing minimal signal overlap are plotted in (D).

Figure 17 shows experiments to confirm ligation specificity of oligonucleotide extension probes. Figure 17(A) shows a schematic diagram of the connections. Figure 17(B) is a bright light image, and Figure 17(C) is the corresponding fluorescent image after ligation of bead populations embedded in a polyacrylamide gel. Fig. 17(D) shows the fluorescence detected from each label before or after ligation.

Figure 18 shows another experiment to confirm ligation specificity and selectivity of oligonucleotide extension probes. Figure 8(A) shows a schematic diagram of the connections. Figure 18(B) is a bright light image, and Figure 18(C) is the corresponding fluorescent image after ligation of bead populations embedded in a polyacrylamide gel. Figure 18(D) shows predicted versus observed junction frequencies, showing that the predicted and observed frequencies are highly correlated based on the proportion of a particular extension probe in the population.

Figure 19 shows experiments confirming that libraries of oligonucleotide extension probes containing degenerate and universal bases can be used to provide specific and selective ligation in a gel. Figure 19(A) shows a schematic of a ligation experiment illustrating four differentially labeled inosine-containing degenerate probe pools after ligation. Figure 19(B) is a bright light image, and Figure 19(C) is the corresponding fluorescent image after ligation of bead populations embedded in a polyacrylamide gel. Figure 19(D) shows predicted versus observed junction frequencies, showing that predicted and observed frequencies are highly correlated based on the proportion of a particular extension probe in the population. Figure 19(E) shows a scatterplot of raw unprocessed data and filtered data representing the top 90% of bead signal values.

Figure 20 is a bar graph showing the signal detected in successive cycles of hybridization stripping of the starting oligonucleotide (primer) to the template. As shown, a small loss of signal occurs beyond 10 cycles.

Figure 21 is a photograph of an automated sequencing system that can be used, for example, to collect sequence information from templates arrayed in or on a substantially flat support. Also shown are dedicated computers that control the operation of the various components of the system, process and store the collected image data, provide the user interface, and more. The lower part of the figure shows a magnified view of the flow chamber used to achieve specific gravity bubble displacement.

Figure 22 shows a schematic diagram of a high-throughput automated sequencing apparatus that can be used to sequence templates arrayed in or on a substantially flat support.

Figure 23 shows a scatterplot of inconsistent alignments, which demonstrates that very few of the 30 frames were inconsistent.

24A-I show schematic diagrams of various views of a flow chamber or portion thereof of the present invention.

Figure 25A shows an exemplary encoding of a preferred probe family set comprising partially defined probes comprising a defined portion of 2 nucleotides in length.

Figure 25B shows a preferred collection of probe families (upper panel) and ligation, detection and cleavage cycle (lower panel).

Figure 26 shows an exemplary encoding of another preferred probe family set comprising partially defined probes comprising defined portions of 2 nucleotides in length.

Figures 27A-27C represent another method for determining the set of 24 preferred probe families defined in Table 1 graphically.

Figure 28 shows a less preferred collection of probe families in which the probes contain a defined portion of 2 nucleotides in length.

Figure 29A shows a diagram of defined portions that can be used to generate probe family collections comprising probes comprising defined portions that are 3 nucleotides in length.

Figure 29B shows a diagram of a mapping scheme that can be used to generate a defined portion of a probe family set comprising probes comprising a defined portion of 3 nucleotides in length from a set of 24 preferred probe families.

Figure 30 shows a method for sequence determination using probe family collections. One embodiment using a set of preferred probe families is described.

Figures 31A-31C show a method for sequence determination using a first set of probe families to generate candidate sequences and decode them with a second set of probe families.

Figure 32 shows a method for sequence determination with less preferred combinations of probe families.

Figure 33A shows a schematic of a slide with beads attached. DNA templates are attached to beads.

Figure 33B shows a population of beads attached to a glass slide. The images below show the same slide area under white light (left) and fluorescence microscopy. The graph above shows the bead density range.

Figures 34A-34C show a scheme for the amplification of two tags of a paired tag present in a nucleic acid fragment (template) as a single nucleic acid population and their capture onto microparticles by the amplification method.

Figures 35A and 35B show details of primer design and amplification for the scheme of Figure 35 . Both strands of the nucleic acid fragment (template) are shown for clarity. Primers and primer binding regions with the same sequence are indicated in the same color. For example, P1 is represented in dark blue, indicating that the sequence of primer P1 present on the microparticle and in solution is identical to the corresponding colored portion of the template strand shown. The dark blue region of the template (labeled P1) may be referred to as the primer binding region, although the corresponding primer (P1) actually binds to the complementary portion of the other strand and is identical in sequence to primer P1.

Figures 35C and 35D show the sequencing of first and second tags, respectively, attached to microparticles produced using the methods shown in Figures 35A and 35B.

definition

For ease of understanding of this specification, the following definitions are provided. It should be understood that, in general, terms not specifically defined are given their usual or generally accepted meanings in the art.

As used herein, an "abasic residue" is a nucleoside that remains after the nitrogenous base has been removed or a significant portion of the nitrogenous base has been removed such that the resulting molecule no longer participates in the hydrogen bonding characteristics of the nucleoside or nucleotides or residues of the nucleotide moiety. Abasic residues can be generated by removal of nitrogenous bases from nucleosides or nucleotides. However, the term "abasic" is used to refer to a structural feature of a residue, independent of the manner in which the residue is generated. The terms "abasic residue" and "abasic site" as used herein refer to a residue in a nucleic acid that lacks a purine or pyrimidine base.

As used herein, an "apurinic/apyrimidinic (AP) endonuclease" refers to an enzyme that cleaves bonds on the 5' side, the 3' side, or both the 5' and 3' sides of abasic residues in a polynucleotide. In certain embodiments of the invention, the AP endonuclease is an AP lyase. Examples of AP endonucleases include, but are not limited to: Escherichia coli (E. coli) endonuclease VIII and its homologues, E. coli endonuclease III and its homologues. It should be understood that when referring to specific enzymes, such as endonucleases such as Escherichia coli Endo VIII, Endo V, etc., it is also intended to include those that are considered homologues in the art and have the ability to remove damaged bases and/or cleave abasic residues. Homologues from other species that have similar biochemical activity with respect to the DNA of the base or other priming residues.

The term "array" as used herein refers to a collection of entities distributed on or in a support substrate; the individual entities are preferably separated by sufficient distances to allow for the identification of discrete features of the array by various techniques. An entity can be, for example, a nucleic acid molecule, a clonal population of nucleic acid molecules, a microparticle (optionally linked to a clonal population of nucleic acid molecules), and the like. When used as a verb, the term "array" and its conjugations refer to any method of forming an array, such as distributing entities on or into a support substrate.

A "damaged base" is a purine or pyrimidine base other than A, G, C, or T that makes it a substrate for removal from DNA by DNA glycosylases. Uracil is considered an impaired base that can be used in the present invention. In some embodiments of the invention, the damaged base is hypoxanthine.

"Degenerate" when referring to a position in a polynucleotide of a population of polynucleotides means that the species of bases that form the moiety of the nucleoside occupying the position differ between different members of the population. Thus, the population contains individual members that differ in sequence at degenerate positions. The term "position" refers to the numerical value assigned to each nucleoside in a polynucleotide, usually relative to the 5' or 3' end. For example, the nucleoside at the 3' end of the extension probe can be designated as position 1. Therefore, N is at position 4 in the library of extension probes of the 3'-XXXNXXXX-5' structure. If the species of N can vary in different members of the library, position 4 is considered a degenerate position. The library of extended probes is also said to be degenerate at position N. A position is said to be k-fold degenerate if it can be occupied by k different kinds of nucleosides. For example, a position that can be occupied by nucleosides containing two different bases is 2-fold degenerate.

"Determining sequence information" includes "sequence determination" and also includes other levels of information, such as one or more possibilities to eliminate the sequence. It should be noted that sequencing a polynucleotide generally yields equivalent information for a fully complementary (100% complementary) polynucleotide and is therefore equivalent to sequencing a fully complementary polynucleotide directly.

When referring to multiple elements, such as nucleosides in an oligonucleotide probe molecule or part thereof, "independently" means that the type of each element is not limited or limited by the type of any other element, such as the choice of the type of each element and The kind of any other element is irrelevant. Thus, knowing the type of one or more elements does not provide any information about the type of any other element. For example, the nucleosides in the sequence NNNN are independent if the species of each N can be A, G, C or T, independent of the species of the other Ns.

"Ligation" refers to the formation of a covalent bond or linkage between the termini of two or more nucleic acids, such as oligonucleotides and/or polynucleotides, in a template-driven reaction. The nature of the bond or linkage can vary widely, and the linkage can be enzymatic or chemical.

The term "particulate" as used herein refers to particles having a smallest cross-sectional dimension of 50 microns or less, preferably 10 microns or less. In certain embodiments, the smallest cross-sectional dimension is about 3 microns or less, about 1 micron or less, about 0.5 microns or less, such as about 0.1, 0.2, 0.3 or 0.4 microns. Microparticles can be made from a variety of inorganic or organic materials including, but not limited to: glass (e.g. pore control glass), silica, zirconia, cross-linked polystyrene, polyacrylic acid, polymethylmethacrylic acid, titanium dioxide, latex , polystyrene, etc. See, eg, US Patent 6,406,848 for various suitable materials and other considerations. Dyna beads available from Dynal, Oslo, Norway are examples of commercially available microparticles that can be used in the present invention. Magnetically responsive microparticles may be used. Magnetic reactivity of certain preferred microparticles facilitates collection and concentration of microparticle-attached templates after amplification and facilitates other steps (eg, washing, reagent removal, etc.). In certain embodiments of the invention, populations of particles having different shapes (eg, some spherical and others non-spherical) are employed.

The term "microsphere" or "bead" as used herein refers to substantially spherical microparticles having a diameter of 50 microns or less, preferably 10 microns or less. In certain embodiments, the diameter is about 3 microns or less, about 1 micron or less, about 0.5 microns or less, such as about 0.1, 0.2, 0.3 or 0.4 microns. In certain embodiments of the present invention, a monodisperse population of microspheres is employed, ie, the microspheres are substantially uniform in size. For example, the coefficient of variation of particle diameter may be less than 5%, such as 2% or less, 1% or less, and the like. However, in other embodiments, the population of microparticles has a coefficient of variation of 5% or greater, such as 5%, 5%-10% (inclusive), 10%-25% (inclusive), etc. In certain embodiments, mixed populations of microparticles are employed. For example, a mixture of two populations with each having a coefficient of variation of less than 5% can be used, resulting in a mixed population that is not monodisperse. For example, mixtures of microspheres with diameters of 1 micron and 3 microns can be used. In certain embodiments of the invention, additional information is provided by microsphere size when sequencing is performed using templates attached to a non-monodisperse population of microspheres. For example, different template libraries can be attached to microspheres of different sizes. Also, since fewer template molecules can be attached to small particles, the signal intensity can vary, which can facilitate multiplexed sequencing.

The term "nucleic acid sequence" as used herein may refer to nucleic acid material itself, and is not limited to sequence information (i.e., selected from the five base letters A, G, C, T, or U) that characterizes the biochemical characteristics of a particular nucleic acid, such as a DNA or RNA molecule. consecutive combinations of letters). Nucleic acids described herein are presented in a 5'→3' orientation unless otherwise stated.

"Nucleoside" includes a nitrogenous base attached to a sugar molecule. The term as used herein includes the 2'-deoxy and 2'-hydroxyl forms of natural nucleosides and nucleoside analogs as described by Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). things. For example, natural nucleosides include adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine. Nucleoside "analogues" refer to synthetic nucleosides containing modified base moieties and/or modified sugar moieties, generally as described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs include synthetic nucleosides designed to have improved binding properties, reduced degeneracy, increased specificity, and the like. Nucleoside analogs include 2-aminoadenosine, 2-thiothymidine, pyrrolo-pyrimidine, 3-methyladenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine Glycoside, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6 )-methylguanine, 2-thiocytidine, etc. Nucleoside analogs can include any of the universal bases described herein.

The term "organism" as used herein refers to any animate or inanimate entity comprising a nucleic acid capable of replication and whose sequence is of interest. It includes plasmids; viruses; prokaryotes, archaea and eukaryotes, cell lines, fungi, protozoa, plants, animals, etc.

"Perfectly matched duplex" when referring to the overhanging strands of the probe and template polynucleotide means that the overhanging strand of one strand forms a duplex structure with the other strand such that each nucleoside in the duplex structure is aligned with the Watson-Crick base pairing occurs with one nucleotide on the opposite strand. The term also includes the pairing of nucleoside analogs useful for reducing probe degeneracy, such as deoxyinosine, nucleosides with 2-aminopurine bases, etc., whether or not such pairing involves hydrogen bond formation.

The term "plurality" refers to more than one species.

The term "polymorphism" has its ordinary meaning in the art and refers to differences in genome sequence among individuals of the same species. "Single nucleotide polymorphism" (SNP) refers to a polymorphism at a single position.

"Polynucleotide", "nucleic acid" or "oligonucleotide" refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) linked by internucleoside linkages. Generally, a polynucleotide includes at least three nucleosides. In certain embodiments of the invention, one or more nucleosides in the extension probe comprise a universal base. Typically, oligonucleotides range in size from a few, eg, 3-4 monomeric units, to several hundred monomeric units. When a letter sequence such as "ATGCCTG" is used to represent a polynucleotide such as an oligonucleotide, it should be understood that the nucleotide sequence is 5'→3' from left to right, "A" refers to deoxyadenosine, and "C" refers to deoxycytidine. Glycoside, "G" refers to deoxyguanosine, and "T" refers to thymidine, unless otherwise stated. In the art, the letters A, C, G and T are generally used to refer to the base itself, the nucleosides or nucleotides comprising the base.

In naturally occurring polynucleotides, the internucleoside linkages are generally phosphodiester bonds and the subunits are termed "nucleotides". However, oligonucleotide probes containing other internucleoside linkages, such as phosphorothioate linkages, are employed in certain embodiments of the invention. It is understood that one or more subunits comprising an oligonucleotide probe having a non-phosphodiester linkage may not include a phosphate group. Such nucleotide analogs are considered to fall within the scope of the term "nucleotide" as used herein, and nucleic acids containing one or more internucleoside linkages other than phosphodiester linkages are still referred to as "polynucleotides", "oligonucleotides", Glycolic Acid" and so on. In other embodiments, polynucleotides such as oligonucleotide probes include linkages comprising AP endonuclease sensitive sites. For example, the oligonucleotide probe may contain an abasic residue, a residue containing a damaged base that is a substrate for DNA glycosylase removal, or another residue that is a substrate for AP endonuclease cleavage or connect. In another embodiment, the oligonucleotide probes contain disaccharide nucleosides.

The term "primer" refers to a short polynucleotide, typically about 10-100 nucleotides in length, that binds to a target polynucleotide or "template" by hybridization to the target. Primers preferably provide a starting point for template-directed synthesis of polynucleotides complementary to the target, which can be synthesized in the presence of appropriate enzymes, cofactors, substrates such as nucleotides, oligonucleotides, and the like. Primers generally provide termini from which extension can occur. In the case of primers for polymerase, such as DNA polymerase catalyzed synthesis (eg, "sequencing by synthesis", polymerase chain reaction (PCR) amplification, etc.), the primers typically contain, or can be modified to contain, a free 3'OH group group. A PCR reaction generally employs a pair of primers (first and second amplification primers), including an "upstream" (or "forward") primer and a "downstream" (or "reverse") primer, which delineate the amplification the boundaries of the region. For primers synthesized for successive cycles of extension, ligation (and optional cleavage), the primers generally contain, or can be modified to contain, a free 5' phosphate group or 3' OH that serve as substrates for DNA ligase group.

A "probe family" as used herein refers to a population of probes each containing the same label.

As used herein, the terms "sequence determination", "determining a nucleotide sequence", "sequencing" and the like, when referring to a polynucleotide, include determining the sequence information of some or all of the polynucleotide. That is, the term includes information on the level of sequence comparison, fingerprinting, etc., of the target polynucleotide, as well as the rapid identification and ranking of individual nucleotides of the target polynucleotide within the region of interest. In certain embodiments of the invention, "sequence determination" includes identification of a single nucleotide, while in other embodiments, identification of more than one nucleotide. In certain embodiments of the invention, sequence information is collected in a single cycle that is insufficient by itself to identify any nucleotides. Identification of nucleosides, nucleotides and/or bases is considered equivalent herein. It should be noted that sequencing polynucleotides generally yields sequence information for equivalent fully complementary (100% complementary) polynucleotides and is therefore equivalent to sequencing directly performed on fully complementary polynucleotides.

A "sequencing reaction" as used herein refers to a set of extension, ligation and detection cycles. When the extended duplex on the template is removed and the template is subjected to a second set of cycles, each set of cycles is considered a separate sequencing reaction, but the resulting sequence information can be combined to generate a single sequence.

As used herein, "semi-solid" refers to a compressible matrix comprising solid and liquid components, wherein the liquid occupies the pores, spaces or other interstices between the solid matrix components. Exemplary semi-solid matrices include matrices made of polyacrylamide, cellulose, polyamide (nylon), and cross-linked agarose, dextran, and polyethylene glycol. The semi-solid support may be provided on a second support, such as a substantially planar rigid support, also referred to as a substrate, which is capable of supporting said semi-solid support.

"Support" as used herein refers to a substrate on or in which nucleic acid molecules, microparticles, etc. in or on substances such that their free diffusion or relative movement is substantially or completely prevented.

A "priming residue" is one that, when present in a nucleic acid, renders the nucleic acid more susceptible to cleavage by a nicking agent (such as an enzyme, silver nitrate, etc.) or combination of nicking agents relative to an otherwise identical nucleic acid that does not contain a priming residue ( such as cleaving the nucleic acid backbone), and/or are susceptible to modification resulting in residues that render the nucleic acid more susceptible to such cleavage. Thus, the presence of a priming residue in a nucleic acid can result in the presence of an easy linkage in the nucleic acid. For example, abasic residues are priming residues because their presence in a nucleic acid renders the nucleic acid susceptible to cleavage by enzymes such as AP endonuclease. Nucleosides containing damaged bases are initiating residues because the presence of nucleosides containing damaged bases in a nucleic acid also makes the nucleic acid more susceptible to cleavage by enzymes such as AP endonuclease, such as after removal of the damaged base by DNA glycosylase . The cleavage site may be a bond between the priming residue and an adjacent residue, or may be a bond shifted by one or more residues from the priming residue. For example, deoxyinosine is the priming residue because the presence of deoxyinosine in a nucleic acid renders the nucleic acid more susceptible to cleavage by E. coli endonuclease V and its homologues. This enzyme cleaves the second phosphodiester bond at the 3' end of deoxyinosine. Any of the probes disclosed herein may contain one or more priming residues. A priming residue can, but need not, contain a ribose or deoxyribose moiety. The nicking agent is preferably one that does not substantially cleave nucleic acids in the absence of a priming residue, but has significant cleavage activity for nucleic acids containing a priming residue under the same conditions, which may include the presence of a nucleic acid modifier such that it is sensitive to cleavage. agent is more sensitive. For example, preferably, if a nicking agent is present in a composition comprising nucleic acids of the same length in which one nucleic acid contains a priming residue and the other nucleic acid does not contain said priming residue, it cleaves the cleavage of the nucleic acid containing the priming residue. 25; 50; 100; 250; 500; 1000; 2500; 5000; 10,000; 25,000; 50,000; 100,000; 250,000; The ratio of the probability of a nucleic acid with a priming residue to the probability of cleaving an otherwise identical nucleic acid without a priming residue is ^10-106 , or any integer subrange therein. It is understood that this ratio may vary for the particular nucleic acid and the position and nucleotide environment of the priming residue.

Preferably, if a nucleic acid containing a priming residue requires modification to render the nucleic acid susceptible to cleavage by a nicking agent, such modification can be readily performed in the presence of a suitable modifying agent, e.g., in reasonable yield and in a reasonable time. For example, in certain embodiments of the invention at least 50%, at least 60%, at least 70%, preferably at least 80% of the %, at least 90%, or more preferably at least 95% of the nucleic acids containing priming residues.

Various suitable priming residues and corresponding cleavage reagents are listed herein. Any priming residues and cleavage agents similar in activity to those described herein can be used. One of ordinary skill in the art can determine whether a particular combination of priming residues and cleavage reagents is suitable for use in the present invention, such as cleavage efficiency and speed, selectivity of cleavage agents for nucleic acids containing priming residues, etc., are suitable for the methods of the present invention. It is important to note that a "priming residue" is distinguished from nucleotides that only form part of a restriction site in that the ability of a priming residue to increase susceptibility to cleavage is generally not significantly dependent on the discovery of the priming residue. specific sequence content, but as noted above, sequence content may have some effect on susceptibility to modification and/or cleavage. Of course, depending on the surrounding nucleotides, the priming residue may form part of a restriction site. Thus, in most cases, the cleavage agent is not a restriction enzyme, but the use of enzymes that are both restriction enzymes and have non-sequence-specific cleavage capabilities is not excluded.

As used herein, a "universal base" is a base that can "pair" with more than one base found in a naturally occurring nucleic acid, such that it can replace a naturally occurring base in a duplex. The base need not be able to pair with every naturally occurring base. For example, certain bases pair selectively only with purines, or only with pyrimidines. Certain preferred universal bases (full universal bases) can pair with any base typically found in naturally occurring nucleic acids and thus can substitute for any of these bases in a duplex. The base does not have to be equally capable of pairing with various naturally occurring bases. If the probe mix contains probes (one or more positions) that contain a universal base that does not pair with all naturally occurring nucleotides, it may be necessary to utilize two or more universal bases at this position of a particular probe in order to At least one universal base pairs with A, at least one universal base pairs with G, at least one universal base pairs with C, and at least one universal base pairs with T.

Various universal bases are known in the art, including but not limited to: hypoxanthine, 3-nitropyrrole, 4-nitroindole, 5-nitroindole, 4-nitrobenzimidazole, 5-nitro Indazole, 8-aza-7-deazaadenine, 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P.Kong Thoo Lin. and D.M.Brown, Nucleic Acids Res., 1989,17,10373-10383), 2-amino-6-methoxyaminopurine (D.M.Brown and P.Kong Thoo Lin, Carbohydrate Research, 1991,216,129- 139) etc. Hypoxanthine is a preferred fully universal base. Nucleosides containing hypoxanthine include, but are not limited to, inosine, isoinosine, 2'-deoxyinosine and 7-deaza-2'-deoxyinosine, 2-aza-2'deoxyinosine.

Other universal bases are known in the art, as described in relevant parts of the following documents: Loakes, D. and Brown, D.M., Nucl. Acids Res. 22:4039-4043, 1994; Ohtsuka, E. et al., J. Biol. Chem.260(5):2605-2608, 1985; Lin, P.K.T. and Brown, D.M., Nucleic Acids Res.20(19):5149-5152, 1992; Nichols, R. et al., Nature 369(6480):492- 493, 1994; Rahmon, M.S. and Humayun, N.Z., Mutation Research 377(2):263-8, 1997; Berger, M. et al., Nucleic Acids Research, 28(15):2911-2914, 2000; Amosova, O. et al., Nucleic Acids Res. 25(10): 1930-1934, 1997; and Loakes, D., Nucleic Acids Res. 29(12): 2437-47, 2001. Universal bases can, but do not necessarily, form hydrogen bonds with bases in opposite positions. Universal bases can form hydrogen bonds through Watson-Crick or non-Watson-Crick interactions such as Hoogsteen interactions.

Rather than employing oligonucleotide probes comprising universal bases, oligonucleotide probes comprising abasic residues are used in certain embodiments of the invention. Abasic residues can occupy the relative positions of the four naturally occurring nucleotides and thus can function in the same way as nucleotides containing universal bases. In some embodiments of the invention, linkages adjacent to abasic residues are cleaved by the AP endonuclease, but in practice of the invention where other easy-cleavable linkages such as phosphorothioates are present and other cleavage reagents are employed Abasic residues (ie, functioning as universal bases) can also be used in the format.

DETAILED DESCRIPTION OF SOME PREFERRED EMBODIMENTS OF THE INVENTION

A. Sequencing by sequential extension, ligation, and cutting cycles

Figure 1A diagrammatically shows the general scheme of one aspect of the present invention, generally similar to the methods described in US Patents 5,740,341 and 6,306,597 issued to Macevicz. For convenience, these patents are collectively referred to herein as "Macevicz." In particular, Macevicz describes a method for identifying nucleotide sequences in a polynucleotide comprising the steps of: (a) extending an initial oligonucleotide along the polynucleotide by ligating an oligonucleotide probe to form an extension duplex; (b) identifying one or more nucleotides of the polynucleotide; and (c) repeating steps (a) and (b) until the nucleotide sequence is determined.

Macevicz also describes a method for determining the nucleotide sequence of a template polynucleotide comprising the steps of: (a) providing a probe-template formed by hybridization of an initial oligonucleotide probe to the template polynucleotide a duplex having an extendable probe end; (b) ligating an extended oligonucleotide probe to the extendable probe end to form an extended duplex containing the extended oligonucleotide probe (c) identifying at least one nucleotide in the extended duplex that is (1) in the template polynucleotide that is complementary to the just-ligated extension probe or (2) that is immediately adjacent to the extended oligonucleotide probe target the nucleotide residue in the template polynucleotide downstream; (d) if the extendable end does not already exist, generate an extendable probe end on the extended probe such that the end generated differs from the end that was joined to the last extension the end of the probe; and (e) repeating steps (b), (c) and (d) until the nucleotide sequence of said target polynucleotide is determined. In certain embodiments of these methods, each extension probe contains a chain-terminating moiety distal to the initial oligonucleotide probe. In certain embodiments, the regeneration step comprises chemically cleaving a scissile internucleoside linkage in the extended oligonucleotide probe.

In FIG. 1A , a polynucleotide template 20 containing a polynucleotide region 50 of unknown sequence and a binding region 40 is attached to a support 10 . Nucleotide 41 distal to binding region 40 is adjacent to nucleotide 51 proximal to polynucleotide region 50 . An initial oligonucleotide 30 is provided which hybridizes to the binding region 40 at the location of the binding region 40 to form a duplex. The starting oligonucleotide 30 is also referred to herein as a "primer", and the binding region 40 may be referred to as a "primer binding region". The duplex can, but need not be, a perfectly matching duplex. The starting oligonucleotide has an extendable end 31 . In FIG. 1A , the starting oligonucleotide is bound to the binding region such that the extendable end 31 is located opposite nucleotide 41 . However, the starting oligonucleotide may bind elsewhere in the binding region, as described below. An extension oligonucleotide probe 60 of length N hybridizes to the template adjacent to the starting oligonucleotide. The terminal nucleotide 61 of the extension oligonucleotide probe is linked to the extendable end 31 .

Terminal nucleotide 61 is complementary to the first unknown nucleotide in polynucleotide region 50 . Thus, the identity of the terminal nucleotide 61 determines the identity of the nucleotide 51 . Preferably, nucleotide 51 is identified by detecting a label (not shown) attached to an extension probe whose terminal nucleotide 61 is known to be A, G, C or T. The marker is removed after detection. Figure 2 shows a scheme for assigning different labels, such as different colored fluorophores, to extension probes with different 3' terminal nucleotides.

After ligation and detection, an extendable probe end is generated on the extension probe 60 if the probe 60 does not have such an end. A second extension probe 70 , also preferably of length N, anneals to the template adjacent to extension probe 60 and ligates to the extendable end of probe 60 . The identity of the terminal nucleotide 71 of the extension probe 70 specifies the identity of the nucleotide 52 at the relative position in the polynucleotide 50 . Thus, terminal nucleotide 71 constitutes the "sequencing portion" of the extension probe, meaning that the hybridization specificity of the portion of the probe is used as a basis for determining one or more nucleotide species in the template. It will be appreciated that other nucleotides in the extension probe will generally hybridize to the template, but only those nucleotides in the probe whose class is associated with a particular label are used to identify nucleotides in the template.

In a preferred embodiment of the invention, generating an extendable terminus comprises cleavage of an internucleoside linkage as described below. Preferably, cleavage also removes the label. Cleavage removes multiple nucleotides M in the extension probe (not shown). Thus, the duplex is extended by N-M nucleotides in each cycle, and the nucleotides located between N-M in the template are identified. It will be appreciated that typically multiple copies of a given template are attached to a support and sequencing reactions are performed simultaneously on these templates.

Macevicz explained that oligonucleotide probes should generally be able to ligate to either the starting oligonucleotide or the extending duplex to generate the extending duplex for the next extension cycle; this ligation should be template-driven because the probe should be Form a duplex with the template before ligation; the probe should have a capping moiety to prevent ligation of multiple probes on the same template in one extension cycle; the probe should be able to be regenerated after ligation by manipulation or modification Extendable ends; the probe should have a signal portion (i.e. a detectable portion) in order to obtain sequence information about the template after successful ligation.

Macevicz describes certain suitable starting oligonucleotides, extended oligonucleotide probes, templates, binding sites and various methods for synthesizing, designing, producing or obtaining these components. Macevicz also describes certain suitable ligases, ligation conditions and various suitable labels. Macevicz also described an alternative method for the incorporation of labeled chain-terminating nucleotides into newly ligated extension probes by polymerase extension for identification. The type of nucleotide added determines the nucleotide at the relative position of the template.

As understood by those of ordinary skill in the art, references to templates, starting oligonucleotides, extension probes, primers, etc., generally refer to a population or library of substantially identical nucleic acid molecules within the relevant region, rather than to a single molecule. Thus, for example, a "template" generally refers to a plurality of substantially identical template molecules; a "probe" generally refers to a plurality of substantially identical probe molecules, and the like. In probes that are degenerate at one or more positions, it is understood that the sequences of the probe molecules comprising a particular probe differ at the degenerate positions, i.e. the sequence of the probe molecules that make up a particular probe may only be present at the non-degenerate positions. Basically the same position. For purposes of illustration, it is understood that the singular form includes both individual molecules and substantially identical groups of molecules. The terms "template molecule", "probe molecule", "primer molecule", etc. are used when it is desired to refer to a single nucleic acid molecule (ie, a molecule). In some cases, the plural nature of populations of substantially identical nucleic acid molecules is specified.

A population of substantially identical nucleic acid molecules can be obtained or produced by a variety of known methods, including chemical synthesis, biosynthesis in cells, enzymatic amplification in vitro from one or more starting nucleic acid molecules, and the like. For example, a nucleic acid of interest can be cloned by inserting a suitable expression vector, such as a DNA or RNA plasmid, followed by introduction into a cell, such as a bacterial cell, capable of replicating therein, using methods well known in the art. Plasmid DNA or RNA containing copies of the nucleic acid of interest is then isolated from the cells. Genomic DNA isolated from viruses, cells, etc., or cDNA produced by reverse transcription of mRNA can also become the source of substantially the same nucleic acid molecule population (such as the template polynucleotide whose sequence is to be tested) without intermediate steps such as cloning or in vitro amplification, However, it is generally preferred to subject it to an intermediate step treatment.

It is to be understood that members of a population are not necessarily 100% identical, as a certain number of "errors" may have occurred during synthesis. Preferably, at least 50% of the population members are at least 90%, or more preferably at least 95%, identical to a reference nucleic acid molecule (ie, the sequence-determined molecule used as the basis for sequence comparison). More preferably, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more of the population members are at least 90%, or more preferably at least 95%, or more Preferably at least 99% identical. Preferably, members of a population having a percent identity of at least 95%, or more preferably at least 99%, to a reference nucleic acid molecule are at least 98%, 99%, 99.9% or more. The percent identity can be calculated by comparing two optimally aligned sequences, determining the number of positions in which a nucleic acid base (such as A, T, C, G, U, or I) is identical in the two sequences to yield the number of matching positions, The percent sequence identity was obtained by dividing the number of matching positions by the total number of positions and multiplying by 100. It is understood that in some cases a nucleic acid molecule such as a template, probe, primer, etc. may be part of a larger nucleic acid molecule that also contains parts that are not part of the template, probe or primer. In this case, these parts of the individual members of the population need not be substantially the same.

Macevicz describes a method of attaching a template to a support, such as a bead, and extending to the end of the template distal to the support, as shown in Figure 1A. Thus, the binding region is closer to the support than the unknown sequence and the extended duplex grows away from the support. However, the inventors have surprisingly found that it is advantageous to carry out the method by an alternative method in which the binding region is located at the end of the template distal to the support, extending inwardly towards the support. Figure 1B depicts such an embodiment, where the various elements are numbered as in Figure 1A. The inventors determined that sequencing "in" from the template distal to the support provided better results. In particular, sequencing from the distal end of the template to a support such as a bead yields higher ligation efficiencies than sequencing out from the support.

As further described by Macevicz, the oligonucleotide probes are preferably added to the template as a mixture of oligonucleotides containing all possible sequences of a predetermined length. For example, a probe mixture containing all possible sequences with the structure NNNNNN (also denoted as (N) _k , where k = 6) with a length of 6 nucleotides (hexamers) contains 4 ⁶ (4096) probes. Needle type. Typically, the structure of the probe is X(N) _k N ^* , where N represents any nucleotide, k is 1-100, * represents a label, and X represents the nucleotide whose species corresponds to the label. In certain embodiments, k is 1-100, 1-50, 1-30, 1-20, such as 4-10. One or more nucleotides may contain a universal base. At the position represented by N, the probe is typically 4-fold degenerate, or contains nucleotides of reduced degeneracy at one or more positions represented by N. If desired, the mixture can be divided into subgroups of probes ("stringency classes") that have similar stability or free energy of binding to a perfectly matched duplex of complementary sequence. These subsets can be used in different hybridization reactions as described by Macevicz.

The complexity (ie the number of different sequences) of a probe mixture can be reduced in a number of ways including the use of so-called reduced degeneracy nucleotides or nucleotide analogs. For example, a library of probes containing all possible sequences of 8 nucleotides contains ⁴⁸ probes. By employing universal bases at two positions the number of probes can be reduced to 4 ⁶ while maintaining various desirable properties of the octamer library, such as length. The invention includes the use of any of the universal bases described above or in the references cited above.

According to this embodiment, the duplex or starting oligonucleotide can be extended with oligonucleotide probes in the 5'→3' direction or in the 3'→5' direction, as described below. In general, oligonucleotide probes do not necessarily form perfectly matched duplexes with the template, but such binding may be preferred. In embodiments where each extension cycle identifies one nucleotide in the template, full base pairing is required to identify that particular nucleotide. For example, in embodiments where oligonucleotide probes are ligated enzymatically to an extending duplex, complete base pairing between the terminal nucleotides of the ligated probe and its template complement is required, i.e. the appropriate Watson -Crick base pairing. Typically, in such embodiments, the remaining nucleotides of the probe serve as "spacers" to ensure that the next ligation occurs at a predetermined site or a certain number of bases shifted along the template. That is, their pairing or unpairing does not provide further sequence information. Also, in embodiments that rely on polymerase extension for base calling, the probe serves primarily as a spacer, so specific hybridization to the template is not important.

The methods described above enable partial sequence determination, ie, the identification of individual nucleotides in a template that are spaced apart from each other. In a preferred embodiment of the invention, in order to gather more complete information, multiple reactions are performed, wherein each reaction utilizes a different starting oligonucleotide i. The starting oligonucleotides i bind to different parts of the binding region. Preferably, the binding position of the starting oligonucleotides should be such that the extendable ends of different starting oligonucleotides are hybridized to the binding region and shifted by 1 nucleotide from each other. For example, as shown in Figure 3, sequencing reactions 1...N are performed. The initial oligonucleotides i ₁ ... i _n have the same length, and after binding to the binding region 40, their terminal nucleotides 31, 32, 33, etc. hybridize to consecutive adjacent positions 41, 42, 43, etc. in the binding region 40 . Thus, extension probes _e1 ... _en bind to consecutive adjacent regions of the template and are ligated to the extendable ends of the starting oligonucleotides. The terminal nucleotide 61 of the _probe en attached to _in is complementary to nucleotide 55 of the polynucleotide region 50, ie the first unknown polynucleotide in the template. In the second cycle of extension, ligation and detection, terminal nucleotide 71 of probe _en is complementary to nucleotide 56 of polynucleotide region 50, the second nucleotide of the unknown sequence. Likewise, the terminal nucleotides of the extension probes attached to the duplex start from the starting oligonucleotides i ₂ , i ₃ , i _{4 ,} etc., with the third, fourth and fifth of the unknown sequence 50 Nucleotides are complementary. It will be appreciated that the starting oligonucleotide may bind to a region that is progressively remote from the polynucleotide region 50, rather than progressively closer to it.

The spacing function of the non-terminal nucleotides of the extension probe allows for template positions that are separated by a certain number of nucleotides from the position where the initial oligonucleotide binds without requiring a correspondingly large number of cycles for any given template. sequence information. For example, by successive cycles of ligating probes of length N followed by cleaving to remove a single terminal nucleotide on the extension probe, nucleotides spaced by N-1 nucleotides apart can be identified in successive cycles. For example, 6 cycles can be used to identify the nucleotides at positions 1, N, 2N-1, 3N-2, 4N-3, and 5N-4 in the template, where the nucleotide at position 1 of the template corresponds to the The nucleotides at the end of the probe can be extended in the duplex formed by the binding of the initial oligonucleotide to the template. Similarly, if cleavage removes two nucleotides of an extension probe of length N, nucleotides at positions N-2 nucleotides apart from each other can be identified in consecutive runs. For example, 6 cycles can be used to identify the nucleotides at positions 1, N-1, 2N-3, 3N-5, 4N-7 in the template. Thus, if the probe is 8 nucleotides in length and 2 nucleotides are removed each cycle, the nucleotides at positions 1, 7, 13, 19 and 25 are identified. Thus, the number of cycles required to identify a nucleotide at a distance X from the first nucleotide in the template is approximately X/M, where M is the length of the extended probe remaining after cleavage, rather than approximately X.

For example, the scheme shown in Figure 3B shows the end result of using the extension, ligation, and cleavage cycle approach with extension probes designed to read the template every 6 bases. The templates were sequentially stripped and sequenced with six starting nucleotides bound to the offset positions of the binding region, and the results were combined to elucidate all template bases over a defined length. For example, if 10 consecutive ligations are performed in each of the 6 reactions, the resulting read length is 60 consecutive base pairs, whereas if 15 consecutive ligations are performed in each reaction, the resulting read length is 90 consecutive base pairs.

While not wishing to be bound by any theory, the inventors propose that, in contrast to this approach, most serial sequencing by synthesis is accompanied by the drawback of error accumulation, which ultimately limits the possibility of long read lengths. An advantageous feature of certain methods described herein is that they identify every n bases (depending on the position of the cleavable moiety in the probe), so that after a given number of cycles (y), the n*y-th (n-1) bases (like the 71st base after 15 cycles in the above example, or the 115th base after 20 cycles with a 6-base probe on the 3' side of the cleavage site base). The ability to "restart" the starting oligonucleotide at position n-1, n-2, etc., greatly reduces the accumulation of consecutive errors (by phase shifting or depletion) over a given length as stripping of the extended strand from the template and The process of hybridizing the fresh starting oligonucleotides effectively resets the background signal to zero. For example, comparing the polymerase synthesis-based sequencing method with the ligation-based method described here, if the signal-to-noise ratio for each extension cycle is 99:1, after 100 cycles of the polymerase-based method, the signal-to-noise ratio is 37:63 , 85:15 for the ligase-based approach. The end result of the ligase-based approach is a large increase in read length over the polymerase-based approach.

The ability to identify nucleotides with fewer cycles than would be required if each preceding nucleotide in the template required one cycle is important for a number of reasons. In particular, the efficiency of each step of the method cannot be 100%. For example, some templates may not be smoothly ligated to extension probes; some extension probes may not be cleaved, etc. Therefore, in each cycle, the reactions occurring on different copies of the template gradually become out of phase, and the number of templates for which useful and accurate information can be obtained decreases. Therefore, there is a particular need to minimize the number of cycles required to read nucleotides located farther from the extendable end of the starting oligonucleotide. However, increasing the extension probe length may lead to increased complexity of the probe mixture, which reduces the effective concentration of each probe sequence. As described herein, nucleotides of reduced degeneracy can be used to reduce complexity, but this may result in reduced hybridization strength and/or reduced ligation efficiency. The inventors have recognized that these competing factors need to be balanced in order to optimize results. Therefore, in a preferred embodiment of the invention, extension probes of 8 nucleotides in length are used, with reduced degeneracy nucleotides at selected positions. In addition, the inventors recognized the importance of selecting appropriate easy-cleavable linkages, as well as cleavage conditions and times, to optimize cleavage step efficiency (ie, the percentage of smoothly cleaved linkages in each cleavage step) and specificity for suitable linkages.

B. Oligonucleotide Extension Probe Design

Although Macevicz mentions that nucleoside analogs with reduced degeneracy can be used in oligonucleotide extension probes, he does not state that specific positions of such residues are specifically required to be included in the extension probes, nor does he state that incorporation of degeneracy Various specific probe structures (ie sequences) of nucleosides with reduced degeneracy. The inventors have recognized that it may be particularly advantageous to employ a particular number of reduced degeneracy nucleosides (eg, nucleosides containing universal bases) at particular positions in an oligonucleotide extension probe. For example, in certain embodiments of the invention, most or all of the nucleotides at position 6 or further away (starting with X) contain a universal base. For example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the nucleotides at position 6 or further away may contain a universal base. These nucleotides do not necessarily all contain the same universal base. In certain embodiments of the invention hypoxanthine and/or nitroindole are used as universal bases. For example, nucleosides such as inosine may be employed.

The inventors have recognized that excellent results can be obtained with extension probes greater than 6 nucleotides in length, where counting from the nucleotide attached to the end of the extendable probe, positions 6 or more from the proximal end of the probe One or more nucleotides at is a nucleotide with reduced degeneracy, such as containing a universal base (i.e. if the most proximal nucleotide is considered to be position 1, then one or more at position 6 or further nucleotides contain universal bases), such as 1, 2 or 3 nucleotides at position 6 or further in the 8-mer probe contain universal bases. For example, in 3'→5' sequencing, a probe with the structure 3'-XNNNNsINI-5' can be used, where X and N represent any nucleotide, and "s" represents an easy-cleavage junction, so that when counting from the 3' end Cleavage occurs between the fifth and sixth residues, and preferably at least one residue between the easy link and the 5' end has a label corresponding to the X species. Another design is 3'-XNNNNsNII-5'. Yet another probe design is 3'-XNNNNsIII-5'. This design yielded a moderately complex probe mixture containing 1024 different probes, of sufficient length to prevent the formation of significant adenylation products (see Example 1), and with extension products obtained after cleavage from unmodified DNA. Advantages of composition. One disadvantage is that this probe only extends the primer 5 bases at a time. Since read length is a function of extension length multiplied by the number of cycles, each base increase in extension length increases read length by 1x cycle number of bases (eg, 20 bases if 20 cycles are used). Another probe design cleavage leaves one or more inosines (or other universal bases) at the end of the extension probe to generate extension duplexes of 6 bases or longer. For example, with the probe 3'-XNNNNIsII-5', the duplex is extended 6 bases at a time, leaving a 5' inosine at the junction. In these designs, it is preferred that at least one residue between the easy link and the 5' end has a label corresponding to the X species. In certain embodiments of the invention, the third nucleotide from the distal end of the probe, counting from the opposite end attached to the terminal nucleotide of the extendable probe, contains a universal base (i.e., if the distal end is considered to be position K, then the nucleotide at position K-2 contains the universal base).

In certain embodiments of the invention, locked nucleic acid (LNA) bases are employed at one or more positions of the initial oligonucleotide probe, the extension probe, or both. Locked nucleic acids are described, for example, in US Patent 6,268,490; Koshkin, AA et al., Tetrahedron, 54:3607-3630, 1998; Singh, SK et al., Chem. Comm., 4:455-456, 1998. LNAs can be synthesized using an automated DNA synthesizer and standard phosphoramidite chemistry and can be incorporated into oligonucleotides that also contain naturally occurring nucleotides and/or nucleotide analogs. They can also be synthesized using labels such as those described below.

C. Template and Support Preparation Methods

Macevicz describes a method of first synthesizing a template comprising a plurality of substantially identical template molecules, as amplified by conventional polymerase chain reaction (PCR) methods in a test tube or other vessel. Macevicz states that the amplified template molecules are preferably attached to supports such as magnetic microparticles (eg beads) after synthesis.

The inventors have recognized that it is desirable to synthesize the templates to be sequenced on or in the support itself, for example, using supports such as microparticles or various semi-solid supports that are linked to one of a pair of amplification primers prior to performing the PCR reaction, as a gel matrix. This method does not require a separate step of attaching the template molecule to the support after synthesis. Thus, multiple templates differing in sequence can be conveniently amplified in parallel. For example, a population of individual particles, each having multiple copies of a particular template molecule (or its complement) attached to it, is produced synthetically on a particle according to the following method, wherein the template molecule attached to each particle is identical to the template molecule attached to other particles. The sequence is different. Therefore, each support is linked with a cloned template group, for example, support A is linked with multiple copies of template X; support B is linked with multiple copies of template Y; support C is linked with multiple copies of template Z, and so on. A "cloned template population", "cloned nucleic acid population", etc. refers to a population of substantially identical template molecules, preferably produced by successive rounds of amplification starting from a single template molecule of interest (the starting template). A substantially identical template molecule may be substantially identical to the starting template or its complement.

Amplification is typically performed by PCR, but other amplification methods may also be used (see below). It is to be understood that members of a clonal population are not necessarily 100% identical, for example, a certain number of "errors" may occur during synthesis such as amplification. Preferably, at least 50% of the members of the clonal population are at least 90%, or more preferably at least 95% identical to the starting template molecule (or its complement). More preferably, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more of the population members are at least 90% identical to the starting template molecule (or its complement), or More preferably at least 95% identical, or more preferably at least 99% identical. Preferably, at least 95%, or more preferably at least 99%, of the population members have a percent identity of at least 98%, 99%, 99.9% or higher to the starting template molecule (or its complement).

Amplification primers can be attached to supports using various techniques. For example, one end (5' end) of the primer can be functionalized with one member of the binding pair (e.g., biotin) and the support can be functionalized with the other member of the binding pair (e.g., streptavidin). Any similar binding pair can be used. For example, a nucleic acid tag of defined sequence can be attached to a support, and a primer containing a complementary nucleic acid tag can hybridize to the nucleic acid tag attached to the support. Various linkers and crosslinkers can also be used.

Methods of performing PCR are well known in the art, see, e.g., U.S. Patents 4,683,195, 4,683,202, and 4,965,188, and Dieffenbach, C. and Dveksler, GS, PCR Primer: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2003. Methods for amplifying nucleic acids on microparticles are well known and described in the art, for example, standard PCR can be performed on beads with attached primers (such as the beads prepared in Example 12) in microtiter plate wells or test tubes. While PCR is a convenient method of amplification, many other methods known in the art can also be used. For example, multiple strand displacement amplification, helicase displacement amplification (HDA), nick translation, Qβ replicase amplification, rolling circle amplification, and other isothermal amplification methods, etc. can be employed.

Template molecules can be obtained from any source. For example, DNA may be isolated from a sample, which may have been obtained or derived from a subject. In a broad sense, the term "sample" refers to any source of templates from which sequencing is performed. By the term "derived from" is meant a sample directly obtained from a subject and/or further processing of nucleic acid in the sample to obtain a template molecule. The sample source can be any viral, eukaryotic, archaeal or eukaryotic species. In certain embodiments of the invention, the source is human. A sample can be, for example, blood or other bodily fluid containing cells; semen; a biopsy sample, and the like. Genomic or mitochondrial DNA from any organism of interest can be sequenced. cDNA can be sequenced. RNA can also be sequenced, for example, first by reverse transcription to generate cDNA using methods well known in the art, such as RT-PCR. DNA mixtures from different samples and/or subjects can be pooled. Samples can be processed in various ways. Nucleic acid can be isolated, purified and/or amplified from a sample using known methods. Of course, completely man-made synthetic nucleic acids, recombinant nucleic acids not derived from an organism can also be sequenced.

Templates can be provided in double-stranded or single-stranded form. Typically, when the template is initially provided in double-stranded form, the two strands are subsequently separated (e.g., denatured DNA) and only one of the two strands is amplified to generate a clonal population of localized template molecules that are (e.g., ligated) On microparticles, immobilized in or on semi-solid supports, etc.

Templates can be selected or processed in various other ways. For example, a template obtained from DNA treated with a methyl-sensitive restriction enzyme such as MspI can be used. This process of generating DNA fragments can be performed prior to amplification. Fragments containing methylated bases are not amplified. Sequence information obtained from a hypermethylated template can be compared to sequence information obtained from a template from the same source that has not been selected for hypermethylation.

Templates can be inserted into a library, or can be provided in a library, or can be derived from a library. For example, hypermethylated libraries are known in the art. Insertion of templates into libraries facilitates the ligation of additional nucleotide sequences, such as tags, primer binding sites, or starting oligonucleotides, to the ends of the templates. For example, certain protocols allow for the addition of tags with multiple binding sites, such as amplification primer binding sites, starting oligonucleotide binding sites, capture agent binding sites, and the like.

Various suitable libraries are known in the art. For example, USSN 10/978,224, PCT publications WO2005042781 and WO2005082098 and Shendure, J. et al., Science, 309(5741):1728-32, 2005, Scienceexpress, Aug. 4, 2005 (www.scienceexpress.org) describe particularly Libraries of interest and methods for their construction. It will of course be understood that other methods of generating such libraries may also be employed. Certain libraries of particular interest contain multiple nucleic acid fragments (typically DNA), each fragment containing two nucleic acid segments of interest separated by sequences complementary to the amplification and/or sequencing primers used in the sequencing step , that is, these sequences serve as primer binding regions (PBR). In embodiments of particular interest, a nucleic acid segment is a contiguous portion of naturally occurring DNA. For example, segments can be derived from the 5' and 3' ends of a contiguous portion of genomic DNA, as described in the above references. Consistent with the aforementioned literature, such nucleic acid segments are referred to herein as "tags" or "terminal tags." Two tags derived from a stretch of contiguous nucleic acid, such as its 5' and 3' ends, are referred to as "paired tags", "paired tags" or "bi-tags". It should be understood that "a pair of tags" includes two tags, that is, expressed in a singular form. The distance separating the two tags is limited by selecting contiguous portions of DNA within predetermined size limits that yield paired tags.

In addition to being separated by sequences complementary to the sequencing and/or amplification primers, the nucleic acid fragments of the library generally also contain sequences complementary to the sequencing and/or amplification primers flanking the tag, i.e. the first such sequence can be Located 5' to the tag that is closer to the 5' end of the fragment, a second such sequence may be located 3' to the tag that is closer to the 3' end of the fragment. It is understood that in various embodiments the positions of the two tags present in the contiguous nucleic acid from which the tags are generated may, but do not necessarily correspond to the positions of the tags in the DNA fragments of the library.

Nucleic acid fragments and tags can be of different size ranges. Nucleic acid fragments generally can be, for example, 80-300 nucleotides in length, such as 100-200, 100-150, about 150 nucleotides, about 200 nucleotides, etc. in length. Tags can be, for example, 15-25 nucleotides in length, such as about 17-18 nucleotides, etc. in length. It should be noted that these lengths are exemplary and not limiting. Shorter or longer fragments and/or tags can be used.

It should also be noted that while obtaining paired tags from a single contiguous nucleic acid provides a convenient method for library construction, the importance of paired tags is that they are separated by a distance from each other ("separation distance") in the nucleic acid from which they were originally generated, Wherein the separation distance belongs to a predetermined distance range. The tags are separated by a separation distance that falls within a predetermined range to enable alignment of the tag sequences with a reference sequence (eg, a reference genomic sequence). Without wishing to be bound by any theory, this may benefit certain applications such as genome resequencing, where it enables the use of shorter read lengths while still being able to map sequences accurately on a reference genome. The 5' and 3' tags of the paired tags represent segments (ie, they have the above sequence) of larger nucleic acid fragments, such as genomic DNA, that are spaced within a predetermined distance from each other in naturally occurring DNA fragments, such as genomic DNA fragments. For example, in certain embodiments of the invention, in naturally occurring DNA fragments, the 5' and 3' tags of a pair of tags represent within 500 nucleotides of each other, within 1 kB of each other, within 2 kB of each other, DNA segments that are within 5 kB of each other, within 10 kB of each other, and within 20 kB of each other. In certain embodiments, the 5' and 3' tags of a pair of tags are separated by 500 nucleotides - 2 kB, such as 700 nucleotides - 1.2 kB, about 1 kB, etc., in a naturally occurring DNA fragment. It should be noted that the exact separation distance of the two tags of a tag pair is not critical and is generally unknown. Furthermore, although tags are initially obtained from larger nucleic acid fragments, the term "tag" is used for any segment of nucleic acid that contains a tag sequence, whether present in the original sequence content or in library fragments, amplification products of library fragments, templates to be sequenced, etc. middle.

Nucleic acid fragments (such as library molecules) may have the following structures:

connector 1-label 1-connector 3-label 1-connector 2

Tag 1 and Adapter 2 can be 5' and 3' tags of a paired tag. Either tag can be a 5' tag or a 3' tag. Adapter 1 and Adapter 2 contain the primer binding regions of one or more primers. In certain embodiments, adapters 1 and 2 each contain the PBR of the amplification primer and the PBR of the sequencing primer. The primers in each adapter may be nested primers such that the sequencing primer PBR is located inside the amplification primer PBR. Adapter 3 may contain a PBR of one or more sequencing primers for sequencing Tag 1 and Tag 2. The term "linker" refers to a nucleic acid sequence present in various nucleic acid fragments of a library, such as substantially all of the fragments of the library. During library construction, linkers may or may not have an actual ligation function, linkers can only be considered as defined sequences shared by most or all members of a given library. Such sequences are also referred to as "universal sequences". Thus, a nucleic acid complementary to an adapter or a portion thereof hybridizes to multiple members of the library and can be used as an amplification primer or sequencing primer for most or all of the molecules in the library.

In some embodiments of the invention, the nucleic acid fragment has the following structure:

adapter1-tag1-internal adapter-tag2-adapter2

Tag 1 and Tag 2 and Adapter 1 and Adapter 2 contain the above PBR. Internal adapters contain two primer binding regions, which may be referred to as IA and IB, as described below. These PBRs can be used to generate microparticles to which two independent populations of substantially identical nucleic acids are attached, one population of nucleic acids comprising tag 1 and the other population of nucleic acids comprising tag 2. Two independent populations of nucleic acids contain at least partially different sequences, eg, different sequences of their tag regions. An internal adapter may contain a spacer between the two primer binding regions. The spacer may contain abasic residues that prevent extension of the polymerase through the spacer. Of course, a spacer containing any other blocking group that prevents extension of the polymerase through the spacer may be used.

In other embodiments, nucleic acid fragments include one or more (eg, 2, 4, 6, etc.) additional tags and one or more additional internal adapters. For example, a nucleic acid fragment can have the following structure:

adapter1-tag1-internaladaptor1-tag2-adapter2-tag3-internaladaptor2-tag4-adapter3

It should be noted that, in addition to the ligation-based sequencing methods described herein, the nucleic acid fragments of the invention, as well as libraries of such fragments, microparticles containing two or more substantially identical nucleic acid populations, and arrays of such microparticles, can be used in a variety of Sequencing method. For example, sequencing methods such as FISSEQ, pyrosequencing, etc. can be used. See eg, WO2005082098. Of course, a connection-based approach can also be advantageously utilized. It is to be understood that in the ligation-based methods described herein, the term "sequencing primer" is to be understood as a "starting oligonucleotide".

In certain embodiments of the invention, PCR is performed in a separate aqueous emulsion chamber (also referred to as a "reactor") to synthesize templates to be sequenced. Preferably, each chamber contains a particulate support such as a bead to which is attached a suitable first amplification primer, a first copy of the template, a second amplification primer and the components necessary to perform the PCR reaction (e.g. nucleotides, polymerase , cofactors, etc.). Methods of making emulsions are found in, eg, US Patents 6,489,103 (Griffiths); 5,830,663 (Embleton); and US Publication No. 20040253731 (Ghadessy). Methods for performing PCR in a single emulsion chamber to generate template clonal populations attached to microparticles are described, for example, in Dressman, D. et al., Proc. .

The methods described in the above references, or modifications thereof, can be used to generate populations of microparticle-attached template clones for sequencing. In a preferred, non-limiting embodiment, short (<500 nucleotides) templates suitable for PCR are generated by ligating universal adapter sequences to each end of a population of different target sequences (templates). ("Universal" here means that the same adapter sequence is ligated to each template, resulting in an "adapter" template that can be amplified with a pair of PCR amplification primers). A bulk PCR reaction is prepared using the adapter template, one free amplification primer, microparticles to which a second amplification primer is attached, and other PCR reagents (eg, polymerase, cofactors, nucleotides, etc.). The aqueous phase PCR reaction was mixed 1:2 with the oil phase (containing light mineral oil and surfactant). Vortex this mixture to produce a water-in-oil emulsion. One milliliter of the mixture is sufficient to generate 4 x ¹⁰⁹ aqueous compartments in this emulsion, each a potential PCR reactor. A sample amount of the emulsion sample is dispensed into wells of a microtiter plate (eg, 96-well plate, 384-well plate, etc.), and thermal cycling is performed to achieve solid-phase PCR amplification on the microparticles. To ensure clonality, the microparticle and template concentrations were carefully controlled so that the reactor contained almost no more than one bead or template molecule. For example, in certain embodiments of the invention, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the reactors contain a bead and a template. Thus, the members of each template clonal population are spatially confined due to attachment to microparticles. Typically, the attachment points of the template can be substantially uniformly distributed on the surface of the particle.

Of particular interest, the PCR emulsion method was used to generate populations of microparticles in which individual microparticles have attached different populations of amplified nucleic acid fragments containing paired 5'-tags and 3'-tags. In other words, it is of particular interest to generate populations of microparticles in which individual particles have different nucleic acid fragments from a library amplified and linked to as described above.

Methods known in the art for amplifying DNA in emulsions (as described in the above references) are limited in terms of the ability to achieve amplification of large nucleic acid molecules and linking these molecules to microparticles. For example, PCR efficiency has been shown to decrease exponentially with longer amplicons. The reduction in PCR efficiency reduces the efficiency with which nucleic acid fragments containing paired tag and primer binding sites (as described above) are amplified in PCR emulsions and attached to microparticles by such amplification. Thus, the method by which a single population of substantially identical nucleic acid fragments containing the first and second tags of a pair of tags are amplified in a PCR emulsion and attached to beads by such amplification suffers from a number of limitations.

The method provided by the present invention enables the use of smaller amplicons while retaining the paired tag information generated when a single nucleic acid fragment containing the 5' and 3' tags of the paired tag is attached to the microparticle through amplification. The invention provides microparticles, such as beads, to which at least two distinct populations of nucleic acids are attached, wherein the at least two populations each consist of a plurality of substantially identical nucleic acids, wherein the first substantially identical populations of nucleic acids comprise a first nucleic acid segment of interest segment, such as a 5' tag, the second nucleic acid population includes a second nucleic acid segment of interest, such as a 3' tag. The first and second nucleic acid populations are amplified from one larger nucleic acid fragment containing both tags, and also containing appropriately distributed primer binding sites flanking and separating the tags, so that in the presence of microparticles and amplification reagents in a single Two amplification reactions are performed consecutively or (preferably) simultaneously in a PCR emulsion reactor. The microparticle has two different primer populations attached, wherein one primer population has a sequence corresponding to a primer binding region other than one tag in the nucleic acid fragment, and the other primer population has a sequence corresponding to a primer binding region other than the other tag in the nucleic acid fragment. The region, the primer binding region, is flanked by the label.

The invention also provides primers that bind to a primer binding region located between two tags so that two different PCR reactions are performed, each amplifying a portion of a nucleic acid fragment containing one tag. The amplified nucleic acid segments contain additional primer binding regions that differ from each other. These other primer binding regions are present in the nucleic acid fragment, within the PBR of the amplification primers, ie they are nested primers. These additional PBRs serve as binding regions for two different sequencing primers. Thus, by applying one or the other of two different sequencing primers to microparticles to which two populations of substantially identical nucleic acid segments are attached, two nucleic acid segments can be sequenced without interference from the presence of the other nucleic acid segment. One or the other of the segments. Each nucleic acid segment is significantly shorter than the nucleic acid fragment from which it is amplified, thereby increasing the efficiency of emulsion-based PCR with fragment libraries containing paired tags, while still preserving the association between the tags of the paired tags.

The method described above can be better understood by referring to the diagrams of Figures 34 and 35, in which portions of nucleic acids having the same sequence are assigned the same color. The foregoing description is for consistent interpretation of Figs. 34 and 35 . Figures 34A and 35A show the same steps, with Figure 35A providing additional detail. As shown in Figures 34A and 35A, paired-end library fragments containing two tags (Tag 1 and Tag 2) were constructed with internal adapter cassettes (IA-IB) and unique flanking adapter sequences (P1 and P2). Both the internal adapter cassette and the flanking linker sequences contain nucleotide sequences for PCR amplification and DNA sequencing. Design PCR primer regions so that nested DNA sequencing primers are used. DNA capture microparticles (beads) are generated by ligating identical two oligonucleotide sequences to unique flanking linker sequences. In PCR amplification, DNA capture particles conjugated to oligonucleotides with P1 and P2 sequences are inoculated into a solution containing a single double-tagged library fragment (i.e., the library fragment contains a 5' tag and a 3' tag of a paired tag) and a solution PCR primers in the reaction.

A limited amount of solution flanking adapter primers (P1 and P2) were added compared to internal adapter primers (IA and IB) to facilitate efficient bead-driven amplification of PCR-generated tagged products (i.e. [P1<< IB], [P2<<IA]). If necessary, properly controlling the amount of primers can also ensure that the nucleic acid populations contain substantially the same amount of nucleic acids, for example, about half of the nucleic acids on a single particle belong to the first population, and about half of the nucleic acids on a single particle belong to the second population. Therefore, if desired, a form of asymmetric PCR can be used to control the ratios of different populations.

During amplification, as shown in Figures 34B and 35B (where Figure 35B again provides additional detail relative to Figure 34B), in the presence of four oligonucleotide primers (P1, P2, IA, and IB), a Paired-end library fragments yield two unique PCR products. One population contains Tag 1 flanked by P1 and IA, and the second population contains Tag 2 flanked by P2 and IB.

After amplification, the microparticles are loaded with two unique PCR populations corresponding to Tag 1 and Tag 2 generated from the starting library fragments. Thus, each tag contains a unique primer block to allow for sequential sequencing of each tag, as shown in Figures 34C, 35C, and 35D. Figures 35C and 35D show sequential sequencing of Tags 1 and 2 with different sequencing primers. A variety of sequencing methods can be employed.

The method described above can be used to produce microparticles with more than two populations such as 4, 6, 8, 12, 16, 20 different nucleic acid sequences linked, for example, wherein the population includes 2, 3, 4, 6, 8, 10 paired tags . Each population can be sequenced individually by providing a unique primer binding region in each sequence, as described above in the Two Tags section.

The present invention includes nucleic acid fragments having the structures shown in Figures 34 and 35 and the structures described above, libraries of such fragments, microparticles to which nucleic acid segments from such fragments are linked, such populations of microparticles (wherein individual microparticles are linked to nucleic acid populations) different from other microparticle-linked nucleic acid populations), microparticle arrays, amplification primers for amplifying nucleic acid segments (tags) from nucleic acid fragments, sequencing primers for sequencing nucleic acid segments attached to microparticles, preparation of such fragments, libraries and Methods of microparticles, and methods of sequencing nucleic acids attached to microparticles. The invention includes kits containing any combination of the above components, optionally also containing one or more enzymes, buffers, or other reagents for amplification, sequencing, and the like.

Various methods can be used to enrich the template-attached microparticles, if desired. For example, hybridization methods can be employed in which an oligonucleotide (capture agent) complementary to a portion of the amplification product (template) attached to the microparticle is attached to a capture entity such as another (preferably larger) microparticle, a microtiter well or other surfaces. This part of the amplified product can be called the targeted region. A targeted region may be incorporated into the template during amplification, such as at one end of a portion of the template containing an unknown sequence. For example, a targeting region may be present in an amplification primer that is not attached to a microparticle such that a complementary portion is present in the amplification template. Thus, multiple different templates can include the same targeting region, and thus one capture agent can hybridize to multiple different templates, which enables the capture of multiple microparticles with only one oligonucleotide sequence, such as a capture agent. Microparticles undergoing amplification are contacted with a capture agent under conditions such that hybridization can occur. As a result, the amplified template-attached microparticle is attached to the capture entity via the capture agent. Unattached microparticles are then removed and residual microparticles are released (eg, by increasing the temperature). In certain embodiments employing particle capture entities, hybridized aggregates consisting of particle-attached capture entities are separated from particle capture entities not attached to particles and particles not attached to capture entities, e.g., by mixing in a viscous solution such as Centrifuge in glycerol. Other methods of separation based on size, density, etc. may also be used. Hybridization is one of many methods available for enrichment. For example, capture agents can be employed that have affinity for a number of different ligands that can be incorporated into the template (eg, during synthesis). Multiple rounds of enrichment may be employed.

Figure 14A shows a chamber image of a water-in-oil emulsion in which a PCR reaction was performed with fluorescently labeled second amplification primers and excess template on beads attached to first amplification primers. Aqueous reactors fluoresce weakly from diffuse free primers, while beads fluoresce strongly from primers aggregated on beads due to solid-phase amplification (ie, incorporation of fluorescent primers into amplification templates attached to beads via a first amplification primer). The bead signal was consistent across reactors of different sizes.

After amplification, the microparticles are collected (eg, using a magnet in the case of magnetic particles) and used for sequencing through repeated cycles of extension, ligation, and cleavage, as described herein. In certain embodiments of the invention, microparticles are arrayed in or on a semi-solid support and then sequenced, as described below. Examples 12, 13, 14 and 15 provide additional details of representative, non-limiting methods that can be used to (i) prepare microparticles with attached amplification primers for template synthesis on microparticles (Example 12) (ii) preparation of an emulsion containing multiple reactors for PCR (Example 13); (iii) PCR amplification in an emulsion chamber (Example 13); (iv) disruption of the emulsion and recovery of microparticles (Example 13); 13); (v) enrichment of microparticles with attached cloned template populations (Example 14); (vi) preparation of glass slides for use as substrates for semi-solid polyacrylamide supports (Example 15); and (vii) Microparticles were mixed with unpolymerized acrylamide to form template-attached microparticle arrays embedded in acrylamide on a substrate (Example 15). Example 15 also describes a polymerase capture protocol that can be used in some methods when PCR is performed on a semi-solid support. Those of ordinary skill in the art recognize that many variations can be made to these methods.

In other embodiments of the invention, PCR is used to amplify templates in a semi-solid support such as a gel in which appropriate amplification primers are immobilized. The templates, other amplification primers and reagents required for the PCR reaction are present on the semi-solid support. One or both primers of the amplification primer pair are attached to the semi-solid support via a suitable linking moiety such as an acrydite group. Joins can be made during aggregation. Other reagents (such as templates, second amplification primers, polymerases, nucleotides, cofactors, etc.) may be present prior to formation of the semisolid support (such as in liquid prior to gel formation), or the semisolid support forms The latter agent or agents may diffuse into the semi-solid support. The pore size of the semi-solid support is chosen so that this diffusion can occur. As is well known in the art, in the case of polyacrylamide gels, the pore size is primarily determined by the concentration of acrylamide monomer, with some influence from the crosslinker. Similar considerations apply in the case of other semi-solid support materials. A suitable cross-linking agent and concentration can be chosen to achieve the desired pore size. In certain embodiments of the invention, the pre-polymerization solution contains additives such as cationic lipids, polyamines, polycations, etc., which form micelles or aggregates surrounding the microparticles in the gel. The methods described in US Pat. Nos. 5,705,628, 5,898,071 and 6,534,262 can also be used. For example, various "encryption reagents" can be used to encrypt the DNA near the beads for clonal PCR. SPRI(R) magnetic bead technology and/or conditions may also be employed. See, eg, US Patent 5,665,572, showing efficient PCR amplification in the presence of 10% polyethylene glycol (PEG). In certain embodiments of the methods of the invention, amplification (eg, PCR), ligation, or amplification and ligation is performed in the presence of certain reagents, such as betaine, polyethylene glycol, PVP-40, and the like. These agents can be added in solution, present in emulsion and/or diffused into a semi-solid support.

A semi-solid support can be positioned or assembled on a substantially planar rigid substrate. In certain preferred embodiments, the substrate is transparent to radiation at excitation and emission wavelengths (e.g., about 400-900 nm) for excitation and detection of typical labels (e.g., fluorescent labels, quantum dots, plasmon resonant particles, nanoclusters) . Certain materials such as glass, plastic, quartz, etc. are suitable. A semi-solid support can be adhered to the substrate and optionally fixed to the substrate by a variety of methods. The substrate may be coated with or without adhesion or bond enhancing substances such as silanes, polylysine, and the like. US Patent 6,511,803 describes methods of using PCR to synthesize clonal template populations in semi-solid supports, methods of preparing semi-solid supports on substantially planar substrates, and the like. A similar approach can be used in the present invention. The substrate may have pores or depressions for receiving liquids prior to forming the semi-solid substrate. Alternatively, raised borders or masks can be used for this purpose.

The method described above provides an alternative method for generating a spatially confined population of clonal templates using a reactor in an emulsion. Clonal populations are present at discrete locations in the semi-solid support such that during sequencing, a signal can be obtained from each population by, for example, imaging, for detection of newly ligated extension probes. In some embodiments of the invention, two or more different clonal populations are amplified from one nucleic acid fragment, present as a mixture at discrete locations on a semi-solid support. Each clonal population in the mixture may contain a tag such that a discrete position contains a fragment containing a 5' tag and a fragment containing a 3' tag. Cloning templates containing 5' and 3' tags contain different sequencing primers, allowing them to be sequenced independently of each other. This method is the same as the above method, and can be used to generate multiple substantially identical nucleic acid populations on microparticles and to obtain sequencing information of two members of a paired tag from one microparticle.

Typically, the semisolid support used in any of the methods of the invention forms a layer having a thickness of about 100 microns or less, such as about 50 microns or less, such as about 20-40 microns. Preferably, prior to polymerization, a coverslip or other similar object having a substantially flat surface can be placed on the semi-solid support material to help create a uniform gel layer, such as to form a gel that is substantially flat and/or substantially uniform in thickness layer.

In other embodiments of the invention, a modified form of the above method may be employed, wherein PCR is used to synthesize templates on microparticles to which appropriate amplification primers are attached, wherein the microparticles are immobilized in or on a semi-solid support prior to template synthesis. on, that is, they are fully or partially embedded in a semi-solid support. Typically, the semisolid support completely surrounds the microparticles, but they may also remain on the underlying substrate. Thus, the microparticles remain in a substantially fixed position relative to each other unless the semi-solid support is disrupted. The method provides an alternative method of using emulsions to generate spatially restricted populations of clonal templates. The microparticles can be mixed with the liquid prior to forming the semi-solid support. Alternatively, the microparticles can be arrayed on a substantially planar substrate, and a liquid added to the microparticle array prior to polymerization, crosslinking, etc. The microparticles are linked with first amplification primers. The second amplification primer can, but need not, be attached to the semi-solid support. Other reagents (e.g., templates, second amplification primers, polymerases, nucleotides, cofactors, etc.) may be present prior to formation of the semisolid support (e.g., in liquid before gel formation), or one One or more reagents can diffuse into the semi-solid support. Typically, a semi-solid substrate is formed on a glass slide as described above.

In certain embodiments of the invention, the gel can be dissolved (eg, digested or disaggregated or melted) to facilitate recovery of the microparticles (eg, using a magnet in the case of magnetic particles) linked populations of clonal templates after template synthesis. Gels that can be solubilized, digested, depolymerized, dissolved, etc. are referred to herein as "reversible" gels. Conventional polyacrylamide polymerization involves the use of N-N' methylene bisacrylamide (BIS) as a crosslinking agent and a suitable catalyst to initiate polymerization (such as N, N, N', N'-tetramethylethylene bis Amines (TEMED). In order to produce reversible gels, another cross-linking agent such as N-N'diallyl tartaric acid diamide (DATD) can be used. This compound is similar in structure to BIS, but has the ability to be (such as a solution containing sodium periodate) cleaved cis-dihydroxyl (Anker, H.S.: F.E.B.S.Lett., 7: 293, 1970). Therefore, it is not difficult to dissolve the DATD gel. The gel prepared with DATD as a crosslinking agent The gel is highly transparent and binds strongly to glass. Another crosslinker with DATD-like properties to form reversible gels is ethylene glycol diacrylate (Choules, G.L. and Zimm, B.S.: Anal. Biochem., 13:336- 339, 1965). N, N'-bispropenylcystamine (BAC) is another cross-linking agent that can be used to form reversible polyacrylamide gels. It can be used to form gels dissolved in periodate Another cross-linking agent is N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA). Various other materials that can form a reversible semi-solid support can also be used. For example, the Thermoreversible polymers such as Pluronic (available from BASF). Pluronic is poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)(PEO-PPO-PEO) family of block copolymers (Nace, V.M. et al., Nonionic Surfactant, Marcel-Dekker, NY, 1996). These materials become semi-solid (gel) at elevated temperature (eg, above room temperature) and liquefy on cooling Pluronic can be chemically derivatized in various ways, e.g. to facilitate ligation of primers (see e.g., Neff, J.A. et al., J. Biomed. Mater. Res., 40:511, 1998; Prud'homme, RK et al., Langmuir, 12:4651, 1996).

After lysis, microparticles can be collected and sequenced with repeated cycles of extension, ligation, and cleavage. Prior to sequencing, microparticles can be arrayed in or on a second semi-solid support (eg, at a higher density than they were present in or on the first semi-solid support). The semi-solid support itself is supported by a substantially planar rigid substrate such as a glass slide.

Thus, two general approaches can be used to generate semi-solid supports with arrays of microparticles carrying clonal template populations embedded in or on the semi-solid support. The first method involves amplification (eg, using emulsion PCR) on microparticles not present in the semisolid support, followed by immobilization of the microparticles in or on the semisolid support. A second general method involves immobilizing microparticles in or on a semi-solid support followed by amplification. In both cases, steps may need to be taken to reduce particle aggregation and/or to align the particles substantially in a focal plane. For example, when immobilizing particles in a polyacrylamide gel, the concentrations of monomer and crosslinker are chosen so that the particles settle to the bottom of the solution, and polymerization is then completed so that they remain on the underlying flat in a plane. In certain embodiments of the invention, an object having a substantially planar surface, such as a coverslip, is placed on liquid acrylamide (or a material capable of forming a semi-solid support) containing microparticles such that the acrylamide is sandwiched between the " between the two layers of the sandwich structure. The sandwich is then inverted so that the microparticles settle and rest on the coverslip (or other object with a substantially flat surface) by gravity. After polymerization, the coverslip is removed. Thus, the microparticles are substantially embedded in the same plane, close to (eg tangent to) the surface of the semi-solid support.

In certain embodiments of the invention, rather than immobilizing a support such as a microparticle in a semisolid matrix as described above, the microparticle is covalently or non-covalently attached to a substantially planar rigid substrate without the use of a semisolid support. things to fix them. Various methods are known in the art for attaching microparticles to substrates such as glass, plastic, quartz, silicon, and the like. The substrate may or may not be coated (eg, spin-coated) or functionalized with certain materials (eg, various polymers) or attachment-promoting substances. Coatings can be thin films, self-assembled monolayers, and the like. A microparticle, a moiety attached to a microparticle, or an oligonucleotide (eg, a template) attached to a microparticle can be attached.

In general, any pair of molecules with mutual affinity to form a binding pair can be used to attach the microparticle or template to the substrate. The first member of the binding pair is covalently or non-covalently attached to the substrate and the second member of the binding pair is covalently or non-covalently attached to the microparticle or template. The first binding member can be attached to the substrate via a linker. The second binding member can be attached to the particle or template via a linker. For example, according to one approach, a glass slide or other suitable substrate is modified with an amine-activated group (eg, using a PEG linker containing an amine-activated group). Under aqueous conditions (eg, pH 8.0), the amine-activating group reacts with amines such as lysine in proteins (eg, streptavidin). Thus, microparticles functionalized with amine-carrying moieties will be immobilized on the substrate. The amine-bearing moiety can be a protein or an appropriately functionalized nucleic acid, such as a DNA template. Multiple moieties can be attached to the bead. For example, a bead can be attached to a protein that reacts with an NHS ester to attach the bead to a substrate, or a DNA template can be attached to which the bead can be attached to a substrate for sequencing. Suitably coated slides with a polymer tether containing an amine-reactive NHS moiety at one end are commercially available from, eg, Schott Nexterion, Schott North America, Inc., Elmsford, NY 10523. Alternatively, coated slides (eg, biotin-coated slides) can be purchased from Accelr8 Technology Corporation, Denver, CO. Their OptiChem ^™ technology represents one method of attaching microparticles to substrates. See, eg, US Patent 6,844,028. Alternatively, the polynucleotides on the beads can be functionalized with biotin, e.g., with terminal transferase and biotin-dideoxyATP and/or biotin-deoxyATP, followed by biotin-streptavidin-promoting binding These beads are conditioned to contact streptavidin-coated slides (commercially available from, for example, Accelr8 Technology Corporation, Denver, CO), thereby attaching the microparticles to the substrate.

In general, nucleic acids such as oligonucleotide primers, probes, templates, etc. can be modified using various methods known in the art to facilitate attachment of such nucleic acids to microparticles or other supports or substrates. In addition, microparticles or other supports can be modified using various methods known in the art to facilitate attachment of nucleic acids, attachment of microparticles to supports or substrates, and the like. Microspheres can be obtained with surface chemistry that facilitates the attachment of desired functional groups. Some examples of these surface chemistries include, but are not limited to, amino groups including aliphatic and aromatic amines, carboxylic acids, aldehydes, amides, chloromethyl groups, hydrazides, hydroxyl groups, sulfonate esters, and sulfate esters. These groups can react with groups present on the nucleic acid, or can modify the nucleic acid by attaching reactive groups. In addition, a large number of stable bifunctional groups, including homobifunctional and heterobifunctional linkers, are well known in the art. See, e.g., Pierce Chemical Technology Library, available from the Web site at URL www.piercenet.com (originally published in the 1994-95 Pierce Directory) and G.T. Hermanson, Bioconjugate Techniques, Academic Press, Inc., 1996. See also US Patent 6,632,655.

Microparticle arrays formed according to the methods described herein are typically random arrays. The terms "randomly patterned" or "randomly" as used herein refer to a disordered, non-Cartesian distribution of entities (features) on a support (in other words, not arranged at predetermined points or positions along the x- and y-axes of the grid or relative to each other). 'Clock position', angle or radius determined at the center of the radiation pattern), which is not achieved by deliberate design (or a program that enables such a design) or placement of a single entity. Such "randomly patterned" or "random" arrays of entities can be obtained by dripping, spraying, plating, spreading, distributing (etc.) a solution, emulsion, aerosol, steam or dry formulation containing a library of entities onto a support or and to allow their settling onto or into the support without intervening in any way in directing them to specific sites in or on the support. For example, the entity can be suspended in a solution containing a semisolid support precursor such as acrylamide monomer. The solution is then distributed on a second support, forming a semi-solid support on the second support. Entities are embedded in or on a semi-solid support. Of course, non-random arrays can also be used. In general, methods of forming arrays as used herein are distinct from methods of synthesizing polynucleotides by sequentially applying individual nucleotide subunits at predetermined locations on a substrate.

Figure 14B (top) shows a fluorescent image of a slide (1 inch x 3 inches) with a polyacrylamide gel on it. Beads (1 micron in diameter) with fluorescently labeled oligonucleotides that hybridize to templates attached to the beads were immobilized in the gel. The graph shows the bead surface density (ie, the number of beads per unit area of substrate within the region where the beads reside), which is sufficient to image approximately 280 million beads per slide. The surface density and imageable area on one slide are sufficient to image at least 500 million beads. For example, Figure 14B (bottom) shows a schematic of a glass slide with a Teflon(R) mask surrounding a clear region where beads are embedded in a layer of a semi-solid support such as a polyacrylamide gel. The area of this mask is 864 mm ² . With 500 million beads, the surface density is 578,000 beads/mm ² . A tightly packed 1 micron hexagonal array contains 1,155,000 beads/mm ² , thus, this embodiment yields an array with a density of 52% of the theoretical maximum. It should be understood that both fewer and higher bead counts, lower or higher bead surface densities than in this particular embodiment may be employed.

Microparticles can be arranged in various densities in or on a substantially planar semi-solid support or another support or substrate, which can be defined in a variety of ways. For example, density can be expressed as the number of particles (eg, spherical particles) per unit area of a substantially planar array. In some embodiments of the invention, the number of particles per unit area on the substantially planar array is at least 80% of the number of particles in the hexagonal array ("hexagonal array" means that each particle in the array contacts at least equal area). A substantially planar microparticle array of another six adjacent microparticles, as described in US Pat. No. 6,406,848). However, in other embodiments of the invention, the particle density is lower, eg, the number of particles per unit area on a substantially planar array is less than 80%, 70%, 60%, or 50% of the number of particles in a hexagonal array. Without wishing to be bound by theory, it is preferable to use lower densities such as those described above to allow sufficient diffusion of reagents such as enzymes, primers, cofactors, etc. reagent distribution effect. This effect can create different reaction conditions at different locations on the array and may even prevent access of these reagents to certain locations on the array. These issues can be more difficult to deal with when reactions are performed in a flow cell because the reagents pass through the cell in a directional fashion. In certain embodiments of the invention, the cells of the flow chamber include mixing means, such as means for effecting fluid mixing by mechanical or acoustic means. Many suitable mixing devices are known in the art.

The sequencing methods of the invention can be performed with templates arranged in arrays of all types, including random and non-random arrays, which can be arrays of microparticles or arrays of the templates themselves. For example, US Patent 5,641,658 and PCT Publication No. WO0018957 describe supports with templates arrayed thereon. Arrays can be located on a variety of substrates such as filter paper, membranes (eg nylon), metal surfaces, and the like. Other examples of array formats that can be sequenced on an array by repeated cycles of extension, ligation, and cleavage are bead arrays in wells positioned at the ends or distal ends of individual optical fibers in a fiber optic bundle. See, eg, US publications and patents such as 6,023,540; 6,429,027, 20040185483, 2002187515, PCT applications US98/05025 and PCT US98/09163 and PCT publication WO0039587 describing bead arrays and "arrays of arrays". The template-attached beads can be arranged as described herein. Amplification is preferably performed prior to formation of the array. Arrays formed on these substrates need not be substantially planar.

In other embodiments, PCR is performed on arrays containing oligonucleotides attached to a substrate or support, (see, e.g., US Patents 5,744,305; 5,800,992; 6,646,243 and related patents (Affymetrix); PCT Publication WO2004029586; WO03065038; WO03040410 (Nimblegen)). Typically, such oligonucleotides contain free 3' or 5' ends. This end can be modified if desired, for example, adding a phosphate or OH group to the 3' end if there is no phosphate or OH group at the 3' end. Template molecules containing regions complementary to oligonucleotides attached to a support or substrate are hybridized to oligonucleotides and in situ PCR is performed on the array to generate a population of clonal templates at each position on the array. An oligonucleotide attached to the array can be used as one of the amplification primers. The templates are then sequenced using the ligation-based methods described herein. Sequencing can also be performed on the templates in the array, as described in US Pub. No. 20030068629.

Other methods of preparing DNA arrays on surfaces can be used. For example, alkanethiols modified with terminal aldehyde groups can be used to prepare self-assembled monolayers (SAMs) on gold surfaces. The aldehyde groups of this monolayer can be reacted with amine-modified oligonucleotides or other amine-bearing biomolecules to form Schiff's, which can then be reduced to stable secondary amines by treatment with sodium cyanoborohydride (Peelen and Smith, Langmuir, 21( 1): 266-71, 2005). PCR amplification of the template can then be performed. Alternatively, microparticles with linked populations of cloned templates can be attached to the surface by reacting amine groups on the microparticle or template or oligonucleotides attached to the particle with the surface.

Another method of obtaining microparticles with linked populations of cloned templates is the "solid phase cloning" method described in U.S. Patent 5,604,097, which uses oligonucleotide tags to sort polynucleotides onto microparticles such that only polynuclei of identical sequence The nucleotides are linked to a specific particle.

In some embodiments of the present invention, by diffusing sequencing reagents (such as extension probes, ligases, phosphatases, etc.) In a semi-solid support such as a gel in spatially independent regions), sequencing is performed in repeated cycles of extension, ligation, and cleavage. In certain embodiments, the template is directly attached to the semi-solid support described above. However, in a preferred embodiment, the template is immobilized on a second support, such as a microparticle, which in turn immobilizes the microparticle in or on a semi-solid support, as described above.

As described in Example 1, the inventors have demonstrated that robust ligation and cleavage can be performed on templates attached to beads immobilized in polyacrylamide gels. Accordingly, the present invention provides a method of linking a first polynucleotide to a second polynucleotide, said method comprising the steps of: (a) providing a first polynucleotide immobilized in or on a semi-solid support. (b) contacting the first polynucleotide with a second polynucleotide and a ligase; and (c) maintaining the first and second polynucleotides in the presence of the ligase under conditions suitable for connection. Suitable conditions include providing buffers, cofactors, temperature, time, etc. appropriate for the particular ligase used. In a preferred embodiment, the semi-solid support is a gel such as an acrylamide gel. In another preferred embodiment, said first species is attached to a support, such as a bead, and the bead itself is immobilized in or on a semi-solid support, such as by partial or complete embedding in the support matrix. The polynucleotides are immobilized in or on a semi-solid support. Alternatively, the first polynucleotide may be attached directly to the semi-solid support by linking moieties such as acrydite. This linking can be covalent or non-covalent (eg, via a biotin-avidin interaction). US Patent 6,511,803 describes various methods that can be used to attach nucleic acid molecules to the preferred support of the present invention, polyacrylamide gels.

The present invention also provides a method for cleaving a polynucleotide, the method comprising the steps of: (a) providing a polynucleotide immobilized in or on a semi-solid support, wherein the polynucleotide contains an easy-cut link; ( b) contacting the polynucleotide with a nicking agent; and (c) maintaining the polynucleotide under conditions suitable for cleavage in the presence of the nicking agent. Suitable conditions include providing a buffer, temperature, time, etc. suitable for a particular cutting agent. In a preferred embodiment, the semi-solid support is a gel such as an acrylamide gel. In another preferred embodiment, said polynucleotide is immobilized in a semi-solid support by attaching to a support such as a bead, and then immobilizing the bead itself in the semi-solid support. Alternatively, the polynucleotide may be attached directly to the semi-solid support by linking moieties such as acrydite. This linking can be covalent or non-covalent (eg, via a biotin-avidin interaction).

Macevicz discloses sequencing a template with a specific sequence. He does not discuss the possibility of parallelizing this approach to simultaneously sequence multiple templates with different sequences. The present inventors have recognized that for efficient sequencing in a high-throughput manner, multiple supports (such as beads) need to be prepared, as described above, so that each support is attached to a template of a specific sequence, and each support is attached to a specific sequence. The templates were carried out simultaneously with the methods described herein. In certain embodiments of the method, multiple supports are arranged in or on a flat substrate, such as a glass slide. In certain embodiments, the support is arranged in or on the gel. The supports can be arranged in a random fashion, ie the position of each support on the substrate is not predetermined. The supports need not be distributed at regular intervals or in an ordered array of rows and columns or the like. Preferably, the support is arranged in such a density that it is possible to detect a single signal emitted by many or most of the supports. In certain preferred embodiments, the supports are distributed predominantly in one focal plane. Multiple supports linked to sequence-identical templates can be included, for example, for quality control. Parallel sequencing reactions are performed on templates attached to each support.

Signals can be collected in a variety of ways, including various imaging modalities. Preferably, in embodiments where sequencing is performed on microparticles arrayed on a substrate (such as beads embedded in a semi-solid support on a substrate) prior to detection, the imaging device has a resolution of 1 μm or less . For example, a scanning microscope equipped with a CCD camera of sufficient resolution or a microarray scanner can be used. Alternatively, beads are passed through a flow cell or fluidic station attached to a microscope equipped for fluorescence detection. Other methods of collecting signals include fiber optic bundles. Appropriate image capture and processing software can be used.

In certain embodiments of the invention, sequencing is performed in a microfluidic device. For example, template-attached beads can be loaded into the device and reagents flowed through. Template synthesis using PCR can also be performed in this device. Examples of suitable microfluidic devices are described in US Patent 6,632,655.

D. Sequencing by reinitiation of different starting oligonucleotides

In a preferred embodiment of the invention, after a sufficient number of cycles, the extended strand generated by extending the first starting oligonucleotide is removed from the template and the second starting oligonucleotide is annealed to the binding region , followed by a cycle of extension, ligation, and detection. This process is repeated with any number of different starting oligonucleotides. In embodiments where the extension probe is cleaved, the number of different starting oligonucleotides used (and thus the number of reactions) is preferably equal to the length of the portion of the extension probe that remains hybridized to the template after releasing the distal portion of the probe. Thus, according to this embodiment, sequence information (such as the order and identity of individual nucleotides) can be obtained from a template attached to a support, in which case using a ratio of the nucleotides identified in each cycle to identify consecutive nucleotides. A much lower number of cycles is required to still read deeply into the sequence.

Embodiments in which starting oligonucleotides are sequentially bound to the same template have certain advantages over methods that require splitting the template into multiple aliquots, such as the method described by Macevicz. For example, application of the starting oligonucleotides to the same template eliminates the need for tracking and subsequent combining of data obtained from multiple aliquots. In embodiments where the supports are arranged in a random fashion such that the position of individual supports cannot be predetermined, it may be difficult or impossible to reliably combine partial sequence information from multiple supports each linked to a template of the same sequence.

E. Identification of multiple nucleotides on a template per cycle

Macevicz described identifying one nucleotide on the template in each cycle of extension, ligation, and detection. However, the inventors realized that the method could be modified to identify multiple nucleotides on the template in each cycle. In this case, the extension probe is labeled such that the identity of two or more (preferably contiguous) nucleotides contiguously extending the duplex can be determined from the label. In other words, the sequence-determining portion of the extension probe is more than one nucleotide, generally comprising the nearest nucleotide, immediately adjacent nucleotides, and possibly one or more additional (preferably contiguous) nucleotides, all of which Nucleotides are capable of specifically hybridizing to a template. For example, in addition to using 4 labels to identify the bases A, G, C, and T, 16 differentially labeled probes or combinations of probes can be used to identify 16 possible dinucleotides AA, AG, AC, AT, GA, GG, GC, GT, CA, CG, CC, CT, TA, TG, TC, and TT. The sequenced portion of each differentially labeled extension probe is complementary to one of these dinucleotides. A similar approach employing more labels enables the identification of longer nucleotide sequences per cycle.

F. mark

In a broad sense, the term "label" as used herein refers to any detectable moiety or detectable moieties attached to a probe that can be used to distinguish between different types of probes (eg, probes containing different terminal nucleotides). Thus, there is not necessarily a one-to-one correspondence between labels and specific detectable moieties. For example, multiple detectable moieties can be attached to one probe, resulting in a combined signal capable of distinguishing that probe from probes to which different detectable moieties or sets of detectable moieties are attached. For example, a combination of detectable moieties according to a labeling scheme called "combinatorial multicolor coding" described in US Pat.

Probes of the invention can be labeled in a variety of ways, including direct or indirect attachment of fluorescent or chemiluminescent moieties, colorimetric moieties, enzymatic moieties that produce a detectable signal upon contact with a substrate, and the like. Macevicz notes that fluorescent dyes can be used to label the probes, as described by Menchen et al., US Patent 5,188,934; Begot et al., PCT Application PCT/US90105565. The terms "fluorochrome" and "fluorophore" as used herein refer to a moiety that absorbs light energy at a particular excitation wavelength and emits light energy at a different wavelength. Preferably, the labels selected for a given probe mix are spectrally resolvable. As used herein, "spectrally resolvable" means that the label can be distinguished under operating conditions based on its spectral characteristics, specifically the wavelength of fluorescence emission. For example, the species of one or more terminal nucleotides may be related to the maximum light emission intensity at a unique wavelength, or may be related to the ratio of intensities at different wavelengths. The spectral signature of the marker used to detect and identify the marker is referred to herein as "color". It will be appreciated that labels are often identified based on specific spectral features, such as the frequency of maximum emission intensity when the label consists of one detectable moiety, or the frequency of emission peaks when the label consists of multiple detectable moieties.

Preferably four probes are provided, with each of the four spectrally resolvable fluorescent dyes in one-to-one correspondence with the four possible terminal nucleotides of the probes. US Patents 4,855,225 and 5,188,934; International Application PCT7US90/05565; and Lee et al., Nucleic Acids Researchss, 20:2471-2483 (1992) disclose sets of spectrally resolvable dyes. In certain embodiments, a dye set consisting of FITC, HEX ^™ , Texas Red and Cy5 is preferred. Many suitable dyes are commercially available from, eg, Molecular Probes, Inc., Eugene OR. Specific examples of fluorescent dyes include, but are not limited to: Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, and Alexa Fluor 680), AMCA , AMCA-S, BODIPY Dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPYTR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), CAL Dye, Carboxyrhodamine 6G, Carboxy-X-Rhodamine (ROX), Cascade Blue, Cascade Yellow, Cyanine Dye (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl , dialkylaminocoumarin, 4',5'-dichloro-2',7'-dimethoxy-fluorescein, DM-NERF, eosin, erythrosine, fluorescein, FAM, hydroxyl Beanin, IRD dyes (IRD40, IRD 700, IRD 800), JOE, Lissamine Rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Oyster Dye, Pacific Blue, PyMPO, Pyrene, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2′,4′,5′,7′-tetrabromosulfone-fluorescein, tetramethyl-rhodamine ( TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X. For further instructions see The Handbook of Fluorescent Probes and Research Products, 9th Edition, Molecular Probes, Inc.

In the process of non-radiative fluorescence resonance energy transfer (FRET), some fluorophores transfer energy to another group, and the detection signal is generated by the second group instead of directly detecting this group. That is, the use of a quencher also falls within the scope of the present invention. The term "quencher" refers to a moiety that absorbs, upon proximity, the energy of an excited fluorescent label and dissipates that energy without emitting visible light. Examples of quenchers include, but are not limited to: DABCYL (4-(4'-dimethylaminophenylazo)benzoic acid) succinimidyl ester, diarylrhodamine carboxylic acid succinimidyl ester ( QSY-7) and 4′,5′-dinitrofluorescein carboxylate succinimidyl ester (QSY-33) (both purchased from Molecular Probes), quencher 1 (Q1; purchased from Epoch) or “black hole Quenchers" BHQ-I, BHQ-2 and BHQ-3 (purchased from BioSearch, Inc.).

In addition to the various detectable moieties described above, the present invention also contemplates the use of spectrally resolvable quantum dots, metal nanoparticles or nanoclusters, etc., which can be directly attached to oligonucleotide probes, or embedded or attached to polymers The matrix is then attached to the probe. As noted above, the detectable moiety itself is not necessarily directly detectable. For example, they may be reactive on the substrate to be detected or they may need to be modified to become detectable.

As noted above, in certain embodiments of the invention, the label consists of multiple detectable moieties. The combined signal of these detectable moieties produces the color used to identify the probe. For example, sequence-specific "purple" probes can be constructed by linking "blue" and "red" detectable moieties. Alternatively, hybrid probes can be produced by mixing two probes of the same sequence but labeled with different detectable moieties, thereby producing a unique color. Thus, a "purple" probe of a particular sequence can be generated by constructing two probes of that sequence. A "red" detectable moiety is attached to the first probe and a "blue" detectable moiety is attached to the second probe. Mix sample amounts of these two probes. Different shades of purple can be produced by mixing sample amounts in different proportions. This approach offers many advantages. First, it enables the generation of multiple distinguishable probes with fewer detectable moieties. Second, the use of mixed probes provides a degree of degeneracy that may help reduce bias that may result from the interaction of specific detectable moieties with specific nucleotides.

In certain embodiments of the invention, a detectable moiety is attached to a nucleotide in an oligonucleotide extension probe by a cleavable linkage such that the detectable moiety is removed after ligation and detection. A variety of different cuttable connections can be used. As used herein, the term "cleavable linkage" refers to a chemical moiety that attaches a detectable moiety to a nucleotide and, if desired, can be cleaved to remove the detectable moiety on the nucleotide without substantially altering the core to which it is attached. nucleotide or nucleic acid molecule. Depending on the nature of the linkage, cleavage can be achieved, for example, by acid or base treatment, or by oxidation or reduction of the linkage, or by phototreatment (photocleavage). For examples of cleavable linkers and cleavage agents see Shirnkus et al., 1985, Proc. Natl. Acad. Sci. USA 82:2593-2597; Soukup et al., 1995, Bioconjug. 5:247-255; and Herman and Fenn, 1990, Meth. Enzymol. 184:584-588.

For example, disulfide linkages can be reduced to be cleaved with a thiol reducing agent such as dithiothreitol (DTT), as described in US Patent No. 6,511,803. Fluorophores are available that contain a thiol (SH) group that can be used for conjugation to reactive arylamino-containing nucleotides (such as dCTP) (eg, SH-containing cyanine 5 or cyanine 3 fluorophores; New England Nuclear—DuPont). Reactive pyridyl dithiols can react with sulfhydryl groups to generate sulfhydryl bonds that can be cleaved by reducing agents such as dithiothreitol. NHS ester heterobifunctional cross-linking agent (Pierce) can be used to connect the deoxynucleotides containing active aryl amino group to pyridyl dithiol group, and then react with SH on the fluorophore to produce the bisphosphonate used in the method of the present invention. Sulfur-linked cleavable nucleotide-fluorophore complexes. Alternatively, the cis-diol linkage between the nucleotide and the fluorophore can be cleaved by periodate. Various cleavable linkages are described in US Patent Nos. 6,664,079 and 6,632,655, US Published Application 20030104437, WO 04/18497 and WO 03/48387.

In other embodiments of the invention, detectable moieties are used that can be rendered undetectable by exposure to electromagnetic energy such as light (photobleaching).

In embodiments of the invention utilizing extended probes containing a label attached to the probe by a cleavable ligation or a label that can be photobleached, the sequencing method generally includes one or more cycles after ligation and label detection have been performed. Steps for cleavage or photobleaching. As noted above, cleavage of easy ligation in oligonucleotide extension probes may not proceed to completion (ie, less than 100% of the newly ligated probe may be cleaved in its cycle of ligation). Since such probes typically contain a non-extendable template or are capped, they cannot be cycled continuously. However, the inability to cleave the probe means that the label remains attached to the probe-attached template molecule, which would generate a background signal (i.e., background fluorescence), potentially increasing noise in subsequent cycles. Adding a cleavage or photobleaching step to remove the label or make it undetectable can reduce this background and improve the signal-to-noise ratio. Cleaving or photobleaching can be performed in every cycle, or less frequently, such as once every two cycles, every three cycles, or every five or more cycles. In certain embodiments of the invention, it is not actually necessary to add an additional step to cleave the cleavable linker. For example, a cleavage agent such as DTT may already be present in the wash buffer and can be used to remove unligated extension probes.

G. Preferred easy-cut connection

The inventors have found that extension probes containing at least one phosphorothioate linkage are particularly useful in methods for sequencing by successive cycles of extension, ligation, detection and cleavage. In this linkage, one of the bridging oxygen atoms of the phosphodiester bond is replaced by a sulfur atom. The phosphorothioate linkage can be a 5'-S-phosphorothioate linkage (3'-OPS-5') as shown in Figure 4A or a 3'-S-phosphorothioate linkage (3'-OPS-5') as shown in Figure 4B -SPO-5'). It will be appreciated that the phosphorus atom in linkages denoted 3'-OPS-5' or 3'-SPO-5' may be linked to two non-bridging oxygen atoms as shown in Figures 4A and 4B (as typical phosphodiester key). Alternatively, the phosphorus atom can be attached to various other atoms or groups, such as S, _CH3 , _BH3 , and the like. Accordingly, one aspect of the invention is a labeled oligonucleotide probe comprising a phosphorothioate linkage. While the probes are particularly useful in the sequencing methods described herein, they can also be used for a variety of other purposes. Specifically, the present invention provides (i) oligonucleotides in the form of ₍ i) 5'-OPOXOPS-(N) _kNB ^* -3'; and (ii) 5'- _NB ^* (N) _k -SPOX-3 ' form of the oligonucleotide. In these probes, N represents any nucleotide, N _B represents a ligase non-extendable portion, * represents a detectable portion, X represents a nucleotide, and k is 1-100. In certain embodiments, k is 1-50, 1-30, 1-20, such _as 4-10, with the proviso that the detectable moiety may be present in place of (N _{) k} instead of, or in addition to, NB on any nucleotide. The terminal nucleotides in these probes may or may not include phosphate groups or hydroxyl groups. It should also be understood that in preferred embodiments the phosphorus atom is generally attached to two other (non-bridging) oxygen atoms.

Methods for synthesizing oligonucleotides containing 5'-S-phosphorothioate or 3'-S-phosphorothioate linkages are known in the art, some of which are suitable for automated solid-phase oligonucleotide synthesis. For synthetic methods see, for example: Cook, AF, J.Am.Chem.Soc., 92:190-195, 1970; Chladek, S. et al., J.Am.Chem.Soc., 94:2079-2084, 1972; Rybakov , VN et al., Nucleic Acids Res., 9:189-201, 1981; Cosstick, R. and Vyle, JS, J.Chem.Soc.CHem.Commun., 992-992, 1988; Mag, M. et al., Nucleic Acids Acids Res., 19(7); 1437-1441, 1991; Xu, Y and Kool, ET, Nucleic Acids Res., 26(13): 3159-3164, 1998; Cosstick, R. and Vyle, JS, Tetrahedron Lett. , 30: 4693-4696, 1989; Cosstick, R. and Vyle, JS, Nucleic Acids Res., 18: 829-835, 1990; Sun, SG and Piccirilli, JA, Nucl.Nucl., 16: 1543-1545, 1997; Sun SG et al., RNA, 3:1352-1363, 1997; Vyle, JS et al., Tetrahedron Lett., 33:3017-3020, 1992; Li, X. et al., J.Chem.Soc.Perkin Trans., 1 : 2123-22129, 1994; Liu, XH and Reese, CB, Tetrahedron Lett., 37: 925-928, 1996; Weinstein, LB et al., J. Am. Chem. Soc., 118: 10341-10350, 1996; and Sabbagh, G. et al., Nucleic Acids Res., 32(2):495-501, 2004. Furthermore, the present inventors developed a new synthesis method. For example, Figure 7 shows a synthetic scheme for the 3'-phosphoramidite of dA. A similar scheme can be used for the synthesis of 3'-phosphoramidites of dG. These phosphoramidites can be used to synthesize oligonucleotides containing 3'-S-phosphorothioate linkages linked to purine nucleosides, e.g., using an automated DNA synthesizer.

Phosphorothioate linkages can be cleaved with various metal-containing substances. The metal may be, for example, Ag, Hg, Cu, Mn, Zn or Cd. Preferably, the species is a water-soluble salt providing anions of Ag ⁺ , Hg ⁺⁺ , Cu ⁺⁺ , Mn ⁺⁺ , Zn ⁺ or Cd ⁺ (salts providing ions in other oxidation states may also be used). _I2 can also be used. Particular preference is given to silver-containing salts such as silver nitrate (AgNO ₃ ) or other salts which provide Ag ⁺ ions. Suitable conditions include, for example: 50 mM AgNO ₃ , about 22-37° C., for 10 minutes or longer, such as 30 minutes. Preferably, the pH is 4.0-10.0, more preferably 5.0-9.0, such as about 6.0-8.0, such as about 7.0. See, eg, Mag, M. et al., Nucleic Acids Res., 19(7):1437-1441,1991. Example 1 provides an exemplary protocol.

Sequencing can be performed in the 5'→3' direction with an extension probe containing a 3'-OPS-5' junction. Figure 5A shows a cycle of hybridization, ligation and cleavage with an extension probe of the form 5'-OPOXOPS-NN _B ^* -3', where N stands for any nucleotide and N _B represents the portion that cannot be extended by the ligase ( For example, _NB is a nucleotide lacking a 3' hydroxyl group or having a blocking moiety attached), * represents a detectable moiety, and X represents a nucleotide whose type corresponds to the detectable moiety. Alternatively, a bulk blocking moiety can be attached to the 3' terminal nucleotide to prevent multiple ligation. For example, attachment of a bulky group to, eg, the 2' or 3' position of the sugar moiety of the nucleotide will prevent attachment. Fluorescent labels can be used as suitable macrogroups.

A template comprising a binding region 40 and a polynucleotide region of unknown sequence 50 is attached to a support such as a bead. In a preferred embodiment, as shown in Figure 5A, the binding region is located at the other end of the junction between the template and the support. An initial oligonucleotide 30 with an extendable end (in this case a free 3'OH group) is annealed to the binding region 40 . The extension probe 60 hybridizes to the polynucleotide region 50 of the template. Nucleotide X forms a complementary base pair with unknown nucleotide Y in the template. The extension probe 60 is ligated to the starting oligonucleotide (eg, using T4 ligase). After ligation, the label (not shown) attached to the extension probe 60 is detected. This label corresponds to the species of nucleotide X. Nucleotide Y is therefore identified as a nucleotide complementary to nucleotide X. Then, extension probe 60 is cleaved at the phosphorothioate linkage (eg, with _AgNO3 or another salt that provides Ag ⁺ ions), generating an extension duplex. Cleavage generates a phosphate group on the 3' end of the extended duplex. Treatment with phosphatase generates extendable probe ends on the extended duplex. Repeat the process for the desired number of cycles.

In a preferred embodiment, sequencing is performed in the 3'→5' direction with an extension probe containing a 3'-SPO-5' junction. Figure 5B shows a cycle of hybridization, ligation, and cleavage with an extension probe of the form 5'- _NB ^* -NNNN-SPOX-3', where N represents any nucleotide and _NB represents the ligase that cannot be extended. moiety (such as _NB is a nucleotide lacking a 5' phosphate group or having a blocking moiety attached), * represents a detectable moiety, and X represents a nucleotide whose type corresponds to the detectable moiety.

A template comprising a binding region 40 and a polynucleotide region of unknown sequence 50 is attached to a support such as a bead. In a preferred embodiment, as shown in Figure 5B, the binding region is located at the other end of the junction between the template and the support. An initial oligonucleotide 30 with an extendable end (in this case a free 5' phosphate group) is annealed to the binding region 40 . The extension probe 60 hybridizes to the polynucleotide region 50 of the template. Nucleotide X forms a complementary base pair with unknown nucleotide Y in the template. The extension probe 60 is ligated to the starting oligonucleotide (eg, using T4 ligase). After ligation, the label (not shown) attached to the extension probe 60 is detected. This label corresponds to the species of nucleotide X. Nucleotide Y is therefore identified as a nucleotide complementary to nucleotide X. Then, extension probe 60 is cleaved at the phosphorothioate linkage (eg, with _AgNO3 or another salt that provides Ag ⁺ ions), generating an extension duplex. Cleavage generates an extendable monophosphate group on the 5' end of the extending duplex, so no additional steps are necessary to generate extendable ends. Repeat the process for the desired number of cycles.

It should be understood that many variations of this scheme may be employed. For example, a probe can be shorter or longer than 6 nucleotides; a label need not be on the 3' terminal nucleotide; a P-S junction can be between any two adjacent nucleotides, etc. In the above embodiments, successive cycles of extension, ligation, detection and cleavage result in the identification of nucleotides at adjacent positions. However, by bringing the P-S junction closer to the distal end of the extension probe (ie, the opposite end from which the ligation occurs), sequentially identified nucleotides will be spaced along the template, as described above and in Figures 1 and 6 .

6A-6F are more detailed schematics of several sequencing reactions performed sequentially on a template. Sequencing was performed in the 3'→5' direction with an extension probe containing a 3'-S-P-O-5' junction. Each sequencing reaction includes multiple cycles of extension, ligation, detection and cleavage. This reaction utilizes starting oligonucleotides that bind to different parts of the template. The extension probe is 8 nucleotides in length and contains a phosphorothioate linkage between the 6th and 7th nucleotides from the 3' end of the probe. Nucleotides 2-6 were used as spacers to allow each reaction to identify multiple nucleotides spaced along the template. The complete sequence of the partial template is determined by performing multiple reactions in succession and appropriately combining the partial sequence information obtained from each reaction.

Figure 6A shows priming with the first starting oligonucleotide (referred to as primer in Figures 6A-6F) that hybridizes to the adapter sequence (referred to above as the binding region) in the template to provide an extendable duplex . Figures 6B-6D show several nucleotide identification cycles in which every 6 bases in the template are read. In FIG. 6B, the first extension probe whose 3' terminal nucleotide is complementary to the first unknown nucleotide in the template sequence binds to the template and connects to the extendable end of the primer. A label attached to the extension probe identifies the 3' terminal nucleotide of the probe as an A, thereby identifying the first unknown nucleotide of the template sequence as an A. Figure 6C shows the cleavage of the extension oligonucleotide with _AgNO3 at the phosphorothioate linkage and releases the part of the extension probe to which the label is attached. Figure 6D shows additional extension, ligation and cleavage cycles. Since the length of the spacer contained in the probe is 5 nucleotides, the sequencing reaction identifies every 6 nucleotides on the template.

After the desired number of cycles, the extended strand comprising the first starting oligonucleotide is removed, binding to a second starting oligonucleotide that binds to a portion of the binding region different from that to which the first starting oligonucleotide binds hybridize to the template. Figure 6E shows a second sequencing reaction, initiated with a second starting oligonucleotide, followed by several cycles of nucleotide identification. Figure 6F shows priming with a third starting oligonucleotide followed by several cycles of nucleotide identification. Extensions from the second starting oligonucleotide can be identified every 6 bases in a "reading frame" different from the nucleotides identified in the first sequencing reaction.

While phosphorothioate-linked extension probes are preferred in certain embodiments of the invention, a variety of other easy-cleavable linkages are also suitable. For example, many variations on O-P-O linkages found in naturally occurring nucleic acids are known (see eg, Micklefield, J. Curr. Med. Chem., 8:1157-1179, 2001). Any structure containing a P-O bond described therein can be modified to contain a scissile P-S bond. For example, NH-P-O bonds can be changed to NH-P-S bonds.

In some embodiments of the invention, the extension probes contain a priming residue that, when optionally modified with a modifier, renders the nucleic acid susceptible to cleavage by a cleavage agent or a combination thereof. In particular, the present inventors have discovered that enzymes involved in DNA repair are advantageous cleavage agents for carrying out methods for sequencing via successive cycles of extension, ligation, detection and cleavage. Typically, following optional DNA glycosylase modification, the presence of priming residues, such as damaged bases or abasic residues, in the extension probe renders the probe susceptible to cleavage by one or more DNA repair enzymes. Thus, extension probes containing ligation that are cleavage substrates for enzymes involved in DNA repair, such as AP endonuclease, are useful in the present invention. Extension probes containing residues that are substrates for modifications by enzymes involved in DNA repair, such as DNA glycosylase, where the modification renders the probe susceptible to cleavage by AP endonucleases are also particularly useful in the present invention. In some embodiments, the extension probe contains an abasic residue, ie, it lacks a purine or pyrimidine base. The linkage between the abasic residue and the adjacent nucleoside is readily cleaved by the AP endonuclease and is therefore an easy linkage. In certain embodiments of the invention, the abasic residue comprises 2' deoxyribose. In some embodiments, the extension probes comprise damaged bases. The damaged base is a substrate for an enzyme that removes the damaged base, such as DNA glycosylase. After removal of damaged bases, the resulting abasic residue and the linkage between adjacent nucleosides are easily cleaved by AP endonuclease, and thus are considered as easy-cleavable linkages in the present invention.

A number of different AP endonucleases can be used as cleavage reagents in the present invention. Two main types of AP endonucleases are distinguished according to the mechanism by which they cleave junctions adjacent to abasic residues. Class I AP endonucleases such as Escherichia coli endonuclease III (Endo III) and endonuclease VIII (Endo VIII) and the human homologues hNTH1, NEIL1, NEIL2 and NEIL3 cleave the 3' side of AP residues AP lyase of DNA, this cleavage produces a 5' portion containing a 3' terminal phosphate and a 3' portion carrying a 5' terminal phosphate. Class II AP endonucleases such as E. coli endonuclease IV (Endo IV) and exonuclease III (Exo III) cleave the DNA 5' to the AP site, and this cleavage produces 3' on the ends of the resulting fragments. 'OH and 5' deoxyribose phosphate moieties. See, for example, Doublie, S. et al., Proc. Natl. Acad Sci. 101(28), 10284-10289, 2004; Haltiwanger, B.M. et al., Biochem J., 345, 85-89, 2000; Levin, J. and Demple , B., Nucl.Acids.Res., 18(17), 1990; and references to all of the above for further discussion of the various Class I and Class II AP endonucleases and their removal of damaged bases on DNA and/or conditions that cleave DNA containing abasic residues. Those of ordinary skill in the art will appreciate that various homologues of these enzymes exist in other organisms, such as yeast, and can be used in the present invention.

Certain enzymes are bifunctional in that they have both glycosylase activity to remove damaged bases to generate AP residues and have been shown to cleave the phosphodiester backbone 3' to the AP site generated by glycosylase activity lyase activity. Thus, these dual activity enzymes are AP endonuclease and DNA glycosylase. For example, Endo VIII acts as N-glycosylase and AP-lyase. N-glycosylase activity releases damaged pyrimidines from double-stranded DNA, generating apurinic bases (AP sites). The AP-lyase activity cleaves the 3' and 5' ends of the AP site, generating a 5' phosphate and a 3' phosphate. Damaged bases recognized and excised by endonuclease VIII include urea, 5,6-dihydroxythymine, thymidinediol, 5-hydroxy-5-methylhydantoin, uracildiol, 6-hydroxy - 5,6-dihydrothymidine and methylpropanol diureide. See, for example, Dizdaroglu, M. et al., Biochemistry, 32, 12105-12111, 1993 and Hatahet, Z. et al., J Biol. Chem., 269, 18814-18820, 1994; Jiang, D. et al., J. Biol. Chem. ., 272(51), 32220-32229, 1997; Jiang, D. et al., J. Bact, 179(11), 3773-3782, 1997.

Fpg (formamidopyrimidine [fapy]-DNA glycosylase) (also known as 8-oxoguanine DNA glycosylase) is also used as N-glycosylase and AP-lyase. N-glycosylase activity can release damaged purines from double-stranded DNA, resulting in apurinic bases (AP sites). The AP-lyase activity cleaves the 3' and 5' ends of the AP site, thereby removing the AP site and creating a 1 base gap. Some of the damaged bases recognized and removed by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxadenine, fapy-guanine, methyl-fapy-guanine, fapy - adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine and 5-hydroxy-uracil. See, for example, Tchou, J. et al., J. Biol. Chem., 269, 15318-15324, 1994; Hatahet, Z. et al., J. Biol. Chem., 269, 18814-18820, 1994; Boiteux, S. et al. , EMBO J., 5, 3177-3183, 1987; Jiang, D. et al., J.Biol.Chem., 272(51), 32220-32229, 1997; Jiang, D. et al., J.Bact, 179(11 ), 3773-3782, 1997.

A number of DNA glycosylases and AP endonucleases are commercially available from, e.g., New England Biolabs, Ipswich, MA.

In some embodiments of the invention, the above-described sequencing methods or sequencing methods AB (see below) for phosphorothioate-linked extension probes employ an extension probe containing a site that is a substrate for AP endonuclease cleavage. Needle. In any of these methods, after ligation of the extension probe to the growing nucleic acid strand, the extension probe is cleaved with AP endonuclease to remove the labeled portion of the probe.

Depending on the specific AP endonuclease, and depending on whether sequencing is performed in the 3'→5' or 5'→3' orientation, it may be necessary or desirable to treat the extended duplex with a polynucleotide kinase or phosphatase after cleavage in order to An extendable probe end is generated on the extension duplex (see Figures 5A and 5B for a description of the extendable probe end). Thus, in certain methods of the invention, polynucleotide kinases or phosphatases are used to generate extendable ends. Those of ordinary skill in the art will appreciate that suitable buffers for the various enzymes may be employed, and that additional washing steps may be included to remove enzymes and provide suitable conditions for subsequent steps in the method.

In other embodiments, the extension probes contain damaged bases that are substrates for removal by DNA glycosylases. Various cytotoxic and mutagenic DNA bases are removed by different DNA glycosylases, thereby initiating the base excision repair pathway after DNA damage (Krokan, H.E. et al., Biochem J, 325(Pt 1): 1-16, 1997). DNA glycosylase cleaves the N-glycosyl bond between the damaged base and the deoxyribose sugar, thereby releasing the free base and creating an apurinic/apyrimidinic (AP) site. In some embodiments, the extension probes contain uracil residues that are removed by uracil-DNA glycosylase (UDG). UDGs are found in all living organisms studied to date, and a large number of such enzymes are known in the art and can be used in the present invention (Frederica et al., Biochemistry, 29, 2353-2537, 1990; Krokan, supra). For example, mammalian cells contain at least 4 types of UDGs: mitochondrial UNG1 and nuclear UNG2, SMUG1, TDG and MBD4 (Krokan et al., Oncogene, 21, 8935-8948, 2002). UNG1 and UNG2 belong to a highly conserved family represented by Escherichia coli Ung.

In embodiments where the extension probe contains a damaged base, after ligation of the extension probe to the end of the extendable probe, the extension duplex is contacted with a glycosylase capable of removing the damaged base, thereby generating an abasic residue . Extension probes containing damaged bases removed by glycosylases are considered "easily modified to contain easy cleavage linkages". The extended duplex is then exposed to AP endonuclease, which cleaves linkages between abasic residues and adjacent nucleosides, as described above. In certain embodiments of the invention, both reactions are performed with a dual activity enzyme that is a DNA glycosylase and an AP endonuclease. In some embodiments, the extended duplex containing the damaged base is contacted with a DNA glycosylase and an AP endonuclease. In various embodiments of the present invention, these enzymes can be used in combination or sequentially (ie, endonuclease is used after glycosylase).

In some embodiments of the invention, the priming residue contained in the extension probe is deoxyinosine. As mentioned above, Escherichia coli endonuclease V (Endo V), also known as deoxyinosine 3' endonuclease and its homologues can detect the second phosphodiester on the 3' side of the deoxyinosine residue Cleaves deoxyinosine-containing nucleic acids at the bond, producing 3'OH and 5' phosphate ends. Therefore, this bond serves as an easy cleavable link for the extension probe. Endo V and its cleavage properties are known in the art (Yao, M. and Kow Y.W., J Biol. Chem., 271, 30672-30673 (1996); Yao, M. and Kow Y.W., J Biol. Chem., 270, 28609-28616 (1995); He, B et al., MiitatRes., 459, 109-114 (2000).In addition to deoxyinosine, Endo V also recognizes deoxyuridine, deoxyxanthine nucleoside and deoxyoxanosine (Hitchcock, T. etc., Nuc.Acids Res., 32(13), 32(13) (2004).Mammalian homologs such as mEndo V also have cleavage activity (Moe, A. etc., Nuc.Acids Res., 31(14) , 3893-3900 (2004). Although Endo V is the preferred cutting agent for probes containing deoxyinosine, other cleavage reagents can also be used to cleave probes containing deoxyinosine. For example, as damaged bases, hypoxanthin Purines can be removed by suitable DNA glycosylases, resulting in extended probes containing abasic residues which are subsequently cleaved by endonucleases.

It will be appreciated that if deoxyinosine is used as the priming residue, it may be desirable to avoid the use of deoxyinosine elsewhere in the probe, particularly at positions that would be attached to the end of the extendable probe and the priming residue. Thus, nucleosides other than deoxyinosine may be used if the probe contains one or more universal bases. It will also be understood that when a priming residue that makes a nucleic acid containing a priming residue susceptible to cleavage by a particular cleavage agent is used to extend a probe, it may be desirable to avoid the presence of probes that initiate cleavage by the same cleavage agent (or that would be used together with the extension probe). Other residues are included in other probes used in sequencing reactions).

The present invention includes the use of any enzyme that cleaves a nucleic acid containing a priming residue. Additional enzymes can be identified by studying catalogs of enzyme suppliers such as New England Biolabs(R), Inc. The New England Biolabs Catalog, 2005 Edition (New England Biolabs, Ipswich, MA 01938-2723) is incorporated herein by reference, and the invention contemplates the use of any enzyme disclosed herein or a homolog of such an enzyme that cleaves a nucleic acid containing a priming residue. things. Other enzymes employed include, for example, hOGG1 and its homologues (Radicella, JP et al., Proc Natl AcadSci USA, 94(15):8010-5, 1997).

Methods for the synthesis of oligonucleotides containing priming residues such as damaged bases, abasic residues, and the like are known in the art. Methods for the synthesis of oligonucleotides containing sites that are substrates for AP endonucleases, such as oligonucleotides containing abasic residues, are known in the art and are generally suitable for automated solid phase oligonucleotide synthesis. In some embodiments, oligonucleotides are synthesized that contain uridine at the desired position of the abasic residue. The oligonucleotide is then treated with an enzyme such as UDG which removes uracil to generate an abasic residue wherever the uridine is present in the oligonucleotide.

In some embodiments of the invention, the oligonucleotide probes contain disaccharide nucleosides as described by Nauwelaerts, K. et al., Nuc. Acids. Res., 31(23), 2003. After ligation, the extended duplex is cleaved with periodate ( _NaIO4 ), followed by treatment with _a base such as NaOH to remove the label, yielding free 3'OH and P5- _OPO3H2 groups. Depending on whether sequencing is performed in the 3'→5' or 5'→3' orientation, it may be necessary or desirable to treat the extended duplex with a polynucleotide kinase or phosphatase to generate extendable ends. Thus, in certain methods of the invention, polynucleotide kinases or phosphatases are used to generate extendable ends.

Disaccharide nucleoside-containing polynucleotides are considered to contain abasic residues. For example, a polynucleotide with a ribose residue interposed between the 3'OH of one nucleotide and the 5' phosphate group of the next nucleotide is said to contain abasic residues.

cap

In some cases, not all probes with extendable ends successfully participated in the ligation reactions of each extension, ligation, and cleavage cycle. It will be appreciated that if such probes are involved in subsequent cycles, the accuracy of each nucleotide identification step will progressively decrease. Although the inventors have demonstrated that high-efficiency ligation can be achieved using extension probes containing phosphorothioate linkages, in certain embodiments of the invention a capping step is included to prevent extensible ends that do not undergo ligation Participate in subsequent cycles. When sequencing in the 5'→3' direction with extension probes containing 3'-O-P-S-5' phosphorothioate linkages, e.g., after a ligation or detection step, a DNA polymerase and a non-extendable moiety, such as a chain-terminating core, can be used. Capping is performed by extending the unlinked extendable terminus with nucleotides such as dideoxynucleotides or nucleotides linked to blocking moieties. For sequencing in the 3'→5' direction with extension probes containing 3'-S-P-O-5' phosphorothioate linkages, for example, capping can be done by treating the template with phosphatase after ligation or detection. Other capping methods can also be used.

H. Sequencing with Oligonucleotide Probe Families

In the sequencing methods described above, collectively referred to as "Method A," one or more nuclei attached to the label of any particular extension probe and to the proximal end of the probe (i.e., the end attached to the extensible probe terminus of the extension duplex) There is a direct and known correspondence between classes of nucleotides. Thus, identification of the label of the newly ligated extension probe is sufficient to identify one or more nucleotides in the template. The present invention provides other sequencing methods employing different methods for nucleotide identification, collectively referred to as "Methods AB", also comprising sequential cycles of extension, ligation and (preferably) cleavage.

The sequencing method AB provided by the present invention employs a collection of at least two differentially labeled oligonucleotide probe families. Assign each probe family a name according to its label, eg "red", "blue", "yellow", "green". Extension is initiated from the duplex formed by the starting oligonucleotide and the template as described above. The starting oligonucleotide is extended by ligation of the oligonucleotide probe to the end of the starting oligonucleotide to form an extension duplex, and the extension is then repeated through successive ligation cycles. The probe contains a non-extendable portion at its terminal position (opposite the end of the probe to the nucleotides attached to the growing duplex nucleic acid strand) so that only one extension occurs in a single cycle of extending the duplex. In each cycle, labels on or attached to successfully ligated probes are detected and non-extendable portions are removed or modified to generate extendable ends. Detection of the label enables the determination of the name of the probe family to which the probe belongs.

Successive cycles of extension, concatenation, and detection produce an ordered list of tag names. These labels correspond to the probe families to which well-ligated probes hybridize to the template at consecutive positions. After ligation, the proximal position of the probe is relative to a different nucleotide in the template. Therefore, there is a corresponding relationship between the sequence of probe family names and the sequence of nucleotides in the template.

In embodiments of the invention where the easy-to-cleavage linkage is located between the proximal nucleoside and the adjacent nucleoside of the extension probe, the probe can be obtained by successive cycles of extension, ligation, detection and cleavage starting from one starting oligonucleotide. An ordered list of needle family names as each cycle extends the extended oligonucleotide probe by one nucleotide. If the easy junction is between two other nucleosides, assemble an ordered list of probe family names from the results obtained from multiple sequencing reactions in which the starting oligo hybridized to a different position in the binding reaction region. Nucleotides, as described in Sequencing Method A.

Knowing which probe family a newly ligated probe belongs to is not, by itself, sufficient to determine the nucleotide species in the template. However, determining the probe family name eliminates the possibility of certain combinations of nucleotides as the sequence of at least a portion of the probe, giving at least two possible species for each nucleotide. Thus, knowing the probe family name in the absence of other information gives at least two possible template nucleotide species at relative positions to the nucleotides of the newly ligated probe. Thus, any one cycle of extension, ligation, detection (and optional cleavage) cannot by itself identify any nucleotide in the template. However, it provides sequence information by eliminating one or more possible sequences of the template. In certain embodiments of the invention, template sequences can still be determined by appropriate design of probes and probe families as described below. In some embodiments of the present invention, the sequencing method AB comprises two stages: the first stage obtains an ordered list of probe family names, and the second stage decodes the ordered list to determine the template sequence.

Unless otherwise stated, sequencing methods A and AB generally employ similar methods to synthesize probes, prepare templates and perform the steps of extension, ligation, cleavage and detection.

Characterization of Oligonucleotide Extension Probes and Probe Families for Sequencing Methods AB

The probe families used in the sequencing method AB are characterized in that each probe family includes a plurality of labeled oligonucleotide probes of different sequences, and at each position in said sequence, one probe family includes the position At least two probes with different bases. Probes within each probe family contain the same label. Preferably, the probe comprises an easy internucleoside linkage. The easy-cut junction can be located anywhere in the probe. One end of the probe preferably contains a ligase non-extendable portion. The probe is preferably labeled at a position between the easy junction and the non-extendable portion of the ligase so that cleavage of the easy junction after ligation of the probe to the end of the extendable probe results in an unlabeled portion attached to the end of the extendable probe and the marked part that is no longer connected to the unmarked part.

The probes in each probe family preferably contain at least j nucleosides X, where j is at least 2, and each X is at least 2-fold degenerate in the probes of each probe family. The probes of each probe family also contain at least k nucleosides N, where k is at least 2, where N represents any nucleoside. Usually, j+k is equal to or less than 100, generally less than or equal to 30. Nucleoside X can be located anywhere in the probe. Nucleoside X does not have to be in contiguous positions. Similarly, nucleosides N need not be in contiguous positions. In other words, nucleosides X and N can be interspersed. Nucleoside X may be considered to have a 5'→3' sequence, although the nucleosides are not necessarily contiguous. For example, the nucleoside X of a probe of the structure X _A NX _G NNX _CN is considered to contain the sequence AGC. Similarly, nucleoside N can be considered to contain sequence.

The nucleosides X may be the same or different, but cannot be independently selected, ie the identity of each X is limited by the identity of one or more other nucleosides X in the probe. Thus, typically only certain combinations of nucleosides X will be present in a particular probe and probes of a particular probe family. In other words, in each probe, the sequence of nucleoside X can represent only a subset of all possible sequences of length j. Thus, the species of one or more nucleotides in X limits the possible species of one or more other nucleosides.

The nucleoside N is preferably independently selected and may be A, G, C or T (or optionally a nucleoside of reduced degeneracy). The sequence of nucleoside N preferably represents all possible sequences of length k, except that one or more N may be nucleosides of reduced degeneracy. Therefore, the probe contains two parts, where the part consisting of nucleoside N is called the unrestricted part and the part consisting of nucleoside X is called the restricted part. As noted above, the moieties need not be contiguous nucleosides. Probes containing restricted and unrestricted moieties are referred to herein as partially defined probes. The one or more nucleosides of the restricted portion are preferably located at the proximal end of the probe, i.e. the end containing the nucleosides that will be attached to the end of the extendable probe, which may be an oligonucleotide probe in various embodiments of the invention 5' or 3' end.

Since the constrained portion of any oligonucleotide probe can only have certain sequences, knowing the identity of one or more nucleosides of the constrained portion of the probe can provide information about one or more other nucleosides. This information may or may not be sufficient to accurately identify the one or more other nucleosides, but it is sufficient to eliminate one or more possibilities for the restricted portion of the one or more other nucleoside species. In certain preferred embodiments of Sequencing Methods AB, knowledge of the identity of one nucleoside of the restricted portion of the probe is sufficient to accurately identify each of the other nucleosides of the restricted portion, ie, to determine the identity and order of the nucleosides containing the restricted portion.

The most proximal nucleoside in the extension probe complementary to the template is ligated to the extendable end of the starting oligonucleotide (in the first cycle of extension, ligation, and detection) and the extended oligonucleotide as described in the sequencing method above. The extendable end of the acid probe (in subsequent extension, ligation and detection cycles). The detection determines the name of the probe family to which the newly ligated probe belongs. Since each position of the restricted portion of the probe is at least 2-fold degenerate, the probe family name itself does not identify any nucleotides of the restricted portion. However, since the sequence of the restricted part is one sequence in the subset of all possible sequences of length j, identifying the probe family does not eliminate some possible restricted part sequences. The restricted portion of the probe constitutes its sequence determination portion. Thus, eliminating one or more possibilities of one or more nucleotide species of the restricted portion of the probe by identifying the probe family to which the probe belongs eliminates one or more of the nucleotide species of the template to which the extension probe hybridizes. Many possibilities. In a preferred embodiment of the invention, partially defined probes contain an easy linkage between any two nucleosides.

In certain embodiments, partially defined probes have the general formula (X) _j (N) _k , where X represents a nucleoside, and (X) _j is at least 2-fold degenerate at each position, so X can be Any of at least 2 nucleosides with different base pairing specificities, N represents any nucleoside, j is at least 2, k is 1-100, at least one N or X other than the X at the end of the probe contains possible detection part. Preferably, (N) _k is independently 4-fold degenerate at each position, so that (N) _k in each probe represents all possible sequences of length k, except that one or more positions in (N) _k may Occupied by nucleotides of reduced degeneracy. The nucleosides in (X) _j can be the same or different, but cannot be chosen independently. In other words, in each probe, (X) _j can only represent a subset of all possible sequences of length j. Thus, the species of one or more nucleotides in (X) _j limits the possible species of one or more other nucleosides. Therefore, the probe contains two parts, where (N) _k is the unrestricted part and (X) _j is the restricted part.

In certain preferred embodiments of the invention, the partially defined probe has the structure 5'-(X) _j ( _{N)kNB *} ^-3 ' or 3'-(X) _j ( _N ) _kNB ^* - 5', where N represents any nucleoside, N _B represents the part that cannot be extended by ligase, * represents the detectable part, (X) _j is the restricted part of the probe that is at least 2 times degenerate at each position, (X) The nucleosides in _j can be the same or different but cannot be independently selected, at least one internucleoside linkage is an easy cleavage linkage, j is at least 2, and k is 1-100, with the proviso that a detectable moiety may be present in the alternative _NB , or on any nucleoside N other than _NB or on X other than X at the end of the probe. An easy-cleavable linkage can be located between two nucleosides in (X) _j , between the most distal nucleotide in (X) _j and the most proximal nucleoside in (N) _k , or a nucleoside within (N) _k Between or between the terminal nucleosides of (N) _k and _NB . The easy-cleavable linkage is preferably a phosphorothioate linkage.

In other more preferred embodiments of the invention, the probe _has the structure 5'-(XY)(N) _kNB ^* -3' or 3'-(XY)( _N ) _kNB ^* -5', wherein N represents any nucleoside, N _B represents the part that cannot be extended by ligase, * represents the detectable part, XY is the restricted part of the probe, where X and Y represent the same or different nucleosides that cannot be independently selected, X and Y are at least 2-fold degenerate, at least one internucleoside linkage is a scissile linkage, and k is 1-100, with the proviso that the detectable moiety can be present in place of _NB , or any nucleotide N other than _NB or on an X other than an X at the end of the probe. The easy-cleavable linkage is preferably a phosphorothioate linkage. Probes with the structure 5'-(XY)(N) _kNB ^* -3' _can be used for sequencing in the 5'→3' direction. Probes with the structure 3'- ₍ XY)(N) _kNB ^* -5' can be used for sequencing in the 3'→5' direction.

The structures of some preferred probes are described in more detail below. For sequencing in the 5' → 3' direction, a partially defined probe with the structure 5'-OPO-(X) _j (N) _k -OPS-(N) _i N _B ^* -3' is used, where N stands for any nucleus glycoside, N _B represents the part that cannot be extended by ligase, ^* represents the detectable part, (X) _j is the restricted part of the probe that is at least 2 times degenerate at each position, and the nucleosides in (X) _j can be the same or different, but not independently selected, j is at least 2, (k+i) is 1-100, k is 1-100, i is 0-99, with the proviso that the detectable moiety may be present in the alternative _NB , or On any nucleoside of (N) _j except _NB . In certain embodiments of the invention, (X) _j is (XY), wherein X and Y are at least 2-fold degenerate and represent identical or different, but not independently selectable, nucleotides. In certain embodiments of the invention, i is O.

Other preferred probes for sequencing in the 5'→3' direction have the structure 5'-OPO-(X) _j -OPS-(N) _iNB ^* -3', where N represents any _nucleoside and _NB represents The part that cannot be extended by ligase, * represents the detectable part, (X) _j is the restricted part of the probe that is at least 2 times degenerate at each position, the nucleotides in (X) _j can be the same or different, but Not independently selectable, j is at least 2 and i is 1-100, with the proviso that the detectable moiety may be present on any nucleoside that replaces _NB or (N) _i in addition to _NB . In certain embodiments of the invention, (X) _j is (XY), wherein positions X and Y are at least 2-fold degenerate, and X and Y represent identical or different nucleosides that cannot be independently selected. Another preferred probe for sequencing in the 5'→3 _' direction has the structure 5'-OPO-(X) _j -OPS-(X) _k (N) _iNB ^* -3', where N represents any nuclear Glycoside, N _B represents the part that cannot be extended by ligase, * represents the detectable part, (X) _j -OPS-(X) _k is the restricted part of the probe that is at least 2 times degenerate at each position, (X) The position of _j -OPS-(X) _k is at least 2 times degenerate, which can be the same or different, but cannot be selected independently, both j and k are at least 1, and (j+k) is at least 2 (such as 2, 3, 4 or 5), i is 1-100, with the proviso that the detectable moiety may be present on any nucleoside of (N) _i that replaces _NB or other than _NB . In certain embodiments of the invention, j and k are both 1.

For sequencing in the 3' → 5' direction, a partially defined probe with the structure 5'-N _B ^* (N) _i -SPO-(N) _k -OPO-(X) _j -3' is used, where N stands for any Nucleosides, N _B represents the part that cannot be extended by ligase, * represents the detectable part, (X) _j is the restricted part of the probe that is at least 2 times degenerate at each position, and the nucleosides in (X) _j can same or different, but not independently selected, j is at least 2, (k+i) is 1-100, k is 1-100, i is 0-99, with the proviso that a detectable moiety may be present in the alternative _NB , Or on any nucleoside of (N) _i other than _NB . In certain embodiments of the invention, (X) _j is (XY), wherein X and Y are at least 2-fold degenerate and represent the same or different nucleosides that cannot be independently selected. In some embodiments of the invention, i is 0.

Other preferred probes for sequencing in the 3'→5' direction have the structure 5'- _NB ^* (N) _i -SPO-(X) _j -3', where N stands for any nucleoside and _NB stands for ligase Non-extendable part, * represents the detectable part, (X) _j is the restricted part of the probe that is at least 2-fold degenerate at each position, the nucleosides in (X) _j can be the same or different, but cannot be independently selected , j is at least 2, i is 1-100, with the proviso that the detectable moiety may be present on any nucleoside that replaces _NB or (N) _i in addition to _NB . In certain embodiments of the invention, (X) _j is (XY), wherein X and Y are at least 2-fold degenerate and represent the same or different nucleosides that cannot be independently selected. In certain embodiments of the invention, j is 2-5, such as 2, 3, 4 or 5, in any moiety-defining probe.

Another preferred probe for sequencing in the 3'→5' direction has the structure 5'- _NB ^* (N) _i -SPO-(X) _k -OPO-(X) _j -3', where N represents any Nucleosides, N _B represents the part that cannot be extended by ligase, * represents the detectable part, -(X) _k -OPO-(X) _j is the restricted part of the probe that is at least 2 times degenerate at each position, - The nucleosides in (X) _k -OPO-(X) _j can be the same or different, but cannot be independently selected, both j and k are at least 1, and (j+k) is at least 2 (such as 2, 3, 4 or 5 ), i is 1-100, with the proviso that the detectable moiety may be present on any nucleoside of (N) _i that replaces _NB , or in addition to _NB . In some embodiments, j=1 and k=1.

In embodiments of the invention where the easy cleavable linkage is between the most proximal nucleoside of (X) _j and the next most proximal nucleoside of (X) _j , it can be achieved by successive extensions from one starting oligonucleotide, ligation The , detection and cleavage cycles obtain an ordered list of probe family names, as each cycle extends the extended oligonucleotide probe by one nucleotide. In embodiments of the invention where the easy link is between two other nucleosides, an ordered list of probe family names is assembled from the results obtained from multiple sequencing reactions in which hybridization to different binding reaction regions is used. The starting oligonucleotide for the position, as described in Sequencing Method A.

It will be appreciated that probes with a wide variety of structures other than those described above may be used in Sequencing Methods AB. For example, a probe may have a structure such as XNY(N) _k where constrained nucleosides X and Y are not adjacent, or XIY(N) _k where I is a universal base. (N) _k X(N) _l , (N) _i X(N) _j Y(N) _k Z(N) _l , (N) _i X(N) _j YIZ(N) _l and (N) _i X (N) _j Y(N) _k Z(I) _l represent other possibilities. As described above for the probes, these probes contain an easily cleavable, detectable portion with a ligase non-extendable portion at one end. Preferably, the probe does not comprise a detectable moiety of nucleotides attached to the opposite end of the probe from the portion of the probe that cannot be extended by the ligase. Probe families comprising probes having any of these structures and others are such that each probe family comprises a plurality of labeled oligonucleotide probes differing in sequence, and at each position in said sequence, one probe A family includes the criterion that at least two probes differ in base at this position. The total number of nucleosides in each probe is preferably 100 or less, such as 30 or less.

Encodes a family of oligonucleotide extension probes.

The sequencing methods of the invention utilize encoded probe families. "Coding" refers to the scheme of associating a specific label with probes containing a portion of one of a defined set of sequences so that probes containing a portion of a member of a defined set of sequences are labeled with that label. Typically, the encoding associates each of multiple distinguishable labels with one or more probes, such that each distinguishable label is associated with a different set of probes, and each probe (which may contain a detectable moiety) is labeled with only one label. The combination). Preferably, the probes of each probe set each contain a portion having the same defined sequence set member sequence. The moiety may be one nucleoside or multiple nucleosides in length, such as 2, 3, 4, 5 or more nucleosides. The length of this portion may constitute only a small fraction of the overall length of the probe, or may constitute the entire probe. The set of defined sequences may contain only one sequence or any number of different sequences, depending on the length of the portion. For example, if the moiety is a nucleotide, then the set of defined sequences may contain up to 4 elements (A, G, C, T). If the portion is two nucleotides in length, the defined sequence group can contain up to 16 elements (AA, AG, AC, AT, GA, GG, GC, GT, CA, CG, CC, CT, TA, TG , TC, TT). Typically, the set of defined sequences will contain fewer elements than the total number of possible sequences, and more than one set of defined sequences will be used for encoding.

Sequencing Method A described herein generally utilizes simply coded probe sets in which the proximal nucleoside of the probe (ie, the nucleoside attached to the end of the extendable probe) corresponds directly to the label species. The proximal nucleoside is complementary to the template nucleotide to which it hybridizes, thus the identity of the proximal nucleoside in the newly ligated probe determines the identity of the template nucleotide at the opposite position in the extending duplex. In a general sense, probes for use in the other sequencing methods described herein have the structure X(N) _k , where X is a proximal nucleoside and each nucleoside N is 4-fold degenerate such that the oligos that make up the probe All possible sequences of length k are represented in the library of nucleotide probe molecules. Thus, for example, some oligonucleotide probe molecules contain A at position k=1, others contain G at position k=1, others contain C at position k=1, others at position k=1 T is contained at the position, and the situation is similar for other positions k, wherein it is considered that the nucleoside adjacent to X in (N) _k occupies position k=1; it is considered that the next nucleoside in (N) _k occupies position k=2, etc. . However, in any given oligonucleotide probe, X represents only one base pairing specificity, which generally corresponds to a specific nucleoside species, such as A, G, C or T. Therefore, X in the library of probe molecules constituting specific probes is generally unified as A, G, C or T. Figure 2 shows a suitable code for a probe of structure X(N) _k . According to this coding, the label "red" is assigned to probes with X=C; the label "yellow" is assigned to probes with X=A; the label "green" is assigned to probes with X=G; The label "blue" is assigned to probes where X=T. Thus, there is a one-to-one correspondence between the sequence-determining portion of a probe and its label.

It will be appreciated that the above-described method of newly ligated extension probes having a label species corresponding to the species of the most-most nucleoside in the extension probe can be extended to include label species corresponding not only to the species of the most-most nucleoside in the extension probe, but also to the species of the most-most nucleoside in the extension probe. The sequence of the most proximal 2 or more nucleotides in the probe encodes the species of multiple nucleotides in the template in one cycle of extension, ligation and detection (typically followed by cleavage). However, this encoding still associates the label with one sequence of the oligonucleotide extension probe in order to identify the species of the complementary nucleotide at the opposite position in the template. As noted above, to identify two nucleotides in one cycle, 16 different oligonucleotide probes are required, each containing a corresponding label (ie, 16 distinguishable labels).

Sequencing Methods AB utilizes another method for associating labels with probes. The same label is assigned to multiple probes having different sequence-determining portions, without a one-to-one correspondence between label species and sequences of the sequence-determining portions of the probes. The probe is a partially constrained probe, the constrained portion of the probe being its sequence determining portion. Thus, the same label is assigned to a plurality of different probes each containing a constrained portion different in sequence, where the sequence is one sequence of a defined set of sequences. As noted above, probes containing the same label form a "probe family". The method employs a plurality of such probe families, each comprising a plurality of probes comprising constrained portions differing in sequence, wherein the sequence is one sequence of a defined set of sequences.

A plurality of probe families is referred to as a probe family "set". The probes of a probe family in the set of probe families are labeled with a label that is distinguishable from the labels used to label other probe families of the set. Each probe family preferably has its own set of defined sequences. Preferably, the lengths of the restricted parts of the probes in each probe family are the same, and preferably, the lengths of the restricted parts of the probe families in the probe family set are the same. Preferably, the combination of the defined sequence set of probe families in the probe family collection includes all possible sequences of the restricted partial length. Preferably, the collection of probe families comprises or consists of 4 differentially labeled probe families. Preferably, the restricted portion of the probe is 2 nucleosides in length.

Collections of various differentially coded, differentially labeled probe families will meet the above criteria and can be used to practice the methods of the invention. However, certain probe family collections are preferred. An exemplary code for a preferred set of four differentially labeled probe families consisting of partially defined probes is shown in Figure 25A. As shown in Figure 25A, the restricted portion consists of the two most 3' nucleosides in the probe. Probe families are labeled "red", "yellow", "green" and "blue". The probes of each probe family include a restricted portion whose sequence is one of a set of defined sequences, the set of defined sequences being different for each probe family. For example, starting from the 3' end of each sequence considered proximal to the probe, the "red" probe family is {CT, AG, GA, TC}; the defined sequence set for the "yellow" probe family is {CC, AT , GG, TA}; the defined sequence set for the "green" probe family is {CA, AC, GT, TG}; the defined sequence set for the "blue" probe family is {CG, AA, GC, TT}. It is a preferred feature that each defined sequence group does not contain any members present in other groups. Furthermore, the combination of a defined sequence set of probe families in the probe family collection includes all possible sequences of length 2, ie all possible dinucleosides. Another feature (preferable but not necessary) of this collection of probe families is that each position of the restricted part of the probe is 4-fold degenerate, ie each position can be occupied by A, G, C or T. Another feature (preferable but not necessary) of this collection of probe families is that, within each defined set of sequences, only one sequence has any particular nucleotide at any position, such as the most proximal position or any other position. It is particularly preferred but not necessary that, if the most proximal nucleoside is considered to be position 1, within each defined sequence group only one sequence has any particular nucleoside at position 2 or higher within the restricted portion. For example, in the defined set of sequences for the red probe family, only one sequence has a T at position 2; only one sequence has a G at position 2; only one sequence has an A at position 2; only one sequence has an A at position 2 have C.

For any of the specific codes shown in Figure 25A, knowledge of the identity of one or more nucleosides of a restricted portion of a probe in a probe family can provide information about the other nucleotides of the restricted portion of the probe. information. In the most general sense, knowledge of one or more nucleoside species in the restricted portion of a probe family probe provides sufficient information to rule out one or more possible nucleoside species at another position, because The defined sequence set for the probe family does not include the sequence for that nucleoside species at that position. In general, knowing the species of one or more nucleosides of a constrained portion of a probe family probe provides sufficient information to rule out multiple nucleosides such as one or more possible species of each of the other nucleosides. In preferred coding, knowledge of the identity of one or more nucleosides of a constrained portion of a probe family probe excludes all but one possibility for each of the other nucleosides in the probe. For example, in the case of the family of encoded probes shown in Figure 25A, if the probe is known to be a member of the red family, and if the most proximal nucleoside is also known to be a C, then the adjacent nucleoside must be a T. Similarly, if the probe is known to be a member of the Green family, and if the most proximal nucleoside is also known to be G, then the adjacent nucleoside must be T. Thus, knowing the species of one nucleoside of the restricted portion is sufficient to rule out all but one possibility of the other nucleosides, thus completely identifying the species of the other nucleosides. But without knowing the identity of at least one nucleoside in the restricted portion of the probe, no information can be obtained on the identity of any particular nucleoside in the probe based solely on the name of the probe family to which it belongs, because each position of the restricted portion The nucleoside on can be A, G, C or T. Figure 25B shows preferred probe family collections (upper panel) and cycles of ligation, detection and cleavage (lower panel) using sequencing method AB.

The present inventors designed 24 probe family sets containing a restricted portion that is 2 nucleosides in length and has the favorable characteristics of the probe family set shown in Figure 25A. These probe families are maximally informative because knowing the name of the probe family to which the probe belongs, and knowing the species of one nucleoside in the probe, is sufficient to accurately identify other nucleosides in the restricted portion. This applies to all probes and to all nucleosides of each restricted moiety. The respective coding schemes of the 24 preferred probe family sets are shown in Table 1. Table 1 assigns coded IDs 1-24 to each probe family set. Each code defines a restricted portion of the collection of preferred probe families of general structure (XY)N _k for sequencing method AB, and thus the collection itself. In Table 1, a value of 1 in the column below "Code ID" indicates that, according to this code, the probes containing nucleosides X and Y as shown in the first and second columns, respectively, are assigned to the first probe family; ( ii) A value of 2 in the column below "Code ID" indicates that, according to this code, the probes containing nucleosides X and Y as shown in the first and second columns, respectively, are assigned to the second probe family; (iii)" A value of 3 in the column below "Code ID" indicates that, according to this code, probes containing nucleosides X and Y, respectively, as shown in the first and second columns, are assigned to the third probe family; and (iv) "Code ID A value of 4 in the lower column indicates that, according to this code, the probes containing nucleosides X and Y as shown in the first and second columns, respectively, are assigned to the fourth probe family. Values 1, 2, 3, and 4 each represent a flag. For example, code 9 defines the set of probe families shown in Figure 25A, where 1 represents blue, 2 represents green, 3 represents red, and 4 represents yellow. It should be understood that the assignment of values to the flags is arbitrary, eg 1 could equally represent green, red or yellow. Changing the association between the values 1, 2, 3, and 4 and the markers does not change the probe sets within each probe family, only a different marker is associated with each probe family.

Table 1: Oligonucleotide probe family codes

Encoded ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 twenty one twenty two twenty three twenty four x Y A A 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 C A 2 4 3 2 2 4 3 2 2 3 4 3 2 3 3 4 2 3 4 4 2 4 3 4 G A 4 3 2 3 3 2 4 4 3 2 2 4 4 2 4 3 4 2 3 2 3 2 4 3 T A 3 2 4 4 4 3 2 3 4 4 3 2 3 4 2 2 3 4 2 3 4 3 2 2 A C 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 C C 1 1 1 1 1 1 1 1 4 4 3 4 4 4 4 3 3 4 3 3 3 3 4 3 G C 3 4 4 4 4 3 3 3 1 1 1 1 3 3 3 4 1 1 1 1 4 4 3 4 T C 4 3 3 3 3 4 4 4 3 3 4 3 1 1 1 1 4 3 4 4 1 1 1 1 A G 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 C G 4 2 4 4 4 2 4 4 1 1 1 1 1 1 1 1 4 2 2 2 4 2 2 2 G G 1 1 1 1 2 4 2 2 4 4 4 2 2 4 2 2 2 4 4 4 1 1 1 1 T G 2 4 2 2 1 1 1 1 2 2 2 4 4 2 4 4 1 1 1 1 2 4 4 4 A T 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 C T 3 3 2 3 3 3 2 3 3 2 2 2 3 2 2 2 1 1 1 1 1 1 1 1 G T 2 2 3 2 1 1 1 1 2 3 3 3 1 1 3 1 3 3 2 3 2 3 2 2 T T 1 1 1 1 2 2 3 2 1 1 1 1 2 3 1 3 2 2 3 2 3 2 3 3

To further illustrate how to use Table 1 to determine a preferred probe family set, consider code 17. Following this coding, probes with restricted moieties AA, GC, TG, and CT are assigned to marker 1 (eg, red); probes with restricted moieties CA, AC, GG, and TT are assigned to marker 2 (eg, yellow); probes with restricted moieties TA, CC, AG, and GT are assigned to marker 3 (eg, green); probes with restricted moieties GA, TC, CG, and AT are assigned to marker 4 (eg, blue) . The resulting collection of probe families is shown in Figure 26.

Figures 27A-27C represent another approach to schematically define a set of 24 preferred probe families. The method utilizes a graph, such as that shown in Figure 27A. The first column of this chart represents the first base. Each marker was linked to four different base sequences given by juxtaposing the bases of the first column with the bases of the selected marker column. For example, if there is an A in the column titled "First Base", then probes containing the restricted portion with the sequence AA are assigned to probe family 1 (label 1); Some probes are assigned to probe family 2 (marker 2); probes containing a restricted portion with the sequence AG are assigned to probe family 3 (marker 3); probes with a restricted portion with the sequence AT are assigned to Assigned to probe family 4 (label 4). For probes containing restricted moieties starting with C, G, or T, probe families were assigned in a similar manner. Therefore, the chart filled with the bases shown in Figure 27A is translated into the code shown in Figure 27B, wherein the probes whose restricted part belongs to the group {AA, CC, GG, TT} are assigned to probe family 1; Probes that partially belong to the group {AC, CA, GT, TG} are assigned to probe family 2; probes that partially belong to the group {AG, CT, GC, TA} are assigned to probe family 3; Probes that partially belonged to the group {AT, CG, GA, TC} were assigned to probe family 4. Figure 27C shows a graph that can be inserted in place of the shaded portion of Figure 27A to generate each of the 24 preferred probe family sets. Methods employing the preferred collection of probe families in Sequencing Methods AB are further described below.

The 24 coded probe family sets identified in Table 1 represent only preferred embodiments of probe family sets for sequencing methods AB. A variety of other coding schemes, probe families and probe structures can be employed with the same basic principles, where knowledge of the probe family name, and knowledge of the identity of one or more nucleosides of the restricted moiety, can provide information about one or more other Nucleoside information. The reasons why less preferred probe family sets are less preferred than preferred probe family sets are usually: (i) at least for some probes, knowing the probe family name and nucleoside species is informative or (ii) at least for some probes, knowing the probe family name is more informative.

In general, a less preferred set of probe families can be used to perform sequencing methods AB in a manner similar to the use of a preferred set of probe families. However, the steps required for decoding may vary. For example, in some cases, comparing candidate sequences to each other may be sufficient to determine at least a portion of the sequence.

An example of a less preferred collection of probe families in which the probe contains a restricted portion of 2 nucleotides in length is shown in FIG. 28 . According to this encoding, the probes whose restricted part belongs to the group {AA, AC, GA, GC} are assigned to probe family 1; the probes whose restricted part belongs to the group {CA, CC, TA, TC} are assigned to Probe family 2; Probes whose restricted part belongs to the group {AG, AT, GG, GT} are assigned to probe family 3; Probes whose restricted part belongs to the group {CG, CT, TG, TT} are assigned to Probe family 4. In this collection of probe families, knowledge of the probe family names can exclude some possibility of template nucleotide species located at relative positions to the proximal nucleosides of the new ligation extension probes that are identified by detection of the new ligation extension probes. The label of the needle is used to determine the probe family name. For example, if the probe family name is 1, then the proximal nucleotide of the newly ligated extension probe must be A or G, so the complementary nucleotide in the template must be T or C. Contrary to when using the preferred pool of probe families, the nucleotides could not be identified exactly because there were at least two possibilities for each position of the restricted portion, but the information obtained from a single cycle was sufficient to rule out some possibilities.

In certain embodiments of the invention, partially defined probes with a constrained portion length of 3 nucleotides are employed. In order to contain probes whose restricted part includes all possible sequences of length 3 (preferred), the collection of probe families should include 4 ³ =64 different probes. Figure 29A shows a diagram of restricted moieties that can be used to generate probe family collections that include probes with a restricted moiety 3 nucleosides long (trinucleosides). The figure shows 4 sets of rows denoted A, G, C and T and 4 columns with probe family names 1, 2, 3 and 4. Groups of 4 rows are opposed to boxes containing nucleoside species inside. To identify probe families for trinucleosides, first select the box containing the last nucleoside of the trinucleoside. Of the 4 rows adjacent to this box, select the row labeled with the letter identifying the first nucleoside of the trinucleotide. Within that row, select the column containing the second nucleoside of the trinucleotide. Assign the trinucleotides to the probe families shown at the top of the column. For example, assign the trinucleotide "TCG" to a probe family as follows: Since the last nucleoside is "G", restrict the focus to the group of 4 lines opposite the box containing "G", i.e. the third Group. Since the first nucleoside is "T", the scope of consideration is further limited to the last row of group 4. Probe family assignment is determined by the heading of the column containing the intermediate nucleoside. Since the middle nucleoside is "C", three nucleosides were assigned to probe family 1. A similar approach yielded the following probe family assignments: AAA=1; ATA=2; AGA=3; GTA=4; GAG=1; TGG=2, etc. Continue this process until all possible trinucleosides have been assigned to probe families.

Figure 29B shows a method for constructing additional restricted portions of probe family collections comprising probes with restricted portions 3 nucleotides long. This method was used to construct a collection from each of the 24 preferred probe family collections described above, wherein the length of the restricted portion is 2 nucleotides, the collection contains 4 probe families. The upper panel of the figure shows an exemplary graph representing a collection of preferred probe families. The columns of the above figure are drawn directly into the figure below, following the colors assigned to the columns in the figure above. Therefore, the columns in the image above are blue, green, yellow, and red from left to right. The entries under column 1 in the figure below are blue, green, yellow and red from top to bottom, and the 4 nucleosides in each group correspond to the columns in the figure above. Columns 2, 3, and 4 in the figure below were generated by gradually shifting down each group of 4 nucleosides of column 1.

It is understood that a "probe family" can be considered to be a "super probe" comprising a plurality of different probes each containing the same label. In such cases, the probe molecules making up the probe are generally not a population of molecules that are substantially identical in any part of the probe. The use of the term "probe family" is not intended to be limiting in any way, but rather for convenience in describing the characteristics of the probes that make up these "super probes".

decoding

As described above, in a sequencing reaction, sequential extension, ligation, detection and cleavage cycles are performed using a probe family pool comprising at least two differentially labeled probe families to generate an ordered list of probe family names, or the Probe family names determined by multiple sequencing reactions initiated at different loci in the array are assembled into an ordered list. The number of cycles performed should be approximately equal to the desired sequence length. Ordered lists contain a lot of information, but do not immediately yield sequences of interest. Additional steps must be performed, at least one of which involves gathering at least one item of additional information about the sequence to obtain the sequence most likely to represent the sequence of interest. The sequence most likely to represent the sequence of interest is referred to herein as the "correct" sequence, and the process of extracting the correct sequence from the ordered list of probe families is referred to as "decoding". It should be understood that elements in the aforementioned "ordered list" may be rearranged during or after sequence generation, as long as the information content, including the correspondence between the elements in the list and the nucleotides in the template, is preserved, and as long as the decoding process (described below ) taking into account rearrangements, fragmentation and/or substitutions as appropriate. Accordingly, the term "ordered list" is intended to include rearranged, fragmented and/or permuted ordered lists produced as described above, so long as such rearranged, fragmented and/or permuted lists comprise substantially the same information content .

Ordered lists can be decoded in various ways. Some of these methods include generating a set of sequences of at least one candidate sequence from an ordered list of probe family names. This set of candidate sequences may provide enough information to achieve the goal. In a preferred embodiment, one or more additional steps are performed to select from a candidate sequence or a group of sequences compared to candidate sequences the sequence most likely to represent the sequence of interest. For example, in one method, at least a portion of at least one candidate sequence is compared to at least one other sequence. Select the correct sequence based on the comparison result. In some embodiments of the invention, decoding comprises repeating the method and obtaining a second ordered list of probe family names using a different set of probe families than the original set of probe families. The information from the second ordered list of probe families was used to determine the correct sequence. In some embodiments, the information obtained from as few as one cycle of extension, ligation, and detection with alternatively encoded probe family pools is sufficient to select the correct sequence. In other words, the first probe family identified with an alternatively encoded probe family provides sufficient information to determine which candidate sequence is correct.

Other decoding methods include using any available sequencing method, such as one-cycle sequencing method A, to specifically identify at least one nucleotide in the template. Information about one or more nucleotides is used as a "key" to decode the ordered list of probe family names. Alternatively, the sequenced portion of the template may include regions of known sequence in addition to regions of unknown sequence. If the sequencing method AB is applied to a portion of the template comprising an unknown sequence and at least one nucleotide of a known sequence, the known sequence can be used as a "key" to decode the ordered list of probe family names. The following sections describe the process of generating candidate sequences. Subsequent sections describe the use of candidate sequences for comparison to known sequences, comparison to a second set of candidate sequences, and use of known nucleotide species to select the correct sequence.

generate candidate sequences

It is understood that the portion of the template to be sequenced is complementary to the extended duplex generated by successive cycles of extension, ligation and cleavage. Thus, generating candidate sequences for the extended duplex is equivalent to generating candidate sequences for the template region to be sequenced. In practice, candidate sequences for the template region to be sequenced can be generated, or candidate sequences for extended duplexes can be generated and their complements used to determine candidate sequences for the template region to be sequenced. The latter method is described in this article. To generate a candidate sequence from a list of probe family names, the first member of the probe family list is considered. The set of restricted parts associated with this probe family limits the possibility of starting nucleotides of the sequence over a length equal to the length of the restricted part. For example, if the constrained moiety is a dinucleotide, then the possible sequences of the first dinucleotide in the extended duplex are limited to those constrained moieties that occur in probes belonging to that probe family (thus the region of the template to be sequenced The possible sequences of the first dinucleotide in are limited to combinations complementary to the restricted portion occurring in probes belonging to this probe family). The probability of the first dinucleotide is generally recorded by computer. Similarly, the possible sequences of the second dinucleotide (i.e., a dinucleotide offset by one nucleotide from the first) in the extended duplex are limited to those belonging to the second probe family. restricted portion of the probe (thus, the possible sequences of the second dinucleotide in the template, i.e., the dinucleotide offset by one nucleotide from the first dinucleotide, are limited to Combinations of restricted partial complementarities that occur in probes of a probe family). The probable sequence of the second dinucleotide is also recorded. Subsequent dinucleotide probabilities are likewise recorded until a dinucleotide probabilities corresponding to the desired length of the sequence to be determined are recorded or there are no more probe families in the list.

A representative example of a method of recording likelihoods is depicted in FIG. 30, where it is assumed that a list of probe family names is generated using the collection of probe families shown in FIG. 25A. The leftmost column of Figure 30 shows a list of probe families in order from top to bottom: yellow, green, red, blue. The sequence likelihoods of the dinucleotides corresponding to each probe family in the list are shown on the right side of the figure. Nucleotide positions are indicated above the sequence likelihood. The sequence starts at position 1, so the first dinucleotide occupies positions 1 and 2; the second dinucleotide occupies positions 2 and 3, etc. For the yellow probe family, the possibilities are CC, AT, GG, and TA, as shown in FIG. 30 . For the green probe family, the possibilities are CA, AC, GT, and TG, etc. The process of recording possible sequences for each dinucleotide continues until the desired sequence length is reached.

After generating the likelihood set, a first hypothesis is made about the identity of the first nucleotide in the candidate sequence, which is assumed to be at the 5' position of the sequence, denoted as position 1 in Figure 30. The first hypothesis can be that the nucleotide is A, the nucleotide is G, the nucleotide is C or the nucleotide is T.

Observe that the possible sequences of each dinucleotide are limited by the possible sequences of adjacent dinucleotides because adjacent dinucleotides overlap, i.e. the second nucleotide of the first dinucleotide is also the second the first nucleotide of a dinucleotide. For example, if the first nucleotide is assumed to be C, then the first nucleotide must be CC. If the first dinucleotide is CC, then the second dinucleotide must have a C in the first position. Since the possible sequence of the second dinucleotide with C at the first position can only be CA, it is proved that the second dinucleotide must be CA. Therefore, the sequence of the first 3 nucleotides must be CCA. Similarly, the possible sequences of the third dinucleotide are limited by the possible sequences of the second dinucleotide. If the second dinucleotide is CA, then the third must be AG, since that is the only possibility for an A in the first position. Therefore the sequence of the first 4 nucleotides must be CCAG. Continuing this process yields the first 5 nucleotides of the sequence 5'-CCAGC-3'. Therefore, CCAGC is the first candidate sequence.

A second candidate sequence is generated by assuming that the first nucleotide is A. This assumption makes the first dinucleotide AT. TG is the only possible sequence of the second dinucleotide that corresponds to the sequence AT of the first dinucleotide. GA is the only possible sequence of the third dinucleotide that coincides with the sequence TG of the second dinucleotide. AA is the only possible sequence of the fourth dinucleotide corresponding to the sequence GA of the third dinucleotide. Assembly of these dinucleotides into full-length candidate sequences generates ATGAA. Similarly, assuming the first nucleotide is G, the resulting candidate sequence is GGTCG, and assuming the first nucleotide is T, the resulting candidate sequence is TACTT. Thus, 4 candidate sequences were generated, each beginning with a different nucleotide that was assumed to be the first nucleotide of the sequence.

There is no requirement to make a hypothesis about the first nucleotide rather than one of the other nucleotides. For example, the same effect can be achieved by making a hypothesis about the species of the fourth nucleotide, in which case a candidate sequence is generated by moving "backwards" along the template (i.e. in the 3'→5' direction). For example, assuming that the fourth nucleotide is T means that the fourth dinucleotide must be TT; the third dinucleotide must be CT; the second dinucleotide must be AC; the first The dinucleotide must be CC. (Nucleotides are written in the 5'→3' direction, although moving through the sequence in the 3'→5' direction generates its species). Alternatively, hypotheses can be made for any nucleotide in the sequence, generating dinucleotide species by shifting in the 5'→3' and 3'→5 directions. It will be appreciated that without making an assumption about one of the nucleotides, the identity of each nucleotide cannot be determined at all, since each position can be occupied by A, G, C or T.

With the preferred collection of probe families, it is assumed that any single nucleotide (eg, first nucleotide) species will generate one and only one candidate sequence. However, when using less preferred probe family pools, it may be necessary to assume more than one nucleotide species, ie the assumed first nucleotide species does not fully define the rest of the sequence. For example, a less preferred collection of probe families might include families whose members' defined sequences are AA and AC. In this case, assuming the first nucleotide is A leaves two possibilities for the second nucleotide. Sequencing with less preferred probe family pools is discussed further below. It will be appreciated that if the constrained portion consists of non-contiguous nucleotides, the above method can still be used with slight modifications.

Sequence identification by comparing candidate sequences with known sequences

Typically, if the candidate sequence for the extended duplex is determined as described above, the corresponding candidate sequence for the region of the template to be sequenced is obtained by taking its complement. In some cases, the candidate sequence itself will provide sufficient information for purposes. For example, if the purpose of sequencing is simply to rule out certain sequence possibilities, it is sufficient to compare candidate sequences to these possibilities. Candidate sequences shown in Figure 30 enable determination, for example, that the sequenced region is not part of the poly A tail. Longer sequences confirm that the sequenced region is not part of the vector.

In many cases, unambiguous determination of the correct sequence is required. According to a preferred embodiment of the present invention, the correct sequence is identified by comparing the candidate sequences of the template region to be sequenced with a set of known sequences. The set of known sequences can be, for example, the set of sequences for a particular organism of interest. For example, if human DNA is sequenced, the candidate sequence can be compared to the draft sequence of the human genome. See the guide to publicly available sources of human genome sequences at URL www.ncbi.nih.gov/genome/guide/human/ . As another example, if nucleic acid derived from an infectious agent, such as a bacterium or virus isolated from a subject, is to be sequenced, a database containing the sequence of a variant strain of the bacterium or virus can be searched. Many such organism-specific databases are known in the art, containing complete or partial sequences, and more will become available as sequencing efforts are accelerated. Some representative examples include the mouse database (see, e.g., the website at URL www.ncbi.nlm.nih.gov/genome/seq/MmHome.html ), the human immunodeficiency virus database (see, e.g., the URL at hiv-web. lanl.gov/content/hiv-db/mainpage.html ), malaria pathogen Plasmodium falciparum database (see e.g. URL at http://www.tigr.org/tdb/edb2/pfal/htmls/ index.shtml 's website), etc. Of course, it is not necessary to employ the set of sequences of a particular organism. Searchable databases such as GenBank (web site at URL http://www.ncbi.nlm.nih.gov/Genbank/ ) contain sequences from various organisms and viruses. The database doesn't even necessarily contain any sequences of the organism or virus that produced the template. Typically, the sequence may be a genomic sequence, cDNA sequence, EST, or the like. Multiple sequences can be searched.

Just doing a search might be enough for the purpose. For example, if a viral nucleic acid is isolated from a patient, comparison of the candidate sequence to a set of known sequences for that virus can determine whether the viral nucleic acid contains a sequence from that virus, even if a matching sequence has never been detected. The presence of a match confirms that the patient is infected with the virus, while the absence of a match indicates that the patient is not infected with the virus.

In certain embodiments, the set of known sequences contains a narrow range of sequences that may be particularly suitable for the purpose of performing the sequencing. Thus, sequenced nucleic acid information can be used to select groups of known sequences. For example, if the known template represents the sequence of a particular gene, the known sequence may represent different alleles, mutant or wild-type sequences, etc. of the gene at a given locus of interest. It may only be necessary to compare the candidate sequence with a known sequence to determine which candidate sequence is the correct one. For example, in certain embodiments of the invention, the template is obtained by amplifying DNA containing the region of interest (eg, using primers flanking the region of interest). A region of interest may include sites of mutations or polymorphisms, such as mutations or polymorphisms associated with a particular base. If the template is known to represent the sequence of a particular region of interest, then it is only necessary to compare the candidate sequence to a reference sequence, such as a wild-type or mutated form of the sequence for this region. In other words, if some or all of the template sequence is known, then it may not be necessary to compare to various known sequences. Instead, a candidate sequence containing all or part of the known sequence is selected as the correct sequence. For example, mutations in the BRCA1 and BRCA2 genes are known to be associated with an increased risk of breast cancer, and it is of interest to determine whether a subject carries such mutations. If the template is known to comprise a sequence from the BRCA1 gene, for example, if primers flanking a region of interest comprising a portion of the gene are used to generate a clonal population of the template, it is only necessary to compare the candidate sequence with wild-type or mutant BRCA1 sequence to determine the correct sequence.

In more general terms, comparing a candidate sequence to a set of known sequences identifies any known sequences that are similar to the candidate sequence. Provided that the candidate sequences are sufficiently long, it is very unlikely that the database will contain sequences that are identical or very similar to more than one candidate sequence. In other words, if the candidate sequences are long enough, it is impossible for more than one candidate sequence to be identical to a sequence in the set of known sequences. The candidate sequence is compared to any sequence considered a "match". It is generally desirable to set an identity threshold required to determine that a match exists. For example, a candidate sequence may be considered to match a known sequence if it is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100% identical to the known sequence . Identity is generally assessed over a window of at least 10 nucleotides in length, such as 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, etc. percentage. The window length can be selected according to various criteria, including but not limited to: the number of sequences in the plurality of known sequences, the type or source of the plurality of known sequences, and the like. For example, if candidate sequences are compared to a large database such as GenBank, the required window length may be longer than when using a database containing fewer sequences. In certain embodiments of the invention, sequences are compared over a plurality of different windows, which windows are not necessarily adjacent to each other. Preferably, the total length of the window is at least 10 nucleotides, such as 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, etc. In some cases, multiple sequences in the set of known sequences can be matched. The sequence may, for example, represent a homologous gene found in the same organism as that which produced the template, a homologous gene from a different organism, a pseudogene, cDNA and genomic sequences, etc.

Typically, the candidate sequence that is the closest sequence in the set of known sequences is selected as the correct sequence. Alternatively, for example, if there is reason to believe that the sequencing method may produce a high error rate, then the corresponding sequence in the database is preferably selected as the correct sequence. For example, if the error rate is known to exceed a predetermined threshold, then preferably a sequence in the database is selected as the correct sequence.

The length required to guarantee the likelihood of finding a match from multiple candidate sequences depends on various factors including, but not limited to: the particular set of known sequences, the threshold for accepting matches, etc. Typically, sequences of about 25-26 nucleotides in length occur only once in the genome of an average organism. Therefore, generating candidate sequences of approximately this length is sufficient to identify the correct sequence. Generally, the length of the candidate sequence should be at least 10 nucleotides, preferably at least 15, at least 20 nucleotides, such as 20-25, 25-30, 30-35, 35-40, 45-50 nucleotides sour or even longer.

Sequence identification by comparing a first set of candidate sequences with a second set of candidate sequences

In certain embodiments of the invention, a first set of probe families encoded according to a first encoding scheme is used to generate a first ordered list of probe families from which a first set of candidate sequences is generated, and then encoded according to the first encoding scheme The second set of probe families encoded by the two encoding schemes generates a second ordered list of probe families from the same template, from which a second set of candidate sequences is generated for decoding. Either remove newly synthesized DNA strands from the template between sequencing reactions, or use a second probe family pool to sequence identically sequenced templates. Compare groups of candidate sequences. It should be understood that no matter which set of probe families is used, one of the candidate sequences is the correct sequence and the other is not (or at most partially correct). Thus, each set of candidate sequences contains the correct sequence, but in most cases the other candidate sequences in any given candidate sequence are different from the sequences found in the other set of candidate sequences. Therefore, the correct sequence can be determined by simply comparing two sets of candidate sequences. It is not necessary to use two sets of probe families with different encodings to generate candidate sequences of equal length. In preferred embodiments of the invention, the candidate sequences generated using the second collection of probe families can be as short as 2 nucleotides, or alternatively, the ordered list of probe families generated using the second collection of probe families can be As short as 1 component (i.e. 1 connection and detection cycle).

Figures 31A-31C show examples of candidate sequence generation and decoding with two differentially labeled preferred probe families. Figure 31A shows a preferred collection of probe families encoded according to the first encoding scheme. Figure 31B shows the generation of 4 candidate sequences from an ordered list of probe families yellow, green, red, blue (may be expressed as "2314", wherein red=1, yellow=2, green=3, blue=4), where the correct sequence is assumed to be CAGGC (indicated in bold). Figure 31C shows a preferred set of probe families encoded according to the second encoding scheme. Since the first dinucleotide in the template is CA, the uppermost probe in the yellow probe family will be ligated to the extendable end in the first extension cycle. This makes the first dinucleotide the following set of candidate sequences: CA, TC, GG, AT. Of the candidate sequences generated with the first set of probe families, only the sequence CAGGC began with either of these dinucleotides. Therefore, it must be the correct sequence. Generally, the first and second probe family collections preferably meet the following conditions: when comparing the first and second probe family collections, (i) among the 4 probes of each probe family in the first collection The 3 new probe families should be assigned to the second pool; and (ii) each of the 3 reassigned probes should be assigned to a different probe family in the second pool.

Decoding the ordered list of probe families with known nucleotide species

As described above, candidate sequences can be generated by assuming the species of one nucleotide in the extended duplex or template. Depending on the particular set of probe families used, it is usually necessary to generate at least 4 candidate sequences. However, the generation of multiple candidate sequences can be avoided if the species of at least one nucleotide in the template (and thus in the extending duplex) is known. In this case, only one candidate sequence needs to be generated. The method of generating candidate sequences is the same as above. The identity of at least one nucleotide in the template can be determined by any sequencing method, including but not limited to: Sequencing Method A, primer extension from a starting oligonucleotide using a set of differentially labeled nucleotides and a polymerase wait. It will be appreciated that one or more nucleotides in a template may first be sequenced using a sequencing method other than Sequencing Method AB, and then the same template may be sequenced using Sequencing Method AB (and vice versa) after the initial oligonucleotide and any extension products may be removed. Of course).

Another approach is to sequence only templates containing one or more nucleotides of known species, except for the portion to be sequenced. For example, the portion between the region where the initial oligonucleotide binds and the beginning of the unknown sequence may include one or more nucleotides of known species. By sequencing method AB on this partial template, one or more nucleotides in the sequence are preidentified and thus can be used to generate a candidate sequence, which will be the correct sequence.

Thus, the method described above comprises the steps of: (i) determining which species is associated with the known nucleotide species and its proximal nucleotides are linked to the relative position of the nucleotides adjacent to the nucleotides of the known species. The probable sequence coincidence of the restricted portion assigns species to nucleotides adjacent to nucleotides of known species on the template; (ii) by determining which species and its proximal nucleotides are linked relative to subsequent nucleotides assigning species to said subsequent nucleotides; and (iii) repeating step (ii) until the sequence is determined. It will be understood that these steps are equivalent to performing the same steps on the extended duplex, since there is an exact correspondence between the extended duplex and the template region to be sequenced.

Sequencing with less preferred probe families

Sequencing methods AB can be performed with less preferred probe family pools in a manner similar to the use of preferred probe family pools. However, results may vary in many ways. For example, certain sequence portions can be fully identified from a candidate sequence without additional information. Figure 32 shows an example of sequence determination using the less preferred set of probe families encoded in Figure 28. Sequencing methods are generally as described in the preferred probe family collections. The template of interest has the sequence "GCATGA", at this point the generated sequence listing for the probe family is "12341". Assuming that the nucleotide at position 1 is A, the resulting candidate sequence is "ACATGA". However, unlike the case of the preferred probe family set, there are two possibilities for the second nucleotide, since the label "1" differs from two different dinucleotides with A as the first nucleotide, namely "AA " is related to "AG". Therefore, assuming that the nucleotide at position 1 is A, the resulting second candidate sequence is "ACATGC". Assuming that the nucleotide at position 1 is G, the resulting candidate sequence is "GCATGA", and "GCATGC" is also generated as a candidate sequence. Since the marker "1" is not associated with any dinucleotide that is C or T at position 1, no candidate sequences starting with "C" or "T" were generated. Figure 32 shows 4 candidate sequences aligned to each other. It should be observed that the middle 4 nucleotides in all candidate sequences are CATG. Therefore, positions 2-5 of the correct sequence must include CATG. If only these nucleotides are of interest, no further decoding steps are required.

As mentioned above, the collection of probe families does not necessarily consist of four different probe families, but may consist of more than 2 and less than 4 ^N species, where N is the length of the constrained section. However, if fewer than 4 families are used, more than 4 candidate sequences may have to be generated, and if more than 4 probe families are used, additional labels are required. For these and other reasons, a set consisting of 4 probe families is preferred.

Sequence identification by comparison of candidate sequences against each other

In some embodiments of the present invention, part or all of the sequences of interest can be determined by comparing candidate sequences with each other. Often this comparison is insufficient to determine which candidate sequence is correct over its entire length. However, if two or more candidate sequences are identical or sufficiently similar over a portion of the sequence, this information may be sufficient to unambiguously identify the nucleotide sequence within that portion of the template.

If desired, the template can be resequenced one or more times with alternately coded probe families to generate additional portions of the identified sequence. These sections can be combined to assemble sequences of desired length.

Correcting Mistakes with Probe Families

It is often desirable to sequence multiple templates representing all or part of the same DNA sequence and to align these sequences. If the template contains only part of the region of interest, longer sequences are obtained by assembling overlapping fragments. For example, when sequencing the genome of an organism, the DNA is typically fragmented and enough fragments are sequenced so that each DNA is extended by several (eg, 4-12) different fragments. Computer software for assembling overlapping sequences into longer sequences is known to those skilled in the art.

With conventional sequencing methods, it is often the case that multiple fragments align perfectly over a region, but one of these fragments (called an outlier fragment) differs from the others at one position in the region. Determining whether an individual difference represents a sequencing error or whether there is a true difference (eg, a single nucleotide polymorphism) at that position can be problematic.

The present invention provides a new method for error checking with sequencing method AB. According to this method, templates comprising fragments representing the same DNA segment are sequenced using the above-described collection of differentially labeled probe families, generating an ordered list of probe families for each template. An ordered list of alignment probe families. If several lists align perfectly over a predetermined length, such as 10, 15, 20, or 25 or more elements in the list, except that one list differs from the other fragments in one position, then attribute the difference to a sequencing error . If an actual polymorphism is present, the ordered probe list generated from the abnormal fragment will differ from the ordered probe list generated from other fragments at two or more adjacent positions.

For example, applying the sequencing method AB using the set of preferred probe families encoded in Table 1 to a template containing the sequence 5'-CAGACGACAAGTATAATG-3' yields an ordered list of the following probe families: "23324322132444142", as follows:

23324322132444142

CAGACGACAAGTATAATG

If there is an actual SNP (such as CAGACGA G AAGTATAATG, where the underlined nucleotide represents the polymorphic site), it will cause changes in two consecutive elements in this list: 233243 33 132444142, where the underlined indicates the change caused by the SNP. The correspondence between the ordered list of probe families and the sequences containing SNPs is as follows:

233243 33 132444142

CAGACGA G AAGTATAATG

However, an error in the identification of the label attached to the ligation extension probe results in an error in the ordered listing of the probe family and a change in the resulting candidate sequence from that point forward. For example, determining errors in markers 233243 3 2132444142 ligated to the 7th ligation extension probe (where underlined numbers represent misidentified markers) would change the resulting candidate sequence to CAGACGA GTTCATATTAC , where the underlined portion represents a sequence error Change. The ordered list of probe families and the correspondence between the sequences are as follows:

233243 3 2132444142

CAGACGA GTTCATATTAC

When using 3 bases and 4 labeling schemes, fragments containing SNPs will produce 3 consecutive differences in the probe family ordered list of abnormal fragments, but only 1 error will be caused by sequencing errors. For example, when using the probe family set coded as shown in Figure 29, the ordered list of the probe family types of the sequence CAGACGACAAGTATAATG is as follows:

2322224132412244

CAGACGACAAGTATAATG

Unusual fragments containing SNPs, such as CAGACGA G AAGTATAATG, will result in an ordered list of probe families that differs at 3 consecutive positions from the ordered list produced by a fragment without a SNP, as follows:

23222 133 32412244

CAGACGA G AAGTATAATG

A sequencing error will produce only one difference in the ordered list of probe families, resulting in candidate sequences that are completely different from the point of error onwards.

Thus, when a sequenced list of probe families produced by a fragment (an abnormal fragment) aligns with ordered lists of probe families produced by other fragments representing the same DNA segment, but differs from the other sequenced lists at a single position, Sequenced lists containing this difference likely represent a sequencing error (misidentification of a probe family). When an ordered list of probe families produced by a fragment (an abnormal fragment) aligns with ordered lists of probe families produced by other fragments representing the same DNA segment, but differs from the other ordered lists at 2 or more consecutive positions , the abnormal fragment may contain SNP. Preferably, the aligned portion of the probe family ordered list is at least 3 or 4 elements in length, preferably at least 6, 8 or more elements in length. Preferably, the aligned portions are at least 66% identical, at least 70% identical, at least 80% identical, at least 90% identical or more, such as 100% identical.

Similarly, a sequencing error may occur when a segment candidate sequence is aligned with other segment candidate sequences representing the same DNA segment on the first part of the sequence, but is significantly different from other segment candidate sequences on the second part of the sequence. When the candidate sequence of a fragment is compared with the candidate sequences of other fragments representing the same DNA fragment on the two parts of the sequence, but only one position is different, the abnormal fragment may contain SNP. Preferably, the aligned portion of the candidate sequence is at least 4 nucleotides in length. Preferably, the aligned portions are at least 66% identical, at least 70% identical, at least 80% identical, at least 90% identical or more, such as 100% identical.

Accordingly, the present invention provides a method for distinguishing single nucleotide polymorphisms from sequencing errors, said method comprising the steps of: (a) sequencing a plurality of templates by sequencing method AB, wherein said templates represent overlapping fragments of a single nucleic acid sequence ; (b) aligning the sequences obtained in step (a); and (c) if the sequences are substantially identical in a first part and significantly different in a second part (each part is at least 3 nucleotides in length ), the difference between the sequences was determined to represent a sequencing error. The present invention also provides a method for distinguishing single nucleotide polymorphisms from sequencing errors, the method comprising the following steps: (a) performing sequencing method AB with various templates representing overlapping fragments of a nucleic acid sequence, thereby obtaining multiple an ordered list of probe families; (b) aligning the ordered lists of probe families obtained in step (a) to obtain aligned regions in which the ordered lists are at least 90% identical; and (c) if the ordered lists are only in A difference between ordered lists of probe families is determined to represent a sequencing error if they differ at one position within the aligned region; or (d) if the ordered lists are at two or more consecutive positions within the aligned region Differences between ordered lists of probe families were determined to represent single nucleotide polymorphisms.

A collection of delocalized information

As is well known in the art, a "bit" (binary number) refers to a number in binary, ie, 1 or 0, which represents the smallest unit of digital data. Since a nucleotide can be of one of four different classes, it is understood that 2 bits are required to define the class of a nucleotide. For example, A, G, C, and T can be represented as 00, 01, 10, and 11, respectively. Two bits are required to define the probe family name in the preferred set of differentially labeled probe families since there are four differentially labeled probe families.

In the most conventional form of sequencing and sequencing method A, each nucleotide is determined as a discrete unit and information corresponding to one nucleotide at a time is collected. Each detection step obtains two bits of information from one nucleotide. In contrast, sequencing method AB obtains less than 2 bits of information each from multiple nucleotides in each detection step, while still obtaining 2 bits of information per detection step with the preferred probe family pool. Each probe family name in the probe family ordered list represents at least 2 nucleotide types in the template, and the exact number is determined by the length of the sequence determination part of the probe. For example, consider the ordered list of probe families obtained from the sequence 5'-CAGACGACAAGTATAATG-3' using the set of probe families coded 4 according to Table 1:

23324322132444142

CAGACGACAAGTATAATG

Probe family 2 is the first probe family in this list because the dinucleotide CA is one of the specified moieties present in probe family 2 probes. Probe family 3 is the second probe family in this list because the dinucleotide AG is one of the specified moieties present in probe family 3 probes. As described above, since there are 4 types of probe families, each probe family type represents 2 bits of information. Thus, each detection step collects 2 bits of information on 2 nucleotides, yielding an average of 1 bit of information per nucleotide.

Therefore, the present invention provides a sequence determination method, wherein said method comprises a plurality of cycles of extension, ligation and detection, wherein said detection step comprises obtaining two bits of information of each of at least two nucleotides in a template simultaneously on average, without Get two bits of information for any single nucleotide. The present invention also provides a method for determining the nucleotide sequence of a template polynucleotide using the first set of oligonucleotide probe families, the method comprising the following steps: (a) performing continuous extension, ligation, detection and cutting cycle, wherein in each cycle the average of at least two nucleotides in the template is simultaneously obtained two bits of information, without obtaining two bits of information for any single nucleotide; and (b) combining the information obtained in step (a) with At least one bit of additional information is incorporated to determine the sequence. In various embodiments of the present invention, the at least one bit of additional information includes information selected from the group consisting of: nucleotide species in the template, information obtained by comparing a candidate sequence with at least one known sequence; Information obtained by repeating the method is repeated for a second set of nucleotide probe families.

Thus, although this method does not obtain 2-bit information for individual nucleotides, it does collect 2-bit information for the template on average in each cycle in a delocalized manner using the preferred probe family pool. With sets of 2 or 3 probe families, less than 2 bits of information were collected per cycle.

Delocalized information collection has many advantages, including the ability to apply error checking methods as described above. Furthermore, since in preferred embodiments each nucleotide in the template is detected more than once, non-localized information collection helps avoid systematic bias in the detection of fluorophores attached to specific nucleotides.

In addition to methods involving sequential cycles of extension, ligation and cleavage of probes, the probe families and collections of probe families described herein can be used in a variety of sequencing methods. The present invention also provides probe families and probe family collections having the above sequences and structures, wherein the probes are optionally free of easy linkages. For example, the probe may contain only phosphodiester backbone linkages and/or may contain no priming residues. In some embodiments of the invention, the family of probes is used for sequencing in which successive cycles of extension and ligation are used, but cleavage is not included in each cycle. For example, the family of probes can be used in ligation-based methods as described in WO2005021786 and elsewhere in the art. In order to employ the probe family in this method, the label on the probe should be attached by a cleavable linker, as described in WO2005021786, so that the label can be removed without cleaving the nucleic acid by scissile ligation. This method can be used to generate an ordered list of probe families, for example, by performing multiple reactions in parallel or sequentially with probe families other than the junction cassette described in WO2005021786 and then assembling the list of probe families. Decode the list as above.

I. Kit

Various kits can be provided to practice different embodiments of the invention. Certain kits include extended oligonucleotide probes containing phosphorothioate linkages. The kit may also include one or more starting oligonucleotides. The kit may contain a cleavage reagent such as _AgNO3 suitable for cleavage of phosphorothioate linkages and a suitable buffer for cleavage. Certain kits include extended oligonucleotide probes containing priming residues such as nucleosides containing damaged bases or abasic residues. The kit may also include one or more starting oligonucleotides. The kit may contain a cleavage reagent suitable for cleaving linkages between nucleosides and adjacent abasic residues and/or a reagent such as a DNA glycosylase suitable for removing damaged bases of a polynucleotide. Certain kits include oligonucleotide probes containing disaccharide nucleotides and include periodate as a cleavage reagent. In certain embodiments, the kit contains a collection of differentially labeled oligonucleotide probe families.

Kits may also include ligation reagents (eg, ligase, buffer, etc.) and instructions for practicing specific embodiments of the invention. Buffers suitable for other enzymes that may be employed such as phosphatases, polymerases may be included. In some cases these buffers may be the same. The kit may also include supports, such as magnetic beads, for anchoring the template. These beads can be functionalized with PCR amplification primers. Other optional components include washing solutions; template-inserted vectors for PCR amplification; PCR reagents such as amplification primers, thermostable polymerases, nucleotides; reagents for preparing emulsions; reagents for preparing gels, and the like.

In certain preferred kits, fluorescently labeled oligonucleotide probes containing phosphorothioate linkages are provided such that probes corresponding to different probe terminal nucleotides carry different spectrally resolved fluorophores. dye. More preferably, four such probes are provided so that there is a one-to-one correspondence between the four spectrally resolvable fluorescent dyes and the four possible probe terminal nucleotides.

Identifiers, such as barcodes, radio frequency ID tags, etc., may be present in or on the kit. For example, an identifier may be used to uniquely identify a kit for quality control, inventory management, tracking, movement between workstations, and the like.

Kits typically include one or more vessels or containers in which certain reagents are kept individually. Kits may also include means for enclosing a single container in a relatively tight seal, such as a plastic box, for commercial distribution, into which instructions, packaging materials such as styrofoam, and the like may be incorporated.

J. Automated Sequencing System

The present invention provides a variety of automated sequencing systems that can be used to collect sequence information for multiple templates in parallel (ie, substantially simultaneously). Preferably, the templates are arranged on a substantially planar substrate. Figure 21 shows a photograph of a system of the present invention. As shown in the photo above, the system of the present invention includes a CCD camera, a fluorescence microscope, a moving stage, a Peltier flow chamber, a temperature controller, a fluid handling device, and a dedicated computer. It should be understood that various substitutions may be made for these components. For example, another image capture device may be used. See Example 9 for additional details of this system.

It should be understood that various sequencing methods, including ligation-based methods and other methods described herein, can be implemented using the automated sequencing system of the present invention and related image processing methods and software, including but not limited to: sequencing by synthesis, such as by synthetic Fluorescence In Situ Sequencing (FISSEQ) (see, e.g., Mitra RD et al., AnalBiochem., 320(1):55-65, 2003). As described herein for ligation-based sequencing methods, templates immobilized directly in or on a semi-solid support, templates immobilized on microparticles in or on a semi-solid support, templates directly attached to a substrate, etc. Implement FISSEQ.

An important aspect of the system of the present invention is the flow chamber. Typically, a flow chamber includes a chamber with input and output ports through which fluid can flow. See, eg, US Patents 6,406,848 and 6,654,505 and PCT Publication No. WO98053300 for a discussion of various flow chambers and materials and methods for their manufacture. Fluid flow enables the addition and removal of various reagents to entities (eg, templates, microparticles, analytes, etc.) located in the flow chamber.

Preferably, a flow cell suitable for use in a sequencing system of the invention includes a location for mounting a substantially flat substrate, such as a glass slide, to allow fluid to flow across the surface of the substrate, and windows to allow for illumination, excitation, signal acquisition, and the like. According to the method of the present invention, entities such as particles are typically arranged on a substrate prior to entering the flow chamber.

In some embodiments of the invention, the flow chamber is positioned vertically so that air bubbles escape from the top of the flow chamber. By arranging the flow chamber, the flow path runs from the bottom end of the flow chamber to the top end, such that the input port is located at the bottom end of the flow chamber and the output port is located at the top end of the flow chamber. Since any air bubbles that can be introduced are buoyant, they float quickly towards the output port without obscuring the lighting window. This method of raising the bubbles to the surface of the liquid due to their lower density than the liquid is referred to herein as "gravitational bubble displacement". Accordingly, the present invention provides a sequencing system in which the orientation of the flow chamber allows for gravitational bubble displacement. Preferably, the substrate is mounted vertically in the flow cell to which the microparticles are directly or indirectly attached (e.g., covalently or non-covalently attached to the substrate) or containing microparticles adhered or immobilized in or on a semi-solid support on the substrate , that is, the largest flat surface of the substrate is perpendicular to the ground plane. Since, in preferred embodiments, the microparticles are immobilized in or on the support or substrate, their relative positions are substantially fixed, which facilitates continuous image acquisition and image recording.

Figures 24A-J show schematic views of flow chambers or portions thereof of the present invention in different orientations. The flow cell of the present invention can be used for various purposes, including but not limited to: analytical methods (such as nucleic acid analysis methods such as sequencing, hybridization experiments, etc.; protein analysis methods, binding experiments, screening experiments, etc.). Flow cells can also be used to perform synthesis, such as generating combinatorial libraries.

Fig. 22 shows a schematic diagram of another automated sequencing system of the present invention. The flow cell is mounted on a temperature-controlled automated bench (similar to that described in Example 9) and connected to a fluid handling system, such as a syringe pump equipped with a multi-port valve. The platform accommodates multiple flow cells in order to image one flow cell while other steps such as extension, connection and cutting are performed on the other flow cell. This approach maximizes the use of expensive optics while increasing throughput.

Fluid lines are equipped with optical and/or conductivity sensors to detect air bubbles and monitor reagent usage. Temperature control and sensors in the fluidic system ensure that the long-term stability of the reagents is maintained at the proper temperature, but raised to the operating temperature as they enter the flow chamber to avoid temperature fluctuations during annealing, ligation, and cleavage steps. Reagents are preferably pre-packaged into the kit to prevent errors when loading samples.

The optics consist of four cameras - each taking a picture through one of four filter sets. To reduce photobleaching effects, illumination optics can be engineered to illuminate only the imaged area, preventing multiple illumination at the edges of the field of view. Imaging optics can be built from standard infinity-corrected microscope objectives along with standard beam splitters and filters. Images can be captured with a standard 2,000 x 2,000 pixel CCD camera. The system incorporates mechanical support for the optics. Light intensity is preferably monitored and recorded for analysis software.

For rapid acquisition of multiple images (eg, about 1800 or more non-overlapping image fields in one representative embodiment), the system preferably employs a fast autofocus system. Autofocus systems based on analysis of the image itself are well known in the art. They usually require at least 5 frames/focus event. This method is slow and expensive due to the additional light (increased photobleaching) required to obtain an in-focus image. In some embodiments of the invention, another autofocus system is used, such as a system based on independent optics, which can focus as fast as the mechanical system can react. Such systems are known in the art, including, for example, focusing systems for consumer CD players that maintain submicron focus in real time while the CD is playing.

In some embodiments of the invention, the system is operated remotely. Scripts to implement specific protocols can be stored in a central database and downloaded for each sequencing run. Samples can be barcoded to maintain the integrity of sample tracking and to associate samples with final data. Central real-time monitoring enables rapid resolution of process errors. In certain embodiments, images collected by the device are uploaded immediately to a central multi-terabyte storage system and one or more processor banks. Using tracking data from the central database, the processor analyzes images and generates sequence data, optionally generating processing specifications, such as background fluorescence levels and bead density, to eg track device performance.

Use the control software to properly arrange pumps, stages, cameras, filters, temperature controllers, and annotate and store image data. A user interface is provided to, eg, assist an operator in setting up and maintaining the device, preferably including functions for determining platform position and activating fluid lines when loading/unloading slides. Display functions may be included to, for example, show an operator various operating parameters such as temperature, platform position, current filter configuration, status of the operating protocol, and the like. An interface to a database that records tracking data such as reagent lot numbers and sample IDs is preferably included.

K. Image and Data Processing Methods

The present invention provides various image and data processing methods implemented at least in part in the form of computer code (ie, software) stored on a computer-readable medium. Further details are set forth in Examples 9 and 10. In addition, generally, sequencing methods A and B typically employ suitable computer software to perform processing steps that include, for example, keeping track of data collected in multiple sequencing reactions, compiling such data, generating candidate sequences, performing sequence comparisons, etc. .

L. Computer-readable media storing sequence information

Additionally, the invention provides computer readable media storing information generated using the sequencing methods of the invention. Information includes raw data (i.e. data that has not been further processed or analyzed), processed or analyzed data, etc. Data includes images, numbers, etc. Such information may be stored in a database, ie, a collection of information (eg, data), typically in an easily searchable arrangement, eg, in a computer's memory. Information includes, for example: sequences and any information about sequences, such as partial sequences, comparisons between sequences and reference sequences, sequence analysis results, genome information such as polymorphism information (such as whether a specific template contains polymorphisms) or mutation information, etc., Linkage information (i.e., information relating to the physical location of a nucleic acid sequence in a chromosome relative to another nucleic acid sequence), disease-associated information (i.e., information relating the presence or susceptibility of a disease to a subject's physical characteristics, such as the subject's alleles) wait. Information may be related to sample ID, subject ID, etc. Other information relating to the sample, subject, etc. may be included, including but not limited to: source of the sample, processing steps performed on the sample, interpretation of the information, characteristics of the sample or subject, etc. The invention also includes a method comprising receiving any of the above information in a computer readable form, such as stored on a computer readable medium. The method may also include the step of providing diagnostic, prognostic or prognostic information based on such information or simply providing information preferably stored on a computer readable medium to a third party.

The following examples are offered for illustration and they do not limit the invention.

Example 1: Efficient Cleavage and Ligation of Phosphorothioated Oligonucleotides

This example describes experiments showing efficient ligation and cleavage of extended oligonucleotides containing 3'-S phosphorothioate linkages.

Materials and methods

ligation sequencing method

Template Preparation: To evaluate the potential for sequencing by oligonucleotide ligation and cleavage cycles and to explore the effect of altering certain aspects of the method, two populations of model bead-based templates were prepared. In a preferred implementation, cycles of oligonucleotide ligation and cleavage extend the strand in the 3'→5' direction, as described in the Examples. Therefore, in order to evaluate the ligation efficiency, the 5' end of the pattern template was bound to beads, and the same binding region was designed at the 3' end. One set consisted of short (70 bp) oligonucleotides bound to streptavidin-coated magnetic beads (1 micron) via a dual biotin moiety. The 3' ends of these short template populations are designed with the same primer binding region (40bp) and unique sequence region (30bp). The population of short oligonucleotide templates is referred to as Ligation Sequencing Templates 1-7 (LST1-7).

A second bead-based template population was designed from PCR-generated long DNA fragments (232-bp) generated by inserting 183-bp spacer sequences (from human p53 exons) into each template population . The template was amplified with a double biotin-containing forward primer and a reverse primer containing a unique 3' end sequence of 30 bases identical to the short template population. Single-stranded templates are generated by unwinding one strand with a buffer containing sodium hydroxide. These long template populations were designed to mimic the species generated from the short fragment paired-end library described in the co-pending patent application, which they termed long-LST1-7.

Primer Hybridization: Pre-mix 2.5 μL of 100 μM FAM-labeled primer with 100 μL of 1X Klenow buffer. After the buffer was removed, this solution was added to a 30 µL sample amount of template-linked magnetic beads (10 ⁶ /µL), and the resulting solution was thoroughly mixed. After allowing the template/primers to hybridize (hybridization reaction at 65°C for 2 min, at 40°C for 2 min, on ice for 2 min), the primer/buffer was removed and the beads were washed with 3× wash 1E buffer, then Resuspended in 300 μL (10 ⁶ /mL) of TENT buffer (containing 10 mM Tris, 2 mM EDTA, 30 mM NaOAc and 0.01% Triton X-100).

Ligation 1: Then, incubate hybridization containing LigSeq in a mixture containing 1 μL of 100 μM LST7-1 nonamer, 4 μL of 5×T4 ligase buffer (Invitrogen), ₁₄ μL of HO, and 1 μL of T4 ligase (1u/μL, Invitrogen) - 2.5 x 10 ⁶ LST7 beads of FAM for 30 min.

Cut 1: The beads were then washed 3 times with 100 μL LS Washl (containing 1XTE, 30 mM sodium acetate, 0.01% TritonX100); a 10 μL aliquot of this solution was removed and stored for analysis. The beads were then washed (1X) with 100 μL of 30 mM sodium acetate. 50 μL of 50 mM AgNO ₃ was added to this solution, and the resulting mixture was incubated at 37° C. for 20 minutes. AgNO ₃ was removed and the beads were washed once with 100 μL of 30 mM sodium acetate. The beads were then washed 3 times with 100 μL LSWash1 and resuspended in 90 μL Wash (TENT) buffer; a 10 μL aliquot of this solution was removed and stored for analysis.

Ligation 2: After removing the TENT buffer, the beads were resuspended in 14 μL H ₂ O and treated with 1 μL 100 μL ST7-5 nonamer, 4 μL 5×T4 ligase buffer (Invitrogen) and 1 μL T4 ligase (1 u/μL, Invitrogen ) mixture was incubated at 37°C for 30 minutes.

Cut 2: The beads were washed 3 times with 100 μL LSWash1 (1XTE, 30 mM sodium acetate, 0.01% Triton X100) and resuspended in 45 μL Wash1E. A 15 [mu]L aliquot of this mixture was withdrawn and stored for analysis. The beads were then washed once with 100 μL of 30 mM sodium acetate and resuspended in 5 μL of 20 mM sodium acetate. 50 μL of 50 mM AgNO ₃ was added to the beads and the mixture was incubated at 37° C. for 20 minutes. After removing the AgNO ₃ , the beads were washed once with 100 μL of 30 mM sodium acetate. The beads were then washed 3 times with 100 μL LSWash1 and resuspended in 30 μL Wash1E. A 20 [mu]L aliquot of this mixture was withdrawn and stored for analysis.

result

The experiment can be better understood with reference to FIG. 8 . The upper part of Figure 8 shows a general overview of the experimental procedure. The starting oligonucleotide (primer) hybridized to a template (labeled LST7) attached to the bead via a biotin linkage. The starting oligonucleotide contains a 5' phosphate and its 3' end is fluorescently labeled with FAM. Synthesis of two 9-mer (nonamers) oligonucleotide probes (cleavable oligonucleotide 1 and cleavable oligonucleotide 2) containing phosphorothioated thymidine bases inside (sT) (underline). The first cleavable probe was ligated to the extensible end of the primer with T4 DNA ligase, and then cleaved with silver nitrate. Cleavage removes the terminal 5 nucleotides of the extension probe and creates an extendable end on the portion of the probe still attached to the primer. Then, a second cleavable probe is ligated to the extensible end, followed by cleavage similarly.

The ligation and cleavage steps were monitored using a fluorescent capillary electrophoresis gel-shift assay. In this experiment, the primer is hybridized to the template strand so that the 5' phosphate can be used as a ligation substrate for the introduction of oligonucleotide probes (the fluorophore is used as a reporter for mobility-based capillary gel electrophoresis). After each step, a sample amount of beads was removed for analysis. After connecting the oligonucleotide probes, collect the magnetic beads with a magnet, release the linker formed by the primer and probe ligation on the template beads by heat denaturation, and use an automatic DNA sequencing equipment (ABI 3730) to label the size standard (lissamine ladder ; size range 15-120 nucleotides; shown as a group of orange peaks in the chromatogram, see Figure 8) for fluorescent capillary electrophoresis. In a typical gel shift, possible peaks include, i) primer peaks (due to no extension or lack of primer extension), ii) adenylation peaks (due to the action of DNA ligase at 5' of non-productive junctions). end with adenosine residues attached - see Figure 8F for mechanism, see also Lehman, I.R., Science, 186:790-797, 1974), and iii) complete peaks (due to ligation of oligonucleotide probes). One advantage of using gel shift experiments to assess ligation efficiency is that the area under the peak is directly related to the concentration of each species.

Figure 8A shows a control ligation with T4 DNA ligase and an exact match probe containing only a phosphodiester ligation (Figure 8A left). Orange peaks represent size markers. The blue peak on the left indicates the position of the primer when not ligated. Ligation of an exact match probe results in a shift to the left (arrow). Figure 8B shows ligation under the same conditions with a probe containing a thiolated T base internally (Figure 8B left). The same shift as the control probe was observed (arrow). The bead-attached template population with attached phosphorothioated probes was then incubated with silver nitrate to induce probe cleavage. Gel shift analysis revealed a left-shifted 4-bp cleavage product, confirming efficient cleavage (Fig. 8C). Figure 8C left shows expected cleavage products. The cleaved bead-based template population was then subjected to a second round of ligation, as demonstrated by a right-shifted 13-bp extension product (Fig. 8D). Figure 8D left shows expected cleavage products. The second round of cleavage confirmed that efficient multiple cleavage steps could be accomplished, as indicated by the expected left-shifted 8-bp cleavage product (Fig. 8E).

These results demonstrate successful ligation and cleavage of probes containing phosphorothioate linkages.

Clearly, ligation did not proceed to 100% completion in these experiments, but a higher degree of completion was observed in other experiments with T4 DNA ligase (see below). While it is indeed desirable that the connection proceed to completion, this is not a requirement. For example, the unligated 5' ends can be effectively "capped" by 5'-phosphatase treatment after the ligation step described above. In this case, however, the number of serial ligations that can be performed may be limited due to the depletion of ligatable molecules. Given the number of contiguous ligations, the read length depends on the probe length remaining after each ligation/cleavage cycle and the number of sequencing reactions, each followed by primer removal and binding to primers that can be performed on a given template. Hybridization of primers that bind to different parts of the site, also known as the "restart" number. This supports the use of longer probes with cleavable linkages close to the 5' end of the probe. In our experiments, hexamer probes produced more non-ligatable adenylation products than octamer and longer probes. Octamers and longer probes ligate to completion (see below). Furthermore, adding a fluorescent moiety to the 5' end of a hexamer probe appears to reduce ligation efficiency, while adding a fluorescent moiety to an octamer probe has little or no effect. For these reasons, it is considered preferable to use octamer or longer probes.

Other experiments (below) have demonstrated ligation and cleavage of probes containing phosphorothioate linkages and nucleotides of reduced degeneracy; 3' end specificity and selectivity of ligated extension probes; medium ligation and cleavage; successive cycles of primer hybridization and removal with only a small loss of signal; 100% fidelity for 3'→5' extension with T4 or Taq ligase; and 4-color spectral resolution of ligated extension probes. An automated system for carrying out the method was constructed.

Example 2: Efficient Cleavage and Ligation of Phosphorothioated Oligonucleotides Containing Nucleotides with Reduced Degeneracy

However, another consideration for probe length is the fidelity of the extended oligo and its impact on subsequent ligation efficiency. The fidelity of T4 DNA ligase has been demonstrated to decrease rapidly after the 5th base after the junction (Luo et al., Nucleic Acids Res., 24:3071-3078 and 3079-3085, 1996). If a mismatch is introduced on the 5' side of the newly ligated junction, ligation efficiency can be reduced by depletion, however, without a phase shift or increase in background signal (a major hurdle encountered in polymerase-based sequencing by synthetic approaches). ).

Preferably, the probe set should be able to hybridize to any DNA sequence in order to resequence uncharacterized DNA. However, the complexity of labeled probe sets increases exponentially with the length and number of 4-fold degenerate bases. Furthermore, complex probe sets are more difficult to synthesize, and more difficult to purify, while maintaining essentially the same representation of all probe species. Higher concentrations of the probe mix are also required to maintain the concentration of each species constant. One way to address this complexity is to employ nucleotides that incorporate universal bases such as deoxyinosine instead of 4-fold degenerate bases at certain positions.

Twelve octanucleotide probes (in Inosine in B-DNA can form a double-dentate hydrogen bond with any of the four typical bases; the order of stability of the inosine base pair is I:C>I:A>I:T≈I: G). One of the goals of evaluating these probe designs was to determine how low octamer complexity could be achieved in the presence of inosine bases and still support efficient ligation.

In a preliminary study, several oligonucleotide probes were ligated to a bead-based template (long-LST1) using T4 DNA ligase. After ligation, the fluorophore-labeled primer (3' FAM primer) shifts to the right in proportion to the amount of oligonucleotide probe attached. Probe design NI8-9 showed the highest level of completion where >99% of the primer population was shifted to the right due to efficient ligation of the probe (see Figure 9). These reactions were performed at 25 °C; when the reaction temperature was increased to 37 °C, ligation was slightly less efficient and completion was more variable.

Further examination of these data revealed that probes with fewer inosine bases within the first five nucleotides (underlined) on the 3' side of the junction showed higher ligation efficiencies. In order to further study and evaluate the possible influence of sequence content on ligation efficiency, four oligonucleotide probe designs with only one inosine residue in the first five bases on the 3' side of the junction were screened in all templates. Figure 10 shows gel shift assays of selected probe compositions on various templates using T4 DNA ligase to assess ligation completeness. Data from these preliminary experiments show that ligation efficiency as well as completion rates are variable and sequence-dependent on the occurrence of inosine residues in the first five 3' positions (underlined) at the ligation. However, efficient ligation of the octamer was consistently observed with oligonucleotide probe designs NI8-9, as evidenced by >99% completion across all templates tested.

While not wishing to be bound by any theory, these data, including the presence of an adenylation intermediate, support the conclusion that the presence of unfavorable inosine base pairs in the core DNA-binding site of T4 DNA ligase renders DNA proteins Complex instability is sufficient to reduce enzyme binding and subsequent ligation. However, an interesting question is whether this destabilizing inosine base pair would affect the fidelity of the ligated oligonucleotide probe.

Example 3: Fidelity of Probe Ligation

Bacterial NAD-dependent ligases such as Taq DNA ligase have been reported to have high sequence fidelity at the ligation, where mismatches on the 3' side have essentially no nick-closing activity, but mismatches on the 5' side are somewhat tolerant (Luo et al., Nucleic Acids Res., 24:3071-3078 and 3079-3085, 1996). On the other hand, T4 DNA ligase has been reported to be slightly less stringent, allowing mismatches at the 3'- and 5'-sides of the junction. Therefore, it was of interest to evaluate the fidelity of probe ligation with T4 DNA ligase compared to Taq DNA ligase in our system.

Using standard ABI sequencing techniques, we developed two methods to assess the sequence fidelity of ligated oligonucleotides. The first approach was designed to clone and sequence the ligation products. In this method, ligated extension products are ligated to adapter sequences, cloned and transformed into bacteria. Individual colonies were picked and sequenced to quantitatively assess the frequency of mismatches at each position of the junction. The second approach was designed to directly sequence the ligation products. In this method, bead-based templates are denatured into single-stranded ligation products, which are sequenced directly with complementary primers. Positions of low accuracy in the resulting sequence trace showed multiple overlapping peaks, at which position the fidelity of the sequence was assessed qualitatively.

The first method was used to assess the relative fidelity of probe ligation using T4 and Taq DNA ligases. A single bead-based template population (LST1) was hybridized to a universal sequencing primer used as the starting oligonucleotide. Then T4 DNA ligase (15U/ ^1x106 beads) or Taq DNA ligase ( ^60U /1x106 beads) in the presence of a degenerate oligonucleotide probe (N7A, 3'ANNNNNNN5', 2000 pmoles) ) for a solution-based ligation reaction at 37° C. for 30 minutes ( FIG. 11 , panel A). Ligation products were cloned and sequenced to assess the positional fidelity of each DNA ligase on the 3' side of its junction (positions 1-8) (Figure 11, panels B and C). The results indicated that the fidelity levels of T4 DNA ligase and Taq DNA ligase were basically the same in the first 5 positions, but the fidelity of T4 DNA ligase was lower in positions 6-8. These results were further confirmed by subsequent cloning experiments that evaluated three degenerate inosine-containing probe designs (3'-NNNNNNIII-5', 3'-NNNNNINI-5' and 3'-NNNINNNI-5') DNA sequences at junctions with all seven templates (LST1-7). This study confirmed that T4 DNA ligase had low sequence fidelity at junction positions 6-8, but high fidelity at the top 5 positions in all templates tested (data not shown).

Evaluation of the fidelity of T4 DNA ligase for degenerate inosine-containing probes by direct sequencing. Oligonucleotide probes were evaluated at 25°C and 37°C in ligation reactions containing T4 DNA ligase and bead-based templates. Oligonucleotide probe ligation efficiency was assessed by gel shift assay (Figure 12, panel A). Ligation reactions were directly sequenced with an ABI3730xl DNA Analyzer to evaluate the fidelity of T4 DNA ligase in oligonucleotide probe ligation (Figure 12, panel B). Ligation of exact-match oligonucleotide probes and two representative degenerate inosine-containing oligonucleotide probes (NI8-9 and NI8-11) was >99% complete with very low frequency of mismatches (No multiple peaks in the sequencing trace). The data indicate that operably linked probes also have high sequence fidelity.

In other experiments, a single bead-based template population (LST1) was hybridized to a universal sequencing primer containing a 5' phosphate used as the starting oligonucleotide. In the presence of degenerate inosine-containing oligonucleotide probes (3'NNNNNiii5', 3'NNNNNiNi5' or 3'NNNNNi5', 600 pmol), use T4 DNA ligase (1U/250,000 beads) at 37°C The solution-based ligation reaction was performed for 30 minutes. The ligation products were cloned, and colonies were picked and sequenced. Sequence fidelity was determined by counting the number of clones representing each position of the junction. The results are tabulated, see Figures 12C-F. These studies demonstrate that degenerate inosine-containing probes are ligated 3' → 5' with T4 DNA ligase with a high level of fidelity in the first 1–5 positions.

Example 4: Ligation and cleavage in a gel

Initial experiments to explore, develop and optimize methods for oligonucleotide ligation cycles were performed with bead-based templates in solution, as described above. In the second set of experiments, bead-based templates embedded in polyacrylamide gels on glass slides were ligated and cleaved.

Slides were prepared by mixing several million beads, each attached to a clonal population of single-stranded DNA templates, with 5% polyacrylamide on the slide and where polymerization occurred. The polyacrylamide solution containing the beads was surrounded with a Teflon(R) mask. Figure 14 (upper panel) shows a fluorescent image of a section of a slide on which beads attached to templates hybridized with Cy3-labeled primers were immobilized in a polyacrylamide gel. (The slide was used in a different experiment and is representative of the slide used in this paper.) Figure 14 (lower panel) shows a schematic of the slide fitted with a Teflon mask to surround the polyacrylamide solution.

Reactants are introduced to the slides by manually dropping the appropriate solution onto the slides or by placing the slides in an automated laminar flow chamber. Preliminary studies have demonstrated that efficient in-gel attachment of templates attached to beads immobilized in the polyacrylamide matrix of such slides is in fact possible. In the experiment shown in Figure 15, single-stranded DNA template beads were immobilized on glass slides containing acrylamide and DATD. After polymerization, a 3' fluorophore-labeled, 5' phosphorylated universal primer (sequencing primer) is diffused into the gel, allowing it to polymerize (panel A). Wash slides to remove unbound sequencing primers, mix with ligation mix containing T4 DNA ligase (10 U) and oligonucleotide probes, and incubate at 37°C for 30 minutes. Slides were then incubated in a buffer containing sodium periodate (0.1 M) to digest the acrylamide polymer and release the bead-based template population. The template strands were denatured by heating to yield ligation products, which were collected and analyzed using the gel shift assay described above. Ligation reactions performed in the gel in the absence of T4 DNA ligase showed a peak representing unligated sequencing primers (panel B). Ligation reactions with the octamer probe in the presence of T4 DNA ligase showed efficient oligonucleotide ligation in the gel with >99% of the bead-based template population ligated efficiently (panel C).

Embodiment 5: four-color detection

To maximize detection efficiency, it is desirable to employ a set of oligonucleotide probes that contain distinctive labels corresponding to each possible base addition product. This method is simulated in an automated sequencing device equipped with appropriate excitation and emission filters, as shown in Figure 15. Three sets of octamer probes were designed to address the issues of probe specificity and selectivity. The first set included four octamers that were complementary to four unique template populations, containing different 3' bases and 5' dye labels. The second group included seven unique octamers containing unique 3' bases and 5' dyes. The third set corresponds to the probe design of four degenerate inosine-containing octamers, each containing a unique 3' terminal base identified with a different 5' dye label.

To validate the four-color spectral species, probe set #1 was used to detect four unique template populations (see Figure 16). Slides were prepared containing four unique populations of single-stranded templates attached to beads embedded in polyacrylamide (Panel A). Each bead is linked to a cloned template population. In situ hybridization with universal sequencing primers containing 5' phosphates, using oligos containing four unique fluorophore probes (Cy5, CAL 610, CAL 560, FAM; 100 pmol each) and T4 DNA ligase (10 U/slide) The nucleotide probe mixture is subjected to a ligation reaction. Incubate slides at 37°C for 30 minutes and wash to remove unbound probes. Slides were imaged under bright light to produce a white-light base image (Panel B), and four bandpass filters (FITC, Cy3, Texas Red, and Cy5) were used for fluorescence excitation. Fluorescent images were captured before and after ligation. A single population produces a false color (Panel C), plotting the image values of the different spectral classes and verifying minimal signal overlap (Panel D).

Example 6: Demonstration of Ligation Specificity and Selectivity in Gels

To verify 3' specificity, a template population was tested with probe set #2 (see Figure 17). Slides were prepared from beads embedded in a polyacrylamide gel linked to a template population (LST1.T) and hybridized in situ with universal sequencing primers (Panel A). The ligation reaction was performed in a gel with T4 DNA ligase (10 U/slide) and an oligonucleotide probe mix consisting of four 5'-labeled probes that differed by only one 3 'base. Incubate slides at 37°C for 30 minutes and wash to remove unbound probe populations. The slides were imaged under white light to generate the base image (Panel B), and four bandpass filters (FITC, Cy3, Texas Red, and Cy5) were used for fluorescence excitation. Fluorescent images captured before and after ligation confirmed the presence of a single population of FAM-based probes (blue dots) after ligation in the gel with T4 DNA ligase, with no spectral overlap (panels C, D). These data show that the probe specificity of T4 DNA ligase is stringent and depends on the first 3' base at the ligation.

To further confirm 3' end specificity and selectivity, probe set #2 was used to identify bead-based template population mixtures containing one base difference and present in different amounts. Slides were prepared with a mixture of beads each attached to one of four template populations, each with a different SNP (LST1; A, G, C, or T), as shown in Figure 18A . These beads are embedded in polyacrylamide gels on slides. Bead-based template populations were used at various frequencies, as indicated in column D. Slides were in situ hybridized with universal sequencing primers. The ligation reaction was performed in a gel with T4 DNA ligase (10 U/slide) and an oligonucleotide probe mix containing equimolar amounts (100 pmol each) of the four 5'-labeled probes. The probes differ by only one 3' base. Incubate slides at 37°C for 30 minutes and wash to remove unbound probe populations. Slides were imaged under white light to generate base images (column B), and four bandpass filters (FITC, Cy3, Texas Red, and Cy5) were used for fluorescence excitation. Individual probe images are superimposed and false-colored (column C). Fluorescent images were counted with bead-calling software. The results, shown in column D, demonstrate that the observed connection frequency (Obs) correlates with the expected frequency (Exp). The data showed high probe specificity and probe selectivity after ligation in the presence of multiple templates and confirmed the detection of single nucleotide polymorphisms (SNPs) by ligation, i.e., a single nucleotide polymorphism in a segment of genomic DNA from different individuals in a population The ability to change nucleotide bases.

Example 7: Demonstration of ligation specificity and selectivity in gels using four-color degenerate inosine-containing extension probes

Another set of experiments was performed with probe set #3 to evaluate the specificity and selectivity of probe ligation using a pool of four-color degenerate inosine-containing oligonucleotide probes. The results are shown in Figure 19. Bead-based slides were prepared as described above, but using four unique single-stranded template populations present in varying amounts on the beads, followed by in situ hybridization with universal sequencing primers (Panel A). In the presence of T4 DNA ligase (10U/slide), the ligation reaction was carried out in the gel with a probe pool whose 3' end consisted of five degenerate bases (N; complexity 4 ⁵ =1024), two universal bases (I, inosine) and an octamer of known nucleotide design, which correspond to specific 5' fluorophores (G-Cy5, A-CAL 610, T-CAL560 , A-FAM; 600 pmol each). Incubate slides at 37°C for 30 minutes and wash to remove unbound probe populations. Slides were imaged under white light to generate base images (column B), and four bandpass filters (FITC, Cy3, Texas Red, and Cy5) were used for fluorescence excitation. Individual probe images are superimposed and false-colored (column C). Fluorescence images were counted with bead-calling software and the frequency of each ligation product was tabulated (column D); spectral scatterplots of raw raw data and filtered data representing the top 90% of bead signal values are shown in column E. The data demonstrate that the observed frequency of ligation (Obs) correlates to the predicted frequency (Exp) based on the known concentrations of each template. This validates that degenerate and universal base-containing probe pools can be used with T4 DNA ligase to provide in-gel specific and selective ligation.

Example 8: Confirmation of repeated cycles of hybridization and removal of starting oligonucleotides in a gel

Experiments with templates immobilized in gels on microscope slides mounted in automated flow cells (see below) demonstrated that multiple cycles of annealing and stripping initiation oligonucleotides can be applied with minimal loss of signal Template for beads embedded in gel on slide. A 44 base fluorescently labeled starting oligonucleotide was used. As shown in Figure 20, minimal signal loss occurs over 10 cycles. The starting oligonucleotides in Figure 20 are referred to as primers. As mentioned above, a major disadvantage of polymerase-based sequencing-by-synthesis methods is the propensity for positive and negative phase shifts to occur on individual template strands. A positive phase shift occurs when nucleotides are misincorporated into a growing strand, thereby causing the base sequence of that particular strand to run ahead of the sequence obtained from the remaining template by n+1 base calls. The more common negative phase shift occurs when the strand is not fully extended, causing background base calls to run (n-1) behind the growing strand. The ability to efficiently strip extension products and "restart" the template by hybridization to position a different starting oligonucleotide enables very long read lengths with little or no loss of signal.

Embodiment 9: automated sequencing system

This example describes a representative automated sequencing system of the invention that can be used to collect sequence information for one or more templates. Preferably, the template is on a substantially flat substrate such as a microscope slide. For example, templates can be attached to beads arrayed on a substrate. A photo of the system is shown in Figure 21. The system is based on an Olympus epifluorescence microscope body (side-mounted) equipped with automation, an autofocus stage, and a CCD camera. Four filter cassettes in a carousel allow for four-color detection at different excitation and emission wavelengths. A flow chamber equipped with a peltier temperature controller is mounted on the platform, which can be opened or closed to accept substrates such as slides (with gaskets to seal the edges of areas containing semi-solid supports such as gels). The vertical orientation of the flow chamber is an important aspect of the system of the present invention, allowing air bubbles to escape from the top of the flow chamber. The flow chamber can be completely filled with air to drive out all reagents prior to each wash step. The flow cell is connected to a fluid handler equipped with two 9-port Cavro syringe pumps capable of pumping 4 differentially labeled probe mixes, cleavage reagents, any other required reagents, enzyme equilibration buffer, wash buffer and air is delivered to the flow chamber through a port. Operation of the system is fully automated and programmable with a dedicated computer with multiple I/O ports through control software. The Cooke Sensicam has a 1.3-megapixel cooled CCD, but less or more sensitive cameras are also available (e.g. 4-megapixel, 8-megapixel, etc.). The flow cell utilizes a 0.25 micron stage with a 1 micron overall dimension.

Embodiment 10: image acquisition and processing method

This example describes a representative method for acquiring and processing images of bead arrays with attached labeled nucleic acids. Accurate feature identification and alignment are important for reliable analysis of each acquired image. All but the most intense pixel for each bead were first discarded to identify features. Make a histogram of the pixel values of the given image; discard the pixels corresponding to the background, and sort the remaining pixel values. In a consistent image where all beads have essentially the same intensity, the algorithm employed removes the bottom 80-90% of pixel values. The top 10-20% of pixels were then scanned to identify pixels that were local maxima within a 4 pixel radius. The average intensity for the area is then recorded as well as the average intensity for the perimeter. The values form a normal distribution, and pixels whose values fall outside that distribution are removed. The percentage of pixels initially ignored, the size of the circular area, and the cutoff to eliminate possible beads in the normal distribution are all parameterized and can be changed if desired. The comparison is accomplished by establishing the feature matrix of each image in the comparison group. The resulting matrix is then searched for the most frequent x,y coordinate offsets to identify optimal alignments.

Bead images were collected in the Cy5 channel (corresponding to the sequencing primers) prior to addition of extension probes. These images were used to create a profile of the marker localization coordinates and raw signal intensity in fluorescence units (RFU) for each bead. For each subsequent duplex extension, sets of images were acquired before and after addition of Cy3-labeled nucleotides. These images were aligned to the original Cy5 images, and RFU values were assigned to each bead and recorded. Baseline correction was performed by subtracting the intensity difference between the unlabeled image (before extension) and the labeled image (fluorescence added) caused by each base addition. These baseline-subtracted values were then normalized to the intensity found in the Cy5 image for each feature to form the basis for determining bead extension (ie, a bead was considered extended if the duplex attached to the bead was extended). Using these methods, it is possible to analyze tens of thousands of features on each of approximately 1,300 images per slide, allowing analysis of five to one hundred million template species per experimental run. The algorithm design makes it easy to import C+ from MATLAB subsequently to further improve efficiency.

Example 11: Bead Alignment and Tracking and Sequence Decoding

This example describes a representative method for processing images of bead arrays with attached labeled nucleic acids and performing sequence determination from the obtained data.

Image analysis was started by crimping the image with a zero-integral circular top-hatkernel whose diameter matched the bead size. This automatically normalizes the background to zero while identifying the center of individual beads through local maxima. Maxima were determined, and those maxima isolated from other local maxima were used as comparison points. Calculate the alignment points of each image in time series. For each pair of images, the alignment points are compared and a displacement vector is calculated from the average displacement of all common alignment points. This provides pairwise image displacement with sub-pixel resolution.

For N images, there are N*(N-1)/2 pairs of displacements, but only N-1 pairs of displacements are independent, since the rest can be computed by independent groups. For example, measuring the displacement between images 1 and 2 and between images 1 and 3 suggests the displacement between images 2 and 3. If the measured displacement between images 2 and 3 is different from the suggested displacement, then the measurements are inconsistent. The magnitude of this inconsistency can be used as a measure of how well the alignment algorithm is performing. Our preliminary tests show that the inconsistency is usually less than 0.1 pixel in all directions (see Figure 23).

Once the image time series is aligned, there are two ways to track individual beads. If the bead density is low, and most beads do not touch other beads, then the optical centroid of each bead can be identified and integrated over the area around the bead to calculate the bead intensity. If the bead density is so high that most of the beads touch each other, it may not be possible to identify individual beads by the dark background band surrounding them. However, after all images are scaled to sub-pixel resolution, it is possible to identify pixels belonging to the same bead by computing the correlation of neighboring pixels in time. Highly correlated pairs of pixels can be reliably assigned to the same bead. A similar technique was applied to lane tracking in DNA sequencing gels with good results (Blanchard, A.P., Sequence-specific effects on the incorporation of dideoxynucleotide incorporation by modified T7 polymerases (Sequence-specific effects on the incorporation of dideoxynucleotides by a modified T7 polymerase), California Institute of Technology, 1993). Once the bead has been tracked through the entire 4-color time series, the sequence can be decoded by knowing which color corresponds to which 3'-terminal base of the probe oligonucleotide.

Example 11: Flux Calculations

Typically, the throughput of a sequencing system is primarily determined by the number of images the machine can generate per day and the number of nucleotides (bases) in the sequence data for each image. Since the machine is preferably designed to keep the camera busy at all times, calculations are based on 100% camera utilization. In embodiments where each bead is imaged in 4 colors to determine the identity of a base, 4 images from one camera, 2 images from 2 cameras, or 1 image from 4 cameras can be used. Four-camera imaging can significantly increase throughput compared to other options, and it is preferred that the system utilize this approach.

Our preliminary tests show that a pixel density of 50 pixels per bead (representing 5.4 square microns) provides a suitable density for standard image analysis. By using a 4 megapixel CCD camera (common now), ~80,000 beads can be captured in one frame of CCD image (based on our existing image data). It takes less than 1.5 seconds to capture four images with different cameras and move to the next field of view on the flow cell. If 75% of the beads yielded useful information, we would be able to collect approximately 80,000 beads * 0.75/1.5 = 40,000 bases/second of raw sequence data.

An important issue in maintaining 100% camera utilization is matching the time it takes to perform one ligation/cutting chemistry cycle with the time required to image the entire flow cell. A reasonable estimate of the elongation, cleavage, and ligation cycle time is 11/2 hours (5,400 seconds). These 5,400 seconds will accommodate 1,800 image fields of view or an area of approximately 15mm by 45mm, which is a suitable size for a flow chamber. A conservative estimate of the throughput for a system utilizing four cameras and a flow cell of 15 mm x 45 mm is 40,000 bases per second. Based on our throughput of 28 rounds per day with a read length of about 650 bases (20 bases/sec) that we achieved with the ABI3730xl sequencers, this equates to about 2,000 ABI3730xl sequencers. A 2.5-fold increase in bead density to 200,000 beads per image increases throughput overall to 100,000 bases/second, equivalent to approximately 5,000 ABI3730xl machines. At this level of throughput, the total output is about 8.6 Gb per day, so the time required to complete a 12X human genome sequence is ~4.2 days.

It should be noted that the inventive sequencing methods described herein may be implemented with a variety of different sequencing systems, image capture and processing methods, and the like. See, eg, US Patents 6,406,848 and 6,654,505 and PCT Publication No. WO98053300 for details.

Example 12: Preparation of Microparticles for Synthesizing Templates thereon

This example describes the preparation of microparticles (in this example magnetic beads) attached to amplification primers to amplify (eg, by PCR) a template to generate a clonal population of template molecules attached to each microparticle. Typically, amplification beads are attached with one of the primers required for the cloning PCR reaction. This primer can be covalently coupled to the bead surface or, for example, labeled with biotin to streptavidin bound to the bead surface. Beads can be used in standard PCR reactions (eg, in microtiter plate wells, test tubes, etc.), emulsion PCR reactions as described in Example 13, etc., to obtain beads with linked template molecule clonal populations.

Material

1xTE : 10mM Tris (pH 8) 1mM EDTA

1xPCR buffer : (ThermoPol buffer, NEB)

20mM Tris-HCl (pH 8.8)

10mM KCl

10mM(NH ₄ ) ₂ SO ₄

2mM _MgSO4

0.1% Triton X-100

1M betaine (add only to 1xPCR-B buffer)

1x binding and washing buffer

5mM Tris HCl (pH 7.5)

0.5mM EDTA

1M NaCl

DNA capture primer (20-mer, 500 μM stock solution)

Bibiotin-(HEG)5-P1: 5’-bibiotin-(HEG)5-CTA AGG TAG CGA CTGTCC TA-3’

(HEG)5 = Hexaethylene glycol linker, contains an 18 carbon spacer, one of many different spacer moieties that can be used. A spacer is included that can be used, for example, to lift the P1 primer portion of the oligonucleotide off the bead surface. Any of the primers described herein can be incorporated into this spacer portion.

Dynal storage magnetic beads (1 μm diameter) = 10 mg/ml (7-12x10 ⁶ beads/μl).

method

1. Remove 50 μl beads (~ ^450x106 beads).

2. Add 200μl 1xTE buffer and mix well. Separate with a magnet.

3. Wash once with 200μl 1xTE buffer. Separate with a magnet.

4. Resuspend in 100μl B/W buffer.

5. Add 3 μl P1 oligonucleotide (500 μM stock solution=1500 pmol).

6. Spin >30 minutes at room temperature.

7. Wash 3 times with 200μl 1x TE buffer.

8. Resuspend in 50 μl (initial volume) of 1×TE buffer.

9. Store the DNA Capture Beads at 4°C or on ice until use. Beads should be used within 1 week (beads tend to clump when stored for >1 week).

Example 13: Method for performing PCR on microparticles in emulsion

This example describes a method that can be used to perform PCR on microparticles in an emulsion to generate microparticles with attached cloning templates. Microparticles (referred to as DNA beads in the nomenclature used below) are first functionalized with a first primer (P1). The second primer (P2) is present in the aqueous phase where the PCR reaction takes place. The aqueous phase may also contain a lower concentration of P1, for example 20 times less, if desired. Doing so enables rapid establishment of the template in the aqueous phase, which is the substrate for continued amplification. As P1 is depleted in solution, the reaction is forced to utilize P1 attached to the microparticle. P1_P2degen10 is an oligonucleotide template (100 bp) with sequences that hybridize to P1 and P2 for amplification by PCR and about 10 degenerate bases (in oligonucleotides) that confer 4 ¹⁰ complexity on this oligonucleotide population. incorporated during acid synthesis).

I. Emulsion protocol (1 μm beads)

1. Prepare the oil phase:

Span 80 (7%)

Tween 80 (0.4%)

Prepared in light mineral oil

Use only freshly prepared oil phase

Total oil phase = 450 μl

2. Prepare the aqueous phase: (estimated to produce ^2x109 drops, each drop 115fL)

Reagent (mother liquor) (μl)/reaction finally _{dH2OMgCl2 buffer} (10X) dNTP (100 mM ea) _MgCl2 (1M) betaine (5M) P1 (primer 1) (10 μM) P2 (primer 2) (200 μM) P1_P2 degen10 (100 pM) DNA beads (8M /μl) Platinum Taq (5U/μl) 156.032.011.37.332.01.640.06.625.09.0 -1X each 3.5 mM23 mM0.5 M11.25 pmol 5625 pmol ^5.9x107 /μl150M/emulsion 0.28U/μl

Total aqueous phase volume = 320 μl

Final reaction = 255 μl water phase: 450 μl oil phase

3. Transfer the aqueous phase tube to ice until the emulsion is added.

4. Add 450 μl of the oil phase to a 2 ml cryovial.

5. Place the cryovial upright into the foam socket attached to the IKA vortexer. Set the vortex to 2500 rpm.

6. Sample volume The aqueous phase (3 sample volumes, 85 μl each = 255 μl) was added to the shaken oil phase. Add the monodisperse aqueous phase to a stirred 2 ml cryovial by inserting the pipette tip into the tube and slowly adding the aqueous phase from the tip to the shaking oil phase. The addition was repeated 2 times with the remaining aqueous phase.

7. Continue to shake the emulsion at 2500rpm for 24 minutes,

8. Transfer ~100 [mu]l aliquots of emulsion into 96-well plates (total = 4 wells). Simultaneously, an aliquot of the remaining aqueous phase (65 [mu]l) was added to a separate well for a solution-based PCR control reaction. Seal the plate and cycle as described in the next section.

II. Emulsion Amplification (1 μm Beads)

PCR cycle parameters for 1.1 μm bead emulsion (primer Tm=62°C):

Procedure: DTB-PCR

94°C, 2 minutes n=1

94°C, 15 seconds

57°C, 30 seconds n=100

70°C, 60 seconds

55°C, 5 minutes n=1

10°C, any time

2. The cycle time is about 6 hours.

3. Observe the emulsion after cycle. A successful emulsion will exhibit a uniform amber color with no visible separate aqueous phase. A "broken" (out of solution) emulsion produces a distinct aqueous phase at the bottom of the tube. Avoid collecting this phase, as the bead population here is not clonogenic.

4. Evaluation of post-cycle emulsions by bright field microscopy. A 2 μl aliquot of the circulating emulsion was removed and dropped onto a glass slide. Emulsion samples were covered with 22 x 60 mm coverslips.

5. Observe the emulsion with a 20X objective lens. Preferably, the beads should be monodisperse, with the majority of droplets containing a single bead.

NOTE: If the emulsion sample contains a large number of multi-beaded droplets, pour the emulsion reaction into a 1.5 mleppendorf tube and centrifuge at 6000 rpm for 15 seconds. Remove the bead suspension that collects at the bottom of the tube. This population consists of free beads and multi-bead droplets that are heavier than single-bead liquid and therefore settle to the bottom of the tube after a brief centrifugation. This bead population is not clonal and should therefore be avoided prior to subsequent processing. Repeat steps 4 and 5 to re-evaluate the emulsion to confirm the integrity of the fluid containing the single beads in the emulsion sample.

6. Break up (break) the emulsion as described in the next section.

III. Emulsion disruption and unzipping (1 μm beads)

Bead Breaking Wash (BBW) Buffer

2% Triton X-100 2% Tween 20; 10mM EDTA

Melting solution 100mM NaOH

1xTE : 10mM Tris (pH8) 1mM EDTA

1x Binding and Washing (B/W) Buffer

5mM Tris-HCl (pH7.5)

0.5mM EDTA

1M NaCl

1. Pour each emulsion set (4 sample sizes) into a 1.5ml eppendorf tube.

2. Add 800 μl BBW buffer. The emulsion was disrupted by vortexing the reaction tube for 10 sec.

3. Centrifuge at 8000 rpm for 2 minutes.

4. Remove the upper 800 μl (mainly the oil phase). The DNA beads will sink to the bottom of the tube.

5. Add 800μl BBW, vortex, and centrifuge at 8000rpm for 2 minutes. Remove the upper 600 μl.

6. Wash twice with 600 μl 1xTE, and exchange each washing solution with a magnet.

8. Add 50 μl of melting solution to the bead pellet and resuspend the sample by vigorous pipetting. Incubate the beads with melting solution for 5 min at room temperature, flicking the tube intermittently.

9. Place the tube in the magnet to remove the melting solution. Wash once with 100 μl melting solution to ensure complete removal of the second strand.

10. Wash the bead pellet twice with 1xTE, resuspend in 20 μl TE buffer and store at 4°C, or resuspend in 20 μl 1xB/W buffer if the next step is enrichment. If beads appear aggregated, switch to 1xPCR-B buffer.

11. Continue enrichment method (optional).

Example 14: Method for Enrichment of Microparticles Linked to Cloned Template Populations

This example describes a method for enriching microparticles that have undergone successful template amplification, eg, in a PCR emulsion. This method utilizes larger microparticles with attached capture oligonucleotides. The capture oligonucleotide comprises a region of nucleotides that is complementary to a region of nucleotides present in the template.

I. Emulsion enrichment (1μm)

A. Preparation of enrichment beads (capture entities)

Enrichment Beads:

Spherotech Streptavidin-Coated Polystyrene Beads (~6.5 µm)

Bead stock solution (0.5% w/v): 33,125 beads/μl

Each protocol: (33,125 beads/μl) (800 μl) = ^{26.5 x 106} beads

application:

119 million beads per emulsion - Emulsion clonality estimate (2%): ~3M template positive beads per emulsion. Add 2-3 enrichment beads per projected template-positive emulsion bead = 10 million enrichment beads per emulsion reaction.

Enrichment oligonucleotides (capture reagents):

P2-enriched (35-mer, Tm=73°C)

5'-bibiotin-18 carbon spacer-ttaggaccgttatagttaggtgatgcattaccctg 3'

(or)

P2-enriched (eg, up to 35-mer, Tm = 52°C)

5'-biotin-18 carbon spacer-ggtgatgcattaccctg 3'

Glycerin Solution - 60% (v/v)

6ml glycerin

4ml nuclease-free _H2O

1. Take out 800 μl of beads and centrifuge at 13,000 rpm for 1 minute to exchange into B/W buffer. Wash once with 500μl B/W buffer and resuspend in 100μl B/W buffer.

2. Add 20 μl enrichment oligonucleotide (500 μM stock solution = 10,000 pmol/rxn).

3. Rotate the beads at room temperature for 1 hour.

4. Wash the beads 3 times with 500 μl 1x TE buffer. Beads were pelleted by centrifugation at 13,000 rpm for 1 minute between washes.

5. Resuspend beads in 25 μl B/W buffer. Concentration = 1 M enriched beads/μl.

NOTE: Pouring the four enriched emulsion populations into 20-30 µl of 1xB/W buffer yields ~40M template-positive beads. Multiple slides can then be run.

B. Enrichment step

1. Add 20 μl of enriched beads to the tube containing the emulsion-derived beads (20 μl). Resuspend the bead mixture by gentle pipetting (or use a ratio of 2-3 enriched beads per projected template-positive emulsion bead).

2. If using enrichment beads coated with biotinylated P2-enrichment primers, incubate the bead mixture at 65°C for 2 minutes. Move the tubes to ice for 10 min.

NOTE: Preliminary experiments suggest that enrichment efficiency may be lower using enrichment beads containing primer sequences for 100-cycle PCR (e.g., P2PCR) because it is able to enrich beads containing primer-dimers, which Aggregates are driven onto beads in template-free droplets. If using enrichment beads loaded with the above P2-enrichment primers, incubate the bead mixture at 50°C for 2 minutes due to the lower Tm of this shorter primer.

3. Add the bead mixture to a 1.5ml eppendorf tube containing 300μl 60% glycerol solution.

4. Centrifuge at 13,000 rpm for 1 minute.

5. After centrifugation, the negative beads sink to the bottom of the tube. The enrichment beads with attached template beads will float above the glycerol phase. Collect the upper bead population and transfer it to a clean 1.5ml eppendorf tube.

NOTE: Beads that sink to the bottom of the tube (template-free beads) can be washed and analyzed with a magnet, followed by washing with the same protocol as described for template-positive beads.

6. Add 1 ml of nuclease-free _H2O to the beads collected from the upper phase to dilute the glycerol concentration. Resuspend the bead mixture by gentle pipetting. Centrifuge at 13,000 rpm for 1 minute.

7. After centrifugation, remove the supernatant and wash twice with 100 μl TE.

8. Add 100 [mu]l melting solution to the washed bead pellet. Rotate the tube for 5 minutes at room temperature.

9. Add another 100 μl melting solution and separate the template beads with a magnet.

10. Wash twice with 100 μl TE to remove the non-magnetic enrichment beads, and separate the DNA beads from the enrichment beads with a magnet.

11. Resuspend template beads in 10-20 μl 1x TE. If beads appear to aggregate, dilute into 1x PCR-B buffer.

12. The template-containing beads can be mixed with other enriched populations and added to slides as described in the next example.

Example 15: Preparation method of microparticle array immobilized in or on semi-solid support

This example describes the preparation of slides on which template-attached microparticles are immobilized (eg, embedded) in a semi-solid support. Such slides may be referred to as polony slides. The semisolid support used in this example was polyacrylamide. One approach employs methods that confine the polymerase molecules near the template to enhance amplification.

slide preparation

A. Glass Slides: Bonding - Silane Treatment

Adhesion - Silanes facilitate adhesion of polyacrylamide gels to coverslip surfaces. Slides should be pretreated with Adhesive-Silane just before use.

Note:

^** Store the bonding-silane solution in a chemical fume hood.

^** Adhesion - Silanes are irritating. Work in chemistry lab while preparing solutions.

^** Guaranteed Adhesive-Silane Master Solution is not expired.

^** Do not touch the surface of the slide when transferring from the stand.

Prepare Adhesive-Silane Solution:

1. Add to a 1-L plastic container:

1L dH ₂ O, 1 stir bar

220 [mu]l concentrated acetic acid was added (to bring the pH to 3.5). Add 4 ml Adhesive-Silane Reagent and mix solution >15 min with a stir plate.

Handling slides:

2. Load slides onto plastic 384-well plates upside down (facing the same direction).

3. Wash the slides with dH ₂ O and drain the dH ₂ O.

4. Wash with 100% ethanol and drain the ethanol.

5. Wash again with _dH2O , drain _dH2O and place in tissue culture incubator with running vents and UV light. Washed slides were allowed to dry (~30 minutes).

6. Place the plate in a plastic container and cover the slide with the adhesive-silane solution.

7. Allow the solution to react with the slide for 1 hour. Shake the container intermittently to ensure that the bonding-silane is evenly coated onto the glass.

8. After incubation, wash slides 3 times with dH ₂ O.

9. Wash once with 100% ethanol, drain the ethanol.

10. Allow slides to dry thoroughly before use.

11. Store the bonded-silane treated slides in a desiccator.

B. Acrylamide-based slide (small mask)

·Non-capture scheme

Keep all reagents on ice. Add the following chilled reagents to a 1.5ml eppendorf tube:

Reagent amt (μl) 2 slides 1 slide 1xTE 13 6.5 Beads (1-3M, diluted in 1xTE) 10 5 Rhinohide 1 0.5 40% Acrylamide: Bisacrylamide (19:1, F/S) 5 2.5 TEMED (5%, formulated with 1xTE) 2 1 APS (0.5%, freshly prepared) 3 1.5 total 34μl 17μl

The mixture was pipetted vigorously to break up the beads.

Add 17 μl per slide under the coverslip.

Polymerize by inverting up and down at room temperature for 60 minutes.

Peel off the coverslip with a clean razor blade.

Slides were soaked and washed twice in 1E buffer (to remove unbound beads) within 15 minutes.

Bead-embedded slides can be stored in Wash IE at 4°C.

2. Hybridize the fluorophore-labeled sequencing primer to the embedded bead population. Slides were equilibrated from wash IE to 1x PCR-B buffer by quick drops into a Coplin jar containing 1x PCR-B buffer.

3. In a 1.5ml eppendort tube, add 1-6μl (100μM stock solution) primers to 99μl 1xPCR buffer. On the acrylamide matrix, drop 100 μl of primer solution and cover with a coverslip or sealing gasket,

4. Heat the slide with <DEVIN> program (65°C for 2 minutes, slowly anneal to 30°C) to allow the primers to hybridize to the embedded beads. Wash slides 2 times with Wash IE for 2 min. Slides are ready for ligation-based sequencing.

·Capture scheme

1. Prepare ssDNA template beads at 1M/μl. [Prepare polony slides with 4-5M beads per slide].

2. Resuspend the bead mixture in 30 μl 1x PCR buffer.

3. Add 1 μl of sequencing primer (100 μM stock solution); mix well.

4. Heat to 65°C for 2 minutes.

5. Move to ice for 5 minutes.

6. Wash 3 times with 80 μl 1xTE.

7. Remove all solution with a magnet.

8. Add the following reagents:

Reagent amt (μl) 2 slides 1x buffer 1.5 10x buffer 2.0 High Concentration (HC) Enzyme 16.0 40% Acrylamide: Bisacrylamide (19:1, F/S) 14.4 Rhinohide 2.0 TEMED (5%, formulated with 1xTE) 2.0 APS (0.5%, freshly prepared) 1.5 total 39.4μl

The mixture was pipetted to disperse the beads.

Add 17 μl per slide under the coverslip.

9. Preferably upside-down polymerization, for example, using <Pol-1> cycle program on MJ Research Tetrad PCR machine.

10. Peel off the coverslip with a clean razor blade. Soak and wash slides twice for 10 minutes with 1E buffer. (to remove unbound beads).

11. Polony slides are ready for ligation-based sequencing.

12. Bead-embedded Polony slides can be stored at 4°C in a spacer in Wash IE.

Example 16: Method for preparing microparticle arrays attached to solid supports

This example describes the preparation of slides on which template-attached microparticles are attached to a solid support.

1. Store slides prepared with polymer tethers with active NHS at -20°C.

(Slide H, Product No. 1070936; Schott Nexterion; Schott North America, Inc., Elmsford, NY)

2. In the presence of a desiccant, equilibrate the slides to room temperature before use.

3. Wash slides with 50ml 1xPBS (300mM sodium phosphate, pH8.7) for 5 minutes. Repeat the wash 2 times.

4. Remove the slide from the solution and cover with an adhesive gasket (for loading).

5. In separate tubes, add 1-400 million protein-coated or DNA-coated aliquots of beads in 1xPBS, pH 8.7. DNA can be, eg, a DNA template for sequencing. DNA may include, for example, an amine linker that reacts with NHS.

6. Wash the bead sample 3 times with 1xPBS, pH 8.7 by buffer exchange.

7. Resuspend beads in 125ml 1xPBS, pH 8.7.

8. Add the bead solution to the slide gasket to evenly coat the slide surface.

9. Mount the slides in a dark room and incubate the reaction for 1-2 hours at room temperature.

10. After incubation, remove unbound bead solution and transfer slides to 50ml 1x TE (10 mM Tris, 1mM EDTA, pH 8).

11. Wash the slides 5 times with 50ml 1x TE, stirring at a constant speed for 15 minutes each time.

12. Slides can be stored in 1xTE at 4°C for several weeks.

13. If desired, bead populations can be evaluated by white light (WL) brightfield image analysis or fluorescence using complementary DNA oligonucleotides attached to fluorophore-based dyes. The DNA template can be sequenced using, for example, ligation-based sequencing.

Figure 33A shows a schematic of a slide with beads attached.

It should be noted that only a small fraction of the DNA template molecules are attached to the slide. One micron beads (Dynabeads MyOne Streptavidin Beads; Dynal Biotech, Inc., Product No. 650.01) were used. However, various beads can also be used.

Figure 33B shows a population of beads attached to a glass slide. The lower panel shows the same area of the slide under white light (left) and under a fluorescent microscope. The upper column shows the bead density range.

Equivalent Form and Range

Those skilled in the art will recognize, or be able to ascertain, using routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not limited to the above description, but also includes the scope listed in the appended claims. In the appended claims articles such as "a", "an" and "the" may refer to one or more than one unless stated otherwise or evidently otherwise from the context. If one, more than one, or all group members are present in, used in, or related to a given product or process, claims or descriptions joined by "or" between one or more members of the group should be used unless otherwise stated Or that's clearly not the case in the text.

Furthermore, it is to be understood that the invention includes all changes, combinations and substitutions in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more listed claims are introduced into another claim. In particular, any claim that is dependent on another claim may be adapted to include one or more limitations that exist in any other claim that is dependent on the same base claim.

Furthermore, it is to be understood that any one or more embodiments may be expressly excluded from the claims, even if a specific exclusion is not expressly recited herein. It should also be understood that when the description and/or claims disclose reagents (such as templates, microspheres, probes, probe families, etc.) Other known methods of sequencing using this reagent, unless a person of ordinary skill in the art can make a different understanding, or there is a different description in the specification. In addition, when the description and/or claims disclose a sequencing method, any one or more of the reagents described herein can be used in the method, unless a person of ordinary skill in the art can make a different understanding, or the description explicitly excludes to use this reagent in this method. It is also to be understood that where specific components for sequencing are disclosed in the specification or claims, the invention also includes methods of preparing such reagents. The term "component" is used broadly to refer to any item for sequencing, including templates, template-linked particles, libraries, and the like. Furthermore, the drawings are an integral part of the description, and the invention includes the structures shown in the drawings, such as template-linked particles, and the methods described in the drawings.

Where ranges are given herein, the endpoints are included. It is also to be understood that values expressed as ranges in various embodiments of the invention presuppose any specific value or subrange within said range, unless otherwise indicated or where the context clearly differs from that understood by one of ordinary skill in the art. One-tenth of the unit of the lower limit, unless the text expressly states otherwise.

Claims (354)

1. method of identifying template polynucleotide inner nucleotide sequence said method comprising the steps of:

(a) by the duplex that oligonucleotide probe and initial few nucleic acid are connected to form prolongation described initial oligonucleotide is extended along described template polynucleotide, wherein said oligonucleotide probe comprises thiophosphatephosphorothioate and connects;

(b) one or more Nucleotide of the described polynucleotide of evaluation; With

(c) repeating step (a) and (b) is up to determining nucleotide sequence.

2. the method for claim 1 is characterized in that, described authentication step comprises the mark that detects the oligonucleotide probe that is connected in nearest connection.

3. the method for claim 1 comprises that also cutting described thiophosphatephosphorothioate with the cutting agent that contains the atom that is selected from Ag, Hg, Cu, Mn, Zn or Cd connects the step that produces extensible probe end.

4. method as claimed in claim 3 is characterized in that described cutting agent is AgNO ₃

5. the method for claim 1 is characterized in that, in semi-solid upholder or on carry out described extension step.

6. method of measuring template polynucleotide inner nucleotide sequence said method comprising the steps of:

(a) probe-template duplex that provides probe and template multi-nucleotide hybrid to form, described probe has extensible end;

(b) will extend oligonucleotide probe and described extensible end and be connected to form and contain the prolongation duplex that prolongs oligonucleotide probe, wherein said extension probes contains thiophosphatephosphorothioate and connects;

(c) in described prolongation duplex, identify at least one (1) and the extension probes complementary Nucleotide that just has been connected or (2) lucky nucleotide residue in the template polynucleotide in described prolongation oligonucleotide probe downstream;

(d) if there is not ready-made extensible end, on described prolongation oligonucleotide probe, produce extensible end, make the end of generation be different from an end that extension probes connected; With

(e) repeating step (b), (c) and (d), the nucleotide sequence in determining described template polynucleotide.

7. method as claimed in claim 6 is characterized in that, an end of described each extension probes contains non-extensible part.

8. method as claimed in claim 6 is characterized in that, described authentication step comprises the mark that detects the extension probes that is connected in nearest connection.

9. method as claimed in claim 6 is characterized in that, described authentication step is included under the existence of chain termination nucleoside triphosphate of one or more marks and removes described non-extensible part and extend the oligonucleotide probe of described extension with nucleic acid polymerase.

10. method as claimed in claim 6 is included in also that not have extension probes to be connected in the described Connection Step described extensible when terminal, adds the step of cap for the oligonucleotide probe that extends.

11. method as claimed in claim 6 is characterized in that, described generation step comprises that cutting described thiophosphatephosphorothioate with the cutting agent that contains the atom that is selected from Ag, Hg, Cu, Mn, Zn or Cd connects.

12. method as claimed in claim 11 is characterized in that, described cutting agent is AgNO ₃

13. method as claimed in claim 6 is characterized in that, in semi-solid upholder or on carry out described connection and produce step.

14. method as claimed in claim 6, it is characterized in that, step (a) is included in a plurality of different probes-template duplex is provided in the independent sample size, each duplex contains the initial oligonucleotide probe of hybridizing in the template polynucleotide, wherein the template polynucleotide in each duplex are identical, but the initial oligonucleotide probe in each duplex is incorporated into the different sequences of described template polynucleotide; Independently each sample size is carried out step (b)-(e).

15. method as claimed in claim 14 is characterized in that, with regard to each sample size, an end of described extension oligonucleotide probe contains non-extensible part.

16. method as claimed in claim 15 is characterized in that, with regard to each sample size, described authentication step comprises the mark that detects the extension probes that is connected in nearest connection.

17. method as claimed in claim 15, it is characterized in that, with regard to each sample size, described authentication step is included in the chain termination nucleoside triphosphate existence of one or more marks and removes described non-extensible part and extend the oligonucleotide probe of described extension with nucleic acid polymerase down.

18. method as claimed in claim 15 is included in also that not have extension probes to be connected in the described Connection Step described extensible when terminal, adds the step of cap for the oligonucleotide probe that extends.

19. method as claimed in claim 15 is characterized in that, described generation step comprises that cutting described thiophosphatephosphorothioate with the cutting agent that contains the atom that is selected from Ag, Hg, Cu, Mn, Zn or Cd connects.

20. method as claimed in claim 19 is characterized in that, described cutting agent is AgNO ₃

21. method as claimed in claim 15 is characterized in that, in semi-solid upholder or on carry out described connection and produce step.

22. method as claimed in claim 6 is further comprising the steps of: (f) described linking probe and the described initial oligonucleotide on the described template of removal; (g) with the not homotactic second kind of oligonucleotide repeating step (a) that is incorporated into described template polynucleotide; (h) repeating step (b)-(e).

23. method as claimed in claim 22 is characterized in that, repeatedly repeats described method with the not homotactic initial oligonucleotide that is incorporated into described template polynucleotide.

24. method as claimed in claim 23 is characterized in that, an end of described extension probes contains non-extensible part.

25. method as claimed in claim 23 is characterized in that, in repeating, described authentication step comprises the mark that detects the extension probes that is connected in nearest connection at every turn.

26. method as claimed in claim 23, it is characterized in that, in repeating, described authentication step is included in the chain termination nucleoside triphosphate existence of one or more marks and removes described non-extensible part and extend the oligonucleotide probe of described extension with nucleic acid polymerase down at every turn.

27. method as claimed in claim 23 is included in also that not have extension probes to be connected in the described Connection Step described extensible when terminal, adds the step of cap for the oligonucleotide probe that extends.

28. method as claimed in claim 23 is characterized in that, described generation step comprises that cutting described thiophosphatephosphorothioate with the cutting agent that contains the atom that is selected from Ag, Hg, Cu, Mn, Zn or Cd connects.

29. method as claimed in claim 28 is characterized in that, described cutting agent is AgNO ₃

30. method as claimed in claim 23 is characterized in that, in semi-solid upholder or on carry out described connection and produce step.

31. method as claimed in claim 22 is characterized in that, described removal step comprises linking probe, initial oligonucleotide and template and contains the 1.0-3.0%SDS that has an appointment, 100-300mM NaCl and 5-15mM sodium pyrosulfate (NaHSO ₄) the aqueous solution contact.

32. method as claimed in claim 22 is characterized in that, described removal step comprises linking probe, initial oligonucleotide and template and contains the 2%SDS that has an appointment, 200mM NaCl and 10mM sodium pyrosulfate (NaHSO ₄), as 2%SDS, 200mM NaCl and 10mM sodium pyrosulfate (NaHSO ₄) solution contact.

33. an evaluation is connected in the method for the template polynucleotide inner nucleotide sequence of upholder on tie point, said method comprising the steps of:

(a) by the duplex that oligonucleotide probe and initial oligonucleotide is connected to form prolongation described initial oligonucleotide is extended along described template polynucleotide, wherein extend along the tie point of described template to it and described upholder;

(b) one or more Nucleotide of the described polynucleotide of evaluation; With

(c) repeating step (a) and (b) is up to determining nucleotide sequence.

34. method as claimed in claim 33 is characterized in that, described authentication step comprises the mark that detects the extension probes that is connected in nearest connection.

35. method as claimed in claim 33 is characterized in that, described oligonucleotide probe contains thiophosphatephosphorothioate and connects, and described generation step comprises that cutting described thiophosphatephosphorothioate with the cutting agent that contains the atom that is selected from Ag, Hg, Cu, Mn, Zn or Cd connects.

36. method as claimed in claim 35 is characterized in that, described cutting agent is AgNO ₃

37. method as claimed in claim 35 is characterized in that, described connection and produce step in semi-solid upholder or on carry out.

38. a method that is determined at the template polynucleotide inner nucleotide sequence that is connected in upholder on the tie point said method comprising the steps of:

(a) probe-template duplex that provides probe and template multi-nucleotide hybrid to form, described primer has extensible end;

(b) will extend oligonucleotide probe and be connected in described extensible end, and form and contain the prolongation duplex that prolongs oligonucleotide probe;

(c) in described prolongation duplex, identify at least one (1) and the extension probes complementary Nucleotide that just has been connected or (2) lucky nucleotide residue in the template polynucleotide in described prolongation oligonucleotide probe downstream;

(d) if there is not ready-made extensible end, on described prolongation oligonucleotide probe, produce extensible end, make the end of generation be different from an end that extension probes connected; With

(e) repeating step (b), (c) and (d), the nucleotide sequence in determining described template polynucleotide.

39. method as claimed in claim 38 is characterized in that, an end of each extension probes contains non-extensible part.

40. method as claimed in claim 38 is characterized in that, described authentication step comprises the mark that detects the extension probes that is connected in nearest connection.

41. method as claimed in claim 38 is characterized in that, described authentication step is included under the existence of chain termination nucleoside triphosphate of one or more marks and removes described non-extensible part and extend the oligonucleotide probe of described extension with nucleic acid polymerase.

42. method as claimed in claim 38 is included in also that not have extension probes to be connected in the described Connection Step described extensible when terminal, adds the step of cap for the oligonucleotide probe that extends.

43. method as claimed in claim 38 is characterized in that, described oligonucleotide probe contains thiophosphatephosphorothioate and connects, and described generation step comprises that cutting described thiophosphatephosphorothioate with the cutting agent that contains the atom that is selected from Ag, Hg, Cu, Mn, Zn or Cd connects.

44. method as claimed in claim 43 is characterized in that, described cutting agent is AgNO ₃

45. method as claimed in claim 38 is further comprising the steps of: (f) described linking probe and the described initial oligonucleotide on the described template of removal; (g) with the not homotactic second kind of oligonucleotide repeating step (a) that is incorporated into described template polynucleotide; (h) repeating step (b)-(e).

46. method as claimed in claim 45 is characterized in that, repeatedly repeats described method with the not homotactic initial oligonucleotide that is incorporated into described template polynucleotide.

47. method as claimed in claim 38 is characterized in that, described connection and produce step in semi-solid upholder or on carry out.

48. method as claimed in claim 38 is characterized in that, described template is connected in the particulate that is connected with the rigid substrate of substantially flat.

49. a method of identifying template polynucleotide inner nucleotide sequence said method comprising the steps of:

(a) provide be connected in be fixed within the semi-solid upholder or on the template polynucleotide of particulate.

(b) by the duplex that oligonucleotide probe and initial oligonucleotide is connected to form prolongation initial oligonucleotide is extended along described template polynucleotide, wherein said oligonucleotide probe contains easily cuts connection;

(c) one or more Nucleotide of the described polynucleotide of evaluation; With

(d) repeating step (b) and (c) is up to determining nucleotide sequence.

50. method as claimed in claim 49 is characterized in that, carries out described extension step on described semi-solid upholder.

51. method as claimed in claim 49 is characterized in that, described template is connected in the particulate that is connected with the rigid substrate of substantially flat.

52. a method of measuring template polynucleotide inner nucleotide sequence said method comprising the steps of:

(a) probe-template duplex that provides probe and template multi-nucleotide hybrid to form, described probe has extensible end, described probe-template duplex be connected in be embedded within the semi-solid upholder or on particulate;

(b) will extend oligonucleotide probe and be connected in described extensible end, and form and contain the prolongation duplex that prolongs oligonucleotide probe, wherein said extension probes contains thiophosphatephosphorothioate and connects;

(c) in described prolongation duplex, identify at least one (1) and the extension probes complementary Nucleotide that just has been connected or (2) lucky nucleotide residue in the template polynucleotide in described prolongation oligonucleotide probe downstream;

(d) if there is not ready-made extensible end, on described prolongation oligonucleotide probe, produce extensible end, make the end of generation be different from an end that extension probes connected; With

(e) repeating step (b), (c) and (d), the nucleotide sequence in determining described template polynucleotide.

53. method as claimed in claim 52 is characterized in that, carries out described connection and produce step in described semi-solid upholder.

54. method as claimed in claim 52 is characterized in that, described template is connected in the particulate that is connected with the rigid substrate of substantially flat.

55. a method of measuring template polynucleotide inner nucleotide sequence said method comprising the steps of:

(a) in the presence of particulate in the emulsion chamber amplification template polynucleotide molecule, produce the particulate of the clonal population be connected with the template polynucleotide;

(b) from described emulsion, reclaim described particulate;

(c) described particulate is embedded within the semi-solid upholder or on;

(d) by the duplex that oligonucleotide probe and initial oligonucleotide is connected to form prolongation initial oligonucleotide is extended along described template polynucleotide, wherein said oligonucleotide probe contains easily cuts connection;

(e) one or more Nucleotide of the described polynucleotide of evaluation; With

(f) repeating step (d) and (e) is up to determining nucleotide sequence.

56. method as claimed in claim 55 is characterized in that, (i) amplification contains not homotactic multiple template polynucleotide molecule in single emulsion chamber; (ii) from described emulsion, reclaim multiple particulate and be embedded in the described upholder or on, each particulate is connected with template polynucleotide clonal population, wherein said clonal population has different sequences, (iii) to the parallel step (d), (e) and (f) of carrying out of the described clonal population that is connected in described embedding particulate, so that a plurality of sequences of replicate(determination).

57. the method for template polynucleotide inner nucleotide sequence information is measured in first set of the oligonucleotide probe family of at least two kinds of distinctive marks of a use, said method comprising the steps of:

(a) by the duplex that oligonucleotide probe and initial oligonucleotide is connected to form prolongation initial oligonucleotide is extended along described template polynucleotide, wherein said oligonucleotide probe is the member of the oligonucleotide probe man family set of described distinctive mark;

(b) detect the mark that is connected with described oligonucleotide; With

(c) repeating step (a) and (b) is up to the ordered list that obtains probe family title; With

(d) adopt the ordered list of probe family title to get rid of one or more possible nucleotide sequences.

58. method as claimed in claim 57 is characterized in that, step (d) comprises the ordered list of the described probe of decoding family title, to determine described sequence.

59. method as claimed in claim 57, it is characterized in that, probe-template the duplex that provides initial oligonucleotide probe and template multi-nucleotide hybrid to form is provided described method, described probe has extensible end, wherein said extension step comprises oligonucleotide probe is connected in described extensible end, formation contains the prolongation duplex that prolongs oligonucleotide probe, be included in also that not have oligonucleotide probe to be connected in the described extension step described extensible when terminal, add the step of cap for all the other extensible ends.

60. method as claimed in claim 57 is characterized in that, an end of oligonucleotide probe contains non-extensible part described in each probe family.

61. method as claimed in claim 57, after detecting step, each also comprises: if (f) extensible end does not exist, just on the oligonucleotide probe of described nearest connection, produce extensible end, so that the end that produces is different from the end that the oligonucleotide probe of described nearest connection connects.

62. method as claimed in claim 61, it is characterized in that, described oligonucleotide probe contains thiophosphatephosphorothioate and connects, and cuts described thiophosphatephosphorothioate with the cutting agent that contains the atom that is selected from Ag, Hg, Cu, Mn, Zn or Cd and connects, thereby produce described extensible probe end.

63. method as claimed in claim 62 is characterized in that, described cutting agent is AgNO ₃

64. method as claimed in claim 57 is characterized in that, in semi-solid upholder or on carry out described extension step.

65. method as claimed in claim 57 is characterized in that, described template is connected in the particulate that is connected with the rigid substrate of substantially flat.

66. method as claimed in claim 57 is characterized in that, described set comprises the probe family of 2 kinds of distinctive marks.

67. method as claimed in claim 57 is characterized in that, described set comprises the probe family of 3 kinds of distinctive marks.

68. method as claimed in claim 57 is characterized in that, described set comprises the probe family of 4 kinds of distinctive marks.

69. method as claimed in claim 57 is characterized in that, described set comprises the probe family of distinctive mark more than 4 kinds.

70. method as claimed in claim 57 is characterized in that, described oligonucleotide probe comprises the limited part that nucleosides is not independently selected, and wherein distributes to probe family according to the encoding scheme oligonucleotide probe that limited partial sequence is different.

71. method as claimed in claim 57 is characterized in that, according to one of 24 kinds of listed encoding schemes of table 1 described oligonucleotide probe is distributed to first, second, third and four point probe family.

72. method as claimed in claim 58 is characterized in that, the kind of at least one Nucleotide is known in the described template, and wherein said decoding step comprises:

(i) by determining that the possible sequence of the limited part of this probe which kind and known nucleotide kind and its near-end Nucleotide are connected in the Nucleotide adjacent nucleotide relative position of known kind conforms to, to the Nucleotide given category adjacent on the described template with the Nucleotide of known kind;

(ii), give described follow-up Nucleotide given category by determining which kind conforms to the possible sequence that its near-end Nucleotide is connected in the limited part of this probe of follow-up Nucleotide relative position; With

(iii) repeating step (ii), up to measuring this sequence.

73. method as claimed in claim 58 is further comprising the steps of:

(a) the Nucleotide kind in the described template of mensuration, so that described Nucleotide has known kind, wherein said decoding step comprises:

(i) by determining that the possible sequence of the limited part of this probe which kind and known nucleotide kind and its near-end Nucleotide are connected in the Nucleotide adjacent nucleotide relative position of known kind conforms to, to the Nucleotide given category adjacent on the described template with the Nucleotide of known kind;

(ii), give described follow-up Nucleotide given category by determining which kind conforms to the possible sequence that its near-end Nucleotide is connected in the limited part of this probe of follow-up Nucleotide relative position; With

(iii) repeating step (ii), up to measuring this sequence.

74. as the described method of claim 73, it is characterized in that, described determination step is included under the certain condition that has polysaccharase template-probe duplex is contacted with labeled nucleotide, if described under the described conditions labeled nucleotide with described duplex position adjacent on described template complementation, just can mix described labeled nucleotide.

75. method as claimed in claim 58 is characterized in that, described decoding step comprises: produce at least a candidate sequence from the ordered list of probe family title; With the nucleotide sequence of selecting candidate sequence as described template.

76., it is characterized in that described generation step comprises at least 4 candidate sequences of generation as the described method of claim 75.

77., it is characterized in that described generation step comprises as the described method of claim 75:

(i) kind of first Nucleotide of the described nucleotide sequence of supposition;

(ii) basis is determined the possible kind of adjacent nucleotide corresponding to the probe family title of described first Nucleotide, thereby specifies the kind of the Nucleotide adjacent with described first Nucleotide;

(iii) basis is determined the possible kind of follow-up Nucleotide corresponding to the probe family title of the Nucleotide of nearest given category, thereby specifies the kind of follow-up Nucleotide;

(iv) repeating step (iii), up to producing candidate sequence; With

(v) repeating step (i)-(iv) wherein, is taken turns in the repetition at each, and described first Nucleotide is assumed to different sorts, up to the candidate sequence that produces desired number.

78., it is characterized in that described selection step comprises at least a candidate sequence and one or more known arrays as the described method of claim 75, and select and one or more known arrays have predetermined homogeny degree or immediate candidate sequence.

79., it is characterized in that described template is derived from interested organism as the described method of claim 78, wherein said comparison step comprises at least a candidate sequence and contains available from the sequence in the database of the sequence of described organism.

80., it is characterized in that described comparison step comprises at least a candidate sequence and the sequence that contains in the database of a plurality of comparative sequences as the described method of claim 78, each sequence contains the difference of polynucleotide sequence to be measured may sequence.

81., it is characterized in that described selection step comprises as the described method of claim 75:

(i) use second set of the coding probe family of distinctive mark to obtain second kind of probe family title ordered list from described template, the man family set middle probe of wherein said second probe family is different with the coding of the man family set middle probe of described first probe family;

(ii) produce at least a comparative sequences from described second kind of probe family title ordered list;

The (iii) part of the part of at least a described candidate sequence and at least a described comparative sequences; With

(iv) be chosen in the step (c) on the part relatively with comparative sequences predetermined homogeny degree or the immediate candidate sequence nucleotide sequence as described template is arranged.

82., it is characterized in that described rating unit is a dinucleotides as the described method of claim 81.

83., it is characterized in that the described second probe family title ordered list only contains an element as the described method of claim 81.

84. method as claimed in claim 57 is characterized in that, oligonucleotide probe has following structure described in each probe family: 5 '-(XY) (N) _kN _B ^*-3 ' or 3 '-(XY) (N) _kN _B ^*-5 ', wherein N represents any nucleosides, N _BThe part that representative can not be extended with ligase enzyme, but * represent the test section, XY is the limited part of described probe, wherein X and Y represent nucleosides identical or different but that can not independently choose separately, X and Y are at least 2 times of degeneracys, connecting between at least one nucleosides is easily to cut connection, and k is 1-100, and restricted condition is: but the test section can be present in Y or (N) _kIn arbitrarily on the Nucleotide and be present in N in addition _BGo up or be not present in N in addition _BOn.

85. as the described method of claim 84, it is characterized in that, describedly cut easily that to connect be that thiophosphatephosphorothioate connects.

86. as the described method of claim 84, it is characterized in that, but described test section connects, can or have this two kinds of features by photobleaching by cutting joint.

87., it is characterized in that the described joint that cuts contains disulfide linkage as the described method of claim 86.

88. as the described method of claim 84, it is characterized in that, adopt the oligonucleotide probe family of 4 kinds of distinctive marks, wherein the different oligonucleotide probe of the limited partial sequence of this probe is distributed to first, second, third and four point probe family according to one of 24 kinds of listed encoding schemes of table 1.

89. method as claimed in claim 57 is characterized in that, described detection step comprises simultaneously from described template at least 2 Nucleotide 2 information of average acquiring separately, and does not obtain two information from any single Nucleotide.

90. method as claimed in claim 57 is characterized in that, described detection step comprises simultaneously that at least 2 Nucleotide obtain separately from described template and is less than 2 information.

91. the method for template polynucleotide inner nucleotide sequence information is measured in first set with the oligonucleotide probe family of at least two kinds of distinctive marks, said method comprising the steps of:

(a) thus probe-template composite contacted with the oligonucleotide probe family of two kinds of distinctive marks at least make oligonucleotide probe hybridization, described probe-template composite contains double-stranded part and the strand part to be checked order with extensible end, and described oligonucleotide probe contains the template part complementary part that partly is close to described duplex;

(b) oligonucleotide probe with hybridization is connected with described extensible end, contains the probe-template composite that prolongs duplex thereby produce;

(c) detect the mark that links to each other with described linking probe;

(d), then on described prolongation duplex, produce extensible probe end if there is not ready-made extensible probe end; With

(e) repeating step (a)-(d) is up to the ordered list that obtains probe family title.

92., it is characterized in that described detection step comprises simultaneously from described template at least 2 Nucleotide 2 information of average acquiring separately, and does not obtain two information from any single Nucleotide as the described method of claim 91.

93., it is characterized in that described detection step comprises simultaneously that at least 2 Nucleotide obtain separately from described template and is less than 2 information as the described method of claim 91.

94. the method with the nucleotide sequence information of the first set mensuration template polynucleotide of oligonucleotide probe family said method comprising the steps of:

(a) carry out orderly continuously extension, connection, detection and cutting circulation, wherein said detection step comprises: at least two Nucleotide respectively obtain average two information from described template simultaneously, and do not obtain two information from any single Nucleotide; With

(b) information that step (a) is obtained and at least one out of Memory merge, to determine described sequence.

95., it is characterized in that described at least one out of Memory comprises an information that is selected from down group as the described method of claim 94: the Nucleotide kind in the described template, by comparing the information that candidate sequence and at least a known array obtain; Repeat the information that described method obtains with second set that utilizes oligonucleotide probe family.

96. a method of distinguishing single nucleotide polymorphism and order-checking mistake said method comprising the steps of:

(a) with the described method of the claim 58 multiple template that checks order, wherein, described template is represented the overlapping fragments of a nucleotide sequence;

(b) sequence of comparison step (a) acquisition; With

(c) represent the sequence mistake if described sequence, is then determined the difference between the sequence basic identical in the first part and significantly different on second section, the length of described each several part is at least 3 Nucleotide.

97. a method of distinguishing single nucleotide polymorphism and order-checking mistake said method comprising the steps of:

(a) carry out the step (a)-(c) of claim 58 with the multiple template of the single nucleotide sequence overlapping fragments of representative, thereby obtain a plurality of probe family ordered list;

(b) the probe family ordered list that obtains of comparison step (a) to be to obtain a comparison area, and is identical in tabulation at least 90% described in the described comparison area; With

(c), then determine the difference representative order-checking mistake between the described probe family ordered list if described tabulation is only different on a position of described comparison area; Or

(d), determine that then the difference between the described probe family ordered list is represented single nucleotide polymorphism if described tabulation is different on two or more consecutive positions of described comparison area.

98. the set of the oligonucleotide probe family of at least two kinds of distinctive marks, wherein the probe of each probe family comprises limited part and not limited part, be at least 2 times of degeneracys on each position of described limited part, the probe of each family contains easily to cut between nucleosides and connects.

99. the oligonucleotide probe man family set as the described distinctive mark of claim 98 is characterized in that each probe contains the inductile end of ligase enzyme.

100. oligonucleotide probe man family set as the described distinctive mark of claim 98, it is characterized in that, each probe contains the inductile end of ligase enzyme, but each probe comprises the test section on a described position of easily cutting between connection and the inductile end of described ligase enzyme.

101. the oligonucleotide probe man family set as the described distinctive mark of claim 98 is characterized in that, describedly cuts easily that to connect be that thiophosphatephosphorothioate connects.

102. the oligonucleotide probe man family set as the described distinctive mark of claim 98 is characterized in that described set comprises 2 kinds of probe families.

103. the oligonucleotide probe man family set as the described distinctive mark of claim 98 is characterized in that described set comprises 3 kinds of probe families.

104. the oligonucleotide probe man family set as the described distinctive mark of claim 98 is characterized in that described set comprises 4 kinds of probe families.

105. the oligonucleotide probe man family set as the described distinctive mark of claim 98 is characterized in that described set comprises probe family more than 4 kinds.

106. the oligonucleotide probe man family set as the described distinctive mark of claim 98 it is characterized in that, but described probe contains the test section, but described test section connects, can or have this two kinds of features by photobleaching by cutting joint.

107. the set of the oligonucleotide probe family of at least two kinds of distinctive marks, wherein the oligonucleotide probe of each probe family has following structure 5 '-(X) _j(N) _kN _B-3 ' or 3 '-(X) _j(N) _kN _B-5 ', wherein N represents any nucleosides, N _BThe part that representative can not be extended with ligase enzyme, (X) _jBe the limited part of described probe, wherein each X represents a nucleosides, (X) _jIn nucleosides identical or different, but can not independently choose separately, each X is at least 2 times of degeneracys, j is 2-5, k is 1-100, but each probe has the test section at its end, this position, test section is not (X) _jIn nucleosides, wherein the probe in each probe family comprises same tag, but the probe of different probe family comprises different separators.

108. the oligonucleotides coding probe man family set as the described distinctive mark of claim 107 is characterized in that connecting between at least one nucleosides is easily to cut connection.

109. the oligonucleotide probe man family set as the described distinctive mark of claim 107 is characterized in that, describedly cuts easily that to connect be that thiophosphatephosphorothioate connects.

110. the oligonucleotide probe man family set as the described distinctive mark of claim 107 is characterized in that, but described test section connects, can or have this two kinds of features by photobleaching by cutting joint.

111. the oligonucleotide probe man family set as the described distinctive mark of claim 110 is characterized in that the described joint that cuts contains disulfide linkage.

112. the oligonucleotide probe man family set as the described distinctive mark of claim 107 is characterized in that, described group by the group composition of four kinds of probes man, and wherein the oligonucleotide probe of each probe family has following structure: 5 '-(XY) (N) _kN _B ^*-3 ' or 3 '-(XY) (N) _kN _B ^*-5 ', wherein N represents any nucleosides, N _BThe part of representing function to extend with ligase enzyme, but * represent the test section, XY is the limited part of described probe, wherein X and Y represent nucleosides identical or different but that can not independently choose separately, X and Y are at least 2 times of degeneracys, connecting between at least one nucleosides is easily to cut connection, and k is 1-100, and restricted condition is: but the test section can be present in Y or (N) _kIn arbitrarily on the Nucleotide and be present in N in addition _BGo up or be not present in N in addition _BOn.

113. the oligonucleotide probe man family set as the described distinctive mark of claim 112 is characterized in that, describedly cuts easily that to connect be that thiophosphatephosphorothioate connects.

114. the oligonucleotide probe man family set as the described distinctive mark of claim 112 is characterized in that, but described test section connects, can or have this two kinds of features by photobleaching by cutting joint.

115. the oligonucleotide probe man family set as the described distinctive mark of claim 114 is characterized in that the described joint that cuts contains disulfide linkage.

116. oligonucleotide probe man family set as the described distinctive mark of claim 112, it is characterized in that, the different oligonucleotide probe of the limited partial sequence of this probe is distributed to first, second, third and four point probe family according to one of 24 kinds of listed encoding schemes of table 1.

117. the set of the oligonucleotide probe family of at least two kinds of distinctive marks, wherein the oligonucleotide probe of each probe family has following structure 5 '-(X) _j(N) _kN _B-3 ' or 3 '-(X) _j(N) _iN _B-5 ', wherein N represents any nucleosides or dealkalize base residue, N _BThe part that representative can not be extended with ligase enzyme, (X) _jBe the limited part of described probe, wherein each X represents nucleosides or dealkalize base residue, and restricted condition is: X ₁Represent a Nucleotide, (X) _jIn nucleosides identical or different, but can not independently choose separately, each X is at least 2 times of degeneracys, j is 2-5, k is 1-100, but each probe comprises the test section at its end, but the position at place, test section is not (X) _jIn nucleosides, wherein the probe in each probe family comprises same tag, but the probe of different probe family comprises different separators.

118. the oligonucleotides coding probe man family set as the described distinctive mark of claim 117 is characterized in that connecting between at least one nucleosides is easily to cut connection.

119. the oligonucleotide probe man family set as the described distinctive mark of claim 117 is characterized in that, describedly easily cuts connection between nucleosides and dealkalize base residue.

120. the oligonucleotides coding probe man family set as the described distinctive mark of claim 117 is characterized in that described oligonucleotide probe contains the initiation residue.

121. the oligonucleotide probe man family set as the described distinctive mark of claim 117 is characterized in that, but described test section connects, can or have this two kinds of features by photobleaching by cutting joint.

122. the oligonucleotide probe man family set as the described distinctive mark of claim 121 is characterized in that the described joint that cuts contains disulfide linkage.

123. the oligonucleotide probe man family set as the described distinctive mark of claim 117 is characterized in that, described group by the group composition of four kinds of probes man, and wherein the oligonucleotide probe of each probe family has following structure: 5 '-(XY) (N) _kN _B ^*-3 ' or 3 '-(XY) (N) _kN _B ^*-5 ', wherein N represents any nucleosides or dealkalize base residue, N _BThe part that representative can not be extended with ligase enzyme, but * represent the test section, XY is the limited part of described probe, wherein X and Y represent nucleosides identical or different but that can not independently choose separately, X and Y are at least 2 times of degeneracys, connecting between at least one nucleosides is easily to cut connection, and k is 1-100, and restricted condition is: but the test section can be present in Y or (N) _kIn arbitrarily on the Nucleotide and be present in N in addition _BGo up or be not present in N in addition _BOn.

124. the oligonucleotide probe man family set as the described distinctive mark of claim 123 is characterized in that, describedly easily cuts connection between nucleosides and dealkalize base residue.

125. the oligonucleotide probe man family set as the described distinctive mark of claim 123 is characterized in that, but described test section connects, can or have this two kinds of features by photobleaching by cutting joint.

126. the oligonucleotide probe man family set as the described distinctive mark of claim 125 is characterized in that the described joint that cuts contains disulfide linkage.

127. the oligonucleotides coding probe man family set as the described distinctive mark of claim 123 is characterized in that described oligonucleotide probe contains the initiation residue.

128. oligonucleotide probe man family set as the described distinctive mark of claim 123, it is characterized in that, the different oligonucleotide probe of the limited partial sequence of this probe is distributed to first, second, third and four point probe family according to one of 24 kinds of listed encoding schemes of table 1.

129. a test kit, it comprises the set of the oligonucleotide probe family of at least two kinds of distinctive marks.

130., it is characterized in that described probe contains easily to cut between nucleosides and connects as the described test kit of claim 129.

131. as the described test kit of claim 130, it is characterized in that, describedly cut easily between nucleosides that to connect be that thiophosphatephosphorothioate connects.

132. as the described test kit of claim 129, also comprise at least one that is selected from down group: the reagent of the reagent of ligase enzyme, the material that can cut the thiophosphatephosphorothioate connection, Phosphoric acid esterase, polysaccharase, upholder, damping fluid, thermostability polysaccharase, Nucleotide, preparation emulsion and preparation gel.

133. a method for preparing multiple template polynucleotide said method comprising the steps of:

(a) a plurality of particulates are embedded within the semi-solid upholder of reversibility or on, form first arrays of microparticles; With

(b) amplification starting template polynucleotide in described semi-solid upholder, like this, after amplification, each particulate is connected with the clonal population of template molecule.

134., further comprising the steps of as the described method of claim 133:

(a) the described semi-solid upholder of dissolving; With

(b) collect described particulate.

135., further comprising the steps of as the described method of claim 134:

In second half solid support or on form second arrays of microparticles, the density of particle in wherein said second array is greater than the density of particle in described first array.

136. a test kit, it comprises the oligonucleotide probe that contains the thiophosphatephosphorothioate connection, but wherein said probe has carried out mark with the test section.

137. as the described test kit of claim 136, it is characterized in that, but described test section is a fluorescence dye.

138., also comprise and to cut the material that thiophosphatephosphorothioate connects as the described test kit of claim 136.

139., also comprise ligase enzyme as the described test kit of claim 136.

140., also comprise ligase enzyme and can cut the material that thiophosphatephosphorothioate is connected as the described test kit of claim 136.

141. as the described test kit of claim 136, also comprise at least one that is selected from down group: the reagent of the reagent of ligase enzyme, the material that can cut the thiophosphatephosphorothioate connection, Phosphoric acid esterase, polysaccharase, upholder, damping fluid, thermostability polysaccharase, Nucleotide, preparation emulsion and preparation gel.

142. as the described test kit of claim 136, it is characterized in that, described test kit comprises and contains the multiple fluorescently-labeled oligonucleotide probe that thiophosphatephosphorothioate connects, so that carry different fluorescence dyes that can be spectrally resolved corresponding to the probe of different probe terminal nucleotide.

143. a form is 5 '-O-P-O-X-O-P-S-(N) _kN _B ^*-3 ' oligonucleotide, wherein N represents any Nucleotide, N _BThe part that representative can not be extended with ligase enzyme, but * representative test section, X represents Nucleotide, and k is 1-100, and restricted condition is: but the test section can be present in (N) _kIn any Nucleotide on and be present in N in addition _BGo up or be not present in N in addition _BOn.

144., it is characterized in that described probe contains the Nucleotide that at least one degeneracy reduces as the described oligonucleotide probe of claim 143.

145., it is characterized in that described group comprises multiple fluorescently-labeled oligonucleotide probe as the described oligonucleotide probe group of claim 143, so that carry different fluorescence dyes that can be spectrally resolved corresponding to the probe of different probe nucleotide X.

146. a form is 5 '-N _B ^*(N) _kThe oligonucleotide probe of C-S-P-O-X-3 ', wherein N represents any Nucleotide, N _BThe part that representative can not be extended with ligase enzyme, but * representative test section, X represents Nucleotide, and k is 1-100, and restricted condition is: but the test section can be present in (N) _kIn any Nucleotide on and be present in N in addition _BGo up or be not present in N in addition _BOn.

147., it is characterized in that described probe contains the Nucleotide that at least one degeneracy reduces as the described oligonucleotide probe of claim 146.

148., it is characterized in that described group comprises multiple fluorescently-labeled oligonucleotide probe as the described oligonucleotide probe group of claim 146, so that carry different fluorescence dyes that can be spectrally resolved corresponding to the probe of different probe nucleotide X.

149. a form is 5 '-O-P-O-X-O-(N) _k-O-P-S-(N) _iN _B ^*-3 ' oligonucleotide probe, wherein N represents any Nucleotide, N _BThe part that representative can not be extended with ligase enzyme, but * representative test section, X represents Nucleotide, (k+i) is 1-100, and k is 1-100, and i is 0-99, and restricted condition is: but the test section can be present in (N) _iIn any Nucleotide on and be present in N in addition _BGo up or be not present in N in addition _BOn.

150., it is characterized in that described probe contains the Nucleotide that at least one degeneracy reduces as the described oligonucleotide probe of claim 149.

151., it is characterized in that i=0 as the described oligonucleotide probe of claim 149.

152., it is characterized in that described group comprises multiple fluorescently-labeled oligonucleotide probe as the described oligonucleotide probe group of claim 149, so that carry different fluorescence dyes that can be spectrally resolved corresponding to the probe of different probe nucleotide X.

153. a form is 5 '-N _B ^*(N) _i-S-P-O-(N) _kThe oligonucleotide probe of-O-P-O-X-3 ', wherein N represents any Nucleotide, N _BThe part that representative can not be extended with ligase enzyme, but * representative test section, X represents Nucleotide, (k+i) is 1-100, and k is 1-100, and i is 0-99, and restricted condition is: but the test section can be present in (N) _iIn any Nucleotide on and be present in N in addition _BGo up or be not present in N in addition _BOn.

154., it is characterized in that described probe contains the Nucleotide that at least one degeneracy reduces as the described oligonucleotide probe of claim 153.

155., it is characterized in that i=0 as the described oligonucleotide probe of claim 153.

156., it is characterized in that described group comprises multiple fluorescently-labeled oligonucleotide probe as the described oligonucleotide probe group of claim 153, so that carry different fluorescence dyes that can be spectrally resolved corresponding to the probe of different probe nucleotide X.

157. a form is selected from down the oligonucleotide probe of group: 3 '-XNNNNsINI-5 ', 3 '-XNNNNsIII-5 ', 3 '-XNNNNsNII-5 ' and 3 '-XNNNNIsII-5 ', wherein X and N represent any Nucleotide, on behalf of Yi Qie, " s " connect, and described at least one residue of easily cutting between connection and the described oligonucleotide 5 ' end contains the mark corresponding to specific X.

158., it is characterized in that on behalf of thiophosphatephosphorothioate, s connect as the described probe of claim 157.

159. one kind is connected in the method for second polynucleotide with first polynucleotide, said method comprising the steps of:

(a) provide be fixed within the semi-solid upholder or on first polynucleotide;

(b) described first polynucleotide are contacted with second polynucleotide and ligase enzyme; With

(c) described first and second polynucleotide are maintained ligase enzyme exist with the condition that is fit to be connected under.

160., it is characterized in that described first kind of polynucleotide are directly connected in described semi-solid upholder by covalently or non-covalently connecting as the described method of claim 159.

161. as the described method of claim 159, it is characterized in that, described first kind of polynucleotide be connected in be fixed in the described semi-solid upholder or on upholder.

162., it is characterized in that described semi-solid upholder is a gel as the described method of claim 161, described upholder is a particulate.

163., it is characterized in that described semi-solid upholder is a gel as the described method of claim 161, described upholder is a magnetic particle.

164. a method of cutting polynucleotide said method comprising the steps of:

(a) provide be fixed within the semi-solid upholder or on polynucleotide, wherein said polynucleotide contain easily cuts connection;

(b) described polynucleotide are contacted with cutting agent; With

(c) described polynucleotide are remained under the condition of described cutting agent existence and suitable cutting.

165., it is characterized in that described first kind of polynucleotide are directly connected in described semi-solid upholder by covalently or non-covalently connecting as the described method of claim 164.

166. as the described method of claim 164, it is characterized in that, described first kind of polynucleotide be connected in be fixed in the described semi-solid upholder or on upholder.

167., it is characterized in that described semi-solid upholder is a gel as the described method of claim 166, described upholder is a particulate.

168., it is characterized in that described semi-solid upholder is a gel as the described method of claim 166, described upholder is a magnetic particle.

169. an automatization sequencing equipment, described equipment comprise that its orientation can provide gravity bubble metathetical flow chamber.

170. automatization sequencing system of realizing 40,000 Nucleotide of per second evaluation.

171. automatization sequencing system that produces the 8.6Gb sequence information every day.

172. automatization sequencing system that produces the 48Gb sequence information every day.

173. a method of identifying template polynucleotide inner nucleotide sequence said method comprising the steps of:

(a) by the duplex that oligonucleotide probe and initial oligonucleotide is connected to form prolongation initial oligonucleotide is extended along described template polynucleotide, wherein said oligonucleotide probe contains the initiation residue;

(b) one or more Nucleotide of the described polynucleotide of evaluation;

(c) thus cutting described oligonucleotide probe with cutting agent produces extensible probe end; With

(d) repeating step (a) and (b) and (c) are up to determining nucleotide sequence.

174., it is characterized in that described authentication step comprises the mark that detects the oligonucleotide probe that is connected in nearest connection as the described method of claim 173.

175., it is characterized in that described oligonucleotide probe contains dealkalize base residue, damage base or Hypoxanthine deoxyriboside as the described method of claim 173.

176., it is characterized in that described oligonucleotide probe contains the damage base as the described method of claim 173, described method also comprises the step of removing described damage base.

177., it is characterized in that described removal step comprises that the duplex with described extension contacts with the DNA glycosylase as the described method of claim 176.

178., also comprise with cutting agent and cut the step that described oligonucleotide probe produces extensible probe end as the described method of claim 173.

179., it is characterized in that described cutting agent is selected from AP endonuclease, Endo V or periodate as the described method of claim 178.

180., it is characterized in that described cutting agent is the AP endonuclease as the described method of claim 178.

181., it is characterized in that described cutting agent is endonuclease V III as the described method of claim 178.

182. as the described method of claim 173, it is characterized in that, in semi-solid upholder or on carry out described extension step.

183. a method of measuring template polynucleotide inner nucleotide sequence said method comprising the steps of:

(a) probe-template duplex that provides probe and template multi-nucleotide hybrid to form, described probe has extensible end;

(b) will extend oligonucleotide probe and be connected in described extensible end, and form and contain the prolongation duplex that prolongs oligonucleotide probe, wherein said extension probes contains the initiation residue;

(c) in described prolongation duplex, identify at least one (1) and the extension probes complementary Nucleotide that just has been connected or (2) lucky nucleotide residue in the template polynucleotide in described prolongation oligonucleotide probe downstream;

(d) if there is not ready-made extensible end, on described prolongation oligonucleotide probe, produce extensible end, make the end of generation be different from an end that extension probes connected; With

(e) repeating step (b), (c) and (d), the nucleotide sequence in determining described template polynucleotide.

184., it is characterized in that described extension probes contains dealkalize base residue, damage base or Hypoxanthine deoxyriboside as the described method of claim 183.

185., it is characterized in that an end of each extension probes contains non-extensible part as the described method of claim 183.

186., it is characterized in that described authentication step comprises the mark that detects the extension probes that is connected in nearest connection as the described method of claim 183.

187., it is characterized in that described authentication step is included under the existence of chain termination nucleoside triphosphate of one or more marks and removes described non-extensible part and extend the oligonucleotide probe of described extension with nucleic acid polymerase as the described method of claim 183.

188. as the described method of claim 183, be included in also that not have extension probes to be connected in the described Connection Step described extensible when terminal, add the step of cap for the oligonucleotide probe that extends.

189., it is characterized in that described generation step comprises with the cutting agent that is selected from AP endonuclease or periodate cuts described oligonucleotide probe as the described method of claim 183.

190., it is characterized in that described cutting agent is the AP endonuclease as the described method of claim 189.

191., it is characterized in that described cutting agent is endonuclease V III as the described method of claim 189.

192. as the described method of claim 183, it is characterized in that, in semi-solid upholder or on carry out described connection and produce step.

193. as the described method of claim 183, it is characterized in that, step (a) comprises with independent sample size provides multiple different probe-template duplex, each different duplex contains the initial oligonucleotide probe of hybridizing in the template polynucleotide, wherein the template polynucleotide are identical described in each duplex, but initial oligonucleotide probe described in each duplex is incorporated into the different sequences of described template polynucleotide; Each sample size is carried out step (b)-(e) independently.

194., it is characterized in that with regard to each sample size, an end of described extension oligonucleotide probe contains non-extensible part as the described method of claim 193.

195., it is characterized in that with regard to each sample size, described authentication step comprises the mark that detects the extension probes that is connected in nearest connection as the described method of claim 194.

196. as the described method of claim 194, it is characterized in that, with regard to each sample size, described authentication step is included under the existence of chain termination nucleoside triphosphate of one or more marks and removes described non-extensible part and extend the oligonucleotide probe of described extension with nucleic acid polymerase.

197. as the described method of claim 194, be included in also that not have extension probes to be connected in the described Connection Step described extensible when terminal, add the step of cap for the oligonucleotide probe that extends.

198., it is characterized in that described generation step comprises with the cutting agent that is selected from AP endonuclease or periodate cuts described oligonucleotide probe as the described method of claim 194.

199., it is characterized in that described cutting agent is the AP endonuclease as the described method of claim 198.

200., it is characterized in that described cutting agent is endonuclease V III as the described method of claim 198.

201. as the described method of claim 194, it is characterized in that, in semi-solid upholder or on carry out described connection and produce step.

202. it is, further comprising the steps of: (f) described linking probe and the described initial oligonucleotide on the described template of removal as the described method of claim 183; (g) with the not homotactic second kind of oligonucleotide repeating step (a) that is incorporated into described template polynucleotide; (h) repeating step (b)-(e).

203. as the described method of claim 202, it is characterized in that, repeatedly repeat described method with the not homotactic initial oligonucleotide that is incorporated into described template polynucleotide.

204., it is characterized in that described removal step comprises described linking probe, initial oligonucleotide and template and contains 1.0-3.0%SDS, 100-300mM NaCl and 5-15mM sodium pyrosulfate (NaHSO as the described method of claim 202 ₄) the aqueous solution contact.

205., it is characterized in that described removal step comprises described linking probe, initial oligonucleotide and template and contains the 2%SDS that has an appointment, about 200mM NaCl and about 10mM sodium pyrosulfate (NaHSO as the described method of claim 202 ₄), as 2%SDS, 200mM NaCl and 10mM sodium pyrosulfate (NaHSO ₄) solution contact.

206., it is characterized in that an end of described extension probes contains non-extensible part as the described method of claim 203.

207., it is characterized in that in repeating, described authentication step comprises the mark that detects the extension probes that is connected in nearest connection as the described method of claim 203 at every turn.

208. as the described method of claim 203, it is characterized in that, in repeating, described authentication step is included under the existence of chain termination nucleoside triphosphate of one or more marks and removes described non-extensible part and extend the oligonucleotide probe of described extension with nucleic acid polymerase at every turn.

209. as the described method of claim 203, be included in also that not have extension probes to be connected in the described Connection Step described extensible when terminal, add the step of cap for the oligonucleotide probe that extends.

210., it is characterized in that described generation step comprises with the cutting agent that is selected from AP endonuclease or periodate cuts described oligonucleotide probe as the described method of claim 203.

211., it is characterized in that described cutting agent is the AP endonuclease as the described method of claim 210.

212., it is characterized in that described cutting agent is endonuclease V III as the described method of claim 210.

213. as the described method of claim 203, it is characterized in that, in semi-solid upholder or on carry out described connection and produce step.

214. an evaluation is connected in the method for the template polynucleotide inner nucleotide sequence of upholder on tie point, said method comprising the steps of:

(a) by the duplex that oligonucleotide probe and initial oligonucleotide is connected to form prolongation initial oligonucleotide is extended along described template polynucleotide, wherein said oligonucleotide probe contains the initiation residue, and the tie point along described extension along template to it and described upholder carries out;

(b) one or more Nucleotide of the described polynucleotide of evaluation; With

(c) repeating step (a) and (b) is up to determining nucleotide sequence.

215., it is characterized in that described authentication step comprises the mark that detects the extension probes that is connected in nearest connection as the described method of claim 214.

216., it is characterized in that described generation step comprises with the cutting agent that is selected from AP endonuclease, Endo V or periodate cuts described oligonucleotide probe as the described method of claim 214.

217., it is characterized in that described cutting agent is the AP endonuclease as the described method of claim 216.

218., it is characterized in that described cutting agent is endonuclease V III as the described method of claim 216.

219. as the described method of claim 216, it is characterized in that, in semi-solid upholder or on carry out described connection and produce step.

220. a method that is determined at the template polynucleotide inner nucleotide sequence that is connected in upholder on the tie point said method comprising the steps of:

(a) probe-template duplex that provides probe and template multi-nucleotide hybrid to form, primer has extensible end;

(b) will extend oligonucleotide probe is connected with described extensible end, formation contains the prolongation duplex that prolongs oligonucleotide probe, wherein said prolongation is carried out along the tie point of described template to it and described upholder, and wherein said oligonucleotide probe contains the initiation residue;

(c) in described prolongation duplex, identify at least one (1) and the extension probes complementary Nucleotide that just has been connected or (2) lucky nucleotide residue in the template polynucleotide in described prolongation oligonucleotide probe downstream;

(d) if there is no ready-made extensible end produces extensible end on described prolongation oligonucleotide probe, make the end of generation be different from an end that extension probes connected; With

(e) repeating step (b), (c) and (d), the nucleotide sequence in determining described template polynucleotide.

221., it is characterized in that an end of each extension probes contains non-extensible part as the described method of claim 220.

222., it is characterized in that described authentication step comprises the mark that detects the extension probes that is connected in nearest connection as the described method of claim 220.

223., it is characterized in that described authentication step is included under the existence of chain termination nucleoside triphosphate of one or more marks and removes described non-extensible part and extend the oligonucleotide probe of described extension with nucleic acid polymerase as the described method of claim 220.

224. as the described method of claim 220, be included in also that not have extension probes to be connected in the described Connection Step described extensible when terminal, add the step of cap for the oligonucleotide probe that extends.

225., it is characterized in that described generation step comprises with cutting agent cuts described oligonucleotide probe as the described method of claim 220.

226., it is characterized in that described cutting agent is selected from AP endonuclease, EndoV or periodate as the described method of claim 225.

227., it is characterized in that described cutting agent is the AP endonuclease as the described method of claim 225.

228., it is characterized in that described cutting agent is endonuclease V III as the described method of claim 225.

229. it is, further comprising the steps of: (f) described linking probe and the described initial oligonucleotide on the described template of removal as the described method of claim 220; (g) with the not homotactic second kind of oligonucleotide repeating step (a) that is incorporated into described template polynucleotide; (h) repeating step (b)-(e).

230. as the described method of claim 229, it is characterized in that, repeatedly repeat described method with the not homotactic initial oligonucleotide that is incorporated into described template polynucleotide.

231. as the described method of claim 220, it is characterized in that, in semi-solid upholder or on carry out described connection and produce step.

232., it is characterized in that described template is connected in the particulate that is connected with the rigid substrate of substantially flat as the described method of claim 220.

233., it is characterized in that described removal step comprises described linking probe, initial oligonucleotide and template and contains the 1.0-3.0%SDS that has an appointment, 100-300mM NaCl and 5-15mM sodium pyrosulfate (NaHSO as the described method of claim 229 ₄) the aqueous solution contact.

234., it is characterized in that described removal step comprises described linking probe, initial oligonucleotide and template and contains the 2%SDS that has an appointment, 200mM NaCl and 10mM sodium pyrosulfate (NaHSO as the described method of claim 229 ₄), as 2%SDS, 200mM NaCl and 10mM sodium pyrosulfate (NaHSO ₄) the aqueous solution contact.

235. a method of identifying template polynucleotide inner nucleotide sequence said method comprising the steps of:

(a) provide be connected in be fixed within the semi-solid upholder or on the template polynucleotide of particulate;

(b) by the duplex that oligonucleotide probe and initial oligonucleotide is connected to form prolongation initial oligonucleotide is extended along described template polynucleotide, wherein said oligonucleotide probe contains the initiation residue;

(c) one or more Nucleotide of the described polynucleotide of evaluation; With

(d) repeating step (b) and (c) is up to determining nucleotide sequence.

236. as the described method of claim 235, it is characterized in that, in semi-solid upholder, carry out described extension step.

237., it is characterized in that described template is connected in the particulate that is connected with the rigid substrate of substantially flat as the described method of claim 235.

238., it is characterized in that described oligonucleotide contains easily cuts connection as the described method of claim 235, or it is easy to modified containing and easily cuts connection, wherein saidly easily cut connection between nucleosides and dealkalize base residue.

239. a method of measuring the nucleotide sequence of template polynucleotide said method comprising the steps of:

(a) probe-template duplex that provides probe and template multi-nucleotide hybrid to form, described probe contains extensible end, described probe-template duplex be connected in be embedded within the semi-solid upholder or on particulate;

(b) will extend oligonucleotide probe and be connected with described extensible end, and form and contain the prolongation duplex that prolongs oligonucleotide probe, wherein said extension probes contains the initiation residue;

(c) in described prolongation duplex, identify at least one (1) and the extension probes complementary Nucleotide that just has been connected or (2) lucky nucleotide residue in the template polynucleotide in described prolongation oligonucleotide probe downstream;

(d) if there is no ready-made extensible end produces extensible end on described prolongation oligonucleotide probe, make the end of generation be different from an end that extension probes connected; With

(e) repeating step (b), (c) and (d), the nucleotide sequence in determining described template polynucleotide.

240., it is characterized in that described connection and generation step are carried out as the described method of claim 239 in semi-solid upholder.

241., it is characterized in that described template is connected in the particulate that is connected with the rigid substrate of substantially flat as the described method of claim 239.

242. a method of measuring template polynucleotide inner nucleotide sequence said method comprising the steps of:

(a) in the presence of particulate in the emulsion chamber amplification template polynucleotide molecule, thereby produce the particulate of the clonal population be connected with the template polynucleotide;

(b) from described emulsion, reclaim described particulate;

(c) described particulate is embedded within the semi-solid upholder or on;

(d) by the duplex that oligonucleotide probe and initial oligonucleotide is connected to form prolongation initial oligonucleotide is extended along described template polynucleotide, wherein said oligonucleotide probe contains the initiation residue;

(e) one or more Nucleotide of the described polynucleotide of evaluation; With

(f) repeating step (d) and (e) is up to determining nucleotide sequence.

243., it is characterized in that (i) amplification contains not homotactic multiple template polynucleotide molecule in single emulsion chamber as the described method of claim 242; (ii) from described emulsion, reclaim multiple particulate and be embedded in the described upholder or on, each particulate is connected with template polynucleotide clonal population, wherein said clonal population has different sequences, (iii) to the parallel step (d), (e) and (f) of carrying out of the described clonal population that is connected in described embedding particulate, so that a plurality of sequences of replicate(determination).

244. the method for the nucleotide sequence information of template polynucleotide is measured in first set with the oligonucleotide probe family of at least two kinds of distinctive marks, said method comprising the steps of:

(a) by the duplex that oligonucleotide probe and initial oligonucleotide is connected to form prolongation initial oligonucleotide is extended along described template polynucleotide, wherein said oligonucleotide probe be described distinctive mark oligonucleotide probe man family set the member and contain the initiation residue;

(b) detect the mark that is connected with described oligonucleotide; With

(c) repeating step (a) and (b) is up to the ordered list that obtains probe family title; With

(d) adopt the ordered list of probe family title to get rid of one or more possible nucleotide sequences.

245., it is characterized in that step (d) comprises the ordered list of the described probe of decoding family title, to determine described sequence as the described method of claim 244.

246. as the described method of claim 244, it is characterized in that, probe-template the duplex that provides initial oligonucleotide probe and template multi-nucleotide hybrid to form is provided described method, described probe has extensible end, wherein said extension step comprises oligonucleotide probe is connected in described extensible end, formation contains the prolongation duplex that prolongs oligonucleotide probe, be included in also that not have oligonucleotide probe to be connected in the described extension step described extensible when terminal, add the step of cap for all the other extensible ends.

247., it is characterized in that an end of oligonucleotide probe contains non-extensible part described in each probe family as the described method of claim 244.

248. as the described method of claim 244, after detecting step, each also comprises: if (f) extensible end does not exist, just on the oligonucleotide probe of described nearest connection, produce extensible end, so that the end that produces is different from the end that the oligonucleotide probe of described nearest connection connects.

249. as the described method of claim 248, it is characterized in that, cut described oligonucleotide with the cutting agent that is selected from AP endonuclease, EndoV or periodate and produce described extensible probe end.

250., it is characterized in that described cutting agent is the AP endonuclease as the described method of claim 249.

251., it is characterized in that described cutting agent is endonuclease V III as the described method of claim 249.

252. as the described method of claim 244, it is characterized in that, in semi-solid upholder or on carry out described extension step.

253., it is characterized in that described template is connected in the particulate that is connected with the rigid substrate of substantially flat as the described method of claim 244.

254., it is characterized in that described set comprises the probe family of 2 kinds of distinctive marks as the described method of claim 244.

255., it is characterized in that described set comprises the probe family of 3 kinds of distinctive marks as the described method of claim 244.

256., it is characterized in that described set comprises the probe family of 4 kinds of distinctive marks as the described method of claim 244.

257., it is characterized in that described set comprises the probe family of distinctive mark more than 4 kinds as the described method of claim 244.

258., it is characterized in that described oligonucleotide probe comprises the limited part that nucleosides is not independently selected as the described method of claim 244, wherein the oligonucleotide probe that will contain the different limited part of sequence according to encoding scheme is distributed to probe family.

259. as the described method of claim 244, it is characterized in that, described oligonucleotide probe distributed to first, second, third and four point probe family according to one of 24 kinds of listed encoding schemes of table 1.

260., it is characterized in that the kind of at least one Nucleotide is known in the described template as the described method of claim 245, wherein said decoding step comprises:

(i) by determining that the possible sequence of the limited part of this probe which kind and known nucleotide kind and its near-end Nucleotide are connected in the Nucleotide adjacent nucleotide relative position of known kind conforms to, to the Nucleotide given category adjacent on the described template with the Nucleotide of known kind;

(ii), give described follow-up Nucleotide given category by determining which kind conforms to the possible sequence that its near-end Nucleotide is connected in the limited part of this probe of follow-up Nucleotide relative position; With

(iii) repeating step (ii), up to measuring this sequence.

261., further comprising the steps of as the described method of claim 245:

(a) the Nucleotide kind in the described template of mensuration, so that described Nucleotide has known kind, wherein said decoding step comprises:

(i) by determining that the possible sequence of the limited part of this probe which kind and known nucleotide kind and its near-end Nucleotide are connected in the Nucleotide adjacent nucleotide relative position of known kind conforms to, to the Nucleotide given category adjacent on the described template with the Nucleotide of known kind;

(ii), give described follow-up Nucleotide given category by determining which kind conforms to the possible sequence that its near-end Nucleotide is connected in the limited part of this probe of follow-up Nucleotide relative position; With

(iii) repeating step (ii), up to measuring this sequence.

262. as the described method of claim 261, it is characterized in that, described determination step is included under the certain condition that has polysaccharase template-probe duplex is contacted with labeled nucleotide, if described under the described conditions labeled nucleotide with described duplex position adjacent on described template complementation, just can mix described labeled nucleotide.

263., it is characterized in that described decoding step comprises as the described method of claim 245: produce at least a candidate sequence from the ordered list of probe family title; With the nucleotide sequence of selecting candidate sequence as described template.

264., it is characterized in that described generation step comprises generation at least 4 candidate sequences as the described method of claim 263.

265., it is characterized in that described generation step comprises as the described method of claim 263:

(i) kind of first Nucleotide of the described nucleotide sequence of supposition;

(ii) basis is determined the possible kind of adjacent nucleotide corresponding to the probe family title of described first Nucleotide, thereby specifies the kind of the Nucleotide adjacent with described first Nucleotide;

(iii) basis is determined the possible kind of follow-up Nucleotide corresponding to the probe family title of the Nucleotide of nearest given category, thereby specifies the kind of follow-up Nucleotide;

(iv) repeating step (iii), up to producing candidate sequence; With

(v) repeating step (i)-(iv) wherein, is taken turns in the repetition at each, and described first Nucleotide is assumed to different sorts, up to the candidate sequence that produces desired number.

266. as the described method of claim 263, it is characterized in that, described selection step comprises at least a candidate sequence and one or more known arrays, and selects and one or more known arrays have predetermined homogeny degree or immediate candidate sequence.

267., it is characterized in that described template is derived from organism interested as the described method of claim 266, wherein said comparison step comprises at least a candidate sequence and contains available from the sequence in the database of the sequence of described organism.

268., it is characterized in that described comparison step comprises at least a candidate sequence and the sequence that contains in the database of a plurality of comparative sequences as the described method of claim 266, each sequence contains the difference of polynucleotide sequence to be measured may sequence.

269., it is characterized in that described selection step comprises as the described method of claim 263:

(i) use second set of the coding probe family of distinctive mark to obtain second kind of probe family title ordered list from described template, the man family set middle probe of wherein said second probe family is different with the coding of the man family set middle probe of described first probe family;

(ii) produce at least a comparative sequences from described second kind of probe family title ordered list;

The (iii) part of the part of at least a described candidate sequence and at least a described comparative sequences; With

(iv) be chosen in the step (c) on the part relatively with comparative sequences predetermined homogeny degree or the immediate candidate sequence nucleotide sequence as described template is arranged.

270., it is characterized in that described rating unit is a dinucleotides as the described method of claim 269.

271., it is characterized in that the described second probe family title ordered list only contains an element as the described method of claim 269.

272., it is characterized in that oligonucleotide probe has following structure described in each probe family as the described method of claim 244: 5 '-(XY) (N) _kN _B ^*-3 ' or 3 '-(XY) (N) _kN _B ^*-5 ', wherein N represents any nucleosides, N _BThe part that representative can not be extended with ligase enzyme, but * represent the test section, XY is the limited part of described probe, wherein X and Y represent nucleosides identical or different but that can not independently choose separately, X and Y are at least 2 times of degeneracys, and connecting between at least one nucleosides is easily to cut connection between nucleosides and the dealkalize base residue, or nucleosides and contain connection between the residue that damages base, k is 1-100, and restricted condition is: but the test section can be present in Y or (N) _kIn arbitrarily on the Nucleotide and be present in N in addition _BGo up or be not present in N in addition _BOn.

273. as the described method of claim 272, it is characterized in that, connect between described at least one nucleosides and easily cut connection between nucleosides and the dealkalize base residue.

274. as the described method of claim 272, it is characterized in that, but described test section connects, can or have this two kinds of features by photobleaching by cutting joint.

275., it is characterized in that the described joint that cuts contains disulfide linkage as the described method of claim 274.

276. as the described method of claim 272, it is characterized in that, adopt the oligonucleotide probe family of 4 kinds of distinctive marks, wherein the different oligonucleotide probe of the limited partial sequence of this probe is distributed to first, second, third and four point probe family according to one of 24 kinds of listed encoding schemes of table 1.

277., it is characterized in that described detection step comprises simultaneously from described template at least 2 Nucleotide 2 information of average acquiring separately, and does not obtain two information from any single Nucleotide as the described method of claim 244.

278., it is characterized in that described detection step comprises simultaneously that at least 2 Nucleotide obtain separately from described template and is less than 2 information as the described method of claim 244.

279. the method for the nucleotide sequence information of template polynucleotide is measured in first set with the oligonucleotide probe family of at least two kinds of distinctive marks, said method comprising the steps of:

(a) probe-template composite is contacted so that oligonucleotide probe hybridization with the oligonucleotide probe family of two kinds of distinctive marks at least, described probe-template composite contains the partly double-stranded and strand part of waiting to check order with extensible end, described oligonucleotide probe contains the template part complementary part that partly is close to described duplex, and the probe of wherein said probe family contains the initiation residue;

(b) oligonucleotide probe with described hybridization is connected with described extensible end, contains the probe-template duplex that prolongs duplex thereby produce;

(c) detect the mark that links to each other with described linking probe;

(d) if there is no ready-made extensible probe end then produces extensible probe end on described prolongation duplex; With

(e) repeating step (a)-(d) is up to the ordered list that obtains probe family title.

280., it is characterized in that described detection step comprises simultaneously from described template at least 2 Nucleotide 2 information of average acquiring separately, and does not obtain two information from any single Nucleotide as the described method of claim 279.

281., it is characterized in that described detection step comprises simultaneously that at least 2 Nucleotide obtain separately from described template and is less than 2 information as the described method of claim 279.

282. measure the method for the nucleotide sequence information of template polynucleotide with first set of oligonucleotide probe family for one kind, the probe of wherein said probe family contains the initiation residue, said method comprising the steps of:

(a) carry out continuously orderly extension, connection, detection and cutting circulation, wherein said detection step comprises that simultaneously at least two Nucleotide respectively obtain average two information from described template, and does not obtain two information from any single Nucleotide; With

(b) information that step (a) is obtained and at least one out of Memory merge, to determine described sequence.

283., it is characterized in that described at least one out of Memory comprises an information that is selected from down group as the described method of claim 282: the Nucleotide kind in the described template, by comparing the information that candidate sequence and at least a known array obtain; Repeat the information that described method obtains with second set that utilizes oligonucleotide probe family.

284. the set of the oligonucleotide probe family of at least two kinds of distinctive marks, wherein the probe of each probe family comprises limited part and not limited part, is at least 2 times of degeneracys on each position of described limited part, and the probe of each family contains the initiation residue.

285. the oligonucleotide probe man family set as the described distinctive mark of claim 284 is characterized in that each probe contains the inductile end of ligase enzyme.

286. oligonucleotide probe man family set as the described distinctive mark of claim 284, it is characterized in that, each probe contains the inductile end of ligase enzyme, but each probe comprises the test section on a described position of easily cutting between connection and the inductile end of described ligase enzyme.

287. the oligonucleotide probe man family set as the described distinctive mark of claim 284 is characterized in that described set comprises 2 kinds of probe families.

288. the oligonucleotide probe man family set as the described distinctive mark of claim 284 is characterized in that described set comprises 3 kinds of probe families.

289. the oligonucleotide probe man family set as the described distinctive mark of claim 284 is characterized in that described set comprises 4 kinds of probe families.

290. the oligonucleotide probe man family set as the described distinctive mark of claim 284 is characterized in that described set comprises probe family more than 4 kinds.

291. the oligonucleotide probe man family set as the described distinctive mark of claim 284 it is characterized in that, but described probe contains the test section, but described test section connects, can or have this two kinds of features by photobleaching by cutting joint.

292. a test kit, it comprises the set of the oligonucleotide probe family of at least two kinds of distinctive marks, and wherein said oligonucleotide probe contains the initiation residue.

293., it is characterized in that described initiation residue is dealkalize base residue, Hypoxanthine deoxyriboside or contains the residue that damages base as the described test kit of claim 292.

294., it is characterized in that described probe contains easily cuts connection between nucleosides and the dealkalize base residue as the described test kit of claim 292.

295., also comprise being selected from down at least one of group: ligase enzyme, can cut described material, Phosphoric acid esterase, polysaccharase, upholder, damping fluid, thermostability polysaccharase, Nucleotide, the reagent of preparation emulsion and the reagent of preparation gel of easily cutting connection as the described test kit of claim 292.

296. a test kit, it comprises and contains the oligonucleotide probe that causes residue, and wherein said probe contains or is not difficult modified and contains easily cuts connection, but wherein said probe has carried out mark with the test section.

297. as the described test kit of claim 296, it is characterized in that, but described test section is a fluorescence dye.

298., also comprise and to cut the described material of easily cutting connection as the described test kit of claim 296.

299., also comprise ligase enzyme as the described test kit of claim 296.

300., also comprise ligase enzyme and can cut the described material that is connected of easily cutting as the described test kit of claim 296.

301., also comprise being selected from down at least one of group: ligase enzyme, can cut described material, Phosphoric acid esterase, polysaccharase, upholder, damping fluid, thermostability polysaccharase, Nucleotide, the reagent of preparation emulsion and the reagent of preparation gel of easily cutting connection as the described test kit of claim 296.

302. as the described test kit of claim 301, it is characterized in that, described test kit comprises multiple fluorescently-labeled oligonucleotide probe, wherein said probe contains nucleosides and causes easily cuts connection between the residue, so that carry different fluorescence dyes that can be spectrally resolved corresponding to the probe of different probe terminal nucleotide.

303. a form is 5 '-O-P-O-X-O-P-O-(N) _kN _B ^*-3 ' oligonucleotide, wherein N represents any Nucleotide or dealkalize base residue, and restricted condition is: at least one N causes residue, N _BThe part that representative can not be extended with ligase enzyme, but * representative test section, X represents Nucleotide, and k is 1-100, and restricted condition is: but the test section can be present in (N) _kIn arbitrarily on the Nucleotide and be present in N in addition _BGo up or be not present in N in addition _BOn.

304., it is characterized in that described probe contains the Nucleotide that at least one degeneracy reduces as the described oligonucleotide probe of claim 303.

305., it is characterized in that described group comprises multiple fluorescently-labeled oligonucleotide probe as the described oligonucleotide probe group of claim 303, so that carry different fluorescence dyes that can be spectrally resolved corresponding to the probe of different probe nucleotide X.

306. a form is 5 '-N _B ^*(N) _kThe oligonucleotide probe of C-O-P-O-X-3 ', wherein N represents any Nucleotide or dealkalize base residue, and restricted condition is: at least one N causes residue, N _BThe part that representative can not be extended with ligase enzyme, but * representative test section, it is 1-100 that X represents Nucleotide and k, restricted condition is: but the test section can be present in (N) _kIn arbitrarily on the Nucleotide and be present in N in addition _BGo up or be not present in N in addition _BOn.

307., it is characterized in that described probe comprises the Nucleotide that at least one degeneracy reduces as the described oligonucleotide probe of claim 306.

308., it is characterized in that described group comprises multiple fluorescently-labeled oligonucleotide probe as the described oligonucleotide probe group of claim 306, so that carry different fluorescence dyes that can be spectrally resolved corresponding to the probe of different probe nucleotide X.

309. a form is selected from down the oligonucleotide probe of group: 3 '-XNNNNRINI-5 ', 3 '-XNNNNRIII-5 ', 3 '-XNNNNRNII-5 ', 3 '-XNNNNIRII-5 ', XNNNNRNI-5 ', 3 '-XNNNNRII-5 ', 3 '-XNNNNRII-5 ', 3 '-XNNNNIRI-5, wherein X and N represent any Nucleotide, " R " representative causes residue, and at least one residue between described initiation residue and the described oligonucleotide 5 ' end contains the mark corresponding to specific X.

310., it is characterized in that R represents the ribodesose residue as the described probe of claim 309.

311. first polynucleotide are connected in the method for second polynucleotide, said method comprising the steps of:

(a) provide be fixed within the semi-solid upholder or on first polynucleotide;

(b) described first polynucleotide are contacted with second polynucleotide and ligase enzyme; With

(c) described first and second polynucleotide are remained on ligase enzyme exist with the condition that is fit to be connected under, wherein at least a described polynucleotide contain the initiation residue.

312., it is characterized in that described first kind of polynucleotide are directly connected in described semi-solid upholder by covalently or non-covalently connecting as the described method of claim 311.

313. as the described method of claim 311, it is characterized in that, described first kind of polynucleotide be connected in be fixed in the described semi-solid upholder or on upholder.

314., it is characterized in that described semi-solid upholder is a gel as the described method of claim 313, described upholder is a particulate.

315., it is characterized in that described semi-solid upholder is a gel as the described method of claim 313, described upholder is a magnetic particle.

316. a method of cutting polynucleotide said method comprising the steps of:

(a) provide be fixed within the semi-solid upholder or on polynucleotide, wherein said polynucleotide contain the initiation residue;

(b) described polynucleotide are contacted with cutting agent; With

(c) described polynucleotide are remained under the condition of described cutting agent existence and suitable cutting.

317., it is characterized in that described initiation residue is dealkalize base residue, Hypoxanthine deoxyriboside or contains the residue that damages base as the described method of claim 316.

318., it is characterized in that described first kind of polynucleotide are directly connected in described semi-solid upholder by covalently or non-covalently connecting as the described method of claim 316.

319. as the described method of claim 316, it is characterized in that, described first kind of polynucleotide be connected in be fixed in the described semi-solid upholder or on upholder.

320., it is characterized in that described semi-solid upholder is a gel as the described method of claim 319, described upholder is a particulate.

321., it is characterized in that described semi-solid upholder is a gel as the described method of claim 320, described upholder is a magnetic particle.

322. a component set that is used to prepare particulate colony, described set comprises:

(a) particulate colony, wherein single particulate is connected with the first primer colony and the second primer colony at least, and the primer of wherein said first colony is different with the sequence of the primer of described second colony; With

(b) nucleic acid fragment library, wherein each nucleic acid fragment contains the interested first and second nucleic acid sections, and wherein said first and second primers are corresponding to the universal sequence that is positioned at outside the described interested first and second nucleic acid sections.

323., it is characterized in that 5 ' and the 3 ' label that the described interested first and second nucleic acid sections are paired labels as the described component set of claim 322.

324. as the set of the described component of claim 322, it is characterized in that described nucleic acid fragment comprises internal cohesion of the one or more primer binding sites that contain amplimer, so that with each nucleic acid sections of pcr amplification.

325., also comprise primer binding site complementary primer with described internal cohesion as the described component set of claim 324.

326. one kind be connected with that basic identical nucleotide sequence constitutes first colony and the particulate of second colony that constitutes of basic identical nucleotide sequence, wherein said first nucleic acid population comprises the interested first nucleic acid sections, and described second nucleic acid population comprises the interested second nucleic acid sections.

327., it is characterized in that 5 ' label and 3 ' label that the described interested first and second nucleic acid sections are paired labels as the described particulate of claim 326.

328., it is characterized in that the described interested first and second nucleic acid sections are at a distance of the 5 ' label and the 3 ' label of predetermined distance in the contiguous nucleic acid of natural generation as the described particulate of claim 326.

329. as claim 327 or 328 described particulates, it is characterized in that, by the single bigger nucleic acid fragment described first and second nucleic acid sections that increase.

330. as the described particulate of claim 329, it is characterized in that, in the single cell of PCR emulsion, carry out described amplification.

331., it is characterized in that the essentially identical first and second nucleotide sequence colonies that single particulate connects are different to small part with the essentially identical first and second nucleotide sequence colonies that are connected in other single particulate as the described particulate of claim 326 colony.

332. as the described particulate of claim 331 colony, it is characterized in that, the interested first nucleic acid sections is contained in the described first nucleotide sequence colony that is connected in single particulate, and the interested second nucleic acid sections is contained in the described second nucleotide sequence colony that is connected in single particulate.

333. as the described particulate of claim 332 colony, it is characterized in that, be connected in 5 ' label and 3 ' label that the interested first and second nucleic acid sections described in the described first and second nucleotide sequence colonies of single particulate are or contain paired label.

334. as the described particulate of claim 332 colony, it is characterized in that, be connected in the interested first and second nucleic acid sections described in the described first and second nucleotide sequence colonies of single particulate by single bigger nucleic acid fragment amplification.

335. as the described particulate of claim 334 colony, it is characterized in that, in the single cell of PCR emulsion, carry out described amplification.

336. as the described particulate of claim 331 colony, it is characterized in that, by in the single cell of PCR emulsion, increasing the described first and second nucleotide sequence colonies are connected in single particulate, wherein, the described single cell of an at least a portion nucleic acid fragment containing a particulate and contain the described first and second nucleic acid sections.

337. an array, it comprises the described particulate of each claim colony among the claim 331-336.

338. as the described array of claim 337, it is characterized in that, described particulate is fixed in the semi-solid upholder or on.

339. a generation is connected with the method for the particulate of first nucleotide sequence colony of basic identical nucleotide sequence formation and the second nucleotide sequence colony that basic identical nucleotide sequence constitutes, and said method comprising the steps of:

(a) provide the particulate that is connected with the first primer colony and the second primer colony;

(b) provide the nucleic acid fragment that contains the first and second nucleic acid sections, the wherein said first and second nucleic acid sections both sides are lands of described first and second primers, the sequence of described PBR is corresponding to first and second primers that are connected in described particulate, and separated by at least one other PBR;

(c) under the condition that has the existence of suitable amplifing reagent and primer to increase, hatch described particulate and nucleic acid fragment, thereby make described first and second nucleotide sequences be amplified and be connected in described particulate.

340., it is characterized in that wherein first and second PBRs contain amplimer land and sequencing primer land separately as the described method of claim 339.

341. as the described method of claim 339, it is characterized in that, wherein at least a other PBR contains the land of two kinds of amplimers, so that each nucleotide sequence side joint is in the primer binding site of a pair of amplimer, so that with these two kinds of nucleotide sequences of pcr amplification.

342. as the described method of claim 339, it is characterized in that, in the single cell of PCR emulsion, carry out described amplification.

343., it is characterized in that described first and second nucleotide sequences contain label separately as the described method of claim 339, wherein said label is 5 ' and a 3 ' label of paired label.

344. a generation is connected with the method for the particulate colony of different IPs acid sequence colony, the intragroup nucleotide sequence of each described nucleotide sequence is basic identical, described method comprises carries out the described method of claim 339, wherein in a plurality of cells of PCR emulsion, carry out described amplification, at least a portion cell nucleic acid fragment containing a particulate and comprise the first and second nucleic acid sections wherein, wherein first and second nucleotide sequences of single nucleic acid fragment are different with the described first and second nucleic acid sections.

345. a method that produces arrays of microparticles, described method comprise the described particulate of claim 344 colony is fixed in the semi-solid upholder or on.

346. a method of carrying out nucleic acid sequencing, described method comprises:

(a) acquisition is connected in the sequence information of the first nucleic acid molecule colony of particulate;

(b) acquisition is connected in the sequence information of the second nucleic acid molecule colony of same particulate, and wherein said first nucleic acid divides in the sequence of the colony and the second nucleic acid molecule colony different to small part.

347. as the described method of claim 346, it is characterized in that, obtain described (a) and sequence information (b) successively.

348., it is characterized in that the described first nucleic acid molecule colony comprises 5 ' label of paired label as the described method of claim 346, the described second nucleic acid molecule colony comprises 3 ' label.

349. a sequence measurement, described method comprise particulate colony is carried out the described method of claim 346, wherein the sequence of first and second nucleic acid molecule of each particulate connection is different to small part with first and second nucleic acid molecule that are connected in other particulate.

350., it is characterized in that the described first nucleic acid molecule colony on the described single particulate of wherein parallel order-checking, and the described second nucleic acid molecule colony on the described single particulate of parallel order-checking as the described method of claim 349.

351. a composition, it contains 1.0-3.0%SDS, 100-300mM NaCl and 5-15 mM sodium pyrosulfate (NaHSO ₄) the aqueous solution.

352. as the described composition of claim 351, its pH is 2.0-3.0.

353. as the described composition of claim 351, it contains the 2%SDS that has an appointment, about 200mM NaCl and about 10mM sodium pyrosulfate (NaHSO ₄) the aqueous solution.

354. as the described composition of claim 353, its pH is 2.0-3.0.

Images