CN1771336A - Methods and means for nucleic acid sequencing - Google Patents

Abstract

Nucleic acid sequencing-by-synthesis. Primed synthesis of a second strand complementary to a template strand in repeated sets of steps, each step comprising providing one or more of the possible nucleotide complementarity classes for incorporation into the synthesized strand, and each set of steps comprising providing all four possible nucleotide complementarity classes. Three of the four possible nucleotide complementarity classes may first be provided for incorporation into the synthesized strand, then separately the fourth nucleotide complementarity class alone. Also, a DNA molecule consisting of a stem portion and first and second loop portions, wherein the stem portion consists of a first strand and a second strand, wherein the first strand and second strand are equal in length, complementary and annealed together, wherein the first loop portion joins the 3' end of the first strand to the 5' end of the second strand and the second loop portion joins the 3' end of the second strand to the 5' end of the first strand so the DNA molecule has no free 5' or 3' ends, and uses thereof, especially in sequencing.

Description

Methods and tools for nucleic acid sequencing

The present invention relates to nucleic acid sequencing. The invention relates in particular to "sequencing by synthesis" (SBS), in which a nucleic acid strand with a free 3' end is annealed to a nucleic acid containing a template whose sequence information is desired, and used to prime second strand synthesis, wherein the determination of nucleotide incorporation Sequence information is provided. The present invention is based in part on an elegant concept that allows the use of unblocked nucleotides in so-called "chroma sequencing", thereby overcoming various problems with existing sequencing technologies and allowing Obtain extremely large numbers of sequences in a single workday using standard reagents and equipment. Preferred embodiments allow additional benefits to be obtained. The invention also relates to algorithms and techniques for sequence analysis, and devices and systems for sequencing. The present invention allows the automation of a large number of sequencing jobs using only standard benchtop equipment readily available in the art.

The present invention involves the primed synthesis of a second strand complementary to a template strand in repeated sets of steps, each step comprising providing one or more, but optionally less than all, possible types of nucleotide complementarity for incorporated into the synthetic strand, and each set of steps includes providing all four possible types of nucleotide complementarity, optionally in two or more steps, at least one of which includes the addition of more than one core Types of nucleotide complementarity. Preferably, this involves first providing three of the four possible nucleotide complementarity types for incorporation into the synthetic strand, and then providing only the fourth nucleotide complementarity type alone. Chain elongation terminates with nucleotide incorporation at the last step, as when a fourth nucleotide is provided, because no other nucleotides are present. Determining the number and optionally the type of nucleotides between the stops allows rapid determination of information about the base composition and/or sequence of the template. Four rounds of each of the four different nucleotides to terminate extension, when using a single "stop nucleotide" at a time, provide information that can be used to determine the entire template sequence very quickly and easily.

Although many different methods are used in genome research, direct sequencing is by far the most valuable. In fact, all three major scientific problems in genomics (sequencing, genotyping, and gene expression analysis) could be solved if sequencing could be made sufficiently efficient. Model species can be sequenced, individuals can be genotyped by whole genome sequencing, and RNA populations can be analyzed in detail by conversion to cDNA and sequencing (directly counting the copy number of each mRNA).

Other examples of scientific and medical questions that can be addressed by sequencing include epigenomics (the study of methylated cytosines in the genome - by converting unmethylated cytosine bisulfite to uracil, and then Comparing the resulting sequence to the untransformed template sequence), protein-protein interactions (by sequencing hits obtained in yeast two-hybrid experiments), protein-DNA interactions (by sequencing The obtained DNA fragments are sequenced) and so on. Thus, highly efficient methods for DNA sequencing are required.

But in order to replace auxiliary methods such as microarray and PCR fragment analysis, extremely high sequencing throughput is required. For example, a living cell contains approximately 300,000 copies of messenger RNA, each approximately 2,000 bases long on average. Thus to fully sequence the RNA in even a single cell, 600 million nucleotides must be probed. In complex tissues composed of many different cell types, the task becomes even more difficult as cell type-specific transcripts are further diluted. A throughput of gigabases per day will be required to meet these demands. The table below shows some estimates of the throughput required for each experiment (persons, unless otherwise noted): experiment required flux Genome sequence (10× de novo) 30Gbp genome-wide polymorphism 3Gbp Complete haplotype map (200 individuals) 600Gbp gene expression 600Mbps Epigenomics 3Gbp 10 million protein interactions 400Mbps Entire biosphere (one species per genus) ~300Tbp

The present invention enables all of the above to be achieved at reasonable expense.

Methods for DNA Sequencing

Sanger sequencing using fluorescent dideoxynucleotides (Sanger et al. PNAS 74 no. 12:5463-5467, 1977) is the most widely used method and has been successfully automated in 96 and even 384 capillary sequencers. However, this method relies on the physical separation of a large number of fragments corresponding to each base position of the template, and thus cannot be easily scaled up to ultra-high-throughput sequencing (currently the best instruments generate ~2 million nucleotide sequences per day) .

Sequences can also be obtained indirectly by probing a target polynucleotide with probes selected from a set of probes.

Sequencing via hybridization uses sequences representing all possible sequences up to a certain length (i.e., a set of all k-mers, where k is limited by the number of probes that can fit onto the microarray surface; for 1 million probes , a set of probes with k=10) can be used and hybridized to the template. Reconstruction of template sequences from this set of probes is complex and made more difficult by the inherently unpredictable nature of hybridization kinetics and the combined explosion of the number of probes required to sequence larger templates. Even if these problems could be overcome, the throughput would necessarily be low, since each template would require a microarray carrying millions of probes, and the arrays are usually not reusable.

Nanopore sequencing (US Genomics, U.S. Patent 6,355,420) takes advantage of the fact that when long DNA molecules are forced through a nanopore separating two reaction chambers, bound probes act as the reaction chambers. The change in conductance between them was detected. By modifying the DNA with a subpopulation of all possible k-mers, it is possible to deduce the partial sequence. To date, no viable strategy has been proposed to obtain full-length sequences via the nanopore route, although in principle, if possible, amazing throughput (on the order of a human genome in 30 minutes) could be achieved.

Various approaches have been devised for sequencing by synthesis (SBS).

To increase sequencing throughput, it would be desirable to be able to visualize the incorporation of each base on a large number of templates in parallel, eg, on a glass surface or similar reaction chamber. This is achieved by SBS (see eg Malamede et al US4863849, Kumar US5908755). There are two routes to SBS: either detection of by-products released by each incorporated nucleotide, or detection of permanently attached labels.

Pyrosequencing (eg WO9323564) determines the template sequence by detecting the by-product of each incorporated monomer in the form of inorganic diphosphate (PPi). To keep the reactions of all template molecules synchronized, monomers are added one at a time and unincorporated monomers are degraded before the next addition. However, homopolymeric subsequences (strings of identical monomers) pose a problem because multiple incorporation cannot be prevented. Synchronization is eventually broken (because the total number of non-incorporated or misincorporated on a small fraction of the template eventually overwhelms the true signal), and the best current systems can only read about 20-30 bases, with a combined throughput of Approximately 200,000 bases/day.

While Sanger sequencing requires delicate instrumentation (i.e., capillaries) for each template, pyrosequencing is easily parallelized within a single reaction chamber. US6274320 describes the use of rolling circle amplification to produce tandemly repeated linear single-stranded DNA molecules attached to optical fibers for analysis in a pyrosequencing reaction which can then be performed in parallel. In principle, the throughput of such systems is only limited by the surface area (number of template molecules), reaction speed and imaging equipment (resolution). However, the need to prevent PPi from diffusing away from the detector before it is converted into a detectable signal means that the number of reactive sites must actually be limited. In US6274320, each reaction is limited to a miniature reaction vessel located at the tip of an optical fiber, thereby limiting the number of sequences to one sequence per optical fiber.

Even more limiting are the short read lengths (<30 bp) achieved by pyrosequencing. Such short sequences are not directly available in whole-genome sequencing, and the complex setup of equilibrium reactions makes it difficult to extend read lengths further. Only occasionally and for specific templates, read lengths of up to 100 bp have been reported.

A similar protocol for detecting released markers is described in US6255083. A protocol for the sequential addition of nucleotides and detection of the label which is subsequently cleaved by an exonuclease is described in WO 01/23610.

The principle advantage of detecting released label or by-products is that the template remains label-free in subsequent steps. However, parallelizing such sequencing schemes on solid surfaces such as microarrays can be difficult due to signal diffusion from the template.

Instead of detecting released by-products, one can detect each incorporated nucleotide as it is added to the growing polymer. In principle, such a protocol would be performed like pyrosequencing (adding one base at a time, cycling between the four natural nucleotides), but instead would use labeled nucleotide analogs (i.e. fluorescent) . As an example, Polony sequencing (Mitra RD, Church GM., Nucleic Acids Res 1999 Dec 15;27(24):e34 "In situ localized amplification and contact replication of many individual DNA molecules") is based on the sequential addition of fluorescently labeled nucleosides on an acid basis.

Detection of labels attached to each incorporated nucleotide presents additional difficulties in that the signal generated at each step must be removed, computationally subtracted, or physically quenched in preparation for the next step. Such removal can be accomplished, for example, by photobleaching or by using a cleavable linker between the nucleotide and the label. For example, Polony sequencing uses specifically designed fluorescent nucleotides that carry a dithiol linker between the nucleotide and the fluorescent dye. According to unpublished observations, the linker is efficiently cleaved using a reducing agent such as dithiothreitol, resulting in at least 99.8% pure nucleotides.

Because the read length in the SBS method is primarily limited by the loss of synchrony that occurs at each step, it would be desirable to be able to add all four nucleotides to the sequencing reaction while retaining the time limit for each base incorporation. The ability to stop the reaction between. That way, all four nucleotides will always be available (thereby limiting the rate of misincorporation), yet it will be possible to monitor each incorporated base.

A number of researchers have independently devised a solution sometimes called the base-addition sequencing strategy (BASS). Reactions are prevented from proceeding more than one step at a time by using a 3'-blocking monomer, but the blocking moiety is labile (eg, photocleavable or chemically degradable), exposing the 3'-OH group in preparation for the next synthetic step.

Bass includes:

1. Provide single-stranded template and annealed primer;

2. Add 3'-OH blocked fluorescent nucleotides;

3. Add a polymerase to incorporate a single nucleotide;

4. Read fluorescence;

5. Removal of blocking groups, e.g. by photocleavage;

6. Repeat steps 2-5.

Variations of this protocol use either permanent 3'-OH blocked nucleotides, which are removed using exonucleases (WO1/23610, WO93/21340), or unstable 3'-OH blocked nucleotides, It can be reverted to a functional 3'-OH group (US5302509, WO00/50642, WO91/06678, WO93/05183).

All BASS programs have the following in common:

• Use blocking or terminating nucleotides to prevent more than one step per synthesis.

• Nucleotides incorporated at each step are also labeled, usually with fluorescent dyes.

• At the end of each cycle, the blocking portion (or the entire terminal nucleotide) is removed in preparation for the next cycle.

Taken together, these demands place formidable demands on the enzymes used in BASS:

- They must accept both a nucleotide that is blocked at their 3' (where modifications are generally not tolerated by the enzyme) and that is fluorescently labeled.

• They must incorporate such nucleotides efficiently enough that only a negligible fraction of all templates fall out of synchronization in each cycle.

• They must be able to strictly discriminate base pairing of such nucleotides.

• They must not prematurely remove blocking groups or terminating nucleotides.

The fact that no one has hitherto been able to make BASS work suggests that these difficulties are insurmountable. For example, in (Metzker et al. "Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates", Nucleic Acids Res 1994:22(20):4259-67), none of the 8 enzymes studied was resistant to Both 3'-blocked dUTP and 3'-blocked dCTP are affected, even without the added complication of fluorescent labeling. The search for an enzyme capable of accepting all four nucleotides in 3&apos;-blocked and fluorescently labeled form thus seemed almost hopeless.

In conclusion, if a sequencing-by-incorporation approach could be made to work, one could convincingly sequence millions of templates attached to a surface in parallel. The main attraction of detecting incorporated rather than released labels is that the reactions can be parallelized across the surface. For example, on a surface of 10×10 cm, such a system would be able to sequence, for example, 37 million templates at about 600,000 bp/s, with each cycle of 60 s (assuming a Poisson distribution of 1 template/10 μm), Thus realizing 50Gb/24 hours. In principle, ten human genomes can be sequenced per day on such a system. The cost of the system would be comparable to that of a fluorescence scanner, and the running costs would be comparable to those of current Sanger sequencers.

The remaining major hurdles to achieving the stated goal are: firstly, the read lengths in SBS are too short to be usable in the sequencing of large genomes, and secondly, no method has been developed to place templates on surfaces at a sufficiently high density. reliable way.

The present invention cleverly solves the problems of the prior art in many aspects.

Brief description of the drawings

Figure 1 illustrates a template (top row, showing sequenced strands) sequenced by chroma sequencing using each natural nucleotide (indicated on the left) as a terminating nucleotide. Each chrominance sequence is represented as a series of dashes (measuring the number of intervening bases) and letters (measuring the number of consecutive terminating nucleotides). From this figure, it is clear that by aligning the reads, the raw sequence can be obtained from the read column.

figure 2

In the nucleotide incorporation assay of Example II, the figure shows the fluorescence after attempted incorporation of dTTP (labeled with Cy3), dATP and dGTP with and without DNA polymerase (Klenow) ( any unit). The expected result is two incorporated dTTPs, and the figure clearly demonstrates that sufficient signal is generated from this incorporation event to reliably detect incorporation above background noise.

Figure 3 illustrates an embodiment of a reaction chamber suitable for use in solid-phase chromatic sequencing in a regular microarray scanner. The diagram shows the reaction chamber assembly using a regular 25 x 75 mm glass slide (1 ) onto which templates can be spotted or randomly attached. During the reaction, the rubber gasket (2) seals the glass to the reaction chamber. The inlet (3) and outlet (4) are connected by a connector (5) to the reagent distribution system as illustrated in Figure 4 .

FIG. 4 illustrates an embodiment of a reagent dispensing system suitable for performing colorimetric sequencing in the reaction chamber of FIG. 3 . A 10-port valve (1) allows reagents to be dispensed into and out of the chamber (2) and waste tube (6), and up to eight reagent containers (3) can accommodate the different Reagents and wash buffers. The syringe pump (4) and valve (1 ) can be easily motorized and computer controlled along with the scanner (5, showing a partial view of the slide holder) for a fully automated system.

The present invention is based on the development of a novel sequencing strategy that improves upon the previously described sequencing-by-synthesis approach while allowing most of its difficulties to be avoided. It is a strategy that is easy to parallelize, directly visualizes the incorporation of each monomer (ie without size fractionation) and offers the possibility of long read lengths.

The present invention is based on the recognition that in the SBS method, contrary to what has been assumed, it is not necessary to stop at every position (as in pyrosequencing or the method of WO1/23610 by adding one base at a time, or Blocked nucleotides were used as in BASS).

Instead, sequencing can proceed in skips, skipping from each occurrence of a particular "stop" nucleotide to the next. Inserted nucleotides can be labeled. Terminating nucleotides can be labeled. This offers an improvement, which may be an ideal compromise between the approach of using blocking groups (where each step is productive, but unblocking is problematic) and A scheme in which bases are synchronized (where unblocking is avoided at the cost of making more steps unproductive, exacerbating the problem of loss of synchrony). Also, in contrast to the case of BASS, the present invention eliminates the need to place a label on the same nucleotide as the blocking group.

One aspect of the invention provides a sequencing-by-synthesis method characterized by the incorporation of nucleotides in a stepwise fashion, with one step potentially allowing the incorporation of more than one nucleotide.

In a preferred embodiment, one step potentially allows the incorporation of three out of four possible nucleotides, depending on the underlying template sequence. Preferably, a different step allows for the incorporation of a fourth possible nucleotide, ie the remaining ones other than the three that could potentially be incorporated in the first step.

In other embodiments, different steps are performed to allow incorporation of all four nucleotides in one set of steps, at least one of which allows incorporation of more than one but less than all possible nucleotides. As discussed further below, prior art methods can be generalized or have a set of four distinct iterative steps capable of cycling, each step allowing in principle the incorporation of only one of the four nucleotides (incorporation depends on the underlying template sequence), or have a single iterative step that includes all four blocked nucleotides, again allowing only one of the four nucleotides to be incorporated in each step Both of these can be summarized as a "1-1-1-1" approach. A single step that would in principle allow the incorporation of all four nucleotides is not available for sequencing, which can be generalized as a "4" approach, since the sequenced strand will immediately polymerize with the template ends. The present invention allows, in various embodiments, to implement a method of sequencing by synthesis, characterized by the incorporation of nucleotides in steps following a pattern other than "4" or "1-1-1-1". Thus, in a preferred embodiment, the nucleotides are incorporated in a set of steps following "3-1", as already mentioned. In other embodiments, a set of steps follows "2-2" or "1-2-1", or follows an irregular pattern where nucleotides may repeat within a set of steps (eg, "2-2-3") . Make step groups cycle on demand. Furthermore, groups of steps with different modes can be combined.

According to one aspect of the present invention, a method for determining nucleic acid sequence and/or base composition information is provided, the method comprising:

(i) providing a nucleic acid comprising a first strand comprising a nucleic acid template, wherein the free 3' end of the nucleic acid strand annealed to the first strand of the nucleic acid template allows extension of the nucleic acid strand complementary to the nucleic acid template, which is It is achieved by template-dependent nucleic acid polymerase through template-sequence-dependent incorporation of nucleotides into a nucleic acid strand complementary to the nucleic acid template;

(ii) performing a set of one or more steps, cycling the set of one or more steps a desired number of times, or performing in conjunction with other sets of one or more steps, to extend a nucleic acid strand complementary to a nucleic acid template, thereby permits access to information representing the base composition or sequence of said nucleic acid,

One of these steps includes:

(a) in the presence of:

nucleic acid comprising a first strand comprising a nucleic acid template,

the free 3' end of the nucleic acid strand annealed to the first strand of the nucleic acid template, and

Template-dependent nucleic acid polymerase;

Nucleotides selected from one, two, three or four nucleotide complementarity types are provided for template-dependent incorporation of said nucleotide into a complementary nucleic acid by said nucleic acid polymerase In the nucleic acid strand of the template, wherein each of said nucleotides is a natural nucleotide or a nucleotide analogue, they can be template-dependently incorporated into the DNA strand by a nucleic acid polymerase at the free 3' end of the nucleic acid strand In, and within each type of nucleotide complementarity, the nucleotides and nucleotide analogs associated with adenosine (A), cytosine (C), thymine (T) and guanine (G) one is complementary;

and

(b) removing or inactivating unincorporated nucleotides;

and

where within a set of steps

providing nucleotides selected from all four nucleotide complementarity types and which can be used for template-dependent incorporation,

In at least one step, nucleotides selected from more than one, optionally two, three or four nucleotide complementarity types are provided and are available for template-dependent incorporation, and at least one Nucleotides of the type of nucleotide complementarity which, if incorporated into a nucleic acid strand complementary to a nucleic acid template, allow further extension of the nucleic acid strand complementary to the nucleic acid template, and

optionally providing no nucleotide complementarity types in more than one step, or providing each nucleotide complementarity type in no more than one step within the set of steps; and

Wherein, if nucleotides selected from all four complementarity types are provided in one step, nucleotides in one, two or three nucleotide complementarity types, if incorporated into a nucleic acid template complementary to In the nucleic acid strand of , prevent further extension of the nucleic acid strand complementary to the nucleic acid template and all copies present, if there are multiple copies;

(iii) performing multiple sets of said steps, cycling said set of steps and/or performing said set of steps in conjunction with a different set of steps;

(iv) determining the nature and/or amount of nucleotides incorporated into the nucleic acid strand complementary to the nucleic acid template in at least one set of steps by determining the incorporation into the nucleic acid strand complementary to the nucleic acid template in at least one step of each set The nature and/or amount of nucleotides in the nucleic acid strands of the group for which the nature and/or amount of nucleotides incorporated are determined.

As noted, the present invention allows sequencing without size fractionation.

The free 3' end of the nucleic acid that anneals to the first strand located 5' to the nucleic acid (e.g., DNA) template (about which sequence information and/or base composition information is desired) can be determined by a primer that anneals to the first strand (such as an oligonucleotide primer), which may be provided by a gap in the second strand that anneals to the first strand (in which case, during extension, the portion of the second strand that originally annealed to the nucleic acid template is displaced or degraded ), or may be provided by the loop itself, an extension of the first strand that allows self-initiated backward looping.

Nucleotides or nucleotide analogs can be defined by their base pairing properties. Thus all nucleotides or nucleotide analogues incorporated complementary to natural adenosine belong to the type of nucleotide complementarity of thymine, and those incorporated complementary to natural guanine belong to the type of nucleotide complementarity of cytosine Types, those incorporated complementary to natural thymine belong to the type of nucleotide complementarity of adenosine, while those incorporated complementary to natural cytosine belong to the type of nucleotide complementarity of guanine. The type of nucleotide complementarity thus describes and defines the logical properties of nucleotides or nucleotide analogs with respect to template-directed polymerization.

Incorporation of nucleotides is potentially allowed by providing the nucleotides in the reaction medium for incorporation by the template-dependent polymerase.

The nucleic acid template can be deoxyribonucleic acid (DNA), the nucleic acid polymerase can be a DNA-dependent DNA polymerase, and the nucleotides can be deoxyribonucleotides or deoxyribonucleotide analogs.

The nucleic acid template can be deoxyribonucleic acid (DNA), the nucleic acid polymerase can be a DNA-dependent ribonucleic acid (RNA) polymerase, and the nucleotides can be ribonucleotides or ribonucleotide analogs.

The nucleic acid template can be ribonucleic acid (RNA), the nucleic acid polymerase can be reverse transcriptase, and the nucleotides can be deoxyribonucleotides or deoxyribonucleotide analogs.

In preferred embodiments of the various aspects of the invention, the nucleotides used in the step wherein potentially incorporating more than one different nucleotide are selected from standard nucleotides.

In some preferred embodiments of the various aspects of the invention, the nucleotides used in the step wherein potentially only one of the different nucleotides are incorporated are nucleotides selected from standard nucleotides.

In other embodiments, modified nucleotides or analogs may be employed, as discussed further elsewhere herein.

Nucleotides employed in the present invention may be labeled, and labels may include fluorescent labels. Different nucleotides (as between complementarity types of A, C, G, and T) can be labeled with different labels, eg, different fluorescent labels, possibly of different colors.

As noted, the present invention provides sequencing-by-synthesis methods characterized by incorporation of nucleotides in a scheme other than 4 or 1-1-1-1.

Thus, preferred incorporation protocols first allow for the potential incorporation of 2 or 3 nucleotides, and then, generally following a washing step to remove unincorporated nucleotides, in a different step, the incorporation protocol allows Potentially incorporate 2 nucleotides or 1 nucleotide. Combinations of groups of steps can be performed to provide an overall reaction scheme.

Of course, appropriate conditions are provided in the reaction medium for template-dependent incorporation of nucleotides at the 3' end of the DNA strand, in accordance with the knowledge and techniques available in the art.

In one embodiment, the present invention proposes a method comprising a cyclical step or set of steps: providing a DNA template in which the dissociation of a nucleic acid strand (such as an annealing primer) that anneals to the first strand located 5' to the DNA template The 3' end allows the synthesis of a DNA strand complementary to the DNA template by adding a set of labeled nucleosides in the first step in the presence of a polymerase under conditions that incorporate nucleotides into the extended strand complementary to the template acid (referred to as "intercalated" nucleotides), followed by a wash to remove unincorporated nucleotides, and then in a second step in the presence of a polymerase, primer-based incorporation of nucleotides into the elongated Under strand conditions, a second set of labeled nucleotides ("stop" nucleotides) is added, followed by a wash to remove unincorporated nucleotides and determine the label for incorporated nucleotides. The set of steps can be repeated as many cycles or times as desired.

The number (but not the order) of incorporated nucleotides is thus determined at each step. If the labels for different nucleotides are distinguishable, the number (but not the order) of each incorporated nucleotide species will be determined.

The information obtained about incorporated nucleotides in this way, ie by determining the label, is called colorimetric. Chroma is not a standard DNA sequence, but:

It can be used as a signature sequence and compared with known DNA sequences;

• A set of four (typically) such sequences can be reassembled into normal DNA sequences (as explained further in the text).

Embodiments of the invention, as well as the concept of chromaticity, can be illustrated by reference to a general sequence obtained using dA, dC and dG as intervening nucleotides and dT as a terminating nucleotide, for example, written as follows:

dT[1A, 2C, 1G, 1T]-[2A, 2C, 1G, 3T]-[2A, 2C, 1G, 1T]-[0A, 1C, 0G, 1T]

where the numbers in parentheses give the abundance of each inserted nucleotide between each occurrence of dT, as measured by its labeling intensity, plus the number of consecutive dTs.

Several DNA sequences are capable of generating this data, such as:

ACCGTGCACATTTACAGCTCT

CAGCTCCAAGTTTCACGATCT

wait…

Provided below is a base-calling strategy that uses information or colorimetrics obtained from four such sequence reads (using each of the four nucleotides sequentially as a terminating nucleotide) to identify determine the original sequence.

In one aspect, preferred embodiments of the present invention provide a method (Scheme I) comprising:

1. A single-stranded template is provided with an annealed DNA strand having a 3' end to function as a primer.

2. Adding a set of one or more labeled nucleotides (called "insertion nucleotides"), selecting them so that at least one nucleotide that is complementary to the template (called "stop nucleotides") ) are excluded from the set of labeled nucleotides. Typically, three nucleotides are added bearing a distinguishable label (the fourth natural nucleotide being the stop nucleotide).

3. Optionally, one or more blocking nucleotides (different from the labeled nucleotides) are added. These are also "terminating nucleotides". Examples include 3'-O-modified nucleotides, which can carry a photocleavable group that leaves a 3'-OH when irradiated, or other modifications, namely acyclic nucleotides and dideoxynucleotides .

4. Optionally, add one or more non-incorporated inhibitor nucleotides (different from labeled and blocked nucleotides) that can act to prevent The role of misincorporation at template positions where there is no complement in . Examples include 5'-bis- and mono-phosphate nucleotides, 5'-(α-β-methylene)triphosphate nucleotides.

5. Incubation with an appropriate polymerase under conditions that result in the addition of nucleotides to the growing strand.

6. Wash away unincorporated nucleotides.

7. If any blocking nucleotides were added in step 3, then

a. Removal of the blocking moiety, eg by photocleavage, enzymatic conversion or chemical reaction.

b. Alternatively, replacement of all nucleotides by exonuclease treatment followed by incorporation of non-blocking nucleotides (see eg WO1/23610, WO93/21340).

8. Add the remaining nucleotides ("stop nucleotides") that are necessary to ensure that all nucleotides present in the template have added complements and interact with the polymerase (not necessarily the same as the one in step 5) same) were incubated under conditions that result in the addition of nucleotides to the growing chain. The terminating nucleotide may optionally be labeled, and/or 3'-blocked (as in BASS).

9. Wash away unincorporated nucleotides.

10. Detecting the presence and/or amount of each labeled nucleotide.

11. Optionally, removing or deactivating the label and/or 3&apos;-blocking group. For example, fluorescent labels can be photobleached.

12. Repeat steps 2-11 until desired number of cycles are completed.

This sequencing method is particularly well suited for parallelization on solid phase, both because of its simplicity and because it provides a robust synchronization method. This protocol can be repeated multiple times by restarting at step 1 with fresh primers.

The nucleotides added in steps 3 and 8 are called stop nucleotides because they prevent (either by being blocked or by absence) polymerization from continuing beyond its complement in step 5. The set of terminating nucleotides can vary. For example, if the reaction from step 1 is performed four times, each of the four natural nucleotides can be used as a stop nucleotide.

The primer anneals to the template by base complementarity, leaving a free 3' end to which nucleotides can be added one by one by a template-dependent DNA polymerase. As noted, a free 3' end can be generated by nipping one strand of a double-stranded DNA molecule, or by allowing the free 3' end of a single strand to loop back for self-priming.

Note: "labeled" molecules should be understood to include pure labeled molecules as well as mixtures of labeled and unlabeled molecules. For example, the labeled dTTP can be pure fluorescein-labeled dTTP, or a mixture of fluorescein-labeled dTTP and conventional unlabeled dTTP. The optimal ratio of marked to unmarked is determined by several factors:

• The need to obtain sufficient signal to overcome equipment noise. For example, on a PerkinElmerScanArray, 2.5 fluorochromes/pixel yields a signal three times the noise level.

• Avoid the need for multiple fluorochromes in close proximity to avoid fluorescence resonance energy transfer (FRET, which causes one fluorochrome to quench the other). FRET decays with distance to the sixth power, but can still be significant over a range of a few nucleotides.

• Avoid the need for multiple fluorochromes in close proximity to avoid inhibiting subsequent incorporation of nucleotides by the polymerase (which may be inhibited by steric effects of bulky fluorochromes).

Alternatively, one can force a labeled nucleotide fraction to terminate the growing chain, for example, by using labeled acyclic or dideoxynucleotides, or by placing a label on the 3'-OH or its vicinity. As long as the labeled nucleotides represent only a small fraction of the total nucleotides, the loss of signal due to termination remains insignificant, while at the same time the loss of synchrony due to the enzyme's lower affinity for modified nucleotides can be avoided entirely.

Work in the inventor's laboratory found that -2.5% or less labeled nucleotides worked well (see Examples below). Assuming that the template is 1000 tandem repeat copies of a 100bp sequence, for each incorporated nucleotide, at least 25 fluorescent dyes per template are obtained (i.e., on a PerkinElmer ScanArra, if each template is within a pixel, is more than 10 times higher than the noise level). Assuming four nucleotides are incorporated in an average cycle, label spacing averages 1000 bases, avoiding both quenching and polymerase inhibition.

In additional embodiments of the invention, Scheme I, for example, allows for BASS variants that ease some of the constraints on the polymerase. If the insert nucleotide set is labeled but not blocked, while the stop nucleotides are not labeled but blocked, then all four nucleotides can be added as a mixture in a single step, then washed and scanned as above. A polymerase that accepts both blocking and labeled nucleotides can be used, or a different polymerase can be used that adds the labeled insert nucleotide in the first step and the blocked termination core in the second step. glycosides. The colorimetric difference of this modified scheme is that homopolymers are detected as adjacent loops without incorporation; they each terminate with the incorporation of a single stop nucleotide, thereby scanning for homopolymers stepwise, rather than Populate it in a single run. In such a protocol, it may be desirable to use a photocleavable fluorescent dye (see below) as well as a photocleavable 3&apos;-blocking group. Alternatively, blocking groups removable by mild chemical treatment can be used, for example, allyl groups as described in Kamal et al. (Tetrahedron Letters 1999, vol. 40, pp. 371-372).

In a particularly simple embodiment, an aspect of the invention provides a method (Scheme II) comprising:

1. Provide the free 3' end on the annealed DNA strand to the single-stranded template to function as a primer.

2. Add three nucleotides carrying a distinguishable label, such as a distinguishable fluorescent label.

3. Optionally, one or more non-incorporated inhibitor nucleotides (other than the labeled nucleotides) are added. Examples include 5'-bis- and mono-phosphate nucleotides, 5'-(α-β-methylene)triphosphate nucleotides.

4. Incubation with an appropriate polymerase under conditions that result in the addition of nucleotides to the growing strand.

5. Wash away unincorporated nucleotides.

6. Add remaining nucleotides (labeled, eg fluorescently) and incubate with polymerase (not necessarily the same as in step 5) under conditions that result in the addition of nucleotides to the growing strand.

7. Wash away unincorporated nucleotides.

8. Detect the presence and amount of each labeled nucleotide.

9. Inactivation of labeling (eg by photobleaching, which is not necessary for every cycle, or by chemical treatment with eg dithiothreitol to cleave disulfide bonds).

10. Repeat steps 2-7 until desired number of cycles are completed.

For example, one can use dA/dG/dC in step 2 (as marked red/green/blue) and then add dT in step 6 (as marked yellow). Step 4 will add any number of dA, dG, and dC until the first occurrence of dA in the template and then terminate because there are no complementary nucleotides. The fluorescence readout for dA/dG/dC (e.g. red/green/blue) from step 8 will be proportional to the number of dA, dG and dC between each dT, while the fluorescence of incorporated dA (e.g. yellow) will be proportional to the The number of consecutive dTs is proportional, and after spectral separation, the various contributions can be quantified. The resulting sequence can generally be written as a sequence of four members, giving the number (but not the order) of dA, dG and dC between each dT.

For example, the sequence ACGCTACGCATCAGACTTC (i.e. template TGCGATGCGTAGTCTGAAG) can be recorded as [1A, 2C, 1G, 1T]-[2A, 2C, 1G, 1T]-[2A, 2C, 1G, 2T]-[0A, 1C, 0G, 0T].

By carrying out four different reactions according to Scheme II, varying the terminating nucleotide between the four possibilities, one can ensure termination at each of the different bases in one of the four reactions.

Although fluorochromes are convenient to use, not all fluorochromes are easily bleached. Other types of markers can be used in the above manipulations as long as they can be removed, inactivated, or deducted computationally for each cycle. However, in additional embodiments, in order to allow a wider selection of labels, removal (such as photobleaching of fluorescent dyes) can optionally be replaced by a complete restart, for example as follows:

First, a cycle is performed with labeled, eg fluorescent, nucleotides. Freshly synthesized DNA strands are removed, such as by formamide treatment, and fresh primers are allowed to anneal to restart the process. This time, one cycle was performed with unlabeled nucleotides, followed by one cycle with labeled nucleotides. This process is repeated, each time with increasing cycles of unlabeled nucleotides. In this way, only the last cycle of each restart is labeled, eliminating the need to remove labels from previous cycles, such as bleached fluorescent dyes.

The same approach can also be used to cross non-intended areas, a bit like moving the read head of a tape recorder.

As an alternative to photobleaching, modified fluorescent nucleotides carrying a cleavable linker between the nucleotide and the fluorescent dye can be used. For example, such nucleotides carrying disulfide bonds have been described, which can be efficiently cleaved by reducing agents such as dithiothreitol (see the work of Rob Mitra and George Church on the polony technique for sequencing and genotype analysis, pp. Details, including chemical structures, can be found on the Internet using a browser, such as http://cbcg.lbl.gov/Genome9/Talks/mitra.pdf. Similarly, Li et al. (PNAS 2003, vol.100 no .2, pp.414-419) describe photocleavable fluorescent nucleotides comprising a photolabile 2-nitrobenzyl linker.

The method according to scheme II allows to achieve a number of advantages:

·Compared to the current SBS method where most cycles are unproductive (because in this method, each time one adds a single base, the chance of being complementary at that position is <50%), since one of the four reactions is in each template Termination at position (ignoring homopolymers), so the number of cycles required to sequence n bases is n.

• Since synthesis is reinitiated by primers for each of the four reactions, factors that depend primarily on cycle number will be problematic to a 4-fold lower degree. In particular, a loss of synchrony will occur after many cycles, but for each of the four reactions, since all templates are effectively resynchronized, it is possible to achieve a similar result under similar conditions compared to SBI or pyrosequencing. , able to read four times as many bases (see Example below).

Applications that do not require complete sequences (i.e., signature sequencing for gene expression, methyl-cytosine sequencing for epigenomics, and SNP analysis for specific SNPs) can use only The partial sequence obtained. The obtained sequence contained information equivalent to 1 base pair per cycle. See Scheme III below. See also Figure 1 for a diagram of the data obtainable from the composition of each of dA, dC, dG and dT in different reactions. Any of those data are sufficient for the desired purpose, eg, determining which of several possible sequences (eg, differences in dA nucleotides) are present in the test sample.

• Homopolymer strands are always measured four times, making them easier to base correctly than in SBI or pyrosequencing methods. See Base Incorporation Algorithm II below.

Base introduction algorithm I (basic strategy)

This section of the disclosure sets forth illustrative embodiments of aspects of the invention related to the identification of sequences based on information obtained by methods including the use of the disclosed termination and insertion nucleotides.

By performing four different reactions according to Scheme II, varying the terminating nucleotide between the four possibilities, one can ensure termination at each of the different bases in one of the four reactions. The table below shows the results or shades that would be obtained from the sequence ACGCTACGCATCAGACTC (template TGCGATGCGTAGTCTGAG) in four cycles using each of the four stop nucleotides: termination The sequence obtained (first 4 cycles): dTdAdGdC [1A, 2C, 1G, 1T] - [2A, 2C, 1G, 1T] - [2A, 2C, 1G, 1T] - [0A, 1C, 0G, 0T] [0C, 0G, 0T, 1A] - [ 2C, 1G, 1T, 1A]-[2C, 1G, 0T, 1A]-[1C, 0G, 1T, 1A][1A, 1C, 0T, 1G]-[1A, 2C, 1T, 1G]-[2A , 2C, 1T, 1G] - [1A, 2C, 1T, 0G] [1A, 0G, 0T, 1C] - [0A, 1G, 0T, 1C] - [1A, 0G, 1T, 1C] - [0A, 1G, 0T, 1C]

Reading from left to right, one can easily see that the first nucleotide must be A (since the first step on A does not produce any fluorescence of other bases, it must therefore be terminated without any incorporation into the nucleus glycosides). Removing the corresponding entry, and logging A, yields: termination The sequence obtained: dTdAdGdC [1A, 2C, 1G, 1T] - [2A, 2C, 1G, 1T] - [2A, 2C, 1G, 1T] - [0A, 1C, 0G, 0T] [2C, 1G, 1T, 1A] - [ 2C, 1G, 0T, 1A]-[1C, 0G, 1T, 1A][1A, 1C, 0T, 1G]-[1A, 2C, 1T, 1G]-[2A, 2C, 1T, 1G]-[1A , 2C, 1T, 0G] [1A, 0G, 0T, 1C] - [0A, 1G, 0T, 1C] - [1A, 0G, 1T, 1C] - [0A, 1G, 0T, 1C]

Sequence: A

Now the only matching entry on the left is for C, since it indicates the presence of only 1 A. Removing the corresponding entry, and logging C, we get: termination The sequence obtained: dTdAdGdC [1A, 2C, 1G, 1T] - [2A, 2C, 1G, 1T] - [2A, 2C, 1G, 1T] - [0A, 1C, 0G, 0T] [2C, 1G, 1T, 1A] - [ 2C, 1G, 0T, 1A]-[1C, 0G, 1T, 1A][1A, 1C, 0T, 1G]-[1A, 2C, 1T, 1G]-[2A, 2C, 1T, 1G]-[1A , 2C, 1T, 0G] [0A, 1G, 0T, 1C] - [1A, 0G, 1T, 1C] - [0A, 1G, 0T, 1C]

Sequence: AC

Now the only matching entry on the left is for G:

terminate The obtained sequence: dTdAdGdC [1A, 2C, 1G, 1T] - [2A, 2C, 1G, 1T] - [2A, 2C, 1G, 1T] - [0A, 1C, 0G, 0T] [2C, 1G, 1T, 1A] - [ 2C, 1G, 0T, 1A] - [1C, 0G, 1T, 1A] [1A, 2C, 1T, 1G] - [2A, 2C, 1T, 1G] - [1A, 2C, 1T, 0G] [0A, 1G, 0T, 1C]-[1A, 0G, 1T, 1C]-[0A, 1G, 0T, 1C]

Sequence: ACG

The only matching entry on the left now is for C, since it shows that there is only 1 G between this and the previous C, consistent with the sequence we have had so far.

Continuing in this way eventually provides the complete sequence: ACGCTACGCATCAGACTC.

In fact, it is easy to see that the total amount of fluorescence obtained from intervening nucleotides at each step measures the total distance between each stop nucleotide, while the fluorescence from stop nucleotides measures the number of consecutive stop nucleotides , and one can thus always determine the sequence from a set of four reactions. This fact is further illustrated with reference to FIG. 1 .

Scanning the four rows in Figure 1 enables the "reading" of the sequence. It is possible to obtain sequences by simply determining the number of stop nucleotides incorporated per cycle (by the magnitude of a measured label such as fluorescence), and the intervening nucleotides incorporated per cycle (again by the magnitude of the label measured) and the results of each of the four runs using each of the four different nucleotides as the stop nucleotide were aligned. Preferably, however, the nature of the inserted nucleotide (which could mean identity) is determined for each run, thereby providing the degeneracy of information that allows for extremely fast and accurate determination of sequence, allowing for errors in marker magnitude measurements such as as discussed further in this paper.

Base Introduction Algorithm II

More sophisticated base calling algorithms can be implemented using, for example, dynamic programming, least squares optimization, and/or regular expressions to find the optimal sequence in the face of measurement error. Such algorithms can also make better use of the redundancy of available information. In other words, instead of just using the measured length between each occurrence of the same nucleotide, such an algorithm will find the optimal sequence that makes each of the expected and observed three intervening nucleotides The difference between the abundances is minimized.

The inventors have provided a working dynamic programming algorithm that works well despite 20-25% noise. It first performs a multiple alignment of four series of measurements using dynamic programming, minimizing at each step the difference between the expected and observed abundance of each of the three intervening nucleotides. Then, based on the four available distance measures, a least squares optimization was used to find the most likely length of each homopolymer chain.

Terms and Definitions

A homopolymer is a contiguous sequence of specific nucleotides. A homopolymer sequence is a DNA sequence in which homopolymers are written as numbers rather than repeated letters, ie ACCGGT is written as ACGT and has a homopolymer length of 1,2,2,1.

Let chromaticity be a set of measurements obtained by repeating the method of the present invention as in Protocol I four times using each of the four natural nucleotides as the stop nucleotide. Thus chroma is a three-dimensional array of measurements indexed by cycle, stop nucleotide, and measured nucleotide. For example, if 10 cycles are performed for each stop nucleotide, the chroma will contain 10 (number of cycles) by 4 (number of stop nucleotides) by 4 (number of nucleotides measured) members , and the number at position {4, 'A', 'C'} will be the fluorescence of cytosine measured at cycle number 4 when adenosine is used as the stop nucleotide. For convenience, let the chromaticity of x be the subset of all chromaticities that include measurements obtained with x as the terminating nucleotide. Thus the chromaticity of A is a quarter of the total chromaticity.

Let N be the number of cycles performed in each repetition. Chromaticity is thus 4*4*N members inferred from marker measurements.

Let the introduced sequence be the nucleotide sequence S ₀ , S ₁ , ... S _k (wherein each S is one of [A, C, G, T]). The purpose of base introduction is to find the optimal introduction sequence given the chromaticity. For convenience, we express homopolymeric chains as quantities rather than repeating the same base, in other words, we associate each position i in the introduced sequence with a quantity q _i which gives an estimate of the base S _i number of repetitions. For consistency, we restrict the sequence such that S _n+1 ≠ S _n for all n.

Base introduction phase I, dynamic programming

The purpose of base introduction is to find the optimal introduction sequence given the chromaticity. However, there are 4*3k ^-1 possible incoming sequences of length k, an enormous number even for fairly small k (with k=20, there are over 4 billion possible incoming sequences). In order to find a usable base introduction algorithm, the complexity of this problem is simplified.

Introduced sequences can be classified by the number of occurrences of each nucleotide. For example, base counts {1, 2, 0, 4} correspond to any sequence containing 1A, 2C, no G, and 4T. An example of such a sequence is TCTATCT.

The algorithm provided according to the invention exploits the fact that in some simple cases we can easily derive the optimal introduction sequence, and by recursion more difficult cases can be deduced from simpler cases.

Some simple cases are easy to solve. Base counts {0, 0, 0, 0} correspond to the empty incoming sequence. The count {1, 0, 0, 0} can only correspond to the incoming sequence 'A', and similarly for C, G and T.

However, base counts {1, 1, 1, 1} may correspond to 'ACGT', 'TCGA', and so on. In such cases, chroma can be used to find the best incoming sequence.

Note that any incoming sequence with base counts {i,j,k,l} must correspond exactly to a specific subgroup of chromas, that is, include i cycles of A's chroma, j cycles of C's chroma , a subgroup of k chromaticities of cycle G and l chromaticities of cycle T. It is thus possible to compare the chromaticity introduced into the sequence prediction with the actually measured chromaticity. The best introduced sequence for {i,j,k,l} will be the one whose predicted chromaticity is most similar to the actual measured chromaticity of the corresponding subgroup. Similarity can be measured in various ways, for example, as sum of differences, sum of square differences, Pearson correlation coefficient, etc. The similarity can be reported as a score, ie as an error score to be minimized or a similarity score to be maximized.

The general case {i, j, k, l} described cannot be solved directly. But the optimal incoming sequence of {i, j, k, l} can be generated from shorter sequences in up to four different ways: by adding 'A ', by adding 'C' to the best sequence of {i,j-1,k,l}, by adding 'G' to the best sequence of {i,j,k-1,l}, or by Add 'T' to the best sequence of {i,j,k,l-1}.

By computing scores (as above, by comparing predicted chromaticity with actual chromaticity) and choosing a minimum (or maximum, depending on the measure used), one is able to find out which of (at most) four extensions is one of the best. The following shows how this is done, but assumes for the moment that such a score has been obtained.

We set q to the actual measured amount of newly introduced bases obtained by chroma. For example, when considering the extension of 'A' (i.e. from {i-1, j, k, l} to {i, j, k, l}), then the positions {i, 'A', ' The chromaticity at A'} obtains q, the amount of labeled adenosine measured when using adenosine as the terminating nucleotide in cycle i.

Thus, the optimal incoming sequence for {i, j, k, l} can always be found by finding the optimal extension of the sequence that contains one of the incoming bases less. This operation can then be repeated for each shorter case until a case of an all zero solution such as {1, 0, 0, 0} is reached. It is therefore always possible to obtain the optimal incoming sequence of any length by recursively applying the same simple operation. As a by-product, the homopolymer length q _i as measured in the colorimeter is obtained.

A few restrictions apply:

• The sequence cannot contain any bases less than zero. Thus we cannot get the optimal introduction sequence of {i, j, k, 0} by extending {i, j, k, -1} with 'T'. Due to this restriction, all recursions must end up in the [0, 0, 0, 0} empty sequence.

· Our constraint on the incoming sequence, that S _n+1 ≠ S _n for all n, means that if the optimal incoming sequence of {i-1,j,k,l} ends at 'A', then we cannot Extend with 'A', and so on for other bases.

• In some cases, no extension is possible. For example, {2, 0, 0, 0} cannot be produced by extending {1, 0, 0, 0} with another 'A'. In such cases, there is no incoming sequence.

The similarity score can be calculated in a stepwise manner. Because they differ by only one cycle, the scores for {i-1, j, k, l} are reused when computing the scores for {i, j, k, l}, etc. This can be achieved by keeping track of the length of the optimal incoming sequence and the running score for each {i, j, k, l}. When studying possible extensions from, say, {i-1,j,k,l} to {i,j,k,l} (i.e., extensions through 'A'), one only needs to compute the corresponding The predicted chroma part of the extra loop. This can be calculated by studying the inserted bases back to the most recent 'A' in the incoming sequence. Since the optimal introduction sequence of {i-1, j, k, l} is known, it is also known how it was obtained. In particular, the measured quantity q of each inserted nucleotide is known. For each 'C', 'G' and 'T', these quantities are added up, all the way back to the most recent 'A', to obtain the predicted value for the missing cycle in the predicted chroma. The difference (or variance, etc.) between these predicted values and the corresponding cycle in the actual measured chromaticity is then added to the running score. A normalized score can then be obtained by calculating the running score divided by the length of the incoming sequence.

Note now that to compute the best incoming sequence for {3, 2, 2, 2}, the scores for {2, 2, 2, 2}, {1, 2, 2, 2}, etc. are still computed. But to find the overall best sequence, one has to systematically study all possibilities up to a certain limit (e.g. {N, N, N, N}), each of which leads to a trace back to {0, 0 , 0, 0} scores are recalculated, so the combinatorial surge remains. However, dynamic programming is a clever way to avoid such combinatorial proliferation.

Algorithms can be used such that whenever a score has been calculated, it is stored in a four-dimensional NxNxNxN matrix for reuse. Thus, when computing the best incoming sequence for {3, 2, 2, 2}, the scores for {2, 2, 2, 2}, {1, 2, 2, 2}, etc. will be stored in the matrix. When the score of say {2, 2, 2, 2} is needed again later, the recursion can be avoided entirely, and the previously computed results are simply fetched from the matrix. This provides extremely efficient execution. As opposed to studying approximately 3 ^4N possible introduction sequences, only N ⁴ possibilities need to be studied. For example, in an actual system with N=20, the problem is reduced from about 10 ³⁸ calculations to 160 000, making the algorithm from infeasible to effective.

The longest sequence that can be reliably introduced by an algorithm as disclosed herein is one that has N homopolymers of one of the bases, more than N of one base, and fewer than N of the other bases . This is evident from the fact that when one of the terminating bases exceeds N, the sequence can still be introduced, since the missing base must fill the gap left by the other three. But when the second base exceeds N, the gap left by the remaining bases cannot be unambiguously filled. The limits are not absolute; partial sequences can still be obtained from full chroma.

Depending on the application, one can choose to report the best sequence of (where) any {i, j, k, l} up to {N, N, N, N}, the best sequence of {N, N, N, N}, or One of the best sequences with index N. In the examples below, the latter is used. The choice depends on factors such as whether read length is preferred over accuracy, and whether partial sequences are acceptable.

Base introduction phase II, least squares (optional)

Phase I results in the introduction of the sequence S ₀ , S ₁ , ... S _n and the corresponding homopolymer lengths q ₀ , q ₁ , ... q _n . We can write this out in the conventional way by rounding each q to the nearest integer and spelling out the resulting DNA sequence. However, there is more information in the chrominance that we can exploit to find a better estimate of q _i . After all, the homopolymer length was measured as a single measurement for each stop base, but each position in the incoming sequence had actually been measured four times (once for each stop base).

An example makes this clear. Consider the following sequence:

ACGCATCAAAAGCCTTACACGGTAAGCATCATC

The 'AAA' triplet occurring at position 8 of the sequence would be measured directly in the third step of the color of A and would be an approximate number such as 3.43. If measurement errors are large, it can be difficult to be sure how to round the measured quantities to whole numbers in every case.

However, the 'AAA' triplet also contributes to the fourth step of the chromaticity of C, the second step of the chromaticity of G, and the second step of the chromaticity of T. In two cases (colors of C and T) the triplet is actually measured alone, while in the third case it is measured together with the preceding single A. Let us assume that for the chromaticities A, C, G and T the corresponding measurements are 3.43, 3.1, 4.2 and 2.9 respectively. We would prefer to use these additional measurements to reduce the effect of random measurement errors.

Consider again the homopolymer lengths q ₀ , q ₁ , . . . q _n . Instead of accepting the mere numbers obtained in Phase I, we can form a set of simultaneous equations that describe additional information about q. The above triplet is q ₈ because it is the eighth homopolymer. Likewise, A preceding it is q ₅ . We can now write the following information based on the previous paragraph:

q ₈ =3.43 (chromaticity from A)

q ₈ =3.1 (chromaticity from C)

q ₅ +q ₈ =4.2 (chroma from G)

q ₈ =2.9 (chromaticity from T)

We can do this in a similar fashion for each position in the incoming sequence. The resulting system of simultaneous equations can be solved using e.g. least squares optimization, and the resulting solution gives the set of homopolymer lengths q ₀ , q ₁ , ... q that best matches all measurements in chroma _n .

Examples of Error Tolerant Base Calling Algorithms

The table below shows the template for 10 cycles for each stop nucleotide

ATGGAGCAGCGTCATTCCTTAGCGGGCAACTGTGACGATGGTGAGAAGTCAGAAAGAGAGGC

TCAGGGATTCGAGCATCGGACCTGTATGGACTCTGGGGA

(Given the sequencing strand) Simulation results of chroma sequencing.

Each group shows the chromaticity of the indicated terminating nucleotide, each row shows the (simulated) measurements obtained in units of one base for the indicated nucleotide on the left, and each column is a cycle, including The first three nucleotides are added, followed by one nucleotide. For example, the four numbers in bold show measurements obtained in the first cycle of chroma with dATP as the terminating nucleotide. Since the template starts with A, only A gives a signal significantly different from zero.

A ACGT 0.78-0.190.20.07 1.09-0.142.170.86 1.070.811.090.03 12.071.861.31 1.031.950.023.57 2.012.083.96-0.14 0.861.171.912.19 1.171.211.010.09 1.03-0.113.052.1 1.990.010.960.08 C ACGT 20.962.951.04 1.0511.010.15 0.20.980.730.95 1.011.950.032.02 0.940.920.91.99 -0.061.043.050.02 1.911.10.12-0.03 1.080.992.032.14 4.081.055.863.07 5.851.144.990.12 G ACGT 0.950.062.061.08 1.010.020.87-0.13 1.151.0110.06 0.011.111.06-0.08 2.0830.975.03 -0.011.082.98-0.03 2.172.121.081.16 0.010.070.920.88 1.141.160.990.04 1.130.092.020.95 T ACGT 0.97-0.07-0.061.04 2.022.014.840.93 1.060.81-0.142.25 01.91-0.22.03 3.053.163.971.19 -0.060.060.960.84 1.910.92.010.91 0.020.072.060.96 2.94-0.12.940.93 6.112.245.370.61

Base calling using the dynamic programming algorithm described above identified the following incoming sequence (which does not show homopolymers): ATGAGCAGCGTCATCTAGCGCACTGTGACGATG, which is correct. Expanding the homopolymer by rounding to the nearest integer yields ATGGAGCAGCGTCATTCCTTAGCGGGCAACTGTGACGATGG, which again is correct and covers the 41 bp template. Thus, in only 10 cycles of chroma sequencing, and in the presence of significant measurement error (in this case, 10% CV), one was able to obtain 41 base pairs of sequence information.

To evaluate the error tolerance of the given algorithm, a series of 100 simulations were run on the given template with random noise equivalent to 10% CV. All 100 incoming sequences and all 100 expanded sequences were correct. 59 of them were 41 bp long, while the rest included an additional T from the template. Thus, the shown algorithm is productive and error tolerant in the face of experimental bias.

Nucleotide Addition Protocol

In SBS, it has always been assumed that nucleotides must be added one at a time, or at least must be incorporated one at a time as in BASS. However, as indicated above, other nucleotide addition schemes are available to obtain DNA sequences, and some are better suited to avoid the limitations of SBS (eg, loss of synchrony). In this part, we investigate all possible nucleotide addition schemes and demonstrate that the conventional scheme is in some respects the least likely.

A nucleotide addition scheme is a rule for adding nucleotides to an SBS reaction. It consists of successive steps involving the addition of one or more nucleotides. In this section we will ignore any nucleotides that are added purely as inhibitors or cannot be incorporated for some other reason. And we will call any nucleotide capable of base pairing with adenine "T" (or similarly G, C, A for cytosine, guanine, thymine). In special applications, analogs or derivatives of natural nucleotides may be used, but for sequencing purposes it is their base-pairing ability that dictates the logic of the nucleotide addition scheme. Nucleotide analogs or derivatives capable of multiple base pairing may be denoted "AC", "GCT", etc. to indicate this fact.

Cycling schemes are nucleotide addition schemes that repeat a basic pattern. A cycle with restart scheme is a nucleotide addition scheme that repeats the basic pattern followed by restarting with fresh primers with a variation of the basic pattern. A natural scheme is one in which no bases are repeated until all four bases have been added.

In the natural cycle scheme, "4" indicates that all four nucleotides were added in the first step, which is degenerate and cannot be used for sequencing.

Protocol "1-1-1-1" is the conventional protocol used for all previously published SBS methods. Note that even though BASS falls into this category, they are forced to be incorporated one by one because of the cleavable blocking group, although all four nucleotides can be added simultaneously.

Option 1-1-1-1 is the least productive option. This can be seen from the fact that after each productive step, the next nucleotide on the template could be one of three possibilities (i.e. different from those three of the bases just sequenced), but only A single base is added. As a result, it is the scenario most affected by loss of synchronicity.

The method according to the invention is Scheme 3-1, as disclosed in the text. It is a fully productive protocol (each step ensures nucleotide incorporation, since missing nucleotides in a given step are added in subsequent steps). There are four variants of 3-1 provided by changing single nucleotides in A, C, G and T. As indicated above, those four variants can be used to reconstruct the target sequence.

Scenario 2-2 is another potentially fully productive scenario. The scheme has only three variants, corresponding to AC-GT, AG-CT, and AT-GC; all other combinations are simple inversions.

What is the minimum necessary for a scheme to ensure that one can always reconstruct the original sequence (possibly restarting it)? Essentially, all that is required is that each homopolymer in the target sequence must be separable from its two neighbors. In other words, each homopolymer must be part of at least one nucleotide incorporation step that excludes its left-hand neighbor, and a step that excludes its right-hand neighbor. In scheme 1-1-1-1, every single step has this property, so the sequence can always be reconstructed.

In scheme 3-1, restarting with all four possible variants ensures that each homopolymer is part of a step that does not include other nucleotides. In principle, only three of the four variants are strictly required, since in that case the three bases will be added separately in some steps, which automatically distinguishes it from the fourth. Thus, scheme 3-1 generates redundant information not present in scheme 1-1-1-1, which can be used to improve base introduction in the face of experimental noise (eg, by dynamic programming, as shown above). It is thus not only more productive than 1-1-1-1, but also more error tolerant.

Scenario 2-2 also produced enough information to introduce the sequence over three restarts. It is readily seen that each pair of nucleotides is separable in at least one of AC-GT, AG-CT and AT-GC. Option 2-2 is thus probably the most concise fully productive option, although the extra information produced by 3-1 may be worth the effort. There is still some redundancy (if the nucleotides are labeled with different labels); thus, the error tolerance of scheme 2-2 is between 1-1-1-1 and 3-1.

Unconventional (acyclic) schemes may also be useful in special occasions. For example, when partial sequences are known, unconventional protocols can be used to get past unintended parts faster than would otherwise be possible, or they can be used to generate even more redundant data in order to further reduce base introductions error.

In conclusion, with respect to nucleotide addition schemes, we have investigated that 3-1 is the most productive and error tolerant, whereas, somewhat surprisingly, the traditional scheme 1-1-1-1 is the least productive And the most error-prone.

signature sequencing

Another embodiment of the aspect of the invention useful for signature sequencing includes a method (Scheme III) comprising:

1. Provide annealing primers for single-stranded templates.

2. Add three nucleotides, one of which carries a label such as a fluorescent label.

3. Optionally add one or more non-incorporated inhibitor nucleotides (other than the labeled nucleotides). Examples include 5'-bis- and mono-phosphate nucleotides, 5'-(α-β-methylene)triphosphate nucleotides.

4. Incubation with an appropriate polymerase under conditions that result in the addition of nucleotides to the growing strand.

5. Detecting the presence and amount of labeled nucleotides.

6. Inactivation of labeling, eg by photobleaching (not necessary in every cycle).

7. Add the remaining nucleotides and incubate with the polymerase (not necessarily the same as in step 5) under conditions that result in the addition of nucleotides to the growing strand.

8. Repeat steps 2-7 until desired number of cycles are completed.

For example, one could use fluorescent dC and conventional dA/dG in step 2, then add dT in step 7. Step 4 would then add any number of dA, dG, and dC until the first occurrence of dA in the template and then terminate because there is no complementary dT nucleotide. Fluorescence readings in step 5 will reveal the presence or absence of dC between each pair of dT. The resulting sequence can generally be written as a sequence of binary digits indicating, for each successive pair of Ts, whether there are one or more Cs between them.

For example, the sequence ACGCTACGCATCAGACTC would be written as 1111 and the sequence ACTCAGCTATATT as 11000. In general, such sequences contain information equivalent to 1/2 base pair per cycle. 24 cycles would be equivalent to a signature sequence of 12 bp, and would be unique within the human transcriptome, for example. Existing sequence databases and sequence alignment algorithms can be easily adapted to accommodate such binary signatures for analysis.

Option III is particularly easy to implement since only qualitative measurements are required. For example, Protocol III may be particularly suitable for sequencing single molecules using fluorescence correlation spectroscopy.

Chromatic sequencing using PPi detection

In another embodiment, an aspect of the present invention provides a method (Scheme IV) comprising (as opposed to using labeled nucleotides) monitoring the release of inorganic pyrophosphate (PPi) (see eg WO93/23564) . Such methods may include:

1. Provide annealing primers for single-stranded templates.

2. Adding a set of intervening nucleotides (ie more than one but less than all four possible nucleotides).

3. Optionally add one or more non-incorporating inhibitor nucleotides (other than the intervening nucleotides). Examples include 5'-bis- and mono-phosphate nucleotides, 5'-(α-β-methylene)triphosphate nucleotides

4. Incubation with an appropriate polymerase under conditions that result in the addition of nucleotides to the growing chain while monitoring incorporation (eg as described in WO93/23564).

5. Add the set of terminating nucleotides and incubate with a polymerase (not necessarily the same as in step 5) under conditions that result in the addition of nucleotides to the growing strand while monitoring incorporation (e.g. as in WO93/23564 as described in).

6. Repeat steps 2-5 until desired number of cycles are completed.

Again, the protocol can be repeated using the four natural nucleotides as stop nucleotides. Compared to standard pyrosequencing, this protocol provides a four-fold increase in read length without modifications to the standard protocol (other than changes in the sequence of nucleotide additions and changes in the required base introduction) .

The following examples show the importance of loss of synchrony and the impact of using a chroma sequencing protocol. It shows the results of target DNA sequenced by both pyrosequencing and chroma sequencing. A fixed fraction of all templates is assumed to lose synchrony at each incorporation step. In SBI, steps are the addition of individual bases. In the jump sequencing step, three or one bases are alternately added. Additionally, chroma sequencing was restarted three times with fresh primers, utilizing each of the four natural nucleotides as stop nucleotides.

Target sequence (for each stop nucleotide, the end nucleotide reached by Chroma sequencing is shown in capital letters):

atggagcagc gtcattccctt agcgggcaac tgtgacgatg gtgagaagtc

agaaagagag gctcaGGGat tcgagcatcg gacctgtAtg gactctgggg

atccTTcctt tgggCaaaat gatcccccta ccattttgcc cattactgct

Pyrosequencing

40 terminations for loss of synchronicity

40 reaction steps

reaction result a c g ta c g ta c g ta c g ta c g ta c g ta c g ta c g ta c g ta c g t a - - t2g - - -a - g -- c - -a - g -- c - -- - g t- c - -a - - 2t- 2c - 2t

Total sequence: 20bp

Chroma sequencing

40 terminations for loss of synchronicity

160 reaction steps (ie 40 for each stop base) reaction result cgt acgt acgt acgt acgt acgt acgt acgt acgt acgt acgt acgt acgt acgt acgt acgt acgt acgt acgt acgt a - at2g agc a2g2ct a4t2c a...etc...

...reboot by [gta c], [tac g] and [acg t] and repeat...

Total sequence: 88bp+27bp partial sequence

Altogether, chroma-sequencing circumvents the loss of synchrony issue and achieves more than four times longer read lengths.

solid-phase chromatic sequencing

To automate and parallelize the method, embodiments according to the present invention provide two main approaches.

The first approach uses arrayed or otherwise arranged templates and is suitable when a large number of templates must be sequenced while preserving their identity.

The second approach uses random attachment to a solid support and can be used when a large number of sequences must be randomly obtained from a library.

A method according to an embodiment of an aspect of the invention for sequencing array templates provides a method (Scheme V) comprising:

1. Provide a solid support that provides a number of active regions or active surfaces, each capable of binding template molecules, wherein the binding is

a. directly, or

b. Indirectly, by binding a primer or adapter that hybridizes to or otherwise has an affinity for the template.

2. Add single-stranded templates to each active region or to the active surface, keeping track of which template is placed at each position. Each region will then consist of a large number of identical ssDNA templates, as in a spotted microarray.

3. Optionally, add primers (or use adapters from solid supports).

4. Sequence all templates in parallel according to the invention, as according to any of schemes I-IV.

5. Obtain the sequence for each identified template.

The linker (step 1b) does not have to be the same in all active regions. Different adapters can be used to fish out specific templates from complex mixtures, offering the possibility to sequence subgroup libraries.

The throughput of Protocol V is limited by the resolution of the device used to add the template. Densities of thousands of templates per square centimeter are possible with standard microarray instrumentation.

Another route can be used when higher throughput is required and template density is not critical.

Another embodiment of an aspect of the invention is provided as a method (Scheme VI) comprising:

1. Provide a solid support carrying at least partially single-stranded template molecules attached at random positions (preferably at a density suitable for the detection instrument), optionally amplifying each template to accommodate multiple copies of Target sequences, which are either attached to or in close proximity to the original template (at least closer than any other template molecule).

2. Using the present invention to sequence templates in parallel, for example any one of schemes I-IV, to detect labeled nucleotides in parallel.

There are many ways to provide amplification templates in high density. For example, rolling circle amplification can be used as follows:

a. Provide primers attached to the surface (such as glass), preferably via covalent bonds, or as opposed to covalent bonds, very strong non-covalent bonds (such as biotin/streptavidin) can be used.

b. Adding the circular template, preferably at a density suitable for the detection instrument.

c. Annealing the template to the primer.

d. Amplification using rolling circle amplification to generate long single-stranded tandem repeat templates attached to the surface at each position.

Lizardi et al describe "Mutation detection and single-molecule counting using isothermal rolling circle amplification": Nature Genetics vol 19, p.225.

Modifications to this method include providing a reverse primer to generate additional replication forks, thereby increasing product yield. Alternatives to RCA include solid-phase PCR (Adessi et al. "Solid phaseDNA Amplification: characterization of primer attachment and amdlification mechanisms" Nucleic Acids Research 2000:28(20):87e) and in-gel PCR ('polonies' , US6485944 and MitraRD, Church GM, -In situ localized amplification and contact replication of many individual DNA molecules", Nucleic Acids Research 1999: 27(24): e 34).

A "suitable density" is preferably a density that maximizes throughput, eg, a limiting dilution that ensures detection of a single template molecule by as many detectors (or pixels in a detector) as possible. On any conventional array, a perfect limiting dilution would be such that 37% of all positions would hold a single template (due to the form of the Poisson distribution); the rest would hold none or more than one template.

For example, on a Typhoon 9200 with a 25 μm pixel size, a 35 x 43 cm chamber holds 240 million pixels. By limiting dilution (Poisson distribution), 37% of them will hold a single template, ie 89 million templates. Sequencing 50 bases on each template yielded 1.7 Gb of sequence in 50 cycles. The scanning time is 45 minutes, and the daily throughput is about 3Gbp, which is equivalent to the entire sequence of the human genome.

Templates suitable for solid-phase RCA should optimize yield (in terms of copy number of the template sequence) while providing sequences suitable for downstream applications. In general, small templates are preferred. In particular, the template may consist of 20-25 bp of the primer binding sequence and 40-150 bp of the insert. Primer binding sequences can be used both to initiate the RCA and to prime the sequencing reaction, or the template can contain separate sequencing primer binding sites. Inserts should be as small as possible while remaining long enough to accommodate the desired sequence. For example, if 10 cycles of sequencing are performed with a single stop nucleotide, an average of 40 bases will be probed, so the template must be at least sufficiently longer than 40 bases to prevent the sequencing primers from binding the sequence.

In order to increase the signal generated by rolling circle amplified templates it may be necessary to concentrate them. Since the RCA product is essentially a single-stranded DNA molecule consisting of as many as 1000 or even 10000 tandem copies of the original circular template, the molecule will be very long. For example, a 100 bp template amplified 1000 times with RCA will be on the order of 30 μm, so its signal will extend across several different pixels (assuming a pixel resolution of 5 μm). Utilizing a low resolution instrument may not be helpful as rare ssDNA products occupy only a very small fraction of the 30 μm pixel area and therefore may not be detectable. Thus, it is desirable to be able to concentrate the signal into a smaller area.

In (Lizardi et al., cited above), RCA products are concentrated by using epitope-tagged nucleotides and multivalent antibodies as crosslinkers. On the other hand, the present invention provides a simple alternative which is particularly convenient when sequencing raw double-stranded DNA.

Regarding the template preparation for the method according to the invention, and as a further aspect of the invention, the dsDNA template (which may be short, such as 80bp) is ligated to an adapter oligonucleotide carrying a hairpin loop to form a dsDNA template Double chain ring structure or dumbbell shape. In such a structure, primer binding sites for both RCA and subsequent sequencing reactions can be placed in the hairpin loop. To avoid sequencing both strands at the same time, by using different primers for RCA amplification and for sequencing, one can ensure that only templates with different hairpin loops at both ends will be sequenced. Thus, only templates with at least one RCA primer binding site will be amplified, and only those templates with at least one sequencing primer binding site will be sequenced.

Since the RCA product of such a template will be partially double stranded throughout, it will fold back into a zigzag structure, condensing into smaller regions. But since the primer binding sites are exposed everywhere as single-stranded DNA, primer access is not a problem. The examples below show that such templates form ~5-10 [mu]m products after RCA.

To immobilize oligonucleotides on surfaces, a number of different approaches have been described (see eg Lindroos et al. "Minisequencing on oligonucleotide arrays: comparison of immobilisation chemistries", Nucleic Acids Research 2001: 29(13)e69). For example, biotinylated oligonucleotides (oligos) can be attached to streptavidin-coated arrays; _NH2 -modified oligonucleotides can be covalently attached to epoxysilane derivatized or iso Thiocyanate-coated glass slides for coupling succinylated oligonucleotides to aminophenyl- or aminopropyl-derivatized glass via peptide bonds and thiol/disulfide The exchange reaction immobilizes disulfide-modified oligonucleotides on mercaptosilanised glass. Many more have been described in the literature.

Apparatus for automated high-throughput sequencing

The methods according to the invention are particularly suitable for automation, since they can be performed simply by circulating a number of reagent solutions, optionally with thermal control, through a reaction chamber placed on or in the detector.

In one example, the detector is a fluorescence scanner, which may be operated, for example, by laser excitation, bandpass filtering, and photomultiplier tube detection. For example, the ScanArrayExpress (PerkinElmer) is one such instrument; it scans microscope slides at a resolution of 5 μm/pixel, is capable of detecting as few as 2 fluorochromes per pixel, and has a scan time of ~20 minutes (in four colors). The maximum daily sequencing throughput on such an instrument will be 1.7Gbp.

The reaction chamber provides:

• Easy access to the scan head.

· Airtight reaction chamber.

• Inlets for injection and removal of reagents from the reaction chamber.

• An outlet to allow air and reagents to enter and exit the reaction chamber.

The reaction chamber can be constructed in the form of a standard microarray slide as shown in Figure 3, suitable for insertion into a standard microarray scanner such as ScanArray Express. Reaction chambers can be inserted into the scanner and remain there during the entire sequencing reaction. Pumps and reagent bottles (eg, as shown in Figure 4) supply reagents according to a fixed protocol, and a computer controls both the pumps and scanner, alternating between reacting and scanning. Optionally, the reaction chamber can be temperature controlled.

Dispenser units can be connected to motorized outlets to direct the flow of reagents, and the entire system operates under computer control. The integrated system will consist of scanners, dispensers, outlets and reservoirs, and a computer for control.

According to another aspect of the present invention, there is provided an apparatus for implementing the method of the present invention, the apparatus comprising:

Imaging elements capable of detecting incorporated or released labels,

a reaction chamber for holding one or more templates attached so that they have access to the imaging element at least once per set of steps,

Reagent distribution system for supplying reagents to reaction chambers.

The reaction chamber may provide, and the imaging element may be able to resolve, a density of at least 100/cm ² , optionally at least 1000/cm ² , at least 10 000/cm ² or at least 100 000/cm ² of attached template.

The imaging element can adopt a system or device selected from the group consisting of photomultiplier tubes, photodiodes, charge-coupled devices, CMOS imaging chips, near-field scanning microscopes, far-field confocal microscopes, and wide-field surface illumination (epi-illumination) microscopes and total internal reflection microscopy.

Imaging elements detect fluorescent labels.

An imaging element detects laser-induced fluorescence.

In one embodiment of the apparatus according to the invention, the reaction chamber is a closed structure comprising a transparent surface, a cover, and ports for attaching the reaction chamber to a reagent distribution system, wherein the transparent surface holds template molecules on its inner surface for imaging Elements can be imaged through transparent surfaces.

Example 1 - In Situ Template Amplification

通过退火4μl 100pmol/μl的两个5'-磷酸化的寡核苷酸(TGGTCATCAGCCTTCATGCAACCAAAGTATGAAATAACCAGCGTAATACGACTCACTATAGGGCGTGGTTATTTCATACT和TTGGTTGCATGAAGGCTGATGACCATCCTTTTCCTTACTAGCGTAATACGACTCACTATAGGGCGTAGTAAGGAAAAGGA)，并添加2μl T4连接缓冲液、0.3μl T4DNA连接酶(1.5 Weiss单位；Fermentas)和7μl水，并Circular single-stranded templates were prepared by incubating at 37°C for 1 hour. The ligase was then inactivated by incubation at 65°C for 10 minutes.

Primer A50T7RC

(AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCCCTATAGTGAGTCGTATTACGC) carrying the 5' terminal amino (-NH) moiety was prepared by incubating 10 μM primers in 100 μl MOPS (0.2 M with sodium acetate and EDTA according to Sambrook et al 'Molecular Cloning', third edition, Cold Spring Harbor Laboratory Press 2001) 5 min, while attached to a Greiner silylated microarray slide, reduce with 2.5 mg NaBH in 1 ml PBS/ethanol (3:1) for 5 min, _then rinse in 0.2% sodium dodecyl sulfate , followed by rinsing with distilled water.

Dried slides were then incubated for use with 2 μl of dUTP-Cy3 (100 μM final concentration, PerkinElmer), 2 μl each of dTTP, dATP, dCTP, and dGTP (all 1 mM final concentration, NEB), 4 μl Sequenase Buffer , 1 μl Sequenase (13u, Amersham Biosciences), 4 μl water and 1 μl template for rolling circle amplification. Labeled nucleotides are thus approximately 2.5% of all nucleotides. After two hours of incubation at 37°C, slides were rinsed in water and scanned on a PerkinElmer ScanArray Express. The result is a large number of bright spots, each representing the amplified template. The results also show that a labeling frequency of 2.5% can be easily detected in this format (in fact, many spots saturate the detector).

Magnification of part of the slide shows that most of the amplified template occupies one or a small number of pixels at the pixel size of the 5 μm image. At this size, a very large proportion of the pixels on the scanner can be used for different template molecules, ensuring maximum throughput. White pixels completely saturate the detector, indicating that less than 2.5% label is sufficiently detectable. Assuming a template size of 160 bp, 2.5% labeling represents approximately 4 incorporated nucleotides per template copy, within the range expected for a chroma sequencing reaction.

Example II-Single-step sequencing reaction

Biotinylated T7 primers (GCGTAATACGACTCACTATAGGGCG) were attached to Greiner streptavidin-coated microarray slides by incubation in Dynal binding/wash buffer (Dynal, Norway) at 10 pmol/μl. Holes were made on glass slides by sticking a rubber membrane containing an array of 5 mm wide holes. TOPO2.1 plasmid (Clontech) was boiled, cooled on ice, and added to each well at 20 fmol/μl. After incubation for 15 minutes at room temperature, the slides were washed in binding/wash buffer for 15 minutes.

Add 4 μl EcoPol buffer, 0.4 μl each of dATP, dTTP, and dGTP (100 μM final concentration, NEB), 0.4 μl dUTP-Cy3 (10 μM final concentration, PerkinElmer), 2 μl Klenow exosomes to two wells. DNA polymerase (NEB) and water were added to 40ul of the reaction mixture, and the same mixture was added to the other two wells with water instead of Klenow. After a 10 min incubation and two 15 min washes in binding/wash buffer, slides were scanned on the Typhoon 9200.

Given the template (Clontech TOPO2.1), the expected result is the incorporation of 2 dTTPs. Figure 2 shows the results, clearly showing the incorporation of labeled dTTP, and the signal obtained was significantly above background (as given by the fluorescence in the reaction omitting Klenow).

Claims (52)

1. determine the sequence of nucleic acid and/or the method for based composition information, described method comprises:

(i) provide the nucleic acid that comprises first chain, described first chain comprises nucleic acid-templated, wherein the free 3 ' end with the described first chain annealed nucleic acid chains allows to be complementary to nucleic acid-templated nucleic acid chains extension, this is by the dependent nucleic acid polymerase of template, by template sequence dependency ground Nucleotide is incorporated into to be complementary to and realizes in the nucleic acid-templated nucleic acid chains;

(ii) implement one or more steps of one group, number of cycles with expectation should be organized one or more steps, or implemented with one or more combination of steps of other groups, was complementary to nucleic acid-templated nucleic acid chains with extension, thereby allow to obtain the based composition of the described nucleic acid of expression or the information of sequence

One of them step comprises:

(a) exist when following:

The nucleic acid that comprises first chain, described first chain comprises nucleic acid-templated,

With the free 3 ' end of the described nucleic acid-templated first chain annealed nucleic acid chains and

Template dependency nucleic acid polymerase;

Provide be selected from a kind of, two kinds, the Nucleotide of three kinds or the four kinds complementary types of Nucleotide, with be used for by described nucleic acid polymerase with described oligonucleotide template dependency be incorporated into and be complementary to nucleic acid-templated nucleic acid chains, wherein each described Nucleotide is natural nucleotide or nucleotide analog, they can the free 3 ' end of nucleic acid chains by nucleic acid polymerase template dependency be incorporated in the nucleic acid chains, and in the complementary type of each Nucleotide, described Nucleotide and nucleotide analog and adenosine (A), cytosine(Cyt) (C), the complementation of one of thymus pyrimidine (T) and guanine (G);

With

(b) remove or the uncorporated Nucleotide of deactivation;

And

Wherein in one group of step

Provide the Nucleotide that is selected from the complementary types of all four kinds of Nucleotide, and it can be used for carrying out that template is dependent mixes,

In at least one step, provide and be selected from the Nucleotide that surpasses a kind of, optional two kinds, three kinds or the four kinds complementary types of Nucleotide, and it can be used for carrying out, and template is dependent mixes, and the Nucleotide in the complementary type of at least a Nucleotide, be complementary in the nucleic acid-templated nucleic acid chains if be incorporated into, then allow to be complementary to nucleic acid-templated nucleic acid chains is further extended and

Randomly in surpassing a step, do not provide Nucleotide complementary type; With

Wherein, if in a step, provide the Nucleotide that is selected from all four kinds of complementary types, Nucleotide in the complementary type of then a kind of, two or three Nucleotide, be complementary in the nucleic acid-templated nucleic acid chains if be incorporated into, then prevent to be complementary to nucleic acid-templated nucleic acid chains and further extend, if there is multiple copied with all copies that exist;

(iii) implement the described steps of many groups, the described step group that circulates and/or with the different described step groups of step group Joint Implementation;

(iv) determine to be incorporated into character and/or the amount that is complementary to the Nucleotide in the nucleic acid-templated nucleic acid chains at least one group of step, this is to realize by being incorporated into the character and/or the amount that are complementary to the Nucleotide in the nucleic acid-templated nucleic acid chains at least one step of determining each group, to described group of character and/or the amount that will determine the Nucleotide that mixed.

2. according to the method for claim 1, wherein in one group of step, the Nucleotide that is selected from three kinds or the two kinds complementary types of Nucleotide is provided in first step, and the Nucleotide of choosing from the complementary type of remaining one or both Nucleotide is provided in second step.

3. according to the method for claim 2, comprise the Nucleotide that mixed in first or second step of determining step group or the amount of multiple Nucleotide, to determine the character and/or the amount of the Nucleotide that mixed to described step group.

4. according to the method for claim 3, comprise the amount of the Nucleotide that is mixed in each step of determining group, to the described group of amount that will determine the Nucleotide that mixed.

5. according to the method for claim 4, wherein in one group of step, three kinds of Nucleotide are provided in first step, and a kind of Nucleotide is provided in second step.

6. according to the method for claim 5, comprise the property quality and quantity of the Nucleotide that is mixed in definite first step.

7. according to each method in the claim 2 to 6, wherein the Nucleotide that is provided in first step is differently carried out mark separately.

8. according to each method in the claim 2 to 7, wherein the Nucleotide that is provided in second step is labeled.

9. according to each method in the claim 1 to 8, four kinds of Nucleotide that wherein are complementary to A, C, T and G are differently carried out mark separately.

10. according to the method for claim 7, claim 8 or claim 9, wherein Nucleotide is by fluorescent mark.

11. according to the method for claim 7, claim 8, claim 9 or claim 10, wherein be incorporated into when being complementary in the nucleic acid-templated nucleic acid chains when Nucleotide, the mark of described Nucleotide lost efficacy.

12. according to the method for claim 7, claim 8, claim 9 or claim 10, wherein be incorporated into when being complementary in the nucleic acid-templated nucleic acid chains when Nucleotide, the mark of described Nucleotide is from described Nucleotide cutting or discharge.

13., comprise and determining from being incorporated into the character and/or the amount of the mark that is complementary to the one or more Nucleotide cuttings the nucleic acid-templated nucleic acid chains or discharges according to the method for claim 12.

14. according to each method in the claim 5 to 13, comprise and implement a round-robin step group, wherein in every group of step of this round-robin, three kinds of Nucleotide are provided in first step, and a kind of Nucleotide is provided in second step.

15. method according to claim 14, comprise described nucleic acid is implemented four round-robin step groups, wherein in each circulation, in steps in all second steps of group a kind of Nucleotide of being provided be identical, and a kind of Nucleotide that wherein institute is provided in all second steps of group in steps in each circulation be different from other three round-robin a kind of Nucleotide of being provided in all second steps of organizing in steps.

16. according to each method in the claim 1 to 15, wherein one group of step additionally comprises provides one or more sealing Nucleotide, its termination is mixed Nucleotide in being complementary to nucleic acid-templated nucleic acid chains.

17. according to each method in the claim 1 to 16, wherein one group of step additionally comprises provides the inhibitor of one or more non-mixing property Nucleotide, it suppresses in being complementary to nucleic acid-templated nucleic acid chains mistake and mixes Nucleotide.

18. according to each method in the claim 1 to 17, wherein nucleic acid-templated is thymus nucleic acid (DNA), nucleic acid polymerase is the dependent archaeal dna polymerase of DNA, and Nucleotide is deoxyribonucleotide or deoxyribonucleotide analogue.

19. according to each method in the claim 1 to 17, wherein nucleic acid-templated is thymus nucleic acid (DNA), nucleic acid polymerase is the dependent Yeast Nucleic Acid of DNA (RNA) polysaccharase, and Nucleotide is ribonucleotide or ribonucleoside acid-like substance.

20. according to each method in the claim 1 to 17, wherein nucleic acid-templated is Yeast Nucleic Acid (RNA), nucleic acid polymerase is a ThermoScript II, and Nucleotide is deoxyribonucleotide or deoxyribonucleotide analogue.

21., wherein nucleic acid-templatedly provide with multiple copied according to each method in the claim 1 to 20.

22., comprise by nucleic acid amplification reaction the nucleic acid-templated of multiple copied is provided according to the method for claim 21.

23. according to the method for claim 22, wherein nucleic acid amplification reaction comprises rolling circle amplification.

24. the method according to claim 23 comprises:

The dna molecular of being made up of the stem and first and second ring portions is provided, wherein said stem is made up of first chain and second chain, wherein said first chain and second chain length equate, complementation and annealing are together, and comprise the zone that needs its sequence and/or based composition information, wherein said first ring portion is connected in 3 ' end of described first chain at 5 ' end of described second chain, and described second ring portion is connected in 3 ' end of described second chain at 5 ' end of described first chain, thereby described dna molecular does not have free 5 ' or 3 ' end, and one of them ring portion comprises the primer binding site that is used for rolling circle amplification and a ring portion comprises the primer binding site that is used to check order;

Implement rolling circle amplification, with nucleic acid that multiple copied is provided as described nucleic acid-templated.

25. it is, wherein nucleic acid-templated attached on the solid support according to each method in the claim 1 to 24.

26. according to the method for claim 25, wherein a plurality of different nucleic acid-templated forms with array are attached on the solid support.

27. according to the method for claim 25 or claim 26, wherein nucleic acid-templated Jie by with attached to the primer annealing on the solid support attached on the solid support.

28., comprise by coming the definite kernel acid sequence to being incorporated into the analysis that the character that is complementary to the Nucleotide in the nucleic acid-templated nucleic acid chains and/or amount determine according to each method in the claim 1 to 27.

29. via the synthetic method for nucleic acid sequencing, be characterised in that in mode progressively and mix Nucleotide that one of them step is mixed with allowing the template dependency and surpassed a kind of different Nucleotide.

30. method according to claim 29, one of them step is mixed three kinds of different Nucleotide with allowing the template dependency, described Nucleotide is selected from the Nucleotide that is complementary to adenosine (A), cytosine(Cyt) (C), thymus pyrimidine (T) and guanine (G), and different steps mixing this organizes remaining Nucleotide with allowing the template dependency.

31. through programdesign with control according to the computer processor of each method in the claim 1 to 30.

32. carry the computer readable device that is used for according to the program of the computer processor of claim 31.

33. through programdesign with by implementing to provide the computer processor of the sequence and/or the based composition information of nucleic acid according to each method in the claim 1 to 30.

34. carry the computer readable device that is used for according to the program of the computer processor of claim 33.

35. be suitable for implementing the test kit according to each method in the claim 1 to 30, described test kit comprises the reagent that one or more groups is pre-mixed in one or more reagent containers, wherein the reagent that is pre-mixed of each group comprises

The Nucleotide of from all four kinds of complementary types, choosing,

The Nucleotide of choosing at least one container contains a kind of from surpassing, optional two kinds, three kinds or the four kinds of complementary types, and the Nucleotide in the complementary type of at least a Nucleotide is complementary in the nucleic acid-templated nucleic acid chains if be incorporated into, then allow to be complementary to nucleic acid-templated nucleic acid chains and further extend, and

Wherein, if the Nucleotide that is selected from all four kinds of complementary types is provided in single container, Nucleotide in then a kind of, the two or three complementary type is complementary in the nucleic acid-templated nucleic acid chains if be incorporated into, and then prevents to be complementary to nucleic acid-templated nucleic acid chains and further extends.

36. be used for implementing comprising according to each the instrument of method of claim 1 to 30:

Can detect the image-forming component of the mark that mixes or discharge,

The reaction chamber that is used for the one or more templates of adhering to of splendid attire, thus every group of step has at least once them can be near image-forming component,

Be used to reaction chamber that the reagent distribution system of reagent is provided.

37. according to the instrument of claim 36, wherein reaction chamber provides, and image-forming component can differentiate, density is at least 100/cm ², 1000/cm at least randomly ², at least 10 000/cm ²Or at least 100 000/cm ²The template of adhering to.

38. according to the instrument of claim 35 or claim 36, wherein image-forming component adopts system or the device that is selected from down group: photomultiplier, photorectifier, charge-coupled device, cmos imaging chip, near-field scan microscope, far field confocal microscope, wide visual field surface illumination microscope and total internal reflectance microscope.

39. according to the instrument of claim 35 or claim 36, wherein image-forming component detects fluorescent mark.

40. according to the instrument of claim 39, the fluorescence that brings out of image-forming component detection laser wherein.

41. according to each instrument in the claim 35 to 40, wherein reaction chamber is a closed structure, it comprises transparent surface, covers and is used to port that reaction chamber and reagent distribution system are adhered to, wherein the surperficial within it splendid attire template molecule of transparent surface forms pixel spare and can pass the transparent surface imaging.

42. the dna molecular of forming by the stem and first and second ring portions, wherein said stem is made up of first chain and second chain, wherein said first chain and second chain length equate, complementary and annealing is in the same place, wherein said first ring portion is connected in 3 ' end of described first chain at 5 ' end of described second chain, and described second ring portion is held the 5 ' end that is connected in described first chain with 3 ' of described second chain, thereby described dna molecular does not have free 5 ' or 3 ' end.

43. according to the dna molecular of claim 42, one of them ring portion comprises the primer binding site that is used for rolling circle amplification.

44. according to the dna molecular of claim 42 or claim 43, one of them ring portion comprises the primer binding site that is used to check order.

45. attached to the array on the solid support according to a plurality of different dna moleculars of claim 42, claim 43 or claim 44, randomly be situated between by with adhere to attached to the primer annealing on the solid support.

46. preparation is according to the method for the dna molecular of claim 42, claim 43 or claim 44, described method comprises:

Provide by first chain and have 5 ' end and double chain DNA molecule that 3 ' second chain of holding is formed with 5 ' end and 3 ' end; And

Connect first joint, be connected in 5 ' end of second chain with 3 ' end with first chain, and connect second joint, be connected in 5 ' of first chain with the end with second chain 3 ' and hold, wherein said joint is a hairpin structure.

47. produce the method for multiple copied dna profiling, described method comprises implements rolling circle amplification to the dna molecular according to claim 43 or claim 44, comprises the dna molecular of the extension of multiple copied dna profiling with production.

48. produce the method for a plurality of dna profilings of multiple copied, described method comprises implements rolling circle amplification to a plurality of dna moleculars according to claim 43 or claim 34, comprises the IDNA molecule of a plurality of extensions of multiple copied dna profiling with production.

49. according to the method for claim 47 or claim 48, wherein rolling circle amplification primer or dna molecular are attached on the solid support.

50., comprise that further the annealing between the complementary strand in the dna profiling of multiple copied in the dna molecular that passes through to be extended concentrates the dna molecular of described extension according to the method for claim 47 or claim 48.

51. according to the method for claim 50, wherein the dna molecular of Yan Shening is concentrated on the solid support.

52., comprise that further dna profiling or a plurality of dna profiling to multiple copied in the dna molecular that is extended checks order according to each method in the claim 47 to 51.

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