FR2778414A1 - PROCESS FOR SEQUENCING POLYMERS OF NUCLEIC ACIDS - Google Patents

Abstract

Sequencing of a selected region of a target nucleic acid polymer in a naturally occurring DNA sample can be accomplished in a single container by combining the sample with a sequencing mix containing a pair of primers , a heat stable polymerase such as Thermo Sequenase TM which incorporates dideoxynucleotides into an expanding nucleic acid polymer at a rate not less than about 0.4 times the rate of deoxynucleotide incorporation, nucleotide charges, and a chain terminating nucleotide. The reaction mixture also includes an unconventional nucleotide and an enzyme suitable for degrading nucleic acid polymers containing the unconventional nucleotide. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.

Description

PROCESS FOR THE SEQUEN AGE OF ACID POLYMERS

NUCLEIC

  TECHNOLOGICAL BACKGROUND OF THE INVENTION

  The present invention relates to DNA sequencing reactions, and in particular to improved sequencing reaction protocols using heat stable enzyme enzymes with

reduced error.

  DNA sequencing can be carried out in two distinct environments: a research environment in which each operation is quite specific and in which the sequence to be determined is generally not known before the determination of the sequences is completed, and an environment diagnostic procedure in which the same operating procedure is repeated on numerous samples and o the sequences to be determined are generally known. Although the basic procedures used in these two environments may be the same, the requirements for speed, cost-effectiveness and low risk of error in the diagnostic environment make many of the techniques used in reality too inconvenient for them to be used effectively. This limited the possibility of using sequencing-based diagnostics, and even raised questions about whether the

  sequencing could never be cost-effective

  efficacy for use in routine diagnosis.

  The ideal DNA sequencing technique for use in a diagnostic environment should have the following characteristics: (1) it should be able to use a sample containing DNA that has been subjected to minimal pretreatment only to render DNA accessible for sequencing, (2) it should require the combination of this sample with only a single reaction mixture, which would reduce the risk of error and contamination, and would make automation of the operative technique easier, and (3) it should require only a short time for the sequence determination to be made, thereby lowering marginal costs for equipment and

  the labor required to carry out the test.

  DNA sequencing, whether for research or diagnosis, is generally carried out using techniques based on the "chain termination" method described by Sanger et al. Proc.

  Nat '1 Acad. Sci. (USA) 74 (12): 5463-5467 (1977).

  Basically, in this process, the DNA to be tested is

  isolated, single-stranded, and placed in four containers.

  In each container are the components

  necessary for DNA strand replication, i.e.

  say a matrix-dependent DNA polymerase, a short primer molecule complementary to a known region of the DNA to be sequenced, and the standard deoxynucleotide triphosphates (dNTPs) commonly represented by the letters A, C, G and T, in a buffer promoting hybridization between the primer and the DNA to be sequenced and the chain extension of the hybridized primer. In addition, each container contains a small amount of a type (i.e. a species) of dideoxynucleotide triphosphate (ddNTP), for example dideoxyadenosine

triphosphate (ddA).

  In each container, the primer hybridizes to a specific site on the isolated DNA. The primers are then elongated, with a base each time to form a new nucleic acid polymer complementary to the isolated pieces of DNA. When a dideoxynucleotide triphosphate is incorporated into the expanded polymer, this ends the strand of polymer and prevents it from being extended further. Therefore, in each container, a set of extended polymers having specific lengths are formed, which indicate the positions of the nucleotide corresponding to the dideoxynucleotide in that container. These sets of polymers are then evaluated using gel electrophoresis to determine their sequence. As Church and Gilbert observed, "in a mammalian cell, DNA corresponding to any gene sequence is surrounded by DNA

  corresponding to several million other sequences. "

  "The Genomic Sequencing Technique" in Medical Genetics:

  Past, Present and Future, Alan R. Liss, Inc., p. 17-21.

  (1991). The same applies, to a greater or lesser extent, to any complex DNA sample, for example containing microbial genetic material, plant genetic material, complete cDNA libraries, etc. In the past, DNA sequencing techniques have taken care of this complexity by adding steps aimed at practically purifying the DNA of interest compared to other DNA species present in the sample. This purification was accomplished by cloning the DNA to be sequenced before its sequencing, or by amplifying a selected portion of the genetic material in a sample to enrich the concentration of a region of interest over other DNA. For example, it is possible to amplify a selected portion of a gene using a polymerase chain reaction (PCR, or "Polymerase Chain Reaction", which is abbreviated to PCR in English), as described

  in U.S. Patent Nos. 4,683,194, 4,683,195 and 4,683,202.

  This method involves the use of primer pairs, one for each strand of duplex or double strand DNA, which hybridizes to a site located near a region of interest in a gene. Chain extension polymerization (without chain terminating nucleotide) is then performed in repetitive cycles to increase the number of copies of the region of interest, many times. The amplified polynucleotides are then separated from the reaction mixture and used as a starting sample for the sequencing reaction. Gelfand et al. have described a thermostable enzyme, "Taq polymerase", derived from the organism Thermus aquaticus, which is useful in the amplification process. (see US patents N 4,889,818, 5,352,600 and 5,079,352). Taq polymerase has also been described as being useful in DNA sequencing when certain special conditions are met. See patent

US N 5,075,216.

  Improvements to the original technique described by Sanger et al. included enhancements to the enzyme used to extend the primer chain. For example, Tabor et al. have described enzymes such as T7 DNA polymerase which has improved processability and increased levels of dideoxynucleotide incorporation. (see US Patent No. 4,795,699 and EP-A-0 386 857). More recently, Reeve et al. have described a preparation of thermostable enzymes, called Thermo SequenaseT, having improved qualities for

  DNA sequencing. See Nature 376: 796-797 (1995) and EP-A-

  0 655 506. For sequencing, the Thermo SequenaseT product is used with an amplified DNA sample containing 0.5-2 gg of single-stranded DNA (or 0.5 to 5 gg of double-stranded DNA) in 4 aliquots , and the combination of each aliquot with the preparation of enzymes Thermo SequenaseT, a mixture of dideoxynucleotide terminations containing a ddNTP and the 4 dNTPs and a primer labeled with a dye which hybridizes to the DNA to be sequenced. The mixture is placed in a thermal cycle apparatus and subjected to 20-30 annealing, extension and denaturation cycles to produce measurable amounts of dye marked extension products having different lengths which are then

  evaluated by gel electrophoresis. Document EP-A-

  0 655 506 further indicates that Thermo SequenaseTM and similar enzymes can be used for

amplification reactions.

  Despite observations by those skilled in the art that enzymes useful for amplification can

  also be used for sequencing, and vice

  conversely, efforts to combine the amplification reaction and the sequencing reaction in a single step have been limited. Ruano and Kidd, Proc. Nat'l. Acad. Sci. (USA) 88: 2815-2819 (1991) and US Patent No. 5,427,911, describe a process they call "coupled amplification and sequencing" (CAS) for DNA sequencing. In this method, a sample is treated in a first reaction phase with two primers and amplified during a number of cycles.

  to achieve amplification by 10,000 to 100,000 times.

  DdNTP is then added during the exponential phase of the amplification reaction, and the reaction is carried out on additional thermal cycles to produce fragments of chain terminated sequence. The CAS method does not meet the criteria indicated above for an ideal diagnostic test, since it requires an intermediate addition of reagents (ddNTP reagents). This introduces a source of errors or contaminations and increases the complexity of the equipment, whatever it is, which would be used for automation. The problem of errors occurring during amplification has been approached in an approach envisaging the incorporation into the extension polymers of unusual nucleotides (for example dUTP) which are subjected

  to an enzymatic attack (for example with uracil-

  N-glycosylase) and degradation. See US Patent No. 5,418,149. Such molecules can be used in almost the same ways as those used for conventional amplification, but can be eliminated, as a contamination product of other reactions, by incorporating a pretreatment step using an appropriate enzyme to degrade the

  modified nucleic acid polymers.

  One of the objectives of the present invention is to provide a method for sequencing DNA samples of high complexity, which is perfectly suited for use in the diagnostic environment and for automation and which provides a means for minimizing errors caused by contamination and the

  transfer of nucleic acid polymers.

  Another object of the invention is to provide a method for DNA sequencing which uses a sample containing DNA, which has been subjected only to minimal pretreatment to make the DNA accessible for sequencing, and which provides a way to minimize errors caused by contamination

  and transfer of nucleic acid polymers.

  Yet another object of the invention is to provide a method for DNA sequencing which requires combining a complex sample containing DNA with only a single reaction mixture, thereby reducing the risk of errors and contamination and increasing the ease with which the procedure can be

automated.

SUMMARY OF THE INVENTION

  The present invention provides a method for sequencing a region of interest in a DNA sample, in which a unique set of reagents is added to a minimally processed sample.

  to produce useful sequencing results.

  The invention is based on the observation and the surprising discovery that the addition of a reaction mixture containing the thermostable polymerase Thermo SequenaseM, two primers which attach to complementary strands of a target DNA molecule on sites framing the

  region of interest, a mixture of nucleotide triphospha-

  tes (A C, G and T) and a dideoxynucleotide triphosphate at

  a DNA sample that contains target DNA and not

  practically natural relative abundance target, including highly complex DNA samples such as human genomic DNA, and treatment of the combination with multiple cycles of annealing, extension and denaturation leads to the production of 'a mixture which can be loaded directly onto a gel for sequence analysis of the region of interest. The reaction mixture also includes an unconventional nucleotide and an enzyme suitable for the degradation of nucleic acid polymers containing the nucleotide.

unconventional.

  One aspect of the present invention is a method for sequencing a selected region of a target nucleic acid polymer comprising the steps of (a) combining a naturally abundant sample containing the target nucleic acid polymer with a reaction mixture comprising three types of

  deoxynucleotide triphosphates, a nucleotide triphospha-

  unconventional te corresponding to the fourth basic type, a dideoxynucleotide triphosphate, a first and a second primer, an enzyme which degrades nucleic acid polymers including the unconventional nucleotide, and a heat stable polymerase enzyme which includes dideoxynucleotides in a polymer of nucleic acids in extension at a rate or rate which is not less than about 0.4 times the rate or rate of incorporation of the dideoxynucleotides to form a reaction mixture, the so-called first and second primers attaching to the sense and antisense strands, respectively, of the target nucleic acid polymer at points framing the selected region, (b) exposing the reaction mixture to an initial phase in which the enzyme which degrades the acid polymers nucleic acid comprising an unconventional nucleotide is active for a sufficient time to degrade the acid polymers es containing the non-conventional nucleic acid which may be present in the sample, (c) exposing the reaction mixture to a plurality of temperature cycles each of which comprises at least one denaturation phase at elevated temperature and one phase d extending to a lower temperature to produce a mixture of products comprising sequencing fragments which are terminated by incorporating the dideoxynucleotide, and (d) evaluating the mixture of products to determine the lengths of the sequencing fragments produced.

BRIEF DESCRIPTION OF THE DRAWINGS

  Fig. 1 illustrates the process according to the invention in schematic form; Figs. 2A and 2B show a comparison of sequencing operations carried out with Thermo SequenaseT as a polymerase in the method according to the invention with the results obtained with the use of other thermostable polymerases in a comparative experiment; Fig. 3 shows the layout of FIG. 2A with more details;

  Fig. 4 illustrates a multi-mode implementation

  dyes of the invention; Fig. 5 illustrates a second embodiment of multi-dyes of the invention; and Figs. 6A and 6B illustrate a third bet mode

  using multi-dyes of the invention.

  DETAILED DESCRIPTION OF THE INVENTION

  The present invention meets the need for a simple and easily automated sequencing technique which can be used directly on samples which contain complex mixtures of DNA. To distinguish such mixtures from DNA preparations which have been sequenced in the past, one uses in the

  description and claims of this application

  the expression "natural abundance sample" to describe such a mixture. As used herein, the term "naturally abundant sample" means a sample which has been processed to make the sample DNA accessible for hybridization with oligonucleotide primers, for example by lysis, centrifugation for remove cellular debris and proteolytic digestion to expose the DNA, but which has not been subjected to a preferential purification or amplification step to increase the quantity of target DNA compared to the non-target DNA present in the initial sample. The expression "in natural abundance" does not, however, require the presence of all of the DNA from the original sample. Thus, a complex sample containing just nuclear DNA, or just mitochondrial DNA or a certain sub-fraction of nuclear or mitochondrial DNA obtained by isolation from a tissue sample, but not subjected to amplification preferential, would be a sample "in natural abundance" within the meaning of the expression concerned

  in the description and claims of this

  request. The expression "in abundance naturally" also covers a DNA sample prepared by conversion, for example by reverse transcription, of a preparation of total mRNA or of the genome of an RNA virus into cDNA, of isolated DNA. from an individual bacterial colony growing on a plate or from an enriched bacterial culture, and a preparation of viral DNA o practically the entire viral genome is isolated. The term "abundantly natural" does not encompass a sample in which the isolated DNA is not a complex combination of DNA molecules, and thus does not include, for example, a purified plasmid preparation containing only a single species of plasmid. Samples with a natural abundance of mammalian DNA can be prepared from fluid samples, for example blood, urine or tissue samples, by any of the many techniques, including, lysis, centrifugation to remove cell debris and proteolytic digestion to expose DNA, salt precipitation or standard extraction of K-phenol by SDS-proteinase. Samples with a natural abundance can also be prepared using kits or sets, for example the DNA isolation kit called

Gentra Pure Gene DNA Isolation Kit.

  The process according to the invention uses the properties of enzymes such as Thermo SequenaseT, namely the ability to incorporate dideoxynucleotides into a polynucleotide in extension at a speed which is not less than about 0.4 times the speed of incorporation of deoxynucleotides, to provide a method for sequencing a nucleic acid polymer from a sample having a natural abundance in a single set of thermocycling reactions which can be carried out in a single container. Thus, the method according to the invention is ideally suited for

be automated.

  Fig. 1 illustrates the fundamental simplicity and the elegance of the process according to the invention in the form of a

  logic diagram. As shown in fig. 1, an exchange

  tillon containing a target nucleic acid polymer, which comprises a region to be sequenced, is combined with a reaction mixture containing two primers, a mixture of dNTP, a chain-terminating nucleoside triphosphate, i.e. a dideoxynucleotide triphosphate , and a thermostable polymerase having a high affinity for incorporating ddNTP into a buffer suitable for hybridization and matrix-dependent polymerization. The mixing is carried out during a sufficient number of thermal cycles to produce detectable quantities of sequencing fragments, generally from 20 to 50 cycles. During each cycle, the primers each give rise to a respective strand annealing of the target DNA present in the sample, and the extension of the primer chain using the polymerase enzymes and the nucleotide charges triphosphates takes place up to which it ends with the incorporation of a chain termination nucleotide triphosphate. This leads to the production of sequencing fragments comparable to those formed in a conventional sequencing reaction. Analysis of these fragments provides information regarding the sequence of the selected region of the target DNA. These extension products which are not completed before reaching the complementary region of the other primer can serve as a template for the production of sequencing fragments in subsequent cycles, although this generally occurs to a very small extent. Finally, the mixture of products containing dideoxy-terminated fragments is loaded onto an electrophoresis gel for the analysis of the positions of the base corresponding to the nucleotide triphosphates of chain termination in the

  target nucleic acid polymer.

  The implementation of the invention can be understood in the context of a DNA fragment of 200 nt

  hypothetical having equal amounts of each base.

  This means that there are 50 potential truncation circumstances during the cycle. For each cycle, part of the products is of total length (and therefore capable of hybridizing with one of the two primers to produce more sequencing fragments) and part is truncated at the points where the ddNTP has been added. If each of the truncation circumstances has a statistical probability of occurring 1 in 500 as a result of the relative concentration of ddNTP compared with dNTP and the relative incorporation by the enzyme, then a total product of truncation is formed. in just under 10% of the reactions. Table 1 shows the relative amounts of products of total length and chain termination theoretically formed after 10, 20 and 30 cycles of a reaction according to the invention using this 200 nt polynucleotide, assuming various ratios

  between the truncated product and the total length product.

- __ TABLE 1

  truncation ratio report ratio = 0.1 truncation = 0.3 truncation = 0.5 cycles _ truncated in length truncated in length truncated in total total length

32 613 86 202 57 57

  41,000 376,000 17,400 40,462 3,300 3,300

  25.6x106 230x106 3.5x106 8.2x106 190,000 190,000 The absolute and relative amounts of nucleotide triphosphates and nucleotide triphosphates of chain termination can be optimized for the particular enzyme employed. In actual practice, it has been found that useful results are obtained with Thermo SequenaseTM when the reaction is carried out for 35 to 45 cycles, using a molar ratio of dideoxy: deoxy from 1: 100 to 1: 300. In general, each nucleotide triphosphate is included in the reaction mixture at concentrations of 250 gM to 1.5 mM and the chain terminating nucleotide triphosphate is included in a content of 0.5 gM to 30 gM to produce compositions in which the molar ratio of the chain terminating nucleotide triphosphate to the corresponding nucleotide triphosphate is from 1:50 to 1: 1000, preferably from 1: 100 to 1: 500. This allows the incorporation of a chain terminating nucleotide triphosphate in a proportion of 30 to almost 100% of the polymer chains in extension formed during the treatment of the reaction mixture in

the thermal cycle.

  A key factor for the process according to the invention to proceed successfully is the use of Thermo SequenaseTM or a comparable enzyme as a thermostable polymerase in the reaction mixture. Such enzymes are characterized by a high affinity for the incorporation of dideoxynucleotides into the extending nucleotide chain. In general, for the purposes of the present invention, the polymerase used should be one of those which incorporate dideoxynucleotides into a polymer of nucleic acids in extension at a rate which is not less than about 0.4 times the rate of incorporation of deoxynucleotides. Thermo SequenaseTm is known to promote the incorporation of dideoxynucleotides, and it is suitable for use according to the invention. Tabor et al. also described enzymes with increased ability to be used, and high and increased levels of incorporation of dideoxynucleotides. (see EP 0 655 506). The Roche company markets a polymerase under the name TAQ-FS

  which also meets these criteria.

  Figs. 2A and 2B and fig. 3 illustrate the importance of this characteristic of the polymerase enzyme used. Figs. 2A and 3 represent a sequencing data plot for a sample of real heterozygous patient with DNA in natural abundance, which was obtained with the use of Thermo SequenaseTM and of primers framing exon 2 of the Von Hippel-Lindau gene in a method according to the invention. Significant, well-defined peaks corresponding to the termination fragments were obtained, allowing very direct sample sequence evaluation. In addition, the peaks for homozygous peaks are all about the same size, and can easily be distinguished from base peaks on heterozygous locations. This was achieved by performing the test in a single reactor, with a single un-increased reaction mixture, in a total of 45 thermal cycles. Comparable results can be obtained if fewer reaction cycles are used, for example 35 cycles, as indicated in

Example 1 below.

  On the contrary, fig. 2B represents the plot obtained when a combination of Vent and SequithermTM was used in place of the Thermo SequenaseTM over a total of 45 thermal cycles. In this plot, the peaks for the termination fragments are much smaller and less well defined. In addition, the peaks are very variable in height and did not allow the identification of heterozygous peaks based on the height of the peaks. Performing the same experiment with Taq polymerase alone led to a plotting of the data.

containing no usable peaks.

  In the method according to the invention, a natural abundance sample containing, or supposed to contain, a target DNA sequence is combined in a reaction mixture with an appropriate polymerase, the four

  types of deoxynucleotide triphosphates, a dideoxy-

  nucleotide triphosphate and first and second primers. The primers used in the method according to the present invention may consist of any pair of primers which hybridize with the sense and antisense strands of the target DNA flanking a selected region which is to be sequenced, and which do not hybridize all the two at neighboring points in human DNA or any other DNA that is potentially in the sample. As used herein, the term "box" should be understood to mean the positioning of the primers on the 5 'ends of the selected region of each DNA strand, so that extension of the primers leads to a replication of the region between the primers. Primers are preferably chosen such that the primer pair frames a region of about 500 bp or less, although primers enclosing larger DNA regions can be used with adjustments to the sequencing mix (typically , an increase in the relative amount of deoxynucleotide triphosphates) to increase the amount of longer sequencing fragments produced. The primers can be chosen to hybridize with highly conserved regions which are the same in all variants of the target DNA or can be prepared as degenerate primers to take into account

  known sequence variations at the primer site.

  Thus, the first and second primers according to the invention may each be a particular oligonucleotide species or may consist of a set of oligonucleotide primers having sequences

similar, but not identical.

  One or both of the primers can be labeled with a detectable label on its 5 'end, in particular a fluorescent label such as fluorescein or a cyanine dye such as Cy 5.5. If markers are used in the two primers, the markers chosen must be spectroscopically distinct, that is to say they must have either a different excitation spectrum or a different emission spectrum, so that one can distinguish one of the primers from the other. When the two primers are labeled with different detectable markers, for example with two different fluorophores, as is the case in the method described by Wiemann et al., "Simultaneous On-Line DNA Sequencing on Both Strands with Two Fluorescent Dyes" , Anal. Biochem 224: 117-121 (1995), the sequence of the two strands of the sample can

  be determined in a single reaction.

  The nucleotide triphosphate charge mixture is a standard mixture of three of the four conventional deoxynucleotide bases (A, C, G and T), plus an unconventional nucleotide corresponding to the fourth base in a buffer suitable for primer extension dependent on the matrix with the enzyme used. As will be readily understood by those skilled in the art, the specific concentrations of nucleotide triphosphates and the nature of the buffer vary depending on the enzyme used. Standard reagents and concentrations of reagents can be used according to the invention

  for various known polymerase enzymes.

  The term "unconventional nucleotides" refers to non-natural or analogous type nucleotide triphosphates which can be polymerized in a matrix dependent manner to give the sequencing fragments. Unnatural forms of the modified nucleotides are in particular alkylated nucleotides and nucleotides modified by alkylhydroxylation. Specific examples of modified nucleotides include, but are not limited to, N-7 methylguanine,

  deoxyuridine, deoxyinosine, deoxy-5,6-

  dihydroxythymine (from DNA treated with 0sO4), 5 ', 6'-

  dihydroxydihydrothymine and deoxy-3'-methyladenosine.

  Unconventional nucleotides may also include natural forms of the nucleotides which do not

  are not conventionally incorporated into DNA.

  Thus, for the purposes of the present invention, uracil and

  hypoxanthine are unconventional nucleotides.

  An important characteristic of the non-conventional nucleotides used according to the invention is the existence of an enzyme which degrades the polynucleotides containing the non-conventional nucleotides to make them unsuitable, as substrates, for the further formation of sequencing fragments at temperatures lower than those normally associated with the production of sequencing fragments using a thermostable polymerase. A reaction mixture according to the invention comprises an appropriate enzyme, usually a glycosylase. Examples of specific enzymes are in particular: uracil-Nglycosylase, hypoxanthine-DNA glycosylase, 3-methyladenine-DNA glycosylase I and II,

  hydroxymethyl uracil-DNA glycosylase and foramido-

  pyrimidine DNA glycosylases. The enzyme is preferably a thermolabile enzyme, so that it is active for the destruction of transport polynucleotides before

  the first sequencing reaction, but inactive after.

  In the selection of the reaction conditions, care must be taken that the temperature is not lowered after the synthesis of the sequencing fragments at a temperature which allows significant degradation of the products.

desired before analysis.

  The reaction mixture used according to the present invention also comprises at least one type (or species) of chain terminating nucleotide triphosphates. Separate reactions for the four different types of bases can be carried out either at the same time or successively. The simultaneous implementation of the four bases is in accordance with conventional practice for sequencing. However, a preferred embodiment of the present invention combines the single container methodology according to the present invention with the "single track sequencing" which is described in US patent application N 08 / 577,858 in the name of the same assignee. . In single-track sequencing, the determination of the positions of only one (or in any case less than four) of the nucleotides of a target sequence is frequently sufficient for establishing the presence or determining the qualitative nature of a target microorganism by providing a fingerprint, or barcode, of the target sequence which may be sufficient to distinguish it from all other varieties of known sequences. The passage is increased by reducing the number of reactions and electrophoresis operations required to identify a sequence. By selecting the order of the bases tested and the intermediate analysis, it may not be necessary to process all four bases to determine the

  presence and specific qualitative nature of a micro-

  any target organism present in the sample.

  The present method can be used in combination with any type of detection system which is compatible with the label used on the primers. For example, an A.L.F. from Pharmacia can be used when using primers labeled with fluorescein, while a Visible Genetics MicroGene

  Blaster is suitable when the marker used is Cy5.5.

  When using multiple markers, the sample can be processed in multiple instruments, or it can be evaluated on an instrument which is capable of detecting signals from multiple markers. An example of such an instrument is the Prism 377 Sequencer (Applied Biosystems Inc.) which detects and distinguishes between four dyes in a single track. Dyes which can be distinguished by spectroscopy which are recognized in the Prism 377 sequencer are the dyes FAM, ROX, TAMRA and

JOE, known to those skilled in the art.

  The possibility of multi-dye detection leads to a wide range of applications for the invention, making it possible to improve the sequencing precision and to improve the performance of the instruments. For example, fig. 4 illustrates a process for obtaining sequences both before ("forward") and reverse ("reverse") of a target nucleic acid sequence using two primers, each with a marker that can be distinguished by spectroscopy. The naturally abundant sample is mixed with forward and reverse primers, each with a distinguishable marker (1 and 2). The reaction is carried out with four termination reactions, one for each of A, C, G and T. Each product reacted is placed in a single well of an automated sequencing instrument which detects and distinguishes at least the two markers used. Detection results from

  of marker 1 are combined to give the front sequence.

  The results of detection from marker 2 are combined for the reverse sequence. The two sequences can be used to be reciprocally controlled and to correct any ambiguities in the designation of the bases. In addition, the opposite sequence can be used to confirm the proximal sequence against a primer, if empirically found to be difficult to determine, on DNA sequencers

  machines available commercially.

  Fig. 5 illustrates the use of multiple markers of a different format. In this case, assuming that the instrument can distinguish between 4 markers, 4 genes or gene fragments in a single sample can be sequenced simultaneously. Four pairs of primers are added to the patient's genomic DNA sample. Each pair is specific for a different target, possibly 4 exons of the same gene of interest (P. Q, R and S). One member of each pair is conjugated to a detectable marker (1, 2, 3 or 4) and each marker can be distinguished from the others. This mixture is subdivided into 4 tubes for termination reaction, respectively for ddA, ddC, ddG and ddT, and heat treated. The products of the termination reaction are loaded into a lane and the method described in US patent application N 08 / 634,284 is then used.

  assigned to the same assignee as the present invention.

  The results of each marker are combined to give the sequence of the exon for which this primer is

specific.

  Another embodiment takes advantage of the fact that a single ddA termination reaction identifies the nucleotides A of each strand, FIG. 6A thus identifying the complementary base (in this case T) in the opposite strand. (i.e., the A termination sites on the opposite strand correspond to the T nucleotide sites in the first strand). The complementary base can be located in the "missing" sites of the opposite strand. Note that the opposite strand sequence must be reversed before it can be added to the missing sites, since it begins at the opposite end of the target gene and the chain extension

  runs in the opposite direction.

  In fig. 6B, a second termination reaction is added for ddC. This allows the identification of C and its complement G in each strand. When these results are added to the first reaction, a complete DNA sequence is obtained. Thus, on the basis of 2 termination reactions each employing a ddNTP chain terminator, the complete 4-band sequence can be obtained. The designation by their bases and the compilation of the sequences illustrated in FIGS. 6A and 6B can be facilitated by the use of GeneObjects software (Visible Genetics Inc., Toronto) and the use of techniques described in US patent applications. Our

08 / 497.202 and 08 / 670.534.

  The method according to the present invention is advantageously applied in numerous contexts, in particular: (1) the detection of mutations having a medical importance, in samples coming from a human patient, from an animal, from a plant or a microorganism, (2) determining the HLA type using transplant techniques, (3) detecting and identifying microorganisms, in particular pathogenic microorganisms, in a sample , and (4) in situ sequencing reactions to produce sequencing fragments in a histological specimen, which are then removed from a selected location on tissue preparation and loaded into a gel for sequence analysis. This latter approach is particularly useful for the evaluation of samples archived in retrospective studies, when the evolution of a disease state is known, but the mutation which is potentially the cause is not. This method can be used with labeled primers for single base sequencing (or multiple base sequencing using

multiple tissue samples).

  The basic process according to the invention can also be reinforced by various modifications without departing from the scope of the present invention. For example, improvements in reproducibility and sensitivity can be achieved by using a combination of an enzyme having a high affinity for incorporating dideoxynucleotide triphosphates into the extending polymer, for example a Thermo SequenaseTM, and an enzyme having a low affinity for the incorporation of dideoxynucleotide triphosphates in the polymer in extension, for example of Taq polymerase, under conditions in which the 2 enzymes actively catalyze the polymerization giving the primer extension dependent on the matrix. As noted above, the high affinity enzyme produces almost entirely termination products, with a very small amount of the polymers which are effectively extended to full length. Furthermore, the low affinity enzyme almost exclusively produces a full-length product, with relatively few termination products. The low affinity addition to the reaction mixture increases the sensitivity of the process by producing more total length material to be sequenced, without increasing the duration of the treatment or without adding processing steps. The increase in sensitivity can be controlled by varying the ratio between the high affinity and low affinity enzymes present

in the mixture.

  It should however be noted that the use of a low affinity enzyme to form a product of total length also results in the formation of a peak of products of marked total length, very intense. This peak can make base analysis difficult near the end of the sequence. In order to maintain the benefit of increased sensitivity, while having fewer full length products, it may be desirable to use a low affinity enzyme which is more thermolabile than Taq polymerase, so that the enzyme low affinity is practically inactivated towards the end of the first 15 to 25 cycles. This could allow the production of longer fragments from the start of the test and the production of more fragments

completed later in the test.

  The reaction mixture according to the invention may also comprise other additives which enhance the formation of the sequencing fragments. For example, a product called TaqStartTM antibody is a monoclonal antibody that binds to and blocks the activities of Taq polymerase. This antibody is added to PCR reactions using Taq polymerase to block enzyme activity, during start-up at room temperature to prevent or reduce the formation of non-specific amplification products. TaqStartT antibody can be used according to the present invention with Thermo SequenaseTM to reduce extension reactions

non-specific primer.

  The method according to the invention can also be used in conjunction with the Johnson & Johnson techniques known under the name "PCR IN A POUCH",

  described in US Patent No. 5,460,780.

  Although the preferred method for sequencing nucleic acid polymers according to the invention involves the use of a single step technique as described above, the invention also encompasses other uses of unconventional nucleotides during the preparation of sequencing fragments to provide a mechanism for reducing the risk of contamination by transport, in laboratories carrying out sequencing reactions by adding a preliminary degradation step before each sequencing operating technique. Thus, in the broadest sense, the invention relates to a method for sequencing nucleic acid polymers in a sample, in which the sample is combined with a reaction mixture containing an unconventional nucleotide, 3 conventional nucleotide bases, at the at least one type of a chain terminating nucleotide triphosphate, an enzyme polymerase, and an enzyme for the degradation of polynucleotides containing the unconventional base. The sequencing reaction can be carried out as a single step on naturally abundant DNA, as described above, or can be carried out on a previously amplified sample (preferably, using an unconventional nucleotide). The sequencing fragments can be obtained by any known method, including sequencing with cycles, the CAS method, bidirectional sequencing or conventional sequencing on an extension. The invention is now described in more detail in

  reference to the nonlimiting examples which follow.

EXAMPLE 1

  DNA amplification by PCR with dUTP and uracil-

  n-glycosylase PCR master mix HK-UNG dilution buffer 10 1l HK-UNG 1 dl OX ACP buffer II 55 gl MgC12 25 mM 66 1l dNTP mixture 66 Bl PCR primers 13 1l Sterile water 284 1 DNA AmpliTaq polymerase at 5 U /, i 2.8, 1 with Tarrpon for diluting HK-UNG in Tris-HC1 50 nM (pH 7.5), NaCl 100

  rrM, 0.1 mM EDTA, 1 nM DIT and 0.1% Triton X-100.

  HK-UN is thermacabile uracil-N-glycosylase, Epicenter Technologies Tanpon II of lOX ACP: Tris-HCl 100 rM pH 8.3 at 25 C; KCl 500 rrM Mixture of dNTP: dATP 2.5 rrM, dCTP 2.5 rarM, dGTP 2.5 rM and dUTP 2.5 mRI PCR primers: 2.0 DM of primer CT1590, 2.0 t of primer KLl- Cy5.5 Arrester KL1-Cy5.55'-TCC GGA GCG AGT TAC GAA GA-3 ', labeled with Cy5.5 Primer CT15905'-ATG CCC GGG ATT GGT TGA TC-3', unlabeled 45 p1 of PCR master mix were placed in a thin-walled PCR tube pl of a reccrbinant plasmid (pUC8 containing the hidden plasmid of Chlarrydia trachcaatis, serovar Li) at 400 copies / p

  The tube was closed and placed in a thermal cycler.

  The PCR was started under the following conditions: 37 C 15 min C 15 min 94 C 5 min 37 following cycles follow 94 C 30 s 61 C 30 s 72 C 45 s then 72 C 5 min hold at 4 C Sequencing of 1 arplicon containing dUTP by CLIP with dUTP CLIP Tanpon masterbatch sequencing 22 pl Primers of CLIP 12 il Thermo Sequenase at 32 U / gl 2, 2 pl Sterile water 84.8 pi with Tanpon sequencing Tris-HCl 260 rrM pH 9.1 to 25 C; MgC12 39 nM in ddH2O CLIP arms: 2.0 W primer Cr1431F-Cy5.5, 2.0 M primer

CT1538R

  Primer CT11431F-Cy5.55 '-GTG CAT AAA C = T CIG A3GG AT-3', labeled with Cy5.5 Primer CT1538R5'-CTA AAC GC CCT CIC AAG TC-3 ', unlabeled 11.l of the masterbatch of CLIP were added to a tube; 2 μl of the amplicon containing dUTP, created by the abovementioned PCR reaction, were added to the 11 l1 of master mix of CLIP, to give the master sample mixture; 20.l of sterile mineral oil were added to four 200-gl thin wall PCR tubes 3.l of the master sample mixture were added to each of the four tubes containing mineral oil 3gl of sequencing mix A (750 pM dATP, 750 gM dCTP, 750 pM dGTP, 750 pM dUTP, 2.5 WM ddATP) were added to the first of four 3 g tubes of C sequencing mixture (750 gM dATP , 750 μM dCTP, 750 μM dGTP, 750 μM dUTP, 2.5 μM ddCTP) were added to the second of the four 3 Al tubes of G sequencing mixture (750 μM dATP, 750 μM dCTP, 750 LM of dGTP, 750 µM of dUTP, 2.5 µM of ddGTP) was added to the third of the four 3 µl tubes of T sequencing mixture (750 IM of dATP, 750 µM of dCTP, 750 µM of dGTP, 750 pM dUTP, 2.5 gM ddTTP) were added to the fourth of the four tubes. The tubes were closed and placed in a thermal cycler. The sequencing reactions were started under the following conditions: 94 C 5 min 37 cycles as follows 94 C 10 s C 15 s C 45 s then C 5 min holding at 4 C The tubes were removed from the thermal cycler, and 6 p1 charge dye was added to each tube (A, C, G and T). 2 Bl of each of the tubes containing the charge dye were migrated on a MICROGENE BLASTER sequencer at 50 ° C. in 20 minutes at a voltage of 1300 volts. After the operation, the basics were determined.

EXAMPLE 2

  Sequencing of an HIV amplicon containing dTTP by CLIP with dUTP Master mix of CLIP Sequencing buffer 18.5 gl HK-UNG 1.0 pl Amplicon containing dTTP 5.0 1l AmpliTAQ FS at 15 U / pl 4.0 pl sterile water 69.5 pl with sequencing Tanpon: Tris-HCl 260 rM pH 8.3 at 25 C; MgC12 32.5 rM in ddH2O Anplicon containing dTTP: a 1.3 kb amplicon at a concentration of 100 ng / pl, containing the HIV-1 protease sequence HK-UNS: uracil-N-glycosylase therfolabile, Epicenter 7 p1 A termination mix technologies (640 nM dATP, 640 rM dCTP, 640 mM dGTP, 640 mM dUTP, 1 gM ddATP, 163 mM primer PR170F-Cy5.5, 163 mM d PR170F-A-Cy5.5 primer, 131 nM PR543R-Cy5.0 primer, 200 nM PR543R assay) were added to a tube

200 µl thin-walled PCR.

  7 p1 of C termination mixture (640 WM of dATP, 640 rM of dCTP, 640 mM of dGTP, 640 rm of dUTP, 2 pM of ddCTP, 163 rrM of primer PR170F-Cy5.5, 163 rM of primer PR170F-A-Cy5.5, 131 nM PR543R-Cy5.0 primer, 200 nM PR543R primer) were added to a tube

200 µl thin-walled PCR.

  7 p1 G termination mix (640 WM dATP, 640 WE dCTP, 640 mM dGTP, 640 rrM dUTP, 21 M ddGTP, 163 nrM primer PR170F-Cy5.5, 163 rM primer PR170F-A-Cy5.5, 131 nM PR543R-Cy5.0 primer, 200 nM PR543R primer) were added to a tube

200 µl thin-walled PCR.

  7 p1 of T termination mixture (640 WM of dATP, 640 Wm of dCTP, 640 WM of dGTP, 640 WM of dUTP, 2 pM of ddTTP, 163 mM of primer PR170F-Cy5.5, 163 nm of primer PR170F-A-Cy5.5, 131 rM primer PR543R-Cy5.0, 200 nM primer PR543R) were added to a 200 µl thin-walled PCR tube, with

  Primer PR170F-Cy5.5: 5'-GAG CCR ATA GAC AAG GAA YTR TAT-

3 ', marked with Cy5.5

  Primer PR17OF-A-Cy5.5: 5'-GAG MCG ATA GAC AAG GRV CTG TAT-

  3 ', labeled with Cy5.5 Primer PR543R-Cy5.5: 5'-ACT TTT GGG CCA TCC ATT CCT-3', labeled with Cy5.0 Primer PR543R-Cy5.5: 5'-ACT TTT GGG CCA TCC ATT CCT-3 ', unlabeled 5 g1 of the CLIP masterbatch were added to each

  of the four tubes containing the termination mixes.

  The tubes were closed and placed in a thermal cycler. The sequencing reactions were started under the following conditions: 37 C 15 min C 15 min 94 C 5 min cycles of the following 94 C 20 s 56 C 20 s C 90 s then C 5 min holding at 4 C The tubes have were removed from the thermal cycler, and 12 1l of charge dye were added to each tube (A, C, G and T). 2 g of each of the tubes containing the charge dye were migrated on a Clipper (automated sequencer with two dyes) at 54 ° C. in 35 minutes at a voltage of 1400 volts. After the operation, the bases were determined in the two orientations (front and reverse).

EXAMPLE 3

  Cycle sequencing M13 sequencing with dUTP HK-UNG master sequencing mixture diluted 1/10 1.0 B1 Sequencing buffer 2.0 Al 3 g primer 1.0 g1 M13 ss DNA 1.0.l Thermo Sequenase at 3 , 2 U / gl 2.5 g1 Sterile water 5.5 1 with Sequencing buffer: Tris-HCl 260 mM pH 8.3 at 25 C: MgC12 39 mM in ddH2O Primer M13 Universal-Cy5.5: 5'-GTA AAA CGA CGG CCA GT-3 ', labeled with Cy5.5 M13 ssDNA: single-stranded DNA (M13) at 200 ng / gl HK-UNG: uracil-thermolabile Nglycosylase, Epicenter Technologies Aliquot fraction of 3 gl of termination mixture of A (750 µM dATP, 750 µM dCTP, 750 µM dGTP, 750 BM dUTP, 2.5 gM ddATP) in a 200 gi thin-walled tube 3 g aliquot fraction of C termination mixture (750 pM dATP, 750 pM dCTP, 750 pM dGTP, 750 IIM dUTP, 2.5 gM ddCTP) in a thin-walled tube of 200 3 µl aliquot fraction of G terminator (750 gM dATP , 750 pM dCTP, 750 gM dGTP, 750 pM dUTP, 2.5 iM d dGTP) in a 200 µl thin-walled tube Aliquot fraction of 3 1 T termination mixture (750 pM dATP, 750 vM dCTP, 750 AM dGTP, 750 pM dUTP, 2.5 gM ddTTP) in a thin walled tube of 200 IL1 Aliquot fraction of 3 Al of the master sequencing mixture in each of the termination tubes. The tubes were sealed and placed in a thermal cycler. The sequencing reaction was started under the following conditions: 37 C 15 min C 15 min 94 C 2 min cycles as follows 94 C 40 s 50 C 20 s 72 C 60 s then 72 C 2 min maintained at 4 C We extracted the thermal cycler tubes and 6 g of charge dye was added to each tube (A, C, G and T). 2 g1 of each of the tubes containing the charge dye were migrated on a MICROGENE BLASTER sequencer at 54 ° C. in 35 minutes at a voltage of 1400 volts. After the operation, the data was analyzed to determine the sequence.

Claims (15)

  1. A method for determining the position of at least one selected species of nucleotides in a region of interest in a target nucleic acid polymer, comprising the steps of combining the sample with a reaction mixture to synthesize products of chain extension indicating the positions of the nucleotide species chosen in the region of interest and the evaluation of the products thus obtained, in which the reaction mixture which is combined with the sample comprises an unconventional nucleotide and a first enzyme, said first enzyme being effective in degrading polynucleotides incorporating the unconventional nucleotide.   2. Method according to claim 1, in which polymers of target and non-target nucleic acids are present in the sample in relative abundance. practically natural.   3. The method of claim 2, wherein the reaction mixture further comprises a heat stable polymerase enzyme which incorporates dideoxynucleotides into a polymer of nucleic acids which is elongating at a rate which doesn is not less than 0.4 times the speed   of incorporation of deoxynucleotides.   4. The method of claim 1, wherein the reaction mixture further comprises at least two oligonucleotide primers which, when hybridized to the target DNA, are oriented so as to allow chain extension towards   each of them on the interesting region.   5. Method according to claim 1, comprising the steps of: (a) combining the sample in the reaction mixture with the first and second primers, of a charge mixture of nucleotide triphosphate, of a terminating nucleotide triphosphate chain and heat stable enzyme polymerase, said first and second primers binding to the sense and antisense strands, respectively, of the target nucleic acid polymer at positions framing the selected region; (b) incubating the reaction mixture for a time sufficient to allow degradation of all nucleic acid polymers, including the unconventional nucleotide, by the first enzyme; (c) exposing the reaction mixture after the incubation step to a plurality of temperature cycles each of which includes at least one denaturation phase at elevated temperature and an elongation phase at lower temperature, thereby producing a plurality of fragments completed, and (d) evaluating the completed fragments produced during the additional cycles to determine the positions of the nucleic acid corresponding to the chain terminating nucleotide triphosphate in the selected region, characterized in that the sample contains an acid polymer target nucleic acids and a polymer of non-target nucleic acids, in natural abundance and that the polymerase is a polymerase which incorporates dideoxynucleotides into a polymer of nucleic acids in elongation at a speed which is not less than 0.4 times speed   of incorporation of deoxynucleotides.   6. The method of claim 5, wherein polymers of target and non-target nucleic acids are present in the sample in relative abundance. practically natural.   7. The method of claim 6, wherein the molar ratio of dideoxynucleotide triphosphate relative to the corresponding deoxynucleotide triphosphate   in the reaction mixture is 1:50 to 1: 1000.   8. The method of claim 6, wherein the molar ratio of dideoxynucleotide triphosphate relative to the corresponding deoxynucleotide triphosphate is from 1: 100 to 1: 300.   9. The method of claim 1, wherein at least one of the primers is labeled with a fluorescent label. 10. The method of claim 9, wherein the primers are each labeled with a label different fluorescent.   11. The method of claim 1, wherein the enzyme is a glycosylase.   12. The method of claim 1, wherein the   unconventional nucleotide is dUTP.   13. The method of claim 12, wherein the enzyme is a glycosylase.   14. The method of claim 1, wherein the reaction mixture further comprises a second polymerase enzyme having a low affinity for the incorporation of dideoxynucleotide triphosphates compared to deoxynucleotide triphosphates. 15. The method of claim 14, wherein the second polymerase is Taq polymerase.