CA2266755A1 - Method for sequencing of nucleic acid polymers - Google Patents

Method for sequencing of nucleic acid polymers Download PDF

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CA2266755A1
CA2266755A1 CA002266755A CA2266755A CA2266755A1 CA 2266755 A1 CA2266755 A1 CA 2266755A1 CA 002266755 A CA002266755 A CA 002266755A CA 2266755 A CA2266755 A CA 2266755A CA 2266755 A1 CA2266755 A1 CA 2266755A1
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nucleic acid
sequencing
sample
nucleotide
reaction mixture
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James M. Dunn
Marina T. Larson
James Leushner
May Hui
Jean-Michel Lacroix
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Bayer AG
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Visible Genetics Inc
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • B01L7/52Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • B01L7/52Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00178Special arrangements of analysers
    • G01N2035/00237Handling microquantities of analyte, e.g. microvalves, capillary networks

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Abstract

Sequencing of a selected region of a target nucleic acid polymer in a natural abundance DNA sample can be performed in a single vessel by combining the sample with a sequencing mixture containing a primer pair, a thermally stable polymerase such as Thermo Sequenase TM which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, nucleotide feedstocks, and a chain terminating nucleotide. The reaction mixture also includes an unconventional nucleotide and an appropriate enzyme for degradation of 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

METHOD FOR SEQLJEI~'CING
OF NUCLEIC ACID POLYMERS
This application is a continuation-in-part of International Patent Application No.
PCT/LJS97/07135 filed April 29. 1997, designating the Lnited States) which is a continuation-in-part of US Patent Applications I~os. 081640,672 Tled May 1. 1996. 08/684,498 filed July 19. 1996 and 08/807.138 filed February 27. 1997. All of these applications are incorporated herein by reference.
>JACKGROUND OF THE INVENTION
This application relates to DNA sequencing reactions, and in particular to improved sequen-cing reaction protocols making use of thermally stable polymerase enzymes having reduced error rates.
DNA sequencing can be performed in two distinct environments: a research environment in which each procedure is fairly unique and in which the sequence being determined is generally not known prior to completion of the sequence determination; and a diagnostic environment in which the same procedure is repeated on many samples and the sequences being determined are generally known. While the basic procedures used in these two environments can be the same) requirements Cor speed) cost-effectiveness and low risk of error in the diagnostic environment make many of the techniques actually employed too cumbersome to permit their effective utilization. This hat limited the availability of sequencing-based diagnostics, and has indeed led some to question w hether sequencing can ever be cost effective for routine diagnostic use.
The ideal DNA sequencing procedure for use in a diagnostic environment would have the following characteristics: ( I ) it would be able to utilize a DNA-containing sample which had been subjected to only minimal pretreatment to make the DNA accessible for sequencing; (2) it would require combining this sample with only a single reaction mixture, thus reducing risk of etTOr and contamination, and increasing the ease with which the procedure can be automated; and (3) it would require a short amount of time to perform the sequence determination. thus decreasing the marginal costs in tenors of equipment and labor for performing the test.
DNA sequencing, whether for research or diagnostics) is generally performed using tech-niques based on the "chain termination" method described by Singer et al..
Proc. Nat'1 Acad Sci.
(USA) 74(12): 5463-5467 (1977). Basically) in this process) DMA to be tested is isolated. rendered single stranded) and placed into four vessels. In each vessel are the necessary components to replicate the DNA strand) i.e.. a template-dependant DNA polymerise) a short primer molecule complementary to a known region of the DNA to be sequenced. and the standard deoxynucleotide triphosphates (dI~TTP's) commonly represented by A, C. G and T) in a buffer conducive to hybridization between the primer and the DNA to be sequenced and chain extension of the hybridized primer. In addition) cach vessel contains a small quantity of one type (i.e.) one species) of dideoxynucleotidc triphosphate (ddNTP). e.g. dideoxyadenosine triphosphate (ddA).
In each vessel, the primer hybridizes to a specific site on the isolated DNA.
The primers are then extended, one base at a time to form a new nucleic acid polymer complementary to the isolated LS pieces of DNA. When a dideoxynucleotide triphosphate is incorporated into the extending polymer.
this terminates the polymer strand and prevents it from being further extended. Accordingly) in each vessel. a set of extended polymers of specific lengths arc formed which are indicative of the positions of the nucleotide corresponding to the dideoxynucleotide in that vessel. These sets of polymers are then evaluated using gel electrophoresis to determine the sequence.
2U As Church and Gilbert observed. ''in a mammalian cell, the Dh'A
corresponding to any gene sequence is surrounded by DNA corresponding to some million other sequences."
''The Genomic Sequencing Technique'' in Medical Ge~tetics: Past , Present and Future) Alan R. Liss) Inc.. pp. 17-21. ( 1991 ). The same is true) to a greater or lesser extent) of any complex DNA sample, c.g:
containing microbial genetic materials. plant genetic materials, complete cDNA
libraries ete. In the 25 past. DNA sequencing procedures have dealt with this complexity by adding steps which substantially purify the DNA of interest relative to other DNA species present in the sample. This purification has been accomplished by cloning of the DNA to be sequenced prior to sequencing. or by amplification of a selected portion of the genetic material in a sample to enrich the concentration of a region of interest relative to other DNA. For example, it is possible to amplify a selected portion of a gene using a polymerise chain reaction (PCR) as described in U.S.
Patents Nos.
4,683,19, 4.683,195 and 4,683,202, which are incorporated herein by reference.
This process involves the use of pairs of primers, one for each strand of the duplex DNA.
that will hybridize at a site located near a region of interest in a gene. Chain extension polymerization (without a chain terminating nucleotide) is then carried out 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 the startin5 sample for the sequencing reaction.
Gelfand et al. have 1 (~ described a thermostable enzyme. "Taq polymerise," derived from the organism Tirernu~s aguaticus, which is useful in this amplification process. (See US Patent Nos. 4,889.818;
5,352.600 and 5.079,352 which arc incorporated herein by reference) Taq polymerise has also been disclosed as useful in sequencing DNA when certain special conditions are met. US Patent No. 5,075,216) incorporated herein by reference.
I S Improvements to the original technique described by Singer ct al. have i ncluded improv e-menu to the enzyme used to extend the primer chain. For example) Tabor et al.
have described enrymes such as T7 DVA polymerise which have increased processivity, and increased levels of incorporation of dideoxynucleotides. (See US Patent No. 4.795.699 and EP-A-0 386 8~7, which are incorporated herein by reference). More recently) Reeve et al. have described a thermostable 20 enzyme preparation, called Thermo SequenaseT'~, with improved qualities for DNA sequencing.
Nature 376: 796-797 ( I 995); EP-A-U 655 506. which is incorporated hereiwby reference. For sequencing, the Thermo SequenaseT'~ product is used with an amplified DNA
sample containing 0.5-2 ~tg of single stranded DNA (or 0.5 to 5 ~tg of double stranded DNA) into four aliquots: and combining each aliquot with the Thermo Sequenase~"' enzyme preparation) one dideoxynucleotide 25 termination mixture containing one ddNTP and all four dNTP't; and one dye-labeled primer which will hybridize to the DNA to be sequenced. The mixture is placed in a thermocycler and run for 20-30 cycles of annealing) extension and denaturation to produce measurable amounts of dye-labeled extension products of varying lengths which are then evaluated by gel electrophoresis. EP-A-U 655 506 further asserts that Thermo SequcnascT" and similar enzymes can be used for amplification reactions.
Notwithstanding the observations in the art that enzymes useful for amplification can also be used for sequencing. and vice versa) efforts to combine the amplification reaction and the sequencing reaction into a single step have been limited. Ruano and Kidd) Proc. Nat'l. Acad Sri.
(USA) 88: 2815-2819 (1991 ) and U.S. Patent No. 5.427,911, which are incorporated herein by reference) describe a process which they call "coupled amplification and sequencing" (CAS) for sequencing of DNA. In this process. a sample is treated in a first reaction stage with two primers la and amplified for a number of cycles to achieve 10.000 to 100,000-fold amplification. A ddNTP is then added during the exponential phase of the amplification reaction, and the reaction is processed for additional thermal cycles to produce chain-terminated sequencing fragments. The CAS process does not achieve the criteria set forth above for an ideal diagnostic assay because it requires an intermediate addition of reagents (the ddI~'TP reagents). This introduces and opportunity for error or contamination and increases the complexity of any apparatus which would be used for automation.
The problem of errors occumng during amplification has been addressed in one approach through the incorporation into the extending polymers of unusual nucleotides (for example dUTP) which are subject to enzymatic attack (for example with uracil-N-glycosylase) and degradation. See US Patent No. 5.418,149, which is incorporated herein by reference. Such molecules can be in utilized in most of the same ways that conventional amplification are used, but can be eliminated as contaminants from other reactions by incorporation of a pre-treatment step utilizing an appropriate enzyme to degrade the modified nucleic acid polymers.
It is an object of the present invention to provide a method for sequencing of high-complexity D~IA samples which is well-suited for use in the diagnostic environment and for automation and which provides a means for minimising errors caused by contamination and nucleic acid polymer carryover.
It is a further object of the invention to provide a method for sequencing of DNA which utilizes a DNA-containing sample which had been subjected to only minimal pretreatment to make the DNA accessible for sequencing and which provides a means for minimizing errors caused by contamination and nucleic acid polymer carryover.
It is still a further object of the invention to provide a method for sequencing of DNA which requires combining a complex DNA-containing sample with only a single reaction mixture, thus reducing risk of error and contamination. and increasing the ease with which the procedure can be automated.

The present invention provides a method for sequencing a region of interest in a DMA
sample in w hick a single set of reagents is added to a minimally-treated sample to produce useful sequencing results. The invention is bated on the surprising observation and discovery that the addition of a reaction mixture containing the thermostable polymerise Thermo SequenaseT~~) two primers which bind to complementary strands of a target DNA molecule at sites flanking the region of interest) a mixture of nucleotide triphosphates (A) C. G and T) and one dideoxynucleotide triphosphate to a DNA sample which contains target and non-target D~VA in substantially natural abundance. including highly coritplex DNA samples such as genomic human DNA, and the processing of the combination through multiple cycles of annealing) extension and denaturation results in 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 appropriate enzyme for degradation of nucleic acid polymers containing the unconventional nucleotide.
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 natural abundance sample containing the target nucleic acid polymer with a reaction mixture comprising three types of deoxynucleotide triphosphates. an unconventional nucleotide triphosphate corresponding to the fourth type of base. a didcoxynucleotide triphosphate.
first and second primers. an enzyme which degrades nucleic acid polymers incorporating the unconventional nucleotide. and a thermally stable polymerise enzyme which incorporate dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides to form a reaction mixture, said first and second primers binding to the sense and antisense strands, respectively, of the target nucleic acid polymer at locations flanking the selected region;
(b) exposing the reaction mixture to an initial stage in which the enzyme that degrades nucleic acid polymers incorporating the unconventional nucleotide is active for a period of time sufficient to degrade nucleic acid polymers containing the unconventional nucleic acid which may be present in the sample;
(c) exposing the reaction mixture to a plurality of temperature cycles each of which includes at least a high temperature denaturation phase and a lower temperature extension phase to produce a product mixture comprising sequencing fragments which arc terminated by incorporation I S of the dideoxynucleotide; and (d) evaluating the product 'mixture to determine the lengths of the sequencing fragments produced.
BBIEF DESCRIPTION OF TH nR a vyl~~ ,~
Fig. 1 illustrates the method of the invention Schematically;
2~ Figs. 2A and 2B show a comparison of sequencing tuns performed using Thetmo Sequenaser'1 as the polymerise in the method of the invention with results obtained using other thermostable polymerises in a comparative experiment:
Fig. 3 shows the data trace of Fig. 2A in greater detail;
Fig. 4 illustrates a mufti-dye embodiment of the invention:
25 Fig. 5 illusiratcs a second mufti-dye embodiment of the invemion; and Figs. 6A and 6B illustrate a third mufti-dye embodiment of the invention.

DETAILED DES('RIPTIfIV OF THE INVENTION
The present invention answers the need for a simple and readily-automated sequencing procedure 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. the specification and claims of this application use the term ''natural abundance sample" to describe such a mixture. As used herein a "natural abundance sample" is a sample which has been treated to make DNA in the sample accessible for hybridization with oligonucleotide primers, Cor example by lysis, centrifugation to 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 0 amount of target DNA relative to non-target DNA present in the initial sample. The term "natural abundance'' does not) however, require the presence of all the DNA from the on«inal sample. Thus) a complex sample containing just nuclear DIVA. or just mitochondria) DMA or some subfraction of nuclear or mitochondria) DNA obtained by isolation from a tissue sample but not subjected to preferential amplification would be a "natural abundance" sample within the meaning of that term in the specification and claims of this application. The term "natural abundance"
would also include a DNA sample prepared by conversion, for example by reverse transcription, of a total mRNA
preparation or the genome of an RNA virus to cDNA; DNA isolated from an individual bacterial colony jrowing on a plate or from an enriched bacterial culture; and a viral DNA preparation where substantially the entire viral genome is isolated. The term ''natural abundance" does not encompass a sample in which the isolated DNA is not a complex combination of DNA
molecules, and thus would not encompass, for example, a purified plasmid preparation containing only a single species of plasmid.
Natural abundance samples of mammalian DNA can be prepared from fluid samples, e.g., blood or urine or tissue samples by any of a number of techniques. including lysis) centrifugation to 2J remove cellular debris and proteolytic digestion to expose the DNA; salt precipitation or standard SDS-proteinase K-phenol extraction. Natural abundance samples can also be prepared using kits, for example the Gentra Pure Gene DNA Isolation Kit.
_7_ The method of the invention utilizes the properties of eniymes like Thermo Sequenaser".
namely the ability to incorporate dideoxynucleotides into an extending polynucieotide at a rate w hich is no less than about 0.4 times the rate of incorporation of deoxynucleotides) to provide a method for the sequencing of a nucleic acid polymer from a natural abundance sample in a single set of thermocycling reactions which can be carried out in a single vessel. Thus, the method of the invention is ideally suited for automation.
Fig. J illustrates the fundamental simplicity and elegance of the method of the invention in flow chart form. As shown in Fig. 1. a sample containing a target nucleic acid polymer which includes a region to be sequenced is combined with a reaction mixture containing two primers) a l0 mixture of dV1'P's. a chain terminating nucleotide triphosphate. i.e.. a dideoxynucleotide triphosphate, and a thermostablc polymerise with a high affinity for ddhTP
incorporation in a buffer suitable for hybridization and template-dependant polymerization. The mixture is processed for a number of thermal cycles sufficient to produce detectable amounts of sequencing fragments.
generally from 2U to 50 cycles. During each cycle) the primers each anneal to the respective strand 1S of target DNA present in the sample, and primer chain extension using the polymerise enzymes and the nucleotide triphosphate feedstocks proceeds until terminated by incorporation of a chain-terminating nucleotide triphosphate. This results in the production of sequencing fragments comparable to those generated in a conventional sequencing reaction. Analysis of these fragments prow ides information concerning the sequence of the selected region of the target DNA. Those 20 extension products which are not terminated prior to reaching the region complementary to the other primer can serve as template for generation of sequencing fragments in later cycles. although this generally occurs to a very small extent. Finally, the product mixture containing dideoxy-terminated fragments is loaded onto an electrophoresis gel for analysis of the positions of the base corresponding to the chain-terminating nucleotide triphosphate with in the target nucleic acid 25 polymer.
The operation of the invention can be understood in the context of a hypothetical 200 nt DNA fragment having equal amounts of each base. This means chat there will be 50 potential _g_ truncation events during the cycle. For each cycle, some of the products would be full length (and thus able to hybridize with one of the two primers to produce more sequencing fragments) and some would be truncated at the points where the ddNTP was added. !f each of these truncation events has a statistical likelihood of occurring 1 time in 500 as a result of the relative concentration of dd:VTP
compared to d:~ITP and the relative incorporation by the enzyme, then overall a truncation product will occur in slightly less than ten percent of the reactions. Table 1 shows the relative amounts of full-length and chain-termination products theoretically formed after 10, 20 and 30 cycles of a reaction according to the invention using this 200 nl polynuclcotide assuming various ratios of truncated to full-length product.
I TABLE
p 1 truncation truncation truncation ratio ratio ration =0. I =0.3 = 0.5 - ~

Cycles truncatedfull-len truncatedfull-Icn~thtruncated full-len th th 2U 41.000 376.000 17,400 40.462 3.300 3.300 tS 30 25.6 X 230 X 3.5 X 8.2 X 190,000 190.000 l0 106 ~ 10~ 10 The absolute and relative amounts of nucleotide triphosphates and chain-terminating nucleotide triphosphates may be optimized for the particular enzyme employed.
In actual practice, it has been found that useful results are obtained with Thermo SequenascT"~
when the reaction is run for 3~ to 45 cycles. using a dideoxy:deoxy mole ratio of 1:100 to 1:300. In general) each nucleotide triphosphate will be included in the reaction mixture at concentrations of from ?SO 1rM to 1.5 mM.
and the chain-terminating nucleotide triphosphate will be included at a lev el of from 0.5 pM to 30 1rM to produce compositions in which the mole ratio of the chain terminating nucleotide triphosphate to the corresponding nucleotide triphosphate is from t :~0 to 1:1000, preferably from 1:100 to 1:500. This will result in incorporation of a chain-terminating nucleotide triphosphate into from 30 to almost 100 percent of the extending polymer chains formed durin' the thermal cycling of the reaction mixture.
_c~_ A key factor in successfully performing the method of the invention is the utilization of Thermo Scquenase~~~'~ or a comparable enzyme as the thermostablc polymerise in the reaction mixture. Such enzymes are characterized by a high affinity for incorporating dideoxynucleotides into the extending nucleotide chain. In general, for purposes of the present invention, the poly-merase used should be one which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides.
Thermo SequenaseT'~t is known to favor the incorporation of dideoxynucleotides. and is suitable for use in the invention.. Tabor et al. have also described enzymes having increased processiv ity and high and increased levels of incorporation of dideoxynucleotides. (See EP 0 655 506) . Roche sells a polymerise under the trademark TAQ-FS which meets these criteria as well.
Figs. 2A and 3B and Fig. 3 illustrate the importance of this characteristic of the polymerise enzyme employed. Figs. 2A and :i shows a sequencing data trace for an actual heterozygous patient sample of natural abundance DNA which was obtained using Thermo Sequenase~'~
and primers flanking exon 2 of the Von Hippcl-Lindau ;ene in a process according to the invention. Large) well-1$ defined peaks corresponding to the termination fragments were obtained which made sequence evaluation of the sample very straightforward. In addition) the peaks for homozygous peaks are all approximately the same size, and are readily distinguishable from peaks for bases at heterozygous locations. This result was obtained performing the test in a single reaction vessel, with a single unaugmented reaction mixture. in a total of 45 thermal cycles. Comparable results could be obtained using fewer reaction cycles, for example 35 cycles as shown in Example 1 herein.
In contrast. Fig. 2B shows the trace obtained when a combination of Vent and Sequitherml"~
were used instead of Thermo SequenaseT~~ for a total of 45 thermal cycles. In this trace, the peaks for the termination fragments are much smaller and less well defined.
Furthermore) the peaks are quite variable in height and did not permit identification of heterozygous peaks based on peak height. Perfornung the same experiment using Taq polymerise alone resulted in a data trace that contained no usable peaks.

in the method of the invention. a natural abundance sample containing) or suspected to contain. a target DNA sequence is combined in a reaction mixture with an appropriate polymerise) all four types of dcoxynucieotide triphosphates, a dideoxynucleotide triphosphate, and tint and second primers. The primers used in the method of the present invention can be any pair of primers which hybridize with the sense and antisense strands of the target DNA
flanking a selected region that is to be sequenced. and which do not both hybridize to neighboring iocations in human DNA or other DNA potentially found in the sample. As used herein, the term ''flanking" will be understood to mean the positioning of primers at the 5'-ends of the selected region on each DNA strand, such that extension of the primers leads to replication of the region between the primers. The primers are preferably selected such that the primer pair flanks a region that is about 500 by or less. although primers spanning largo regions of DNA can be utilized with adjustments to the sequencing mixture (generally an increase in the relative amount of deoxynucleotide triphosphates) to increase the amount of longer sequencing fragments produced.
Primers can be selected to hybridize with highly conser~~ed regions which arc the same in all variants of the target DNA or can be prepared as degenerate primers to take known sequence variations at the primer site into account. Thus, the first and second primers of the invention may each be a discrete oligonucleotide species, or may be a set of oligonucleotide primers with similar but not identical sequences.
One or both of the primers may be labeled with a detectable label at the 5'-end thereof) particularly a fluorescent label such as fluorescein or a cyanine dye such as Cy 5.5. If labels are used on both primers, the labels selected should be spectroscopically-distinct. i.e.. they should have either a different excitation spectrum or a different emission spectrum such that one primer can be distinguished from the other. When both primers are labeled with different detectable labels) for example with two different fluorophores as in the process described by Wiemann et al.) "Simultaneous On-Line DNA Sequencing on Both Stands with Two Fluorescent Dyes," Anal.
Binchem 224: 117-121 ( 1995)) the sequence of both strands of the sample can be determined in a single reaction.

The nucicotide triphosphate feedstock mixture is a standard mixture of three of the four con-ventional deoxynucleotide bases (A, C. G and T), plus an unconventional nucleotide corresponding to the fourth base in a buffer suitable for template-dependent primer extension with the enzyme employed. As will be appreciated by persons skilled in the art, the specific concentrations of the nucleotide triphosphates and the nature of the buffer will vary depending on the enzyme employed.
Standard buffers and reagent concentrations for v arious known polymerase enzymes may be employed in the invention.
'The term "unconventional nucleotides" refers to unnatural or analog type nucleotide triphosphates that can be polymerized in a template dependent manner into the sequencing fragments. Unnatural forms of modified nucleotides include alkylated nucleotides and nucleotides modified by aikylhydroxylation. Specific examples of modified nucleotides includes but are not limited to V-7 methylguanine, deoxyuridine. dcoxyinosine, deoxy-5.6-dihydroxythymine (from OsO,.treated DhiA). S'.6'-dihydroxydihydrothymine. and deoxy-3'-methyladenosine.
Unconventional nucleotides may also include natural fot'rrts of nucleotides which arc not i$ conventionally incorporated in DNA. Thus, for purposes of the present invention) uracii and hypoxanthine are unconventional nucleotides.
An important characteristic of the unconventional nucleotides use din the invention is existence of an enzyme which will degrade polynucleotides containing the unconventional nucleotides to make them unsuitable as substrates for further gcneratio of sequencing fragments at temperatures below those normally associated with generation of sequencing fragments using a thermostabie polymerace. The reaction mixture in accordance with the invention includes an appropriate enzyme. frequently a glycosylase. Examples of specif c cn~ymes include: uracil N-glycosylase, hypoxanthine-DNA ~lycosylase. 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 destruction of carry-over polynucleotides prior to the first sequencing reaction, but inactive thereafter. Care should be taken in the selection - l2-of reaction conditions so that the temperature is not lowered after synthesis of sequencing fragments to a temperature which will permit significant degradation of the desired products prior to analysis.
The reaction mixture used in the present invention also includes at least one type (or one species) of chain-terminating nucleotide triphosphatc. Separate reactions for the four different types of bases may be run either concurrently or successively. Running all four bases concurrently comports with conventional sequencing practice. However. a preferred embodiment of the present invention combines the single vessel methodology of this application with "single track sequencing'' which is described in commonly assigned US Patent Application No. 08/577.858.
In single track sequencing, the determination of the positions of only one (or in any event less than 4) nucleotides) of a target sequence is frequently sufficient to establish the presence of and determine the qualitative nature of a target microorganism by providing a finger-print or bar-code of the target sequence that may be sufficient to distinguish it from all other known varieties of the sequence.
Throughput is increased by rcducinb the number of reactions and electrophoresis runs required to identify a sequence. By selection of the order of bases teued, and intermediate analysis) it may be l5 unnecessary to run all four bases to determine the presence and specific qualitative nature of any target microorganism present in the sample.
The present method can be used in combination with any type of detection system that is compatible with the label employed on the primers. For example. a Pharmacia A.L.F. sequences may be employed when fluorescein-labeled primers are used, while a Visible Genetics MicroGene Blaster is appropriate when the label used is Cy~.S. When multiple labels are used) the sample can be processed on multiple instruments, or it can be evaluated on an instrument which is capable of detecting signals from multiple labels. An example of such an instrument is the Prism 377 Sequences (Applied Biosystems lnc.) which detects and distinguishes between 4 dyes in a single lane. Spectroscopically distinouishablc dyes which are recognized by the Prism 377 are the FAM) ROX. TAMRA and JOE dyes known in the art.
The possibility of multi-dye detection leads to a wide range of applications for the invention which lead to improved accuracy in sequencing and to improved instrumental throughput. For - l3-example. Fig. 4 illustrates a method for obtaining both the forward and reverse sequences of a target nucleic acid sequence using two primers, each with a spectroscopically-distinguishable label. The natural abundance sample is mixed with forward and reverse primers) each with a distinguishable label (1 and 2). The reaction is performed with four termination reactions, one each for A. C. G and T. Each reaction is loaded into a single well of an automated sequencing instrument that detects and distinguishes at least the two labels employed. The results detected from label 1 are combined to give the forward sequence. The results detected from label 2 are combined to give the reverse sequence. The two sequences can be used to check each other and correct any ambiguities in base calling. In addition, the opposite sequence can be used to confirm sequence proximal to a primer which is found empirically to be difficult to determine on commercially available automated DNA
sequencers.
Fig. S demonstrates the use of multiple labels in a different format. In this cast, assuming the instrument can distinguish between 4 labels. 4 genes or gene fragments in a single sample can be sequenced contemporaneously. 4 pairs of primers are added to patient sample genomic DNA. Each I S 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 label (l. 2. 3) or 4) and each label is distinguishable from the others. This mixture is divided into tour termination reaction tubes. one each for ddA, ddC, ddG and ddT) and thermally processed. Termination reaction products are loaded in one lane) thus employing the method as disclosed in US Patent Application Setial No.
08/634.284, assigned to the assignee of the instant invention and incorporated herein by reference.
The results from each label arc combined to give the sequence of the exon for which that primer is specific.
Another embodiment takes advantage of the Cact that a single ddA termination reaction identifies the A nucleotides from 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 T nucleotide sites in the first strand). The complementary base can be located in the "missing" sites of the opposite strand. Note that sequence from the opposite strand must be inverted before it can be added in to the missing sites because it starts at the opposite end of the target gene and chain extension is in the opposite direction.
In Fig. 6B. a second termination reaction for ddC is added. This allows identification of C
and its complement G in each strand. When these results are added to the first reaction. ~ full DNA
sequence is obtained. Thus on the basis of 2 termination reactions employing one ddNTP chain terminator each, the full 4 lane sequence of a gene can be obtained.
The base-calling and compiling of sequences illustrated in Fig. 6A and 6B can be facilitated using GeneObjects software (Visible Genetics Inc.) Toronto) and employing techniques disclosed in US Patent Application Nos. 08/497,202 and 08/670.534) incorporated herein by reference.
The method of the present invention is advantageously applied in many contexts including:
(1) detection of mutations) particularly mutations of medical significance. in samples derived from a human patient, animal. plant or microorganism: (2) determination of HLA type ancillary to transplant procedures: (3) detection and identification of microorganisms, particularly pathogenic microorganisms. in a sample: and (4) in-situ sequencing reactions to produce sequencing fragments within a histological specimen which are then removed from a selected location on the tissue preparation and loaded onto a gel for sequence analysis. 1"hi~ latter approach is particularly useful for evaluation of archived samples in retrospective studies where the outcome of a disease condition is known) but the potentially causative mutation is not. This method can be used with labeled primers for single base sequencing (or multiple-base sequencing using multiple tissue samples).
The basic method of the invention can also be enhanced by various modifications without departing from the scope of the present invention. For example. improvements in reproducibility and sensitivity can be obtained by using a combination of an enzyme having a high affinity for incorporation of dideoxynucleotide triphosphates into the extending polymer, c.g.) Thermo Sequenase~"'. and one having a low affinity for incorporation of dideoxynucleotide triphosphates into the extending polymer) e.g.. Taq polymerase. under conditions where both enzymes are actively catalyzing template-dependent primer extension polymerization. As noted above) the high affinity enzyme produces almost entirely termination products, with very few of the polymers actually being - IS -extended to full length. On the other hand, the low affinity enzyme produces almost exclusively full Icngth product) with relatively few termination products. Addition of the low aftinity enzyme to the reaction mixture increases the sensitivity of the method by producing more full length material to be sequenced without increasing the processing time or adding processing steps.
The increase in sensitivity can be controlled by varying the ratio of high affinity to low affinity encyme present in the mixture.
It will be noted. however. that including oC low affinity enzyme to produce full length product will also result in the formation of a very intense labeled full-length product peak. This peak may make analysis of the bases near the end of the sequence difficult. To obtain the benefits of increased sensitivity while making less full length product) it may be desirable to utilize a low affinity enzyme which is more thermolabile than Taq polymerise. such that the low affinity enzyme is essentially inactivated by the end of the first 15 to 25 cycles. This would allow the production of longer fragments early in the assay and the generation of more terminated fragments late in the assay.
1S The reaction mixture of the invention may also incorporate other additives which enhance the formation of sequencing fragments'. For example. a product called TaqStartT"' Antibody is a monoclonal antibody which hinds to and blocks the activities of Taq polymerise. This antibody is added to PCR reactions using Taq polymerise to block encyme activity during set-up at ambient temperature to prevent or reduce the Cormation of non-specific amplification products. TaqStartr"
Antibody can be used in the present invention with Thermo SequenaseT" to reduce nonspecific primer extension reactions.
The method of the invention may also be used in conjunction with Johnson &
Johnson techniques known as "PCR In' A POUCH" which is described in US patent No.
5.460,780 incorporated herein by reference.
While the preferred method for sequencing nucleic acid polymers in accordance with the invention involves the use of a single step procedure 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 carry-over contamination in laboratories performing sequencing reactions through the addition of a preliminary degradation step prior to each sequencing procedure. Thus, in the broadest sense. the invention is a method for sequencing a nucleic acid polymer in a sample. in which the sample is combined with a reaction mixture containing an unconventional nucleotide, three conventional nucleotide bases.
at least one type of one chain-terminating nucleotide triphosphate, a polymerise enzyme. and an enzyme for degrading polynucleotides containing the unconventional base. The sequencing reaction can be done as a single step from natural abundance DNA, as described above. or can be performed on a previously amplified (preferably using an unconventional nucleotide) sample. The sequencing fragments can be obtained using any known method, including cycle sequencing. CAS, bidirectional sequencing or conventional one extension sequencing.
The invention will now be further described with reference to the following non-limiting examples.

DNA Amplification by PCR with dUTP and uracyl-n-glycosylase PCR Vtastcr Mix HK-UNG Dilution Buffer 10 lrl HK-UNG 1 pl lUX PCR Buffer II 55 ~.1 rrtM MgCl2 6b lr 1 dNTP Mix 66 lrl PCR Primers 13 pl Sterile water 284 pl ZS AmpliTaq DNA Polymerise C~ 5 U/pl 2.8 pl Where HK-UVG Dilution Buffer is 50 mM Tris-HCI (pH 7.5)) 100 mM NaCI. O.l m-M EDTA.
1 mM DTT, and 0.1 % Triton X-100 HK-C; NG is Thermolabile Uracyl-N-Glycosylase, Epicentre Technologies 1 UX PCR Buffer II: 100 mM Tris-HCl pH 8.3 C 25°C; 500 mM KCl dl~'TP Mix: ?.5 mM dATP) 2.5 mM dCTP. 2.5 mM dGTP. and 2.5 mM dUTP
PCR Primers: 2.0 IrM primer CT1590, 2.0 ~tM primer KLl-Cy5.5 Primer KL1-Cy5.5~'-TCC GGA GCG AGT TAC GAA GA-3', labeled with Cy5.5 Primer CT15905'-ATG CCC GGG ATT GGT TGA TC-3', unlabeled 45 lrl of the PCR Master Mix was put in a 200.1r1 thin-walled PCR tube.
5 ul of a recombinant plasmid (pL'C8 containing the cryptic plasmid of Chlamydia trachomatis, serovar LI) at 400 copies/pl.
The tube was closed and put in the thermal eyelet. The PCR was started with the following conditions 37°C 1 ~ min 75°C 15 min 94°C 5 min 37 cycles of the following 94°C 30 sec 61 °C 30 sec z0 72°C 45 sec Then 72°C ~ min hold at 4°C
Sequencing of the dUTP-containing amplicon by CLIP with dC;TP
CLIP'.Vlaster Mix Sequencing Buffer 22 pl CLIP Primers 12 Irl ThermoSequenase @ 32 U/pl 2.2 pl Sterile water 84.8 pl Where Sequencing BufferTris-HCI 260 mM pH 9.1 @ 25'C; MgCl2 39 mM in ddH20 CLIP Primers: 2.U ~tM primer CT1431 F-Cy5.5, 2.0 ~tM primer CT1538R
Primer CT1431F-Cy~.55'-GTG CAT AAA CTT CTG AGG AT-3'. labeled with Cy5.5 Primer CT1538R5'-GTA AAC GCT CCT CTG AAG TC-3') unlabeled 11 ftl of the CLIP Master Mix was added to a tube 2 pl of the dUTP-containing amplicon, created by the PCR reaction above, was added to the 11 pi of CLIP Master Mix) this is the Sample Master Mix 20 ~tl of sterile mineral oil was added to four 200 lrl thin-walled PCR
tubes 3 ~t1 of the Sample Master Mix was added to each of the four tubes containing the mineral oil 3 ltl of A-Sequencing Mix (75U pM dATP. 750 p.Vt dCTP, 750 pM dGTP. 750 pM
dUTP, 2.5 uM
ddATP) was added to first of the four tubes 3 ~tl of C-Sequencing Mix (75U NM dATP. 75U 1rM dCTP. 750 lrM dGTP. 750 pM
dUTP, 2.5 NM
ddCTP) was added to second of the four tubes 3 Itl of G-Sequencing Mix (750 pM dATP. 750 NM dCTP. 750 ~M dGTP. 750 uM
dUTP, 2.5 pM ddGTP) was added to third of the four tubes 3 NI of T-Sequencing Mix (75U ~M dATP. 75U lrM dCTP. 750 pM dGTP. 750 lr VI
dUTP. 2.5 pM ddTTP) was added to fourth of the four tubes The tubes were closed and put in the thermal eyelet. The sequencing reactions were started with the following conditions 94°C 5 min 37 cycles of the following 94°C 1 U sec 60°C 15 sec 70°C 45 sec Then 70°C J min hold at 4°C
The tubes were taken out of the thermal cycler, and 6 pl of loading dye was added to each tube (A) C, G) and T). 2 pl of each tubes containing the loading dye were migrated on a MICROGENE BLASTER sequencer at 50°C for 2U minutes at a voltage of 1,300 volts.
After the run) the bases where determined.
Sequencing an H1V dTTP-containing amplicon by CLIP with dUTP
CLIP Master Mix Sequencing Buffer 18.5 irl HK-UNG 1.0 pi dTTP-Containing Amplicon 5.0 pl AmpIiTAQ FS @ 15 U/pl =1.0 irl Sterile water 69.5 pl Where Sequencing Buffer: Tris-HCI 260 mM pH 8.3 @ 25°C; MgCl2 32.5 mM in ddH20 dTTP-Containing Amplicon: a 1.3-kb amplicon at a concentration of 100 ng/pl, containing the sequence of the protease of H1V-1.
HK-UNG: Thermolabile Uracyl-N-Glycosylase, Epicentre Technologies 7 pl of A-Termination Mix (64U mM dATP, 640 mM dCTP. 640 mM dGTP, 640 mM
dUTP, 1 pM ddATP. 163 mM primer PR 170F-Cy5.5. I 63 nM primer PR 170F-A-Cy5.5.
131 nM primer PR543R-Cy5.0, 200 nM primer PR543R) was added to a 200 pl thin-walled PCR tube.
7 pl of C-Termination Mix (64U mM dATP. 640 mM dCTP. 640 mM dGTP, 640 m.VI
dUTP. 21tM ddCTP. 163 mM primer PR170F-Cy5.5. 163 nM primer PR170F-A-Cy5.5, 131 nM primer PR543R-Cy5Ø 200 nM primer PR543R) was added to a 2001rl -..
'0 -thin-walled PCR tube.
7 ~tl of G-Termination Mix (640 mM dATP) 640 m:'Vl dCTP) 640 mM dGTP. 640 mM
dUTP, 21rM ddGTP) 163 mM primer PR 17UF-Cy5.5. I 63 nV1 primer PR 170F-A-Cy5.5, 131 nM primer PR543R-CyS.U. 2U0 nVI primer PR543R) was added to a 200 ftl thin-walled PCR tube.
7 ~tl of T-Termination Mix (640 mM dATP) 64U mM dCTP. 640 mM dGTP. 64U mM
dUTP. 2 ~tM ddTTP. 163 m.M primer PR170F-Cy5.5. 163 nM primer PR170F-A-Cy5.5.
131 nM primer PR543R-Cy5.0) 200 nM primer PR543R) was added to a 2UU ftl thin-walled PCR tube.
Where Primer PR170F-Cy5.5: 5'-GAG CCR ATA GAC AAG GAA YTR TAT-3') labeled with Cy5.5 Primer PR170F-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 lrl of the CL,1P Master Mix was added to each of the four tubes containing the TetirtinatioWlixes.
The tubes were closed and put in the thermal cycler. The sequencing reactions were started with the following conditions 37°C 15 min 2U 75°C 15 min 94°C 5 min 30 cycles of the following 94°C 2U sec 56°C 20 sec 70°C 90 sec Then 70°C 5 min hold at 4°C
The tubes were taken out of the thermal cycler, and l2 pl of loading dye was added to each tube (A.
C) G, and T). 2 ~tl of each cubes containing the loading dye were migrated on a Clipper (two dye automated scquencer) at 54°C for 35 minutes at a voltage of 1.400 volts. After the run) the bases where determined on both orientation (forward and reverse).

Cycle Sequencing Sequencing M13 with dUTP
Sequencing Master Mix HK-UNG diluted 1/10 1.0 pl Sequencing Buffer 2.U lrl Primer at 3 uM 1.0 lrl M13 ssDI~A I.0 pl ThermoSequenase @ 3.2 U/ltl 2.5 ~tl Sterile w ~ater 5.5 lrl Where Sequencing BuCCer: Tris-HCl 260 mM pH 8.3 C~ 25°C: YIgCI? 39 mM in ddH20 PrimerM 13 Universal-CyS.~: 5'-GTA AAA CGA CGG CCA GT-3', labeled with Cy5.5 M13 ssDNA: single stranded DNA (M13) at 200 ng/pl HK-UNG: Thermolabile LTracyl-N-Glycosylase, Epicentre Technologies Aliquot 3 pl of A-Termination Mix (750 pM dATP) 750 pM dCTP. 750 NM Dgtp. 750 pM dUTP) 2.5 ~tM ddATP) in a 2(l0 ul thin-walled PCR tube.
Aliquot 3 pl of C-Termination Mix (750 pM dATP. 75U lrM dCTP, 75U NM dGTP.
750 pM dUTP) 2.5 ttM ddCTP) in a 200 lrl thin-walled PCR tube.
Aliquot 3 a l of G-Termination M ix (750 N M dATP. 7501r M dCTP. 75011 M dGTP.
7501rM dUTP. 2.S 1tM ddGTP) in a 200 Nl thin-walled PCR tube.

Aliquot 3 ~1 of T-Termination Mix (750 ~'.vI dATP. 750 ~M dCTP, 750 IrM dGTP.
75U ~M dUTP. 2.5 ~rM ddTTP) in a 20U ~1 thin-walled PCR tubc.
Aliquot 3 ul of the Sequencing Master Mix in each of the termination tubes.
Close the tubes and put them in the thermal cylcer. Stan the sequencing reaction with the following conditions 37°C 1 S min 75°C 15 min 94°C 2 min 35 cycles of the following 94°C d0 sec 50°C 20 sec 72°C 60 sec Then 72°C 2 min hold at 4°C
Take the tubes out of the thermal cycler. and add 6 ul of loading dye to each tube (A) C. G) and T).
Migrate 2 ~1 of each tubes containing the loading dye a MICROGENE BLASTER
sequencer at 54°C for 35 minutes at a voltage of 1,4(>n volts. After the run) analyse the data to determine the sequence.

Claims (15)

1. A method for determining the position of at least one selected species of nucleotide within a region of interest in a target nucleic acid polymer in a sample comprising the steps of combining the sample with a reaction mixture to synthesize chain-extension products indicative of the positions of the selected species of nucleotide within the region of interest and evaluating the products thus produced, wherein the reaction mixture which is combined with the sample comprises an unconventional nucleotide and a first enzyme said first enzyme being effective to degrade polynucleotides incorporating the unconventional nucleotide.
2. The method according to claim 1, wherein target and non-target nucleic acid polymers are present in the sample in substantially natural relative abundance.
3. The method according to claim 2, wherein the reaction mixture further comprises a thermally-stable polymerise enzyme which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than 0.4 times the rate of incorporation of deoxynucleotides.
4. The method according to claim 1, wherein the reaction mixture further comprises at least two oligonucleotide primers which, when hybridized to the target DNA, are oriented to allow chain extension towards each other across the region of interest.
5. A method according to claim 1, comprising the steps of:
(a) combining the sample in the reaction mixture with first and second primers, a nucleotide triphosphate feedstock mixture, a chain-terminating nucleotide triphosphate and a thermally stable polymerise enzyme, said first and second primers binding to the sense and antisense strands, respectively, of the target nucleic acid polymer at locations flanking the selected region;

(b) incubating the reaction mixture for a period of time sufficient to permit degradation of any 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 a high temperature denaturation phase and a lower temperature extension phase, thereby producing a plurality of terminated fragments; and (c) evaluating terminated fragments produced during the additional cycles to determine the positions of the nucleic acid corresponding to the chain-terminating nucleotide triphosphate within the selected region. characterized in that the sample contains target nucleic acid polymer and non-target nucleic acid polymer in natural abundance and that the polymerise is one which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than 0.4 times the rate of incorporation of deoxynucleotides.
6. The method according to claim 5, wherein target and non-target nucleic acid polymers are present in the sample in substantially natural relative abundance.
7. The method of claim 6, wherein the mole ratio of the dideoxynucleotide triphosphate to the corresponding deoxy nucleotide triphosphate in the reaction mixture is from 1:50 to 1:1000.
8. The method of claim 6, wherein the mole ratio of the dideoxynucleotide triphosphate 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 different fluorescent label.
11. The method of claim 1, wherein the enzyme is a glycosylase.
12. The method according to claim 1, wherein the unconventional nucleotide is dUTP.
13. The method of claim 12, wherein the enzyme is a glycosylase.
14. The method according to claim 1, wherein the reaction mixture further comprises a second polymerise enzyme having a low affinity for incorporation of dideoxynucleotide triphosphates compared to deoxynucleotide triphosphates.
15. The method according to claim 14, wherein the second polymerise is Taq polymerise.
CA002266755A 1998-04-24 1999-03-25 Method for sequencing of nucleic acid polymers Abandoned CA2266755A1 (en)

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DK0655506T3 (en) * 1994-10-17 1997-03-10 Harvard College DNA polymerase with modified nucleotide binding site for DNA sequence analysis
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