CA2252795A1 - Process for direct sequencing during template amplification - Google Patents
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- CA2252795A1 CA2252795A1 CA002252795A CA2252795A CA2252795A1 CA 2252795 A1 CA2252795 A1 CA 2252795A1 CA 002252795 A CA002252795 A CA 002252795A CA 2252795 A CA2252795 A CA 2252795A CA 2252795 A1 CA2252795 A1 CA 2252795A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6872—Methods for sequencing involving mass spectrometry
Abstract
Processes and kits for simultaneously amplifying and sequencing nucleic acid molecules, and performing high throughput DNA sequencing are described.
Description
PROCESS FOR DIRECT SEQUENCING DVRING TEMPLATE AMPLIFICATION
5 Back~round of the Inv~ntio ..
DNA .S~q n"~i,.,~
Current knowledge I egal di~g gene structure, the control of gene activity and the function of cells on a molecular level all arose based on the determination of the 10 base sequence of millions of DNA molecules. DNA seq~len~in~ is still criticaJly important in research and for genetic the. apics and di~ostiçs, (e.g., to verify~ecol..b:nant clones and mutations).
DNA, a polyrner of deoxyribon~ eotides, is found in all living cells and 15 some viruses. DNA is the carrier of genetic info.-na~ion, which is passed from one gene.iltion to the next by homologous replication ofthe DNA molecule. Illru~ lion for the synthesis of all proteins is encoded in the seqUçnce of bases in the DNA.
To obtain the genetic information and therefore to reveal the base 20 sequence of a given DNA molecule, çh~m;c~l and el~malic seq~lçncin~ metho~s have been developed. DNA-sequ~n~.ing as proposed by Maxam-Gilbert (Maxam, A.M., W.
Gilbert, Proc. Natl. Acad. Sci. USA, 74:560-564 1977) is a chemical method of delel,nilf..lg base composition of a nucleic acid mole ~ e A single stranded DNAmolecule with ra~ioaGtive label at its 5' end is r.hçmi~lly modified in four base specific reactions and then cleaved at the modified positionC The cleavage products are sepa~ted on a polyacrylamide gel and typically are detected by autoradiography.
Currently favored is the el~yll,alic chain t~ ;oll reaction accolding to the Sanger-sequ~n~.ing method (Sanger, F. et. al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 1977). In the Sanger method, the four base specific sets of DNA fr~em~nts are - formed by starting with a p.;",el1lemrl~te system elong~ling the primer into the unknown DNA sequence area and thereby copying the t~mpl~te and s~ l.f.ci~ complçm~nt~ry strands using a DNA polymerase in the presence of chain-te....;.~ r~aef nlc The ~ . . ~ .
CA 022~279~ 1998-10-27 chain-tel,.,i.-~';,~g event is achieved by incorporating into the four separate reaction mixtures in addition to the four normal deoxynucleoside triphosphates, dATP, dGTP, dTTP and dCTP, only one ofthe chain-term~ tin~ dideoxyn.~cleosi~le triphosphates, ddATP, ddGTP, ddTTP or ddCTP, respectively, in a limiting small concentration. The incorporation of a ddNlP lacking the 3' hydroxyl function into the growing DNA strand by the enzyme DNA polymerase leads to chain termination through preventing the formation of a 3'-5'-phosphodiester bond by DNA polymerase. Due to the random incorporation ofthe ddNlPs, each reaction leads to a population of base specifictermin~ted fr~m~tlt~ of di~erent Iengths, which all together I epresenl the sequenced DNA-molecule.
A recent modification of the Sanger sequencing strategy involves the degradation of phosphorothioate-con~inine DNA fr~m~nts oblained by using alpha-thio dNTP instead of the normally used ddNTPs during the primer extension reaction medi~ted by DNA polymerase (Labeit et al., DNA 5, 173-177 (1986); Amel~llalll, PCT-Application GB86/00349; F.c~tein et al., Nucleic Acids Res. 16, 9947 (1988)).
Here, the four sets of base-specific sequencing ladders are obtained by limited digestion with exonuclease III or snake venom phosphodiesterase, subsequent separation on PAGE and vi.cll~li7~tion by radioisotopic labeling of either the primer or one of the dNTPs. In a further modification, the base-specific cleavage is achieved by alkylating the sulphur atom in the modified phosphodiester bond followed by a heat tre.~tment (Max-Planck-Gesçll~ch~, DE 3930312 Al).
DNA Amplification 2S DNA can be amplified by a variety of procedures insl~tlin~ cloning (Sambrook et al., Molecular Cloning: A ~aboratory Manual, Cold Spling Harbor Laboratory Press, 1989), polymerase chain reaction (PCR) (C.R. Newton and A.
Graham, PCR, BIOS Publishers, 1994), ligase chain reaction (LCR) (F. Barany Proc.
Na~l. Aca~ Sci USA 88, 189-93 (1991), strand ~i~pl;~cement amplification (SDA) (G.
Terrance Walker et al., Nucleic Acids Res. 22, 2670-77 (1994)) and variations such as RT-PCR, allele-specific amplification (ASA) etc.
CA 022~279~ 1998-10-27 The polymerase chain reaction (Mullis, K. et al., Methods Enzymol., 155:335-350 1987) permits the selective in vitro amplification of a particular DNA region by mimic~in~ the phenomena of in vivo DNA replicvtiQn Required reaction components S are single stranded DNA, primers (oligo~ cleotide sequences complemf nt~ry to the 5' and 3' ends of a defined sequence of the DNA template), deoxyn..c~eotidetriphosphates and a DNA polymerase enzyme. Typically, the single stranded DNA is generated by heat denaturation of provided double strand DNA. The reaction buffers contain m~gn~; .m ions and co-solvents for optimum enzyme stability and activity.
The amplification results from a repetition of such cycles in the following manner: The two di~rel~t primers, which bind selectively each to one of the comple .~ . y strands, are pytcn~led in the first cycle of amp!ificAti~ n. Each newly synth~i7ed DNA then co~ nc a binding site for the other primer. Thc.erore each new 15 DNA strand becol"f s a templAte for any further cycle of amplification e nl~ng the tçmplqte pool from cycle to cycle. ~peqted cycles theoretically lead to exponential synthesis of a DNA-fragment with a length defined by the 5' termini of the primer.
Initial PCR expe,i,-,ti--Ls used thermolabile DNA polymerase. However, 20 thermolabile DNA polymerase must be continually added to the reaction mixture after each denaturation cycle. Major advances in PCR practice were the development of a polymerase, which is stable at the near-boiling te.,.pe~ alLlre (Saiki, R. et al., Science 239:487-491 1988) and the development of a~ltomqted thermal cyclers.
The discovery of thermostable polymerases also allowed modifir.q~ion ofthe Sanger sequçnring reaction with si~nifirqnt advAntA~es The pol~",e.~aLion reaction could be carried out at high tc."pe, ~ re with the use of thermostable DNA
polymerase in a cyclic manner (cycle sequçncing). The con~itions of the cycles are similar to those of the PCR technique and COlllpl ;Sf della~ul a~ion~ ,~qnne~'ing~ and extension steps. Depf ~-~ing on the length of the primers only one anneAlin~ step at the beg;~ g of the reaction may be sufficient. Carrying out a sequenring reaction at ... ... . .
CA 022~279~ 1998-10-27 high temperature in a cyclic manner provides the advantage that each DNA strand can serve as template in every new cycle of extension which reduces the amount of DNA
necçs~ry for sequçn~ing~ thereby providing access to minim~l volumes of DNA, as well as res -lting in improved specificity of primer hybriAi~tion at higher te"~l)e~al~lle 5 and the reduction of secondary structures of the tçmpl~te strand.
However, amplification ofthe te,.~ ted fr~ onts is linear in conventional cycle seqllenrine approaches. A recently developed method, called semi-exponential cycle seq~l~ncing shortens the time ~ hed and incr~,ar,es the extent 10 of amplification obtained from conventional cycle sequçn~.in~e by using a second reverse primer in the sequçn~ing reaction. However, the reverse primer only generates additional temrl~te strands if it avoids being te, ...;.~ d prior to reaching the sequencing primer binding site. Needless to say, tel ...;..~led fr~mçnt~ generated by the reverse primer can not serve as a sufficient tçmp!~te. Therefore, in practice, 15 amplification by the semi-exponential approach is not entirely exponential. (Sarkat, G.
and Bolander Mark E., Semi Exponential Cycle Sequ~nt ing Nucleic Acids Research,1995, Vol. 23, No. 7, p. 1269-1270).
As pointed out above, current nucleic acid sequPn~i~ methods require 20 relatively large ~mo~lnt~ (typically about 1 g) of highly purified DNA template. Often, however, only a small amount of templ~te DNA is available. Although amplifications may be pe,ro.,ned, amplification procedures are typically time con~1ming can be limited in the amount of amplified template produced and the amplified DNA must be purified prior to seq~lçn~i~ A stre~mlined process for amplifying and sequerlcing DNA is 25 needed, particularly to f~.ilit~te highthroughput nucleic acid sequP.ncin~
Summary of the Invention In general, the instant invention provides a one-step process for 30 generating from a DNA template, base spec.ific?lly termin~ted fragmçnts of sllfficient quantity to enable DNA sequenci~
CA 022~279~ 1998-10-27 According to the process of the invention, a combined amplification and tel., inalion reaction is pelroll.lcd using at least two di~ren~ polymerase enzymes, each having a dillerenL affinity for the chain tel..~ -g nucleotide, so that polymerization by 5 an enzyme with relatively low affinity for the chain ttl~ ;llg nucleotide leads to exponential amplification whereas an enzyme with relatively high afflnity for the chain termin~ting nucleotide terminates the polymerization and yields sequencing products.
In another aspect, the invention features kits for directly amplifying 10 nucleic acid t~mp!~tes and generating base specifically termin~ted rlng...~..ls In one embodiment, the kit can comprise an approp.;ate amount of: i) a colllpl.,le set of chain-elongating nucleotides; ii) at least one chain-tel ~ nucleotide; (iii) a first DNA
polymerase, which has a relatively low affinity towards the chain tel...;~ nucleotide;
and (iv) a second DNA polymerase, which has a relatively high affinity towards the chain 15 te~ nucleotide. The kit can also optionally include an applol)liate primer or primers, appropliate buffers as well as instructions for use.
The instant invention allows DNA amplification and tel~l inalion to be pelrollned in one reaction vessel. Due to the use of two polyl,lel~ses with dif~re.ll 20 affinities for dideoxy nucleotide triphosphates, exponential amplification of the target sequence can be accompli~hed in col--bil-~lion with a tel.l..nalion reaction nucleotide. In addition, the process obviates the purification procedures, which are required when amplification is l~-ru--"ed separately f~om base termin?ted ~agment gen~;lalion. Further, the instant process requires less time to accQmrlish than separate amrlific~tion and base 25 specific te-., ina~ion reactions.
When combil-ed with a detection means, the process can be used to detect and/ or qu~ntit~te a particular nucleic acid sequence where only small amounts of template are available and fast and accurate sequence data acquisition is desirable. For 30 example, when comhined with a detection means, the process is useful for seq~lencing unknown genes or other nucleic acid sequences and for ~ grlosing or monitoring certain CA 022~279~ 1998-10-27 dice~es or contlition~, such as genetic ~ice~ces, chromosomal abno~ ;es, geneticpredispositions to certain ~ice~ces (e.g. cancer, obesity, artherosclerosis) and pathogenic (e.g. bacterial, viral, fungal, protistal) infections. Further, when double stranded DNA
molecllles are used as the starting material, the instant process provides an opportunity to 5 simlllt~neously sequence both strands, thereby providing greater cel ~aillLy of the sequ~nce data obtained or acquiring sequence i~ alion from both ends of a longer tP.mpl~te.
The above and further features and advantages of the instant invention will become clearer from the following Detailed Description and Claims.
Brief Description of the Fi~g ~reS
FIGURE 1 shows sequence data from an ABI-Prism ~utomqted sequencer (Model 373A) using a pOM8 derived ~t;co...binalll plasmid with a 400 base pair (BP) insert from the rrnB gene of E. coli as the tçmp!nte in the reaction described in the following Example l. The figure shows a reliable sequence readable to about 440 BP
which is the length of the PCR product.
FIGURE 2 shows sequence data from an ABI-Prism n~tQIn~ted sequçncer (Model 373A) again using the pOM8 derived recombinant plasmid with a 400 BP insert of from the rrnB gene of E. coli. However, because the sequencing reaction was carried out using standard sequencing protocols on a small amount of template (50ng), noreliable sequence was obtained.
FIGURE 3 is a srhPm~tic of a co-l~bined ~rnrlific~ti~n and sequçnring reaction using two polymerases and dye-labeled dideoxynucleotide triphosphate for detection and two reverse oriented primers.
FIGURE 4 is a fluol escenl chromotagram plot of a forward sequence ladder generated in a sim~1lt~neous amplification and sequencing reaction.
FIGllRE 5 is a fluorescent chromatogram plot of a reverse sequence ladder generated in a sirnlllt~neous amplification and seql~ncing reaction.
W O 97/42348 PCT~US97/07966 Detailed Description of the Invention In general, the invention features a process for directly amplifying and base specifically te, ...;l.~ a nucleic acid molecule. Accolding to the process of the 5 invention, a co,nbined amplification and tel" i"a~ion reaction is pe,ro.-..cd on a nucleic acid template using: i) a complete set of chain-elon~,qtine nucleotides; ii) at least one chain-termin~ting nucleotide; and (iii) a first DNA polymerase, which has a relatively low affinity towards the chain tel...;.~ g nucleotide; and (iv) a second DNA poly"~clase, which has a relatively high affinity towards the chain terminqtin~ nucleotide, so that 10 poly",eli~alion by the enzyme with relatively low affinity for the chain te~...;..~li.~g nucleotide leads to amplification of the tçrnrlqt~, whereas the enzyme with relatively high affinity for the chain t~....;.~1;.-g nucleotide terminates the polymerization and yields sequçnclng products.
The combined amplification and sequçncing can be based on any amplification procedure that employs an enzyme with polynucleotide synthetic ability (e.g. polyn.erase). One p.ere"ed process, based on the polymerase chain reaction(PCR), is comprised ofthe following three thermal steps: 1) denaturing a double stranded (ds) DNA molecule at an approp-iate telllpc~al~lre and for an approp,iale period 20 of time to obtain the two single stranded (ss) DNA molecules (the template: sense and ~q.nti~en~e strand); 2) contqctin~ the template with at least one primer that hybridizes to at least one ss DNA template at an app, op,iate temperature and for an approp, ;ate period of time to obtain a primer co.-l ~q,;~ g ss DNA tçmrl~te; 3) cont~ctin~ the primer co~ ni.~ template at an approp,iate temperature and for an appJop,iate period oftime 25 with: (i) a comrlete set of chain elongating nucleotides, (ii) at least one chain te. ".;nAI ;~g nucleotide, (iii) a first DNA polymerase, which has a relatively low affinity towards the chain terminqtin~ nucleotide; and (iv) a second DNA polymerase, which has a relatively high affinity towards the chain terminqting nucleotide.
Steps 1)- 3) can be sequentially pe.ru.. ed for an app.op.ia~e number of times (cycles) to obtain the desired amount of amplified seqUçnri~ Iadders. The CA 022~279~ 1998-10-27 quantity ofthe base spec.ific~lly tt,..~ led rla~ l desired dictates how many cycles are performed. Altho~lgh an increased number of cycles results in an increased level of amplification, it may also detract from the sensitivity of a subsequent detection. It is therero~ generally undesirable to perform more than about S0 cycles, and is morepreferable to pelrolll' less than about 40 cycles (e.g. about 20-30 cycles).
In a p-ere"ed embodiment, the first denaturation step is ~e.rullned at a temperature in the range of about 85~C to about 100~C (most prerel~bly about 92~C to about 96~C) for about 20 seconds (s) to about 2 mimltes (most preferably about 30s- 1 10 minute). The second hybridization step is prefelably pe-~,llled at a tel-lptlalure, which is in the range of about 40~C to about 80~C (most prererably about 45~C to about 72~C) for about 20s to about 2 mimltes (most p-erel~bly about 30s- 1 minute). The third, primer eYt~n.~ion step is preferably pe.~.lned at about 65~C to about 80~C (mostpreferably about 70~C to about 74~C) for about 30 s to about 3 minutes (most pl~relably 15 about 1 to about 2 minlltes) In order to obtain sequence ;l~l ..-alion on both the sense and antisense strands of a DNA molecule ~imlllt~neously, each of the single stranded sense and~ntic~n~e templates generated from the denaturing step can be cont~cted with app-op-iate 20 primers in step 2), so that amplified and chain termin~ted nucleic acid msleclllçs generated in step 3), are co...~ A~y to both strands.
Another pl~re--ed process for sim-~l~t~neously amplifying and chain tellllinA~ a nucleic acid sequ~nce is based on strand tlicrl~cemçnt a...pl;l'lcalion (SDA) (G. Terrance Walker et al., Nucleic Acids Res. 22, 2670-77 ( 1994); European Patent Publication Number 0 684 315 entitled Strand Disp.'~rement Amplif cation Using Therrnophilic Enzymes ). In essence, this process involves the following three steps, which altogether comprise a cycle: 1) denaturing a double stranded (ds) DNA m~lec~lle cont~ining the seqllence to be amplified at an applop.iale te.npel~ re and for an approp. iate period of time to obtain the two single stranded (ss) DNA molecules (the template: sense and ~ spl~ee strand); 2) cont~cting the tçmrl~te with at least one primer CA 022~279~ 1998-10-27 WO 97/4~348 PCT/US97/07966 _ 9 (P), that cQ~ ;...c a recognition/cleavage site for a restriction end~ n--c~e~e (RE) and that hybridizes to at least one ss DNA temr!~te at an app~up.;ate te...~ L.Ire and for an apploplid~e period oftime to obtain a primer CO..~ ss DNA templ~te; 3) cont~ctine the primer co.ll~;,.;ng template at an applop,iale telll~)e,~ re and for an app-opliale 5 period of time with: (i) a col..pl~te set of chain çlong~ti~ nucleotides; (ii) at least one chain te~ nucleotide, (iii) a first DNA polymerase, which has a relatively low affinity towards the chain te....;l-~ -g nucleotide; (iv) a second DNA polymerase~ which has a relatively high affinity towards the chain tc....;~ ;ne nucleotide; and v) an RE that nicks the primer recognition/cleavage site.
Steps 1) - 3) can be sequentially performed for an applopliale number of times (cycles~ to obtain the desired amount of arnplified sequçnrin~ ladders. As with the PCR based process, the quantity of the base specifically ttl ...;I-~ted fragment desired dictates how many cycles are pelrolllled. Plerelably, less than 50 cycles, more preferably less than about 40 cycles and most preferably about 20 to 30 cycles are pelrulllled.
The amplified sequçnr~ing ladders obtained as described above, can be separated and detected and/or ql~ntitated using well established methods, such as polyacrylamide gel ele~ opholèsis (PAGE), or capillary zone electrophoresis (CZE) (Jorgensonetal., J. Chrom~toyr~hy~, 337 (1986); Gestel~nd etal., Nucleic Acids Res. 18, 1415-1419 (1990)); or direct blotting electrophoresis (~BE) (Beck and Pohl, E~IBO J., vol. 3: Pp. 2905-2909 (1984)) in conjunction with, forexample, colorimetry, fluoli.,l.,lly, c~mil-lminescrnce and radioactivity.
Dye-terminator r~ try can be employed in the combined amplification and sequçncine reaction to enable the simultaneous generation of rul vvald and reverse sequence ladders, which can be separated based on the streptavidin-biotin system when one biotinylated primer is provided.
Figure 3 depicts a scheme for the co-nbined amplification and sequencing using two polymerases and dye-labeled chain tel ~ e nucleotide (ddNTP) for detection and two reverse oriented primers. A means of separation for the s~ t~neously generated forward and reverse sequence ladders is shown. Step A
represents the exponential amplification of a target sequence by the polymerase with a low affinity for ddNTPs. One ofthe sequence specific oligonl~cleotide primers isbiotinylated. Step B, ~prese,~ls the gcnc~ ~lion of a sequence ladder either from the S original template or the s;-nllh~neously generated arnrlific~tiQn product carried out by the polymerase with a high affinity for ddNTPs. After cc ~ on of the ~ tiQn, the products are incubated with a streptavidin coated solid support (Step C). Biotinylated forward sequenring products and reverse products hybridized to the r(" v~ralJ templâte are immobilized. In order to obtain readable sequ~.nce info"~-alion, the rolv~ald and 10 reverse sequence ladders are separated in Step D. The imm--bili7ed strands are washed and separated by denaturation with a",.,.ol-ium hydroxide at room temperature. The non-biotinylated reverse sequenrin~ products are removed from the beads with ammonium hydroxide sup~l ~,alanl during this procedure. The biotinylated forwardseq~lçnring products remain immobilized to the beads and are re-solubili7ed with15 ammonium hydroxide at 60~C. After ethanol prtc;y;lalion, both sequencing species can be resuspended in loading dye and run on an automated sequencer, for example.
When mass ~pe~ o",et~ y is used in conjunction with the direct ~mplific~tion and chain t~""-nalion processes, the sequPnci~ ladders can be directly 20 detected without first being separated using several mass s,~,e~;t, om~,ter formats.
Amenable formats for use in the invention include ionization te~hniq~les such as matrix-a~ ted laser desorption (MALDI), continuous or pulsed ele~,l.osp,~ (ESI) and related methods (e.g. Ionspray or Thermospray), and massive cluster impact (MSI); these ion sources can be m~tched with a detection format, such as linear or reflectron time-of-flight 25 (TOF), single or multiple quadrupole, single or multiple m~gn.o,tic sector, Fourier Transform ion cyclotron reSon~nre (FTICR), ion trap, or co,.lbin~l;on~ ofthese to give a hybrid detector (e.g. ion trap-TOF). For ionization, numerous matrix/wavelength combinalions (MALDI) or solvent co.,.bi~lalions (ESI) can be employed.
The above-described process can be pti,ru",.ed using virtually any nucleic acid molecule as the source of the DNA template. For example, the nucleic acid ...... . . . .. . .
CA 022~279~ 1998-10-27 molecule can be: a) single stranded or double stranded; b) linear or covalently closed circular in supe~ coiled or relaxed form; or c) RNA if combined with reverse transcription to generate a cDNA. For example, reverse transcription can be pc.ru~",cd using asuitable reverse transcriptase (e.g. Moloney murine leuk~mi~ virus reverse l,~nsc-i~lase) 5 using standard techniques (e.g. Kawasaki ( 1990) in PCR Protocols: A Gu~de to Methods and Applications, Ir~is et al., eds., ~c~dçmic Press, Berkeley, CA pp21-27).
Sources of nucleic acid t~ pl~es can include: a) pl~mi~is (naturally occurring or reco",bin~,l); b) RNA- or DNA- viruses and bacteriophages (naturally 10 occurring or recombil1a--~); c) chromosomal or episo-"al rep!ir~ting DNA (e.g. from tissue, a blood sample, or a biopsy); d) a nucleic acid fragment (e.g. derived by exonuclease, unspeeific endonuclease or restriction endonuclease digestion or by physical disruption (e.g. sonication or nebulization)); and e) RNA or RNA transcripts like mRNAs.
The nucleic acid to be ~mplified and sequenced can be oblained from virtually any biologic~l sample. As used herein, the term "biological sample" refers to any material obtained from any living source (e.g. human, animal, plant, bacteria, fungi, protist, virus). E~".~les of approp. ;ate biological samples for use in the instant 20 invention include: solid materials (e.g tissue, cell pellets, biopsies) and biological fluids (e.g. urine, blood, saliva, amniotic fluid, mouth wash, spinal fluid). The nucleic acid to be amplified and sequenced can be provided by unpurified whole cells, bacteria or virus.
Alternatively, the nucleic acid can first be purified from a sample using standard techniques, such as: a) cesium chloride gradient centrifi.~tiQn; b) alkaline Iysis with or 2S without RNAse lle<.~ c) ion exchange chlo.~ o~raphy; d) phenol/chlolo~ull.l extraction; e) isolation by hybridization to bound oligonucleotides; f) gel electrophoresis and elution; alcohol precipitation and h) co-..l,inaLions of the above.
As used herein, the phrases "chain-elongating nucleotides" and "chain-30 termin~ting nucleotides" are used in accordance with their art recognized m~ning Fort;~a"lple, for DNA, chain-elongating nucleotides include 2'-deoxyribonucleotides (e.g.
dATP, dCTP, dGTP and dTTP) and chain-te~ in~ g nucleotides include 2', 3'-dideoxyribonucleotides (e.g. ddATP, ddCTP, ddGTP, ddTTP). For RNA, chain-elongating nucleotides include ribonucleotides (e.g., ATP, CTP, GTP and UTP) andchain-te,...;~ nllcleotitles include 3'-deoxyribonucleotides (e.g. 3'dA, 3'dC, 3'dG and 5 3'dU). A complete set of chain elongating nucleotides refers to dATP, dCTP, dGl P and dTTP. The term "nucleotide" is also well known in the art. For the purposes of this invention, nucleotides include nucleoside mono-, di-, and triphosphates. Nucleotides also include modified nucleotides such as phosphorothioate nucleotides and deazapurine nucleotides. A complete set of chain-elongating nucleotides refers to four di~re"l 10 nucleotides that can hybridize to each of the four di~relll bases comprising the DNA
template.
If the ~mrlified seque1 çing ladders are to be detected by mass specllul~cLlic analysis, it may be useful to "condition" nucleic acid molçcllles, for 15 example to decrease the laser energy required for vol~ti7~tion and/or to ...;~
fragm~nl~l;on. Condiliûl~ing is preferably pc.r llllcd while the sequ~ncing ladders are immobilized. An e,.~.llple of conditioning is modification of the phos~horli~cter bac~hon~
ofthe nucleic acid molecule (e.g. cation eyrh~nee)7 which can be useful for çl;~il~ p peak brol~ening due to a heterogeneity in the cations bound per nucleotide unit.20 Cont~cting a nucleic acid molecule, which conlains an -thio-nucleoside-triphosph~te during pol~meli~lion with an alkylating agent such as akyliodide, iodoacet~m;de, -iodoethanol, or 2,3-epoxy-1-propanol, the monothio phosphodie~ter bonds of a nucleic acid molecule can be tran~Çull.,ed into a phosphotriester bond. Further condition;~
involves incorporating nucleotides which reduce sensitivity for depurination 25 (fr~g...~ ion during MS), e.g. a purine analog such as N7- or N9-dea_apurine nucleotides, and partial RNA con~ e oligodeoxynucleotide to be able to remove the unrnodified primer from the amplified and modified sequ~n~ g ladders by RNAse orine lle~ In DNA sequçnlin~ using fluoresce.lt cletection and ~ ~1 electrophoretic sepa.~lion, the N7 de~7.~rl-rine nucleotides reduce the formation of 30 secondary structure resulting in band coll.~)fess;on from which no sequ~ncing ;I~l..lation can be generated.
CA 022~279~ 1998-10-27 Critical to the novel process of the invention is the use of approp-iale amounts of two di~le"l polymerase c~ lles, each having a dil~lent affinity for the particular chain tel...;~ g nucleotide, so that pol~llllcl;,.alion by the enzyme with 5 relatively low affinity for the chain termin~ting nucleotide leads to amplification whereas the enzyme with relatively high affinity for the chain tel ..-in~ g nucleotide terminates the polymerization and yields sequen~ing products. PYererably about 0.5 to about 3 units of polymerase is used in the combined amplification and chain te,llfinalion re a~tion Most preferably about 1 to 2 units is used. Particularly pl~lled polylneld~es for use in 10 conjunction with PCR or other thermal amplification process are thermostable polymerases, such as Taq DNA polymerase (Boehringer ~vl~nnh~im), AmpliTaq FS DNApolymerase (Perkin-Elmer), Deep Vent (exo ), Vent, Vent (exo-) and Deep Vent DNApolymerases (New Fngl~nd Biolabs), Thermo Sequenase (Allle~l,an~) or exo(-) Pseudococcusfuriosus (Pfu) DNA poly"lc.asc (Stratagene, Heidelberg Germany).
15 AmpliTaq, Ultman, 9 degree Nm, Tth, Hot Tub, and Pyrococcus furiosus. In addition, preferably the polymerase does not have 5'-3' PYonllcle~se activity.
As sho~,vn in Example 1, the process of the invention can be carried out using AmpliTaq FS DNA polymerase (Perkin-Elmer), which has a relatively high affinity 20 and Taq DNA polymerase, which has a relatively low affinity for chain terrnin~ting nucleotides. Other appropriate polymerase pairs for use in the instant invention can be determined by one of skill in the art. (See e.g. S. Tabor and C.C. Richardsoll (1995) Proc. Na~. Acad. Sci. (USA), vol. 92: Pp. 6339-6343.) In addition to polymerases, which have a relatively high and a relatively low affinity to the chain t~ g nucleotide, a third polymerase, which has proofreading capacity (e.g. Pyrococcus woesei (Pwo)) DNA polymerase may also be added to the amplification mixture to enhance the fidelity of amplification.
Oligonucleotide primers, for use in the invention, can be desi~ed based on knowledge of the 5' and/or 3' regions of the nucleotide seq~lence to be CA 022~279~ 1998-10-27 WO 97/42348 PCT/US97107g66 amplified and sequenced, e.g., insert fl~r~ ng regions of cloning and sequenringvectors (such as M13, pUC, phagemid, cosmid). Optionally, at least one primer used in the chain extension and te,..~nalion reaction can be linked to a solid support to f~çilit~te purification of amplified product from primers and other react~nts, thereby S increasing yield or to separate the Sanger ladders from the sense and A~ n.~e template strand where ~imult~neous amplification-sequ~nring of both a sense and ~nti~e.n~e strand ofthe template DNA has been pelÇoll"cd.
Examples of appropliate solid supports include beads (silica gel, 10 controlled pore glass, m~gnetic beads, Seph~dçY/Sepharose beads, cçll-llose beads, etc.), capillaries, flat supports such as glass fiber filters, glass surfaces, metal surfaces (steel, gold, silver, ~ minllm, and copper), plastic materials or ,.le.llb.~.nes(polyethylene, polypropylene, polyamide, polyvinylide~-e~1ln.1oride) or beads in pits of flat surfaces such as wafers (e.g. silicon wafers), with or without filter plates.
Immobilization can be accomplished, for ~,,.a",plc, based on hybridization between a capture nucleic acid sequence, which has already been immobilized to the support and a co",pl~,l..e,~la.y nucleic acid sequence, which is also coll~ained within the nucleic acid molecule CGI-~ ~ the nucleic acid sequçnce to be 20 detecte~ So that hybrir~i7~tion between the col"ple .~F n~ r nucleic acid mnl e-nlles is not hindered by the support, the capture nucleic acid can include a spacer region of at least about five nucleotides in length between the solid support and the capture nucleic acid seqUçnce The duplex formed will be cleaved under the inflll~nee of the laser pulse and desorption can be initi~ted. The solid support-bound base sequence can be 25 presented through natural oligoribo- or oligodeoxylibo- nucleotide as well as analogs (e.g. thio-modified phosphndiester or phosphotriester backbone) or employing oligonucleotide mimetics such as PNA analogs (see e.g. Nielsen et al., Science, 254, 1497 (1991)) which render the base sequence less susceptible to enzymatic degradation and hence h~c".ases overall stability of the solid support-bound capture 30 base sequçnce CA 022~279~ 1998-10-27 WO 97/42348 PCTIUS97/0~966 Alternatively, a target detection site can be directly linked to a solid support via a reversible or irreversible bond between an appr~pliate functionality (L') on the target nucleic acid molecule and an applopliate filnctionqlity (L) on the capture molecule. A reversible linkage can be such that it is cleaved under the conditions of 5 mass spectrometry (i.e., a photocleavable bond such as a trityl ether bond or a charge transfer comrleY or a labile bond being formed between relatively stable organicradicals). Furthermore, the linkage can be formed with L' being a quaternary ammonium group, in which case, preferably, the surface of the solid support carries negative charges which repel the negatively charged nucleic acid backbone and thus 10 fq~ilitq~te the desorption required for analysis by a mass spe~ ,r.leler. Desorption can occur either by the heat created by the laser pulse and/or, depending on L,' by specific absorption of laser energy which is in resonance with the L' chromophore.
By way of ~ lllple, the L-L' çh~ni~try can be of a type of rliculfide 15 bond (chemically cleavable, for example, by lllercaploethanol or dithioerythrol), a biotin/streptavidin system, a heterobifunctional derivative of a trityl ether group (Koster et al., "A Versatile Acid-Labile Linker for Mo-lific~qtiQn of Synthetic Biomolecules," Tetrahedron Letters 31, 7095 (1990)) which can be cleaved under mild}y acidic conditions as well as under conriitiQnC of mass spec~ou~etry, a levulinyl 20 group cleavable under almost neutral con.~itions with a hydrq.7ini~lm/acetate buffer, an arginine-arginine or Iysine-lysine bond cleavable by an endopeptidase enzyme like trypsin or a pyrophosphate bond cleavable by a pyrophosphqtq~e or a ribonucleotide in between a deoxynucleotide sequence cleavable by an RNAse or alkali.
The functionalities, L and L,' can also form a charge llallsrer complex and thereby form the telllpol~ly L-L' linkage. Since in many cases the "charge-transfer band" can be determined by W/vis spectrometry (see e.g. Or~-q-ni~ Char~e Transfer Complexes by R. Foster, .Acq~de.mic Press, 1969), the laser energy can be tuned to the corresponding energy of the charge-lldnsrer wavelength and, thus, aspecific desorption offthe solid support can be initi-q~ted Those skilled in the art will recognize that several combinations can serve this purpose and that the donor .. .. ... .
CA 022~279~ 1998-10-27 functionality can be either on the solid support or coupled to the nucleic acid molecule to be detected or vice versa.
In yet another approach, a reversible L-L' linkage can be generated by 5 homolytically forming relatively stable radicals. Under the inflll~nce of the laser pulse, desorption (as ~ cussed above) as well as ionization will take place at the radical position. Those skilled in the art will recognize that other organic radicals can be selected and that, in relation to the dissociation energies needed to homolytically cleave the bond between them, a corresponding laser wavelength can be sPlected (see 10 e.g. Reactive Molecules by C. Wentrup, John Wiley & Sons, 1984).
An anchoring fimction L' can also be incorporated into a target capturing sequence by using appropliate primers during an amplification procedure, such as PCR, LCR or transcription amplification.
For certain applications, it may be useful to Qim~lt~neQusly amplify and chain terminate more than one (mutAted) loci on a particular captured nucleic acid fragment (on one spot of an array) or it may be useful to pelru---- parallel processi~ by using oli~on--cleotide or oligonucleotide mimetic arrays on various solid supports.
20 "Multiplexing" can be achieved either by the sequence itself (composition or length) or by the introduction of mass-modifying fi~nction~lities into the primer oligonucleotide. Such multiplexing is particularly useful in conjunction with mass spe-;l.on-cl,ic DNAsequrncing or mobility modified gel based fluolescence sequ~nr.in~
Without limiting the scope of the invention, the mass or mobility mo~ification can be introduced by using oligo-/polyethylene glycol derivatives. The oligo/polyethylene glycols can also be monoAlkylated by a lower alkyl such as methyl, ethyl, propyl, isopropyl, t-butyl and the like. Other cl-e~ ies can be used in the mass-modified compounds, as for example, those described recently in Olu~on..~leotides ~n-l 30 AnaloPues, A P~ .Al Approach, F. Fckctein, editor IRL Press, Oxford, 1991.
CA 022~279~ 1998-10-27 In yet another embodiment, various mass or mobility modifying fimctionalities, other than oligo/polyethylene glycolst can be selected and att~.h~d via apprupliate linking ch~mie~ries A simple modification can be achieved by using di~.enl alkyl, aryl or aralkyl moieties such as methyl, ethyl, propyl, isoplol.yl, t-butyl, hexyl, phenyl, substituted phenyl or benzyl. Yet another modification can be obtained by att~rhin~ homo- or heteropeptides to the nucleic acid molecule (e.g., primer) ornucleoside triphosphates. Simple olig~mides also can be used. Numerous other possibilities, in addition to those mentioned above, can be performed by one skilled in the art.
Different mass or mobility modified primers allow for multiplex sequçn~.in~ via sim~llt~neQus detection of primer-modified Sanger sequ~nrin~ ladders.
Mass or mobility modifications can be incorporated during the amplification process through nucleoside triphosphates or modified primers.
For use with certain detection means, such as polyacrylamide gel electrophoresis (PAGE), detect~hle labels must be used in either the primer (typically at the 5'-end) or in one of the chain e~rten-lin~ nucleotides, or chain termi~ting nucleotides.
Using radioisotopes such as 32p, 33p, or 35S iS still the most frequently used teçhniq~le.
After PAGE, the gels are exposed to X-ray films and silver grain exposure is analyzed.
Non-radioactive labeling techniques have been explored and, in recent years, integrated into partly ~ltom~ed DNA seqllen( ir~ procedures. All these improvements utilize the Sanger sequencing strategy. The label (e.g. fluorescenl dye) can be tagged to the primer (Smith et aL, Nature ~1, 674-679 (1986) and EPO
Patent No. 87300998.9; Du Pont De Nemours EPO Application No. 0359225;
Ansorge et al. J. Biochem. Biophys. Methods 13, 325-32 (1986)) or to the chain-tel..~ g dideoxynucloside triphosphates (Prober et aL Science ~, 336-41 (1987);
Applied Biosystems, PCT Application WO 91/05060). Based on either labeling the 30 primer or the ddNTP, systems have been developed by Applied Biosystems (Smith et al., Science ~, G89 (1987); U.S. Patent Nos. 570973 and 689013), Du Pont De , . .... , .. .. ,.. , . ~
Nemours (Proberetal. Science~, 336-341 (1987); U.S. PatentsNos. 881372 and 57566), Pharmacia-LKB (Ansorge et al. Nucleic Acids Res. 15, 4593-4602 (1987) and EMBL Patent Application DE P3724442 and P3805808.1) and Hitachi (JP 1-90844 and DE 4011991 A1). A somewhat similar approach was developed by S Brnmb~ugh e~ al. (Proc. Natl. Sci. USA 85, 5610-14 (1988) and U.S. Patent No.
4,729,947). An improved method for the Du Pont system using two clecl.ophol~lic lanes with two dif~lenL specific labels per lane is described (PCT Application W092/02635). A di~lenl approach uses fluorescelllly labeled avidin and biotin labeled primers. Here, the sequencing ladders ending with biotin are reacted during electrophoresis with the labeled avidin which results in the detection ofthe individual sequçnring bands (Br~.mb~lgh et al, U.s. Patent No. 594676).
More recently even more sensitive non-r~~ioac.tive labeling techniques for DNA using ~h~milllminescence triggerable and amplifyable by enzymes have been developed (Beck, O~eefe, Coull and Koster, Nucleic Acids Res. 17, 5115-5123 (1989) and Beck and Koster, An~l. Ch~ , 2258-2270 (1990)). These labeling methods were co---bined with multiplex DNA seq~lenring (Church et al. Scien~ 240, 185-188 (1988) and direct blotting electrophoresis (DBE) (Beck and Pohl, EMBO J
Vol. 3: p 2905-2909 (1984)) to provide for a strategy aimed at high throughput DNA
sequen~ing (Koster et al., Nucleic Acids Res. Symposium Ser. No. 24, 318-321 (1991), University of Utah, PCT Application No. WO 90/15883). However, this strategy still suffers from the disadvantage of being very laborious and difficult to automate.
In a.llon-aled sequ~on~ fluo. csce~ce labeled DNA fM~nent~ are cletected during migration through the seq-len~in~ gel by laser excitation.
Fluorescence label is incorporated during the sequ~nçing reaction via labeled primers or chain e~t~n-1ing nucleotides (Smith, L. et. al., Fluorescence detection in automated DNA seq-l~nce analysis, Nature 321:674-89 1986), (Knight, P., Automated DNA
sequencers, Biotechnology 6: 1095-96 1988).
.
CA 022~279~ 1998-10-27 Multiple ~ clly labeled primers can be used to dis~" s~e sequencing patterns. For example, four differently labeled sequenrin~ primers specific for the single termination reactions, e.g. with fluorescent dyes and online detection using laser excitation in an automated sequençin~ device. The use of eight Ji~. elllly labeled S primers allow the disc-il.l,nalion ofthe seq~lencin~ pattern from both strands. Instead of labeled primers, labeled ddNTP may be used for detection, if separation of the sequencing ~a~ment~ derived from both strand is provided. With one biotin labeled primer, sequçn~in~ fr~mPnts from one strand can be isolated for example via biotin-streptavidin coated magnetic beads. Possible is also the isolation via imm~lnoaffinity 10 chromatography in the case of a digoxigenin labeled primer or with affinity chromatography in case of complementary oligonucleotides bound to a solid support.
Another aspect of this invention concerns kits for directly generating from a nucleic acid template, amplified base specifically te..~ ed fragments. Such 15 kits include co-nl~ a~ions ofthe above-described re~ct~nts For in~ cç~ in oneembodiment, the kit can comprise: i) a set of chain-elongaliilg nucleotides; ii) a set of chain-tel...;.~ ;..g nucleotides; and (iii) a first DNA polymerase, which has a relatively low affinity towards the chain tt-~ n~ g nucleotide; and (iv) a second DNA
polymerase, which has a relatively high affinity towards the chain te~ p 20 nucleotide. The kit can also include appropriate solid supports for capture/purification and buffers as well as instructions for use.
The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited 25 references (in~.lu~iin~ literature references, issued patents, published patent applications (inclu~lin~ international patent application Publication Number WO
94/16101, entitled DNA Sequencing byMass Spectrometry by H. Koester; and international patent application Publication Number WO 94/21822 entitled "DNA
Sequ~ncing by Mass Spe.;l.ol.letry Via Exonuclease Degradation" by H. Koster), and - 30 co-pending patent applications, (incluclinp U.S Patent Application Serial No.
08/406,199, entitled DNA Diagnostics Based on Mass Spectromet7y by H. Koester), , CA 022~279~ 1998-10-27 and continuations-in-part of co-pending patent applications (published or unpublished) are hereby express}y incorporated by l ercrence.
Example 1: Direct Nucleic Acid Amp'ifi~ion and Sequencing s Materials and Metho~
Bacterial Strains and Plasmids E. coli K12 strains HB101 and XL1-Blue (Str~tag~n~, California) were grown in LB broth (Miller, J. (1972) Expe,imenls in Molecular G~ne~ics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.) suppl~mente~l with 100 g ~mpicillin/mL
(Binotal, Bayer, Germany). DNA-template used for sequçnr.ing was a pOM8 derived reco",binalll plasmid (Oberbaumer, I. (1986) Gene 49: 81-91 1986) with an 400 BPinsert from the rrnB gene of E. coli. The plasmid DNA was purified via Diagen plasmid purification tips-100 (Diagen, Hilden, Germany).
DNA Isolation The DNA template used in the sequencing reaction was isolated according to the method described by Birnboim and Doly (Birnboim, H.C., and Doly, J. Nucleic Acid Res. 7: 1513-1523 1979) with the improvements of Ish-Horowiczs and Burke (Ish-Horowicz, D., and Burke, J.F. Nucleic Acid Res. 9: 2989-2998 1981).
Sequencing reac~ions A control sequencinE reaction was pc.~-l.cd in accordance with the Perkin-Elmer, AmpliTaq FS kit protocol.
A combined nucleic acid amplification and sequencing reaction was performed as follows. The base specific reaction mixtures (A, C, G and T, as described below) each conlained a buffered solution of: i) DNA template; ii) two thermostrl le DNA polymerases, each with a di~rerc..l affinity to the ddNTP; iii) the ddNTP; iv) all 4 30 dNTPs (Pharmacia, Freiburg, Germany); a pair of sequ~nce specific oligonucleotide primers, one of them labeled with a fluorcsce--l dye. The reaction mixture was overlayed with wax.
.. ... . . . . . .
CA 022~279~ 1998-10-27 WO 97/42348 PCT/US97tO7966 The sequencin~ reaction was calTied out as follows:
A-reaction: 1 8 M ddATP, 80 M each dATP, dCTP, 7-deaza-dGTP, dTTP, 500 mM Tris-HCl (pH 9.0 at 25~C), 25 mM MgCI2 co.. ~ g lpmol JOE dye primer, thermal stable pyrophosphatase and 1.6 U AmpliTaq DNA polymerase, FS. Tothis 50 ng of the DNA template as pl epa. ed above (1 1), 1 U of Taq DNA polymerase (1 I) and 10 pmol reverse primer (0.5 1) were pipetted.
C-reaction: 4 1 8 M ddCTP, 80 M each dATP, dCTP, 7-deaza-dGTP, dTTP, 500 mM Tris-HC1 (pH 9.0 at 25~C), 25 mM MgCI2 co..~ g lpmol FAM dye primer, thermal stable pyrophosphatase and 1.6 U AmpliTaq DNA polymerase, FS. Tothis50ngoftheDNAtemplate(l 1), 1 UofTaqDNApolymerase(l I)and 10pmol reverse primer (0.5 1) were pipetted.
G-reactior~: 8 1 8 M ddGTP, 80 M each dATP, dCTP, 7-deaza-dGTP, dTTP, 500 mM Tris-HCI (pH 9.0 at 25~C) 25mM MgC12 C~ ,g 1 pmol TAMRA dye primer, thermal stable pyrophosphatase and 1.6 U AmpliTaq DNA polymerase, FS. Tothis 50 ng of the DNA template (1 1), 1 U of Taq DNA polymerase (1 1) and 10 pmol reverse primer (0.5 1) were pipetted.
T-reaction: 8 1 8 M ddTTP, 80 M each dATP, dCTP, 7-deaza-dGTP, dTTP, 500 mM Tris-HCl (pH 9.0 at 25~C), 25mM MgCI2 cont~ining 1 pmol ROX dye primer, thermal stable pyrophl-sph~t~se and 1.6 U AmpliTaq DNA polymerase, FS. To this 50 ng of the DNA t~n~pl?te (1 1), 1 U of Taq DNA polymerase (1 1) and 10 pmol reverse primer (0.5 1) were pipetted.
The incubation conditions inr.lllded an initial denaturation step of 4 min.
95~C, followed by 15 cycles of 30 sec. 95~C, 30 sec. 52~C, 60 sec. ?2~C. The reaction is completed by additional 15 cycles of 30 sec. 95~C, 30 sec. 52~C, 60 sec. 72~C.
The reaction mixture was separated from the wax by pipetting and ethanol precipitated. The samples were run on an ~--tom~tçd ABI prism sequencer model 377.
As can be seen from Figures 1 and 2, while the combined amplification and sequ~ncing reaction yielded a 440 BP readable sequence with 15 ng. of template DNA (Figure 1), no sequence data was obtained employing the same amount oftemplate 10 DNA and using the standard cycle sequencing protocol (Figure 2).
Example 2: Direct Nucleic Acid Amplification and Sequencing A combined nucleic acid amplification and sequencing reaction was performed as follows. The reaction contained a buffered solution of i) DNA template, 15 ii) two thermnst~hle DNA polylllel ~s~ s, each with a d~i enl affinity for the ddN~P; iii) dye-labeled ddNTP; iv) all 4 dNTP s (Pha~l.lacia, Freiburg, C~ .m~ly); a pair of sequence specific oligonucleotide primers, one of them labeled with a biotin group.
Combined amplifica~on and sequencing protocol using two DNA
pol~,,,.~l QS~S.
4ng (1 ~11) of p9G551D (pUC-derivative harbouring a 419 BP insert from human mutant CFTR gene) was subjected to seq~len~ by adding 4 ~,11 of 5 x TACS
buffer, 0.5 ~l of each dye ddNTP solution, 1 ~l dNTP mix (5mM dATP, dCTP, dTTP
and 10 mM N' deaza dGTP), I ,ul AmpliTaq FS, 0.75 U (0.3 ~11) (exo-) PJi( polymerase 25 and 40 pmol of each of the standard Ml 3 primers (for sequence see above) to a final volume of 20 ,LI. The cycling profile comprised one cycle of 95 ~C for 4 min., 20 cycles of 95 ~C for 45 sec, 52~C for 45 sec and 72~C for 45 sec. followed by 28 cycles of 96~C
for 50 sec, 55~C for 60 sec and 60~C for 4 min. The reaction was carried out using an EppendorfMastercycler 5330 with a heated lid.
Separation of the forward and reverse sequenc7ngJ~, ~.u nt~.
CA 022~279~ 1998-10-27 The reaction was mixed with an equal volume of 2x B/W-buffer (lOmM
Tris-HCl, pH 7.5, lmM EDTA, 2 M NaCl) cont~inin~ 300 ~lg of prewashed streptavidin M -280 Dynabeads (Dynal). After inc~lbation for 30 min. at room temperature the beads were washed twice with 50 ~,11 of lx B/W buffer in order to remove unwanted reaction 5 components and unincorporated dye terminators.
The reverse seqllencin~ products were recovered from immobilized biotinylated forward products by addition of 20 ~1 of a 20 % aqueouc ~m m onillmhydroxide solution (freshly prepared) and incub~tion at room telllpeldlllre for two 10 minutes. This step was repeated once the products were pooled.
The beads were then washed with 20 11l 25 % aqueous ammonium hydroxide solution for one minute at room te~")e, ~ re and the supernatant was discarded.
The forward seql~çnring reaction was removed from the beads using 30 ~11 of a 25% aqueous ammonium hydroxide solution and incubation at 60~C for 8 minlltçe This step was also repeated once and the products were pooled.
The separated forward and reverse sequrnçing reactions were finally precipiLaled each by addition of 200 ~ll ethanol to the a ....,o~ lm hydroxide solution, resuspended in 4 1ll loading dye (5 :1 ratio of deionized fo, .. .~. . .ide to 25mM EDTA (pH
8.0) /50 mg/ml Blue dextran) and analyzed with an ABI Prism sequencer (model 377).
RESULTS
Figure 4 and 5 depict the fluorescent chronlalogram plots of a forward (Fig. 4) and reverse (Fig. 5) sequence ladder generated in a siml~lt~neous amplification and seqUçnring reaction. In this experiment 1.5 units of Taq polymerase were added to a 30 dye terminator cycle seqllçnring mix cont~ininp AmpliTaq FS, and only 4 ng of plasmid DNA, a biotinylated fo, vvald primer and a reverse primer. A~er the cycling reaction, CA 022~279~ 1998-10-27 biotinylated fo- w~rd sequçncing products and reverse products hybridized to the forward tçmp!~te were immobilized on streptavidin coated m~netic beads. A separation of reverse and forward seql1çnl~.in~ ladders was achieved by incub~tion of the beads with ammonium hydroxide. Ammonium hydroxide acted as a denaturing agent for double 5 stranded DNA at room temperature, therefore r~le~cing the reverse sequçnr.ir.g products in the solution. The biotinylated products remained bound to the streptavidin, whereas the reverse products were removed with the supe,llata.,l. A recovery ofthe biotinylated products was carried out using arnmonium hydroxide at an incllb~tion temperature of 60~C. At elevated te"-pe~al-lre, ammonium hydroxide acted as a denaturing agent for 10 the streptavidin biotin interaction. The thus separated sequçn~ing ladders were then ethanol precipitated and separately analyzed on a ABI Prism sequencer.
CA 022~279~ 1998-10-27 F.quivalell~
Those skilled in the art will recognize or be able to ascertain using no more than routine e.~pe,;,~nlnl;on numerous equivalents to the specific procedures 5 described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.
5 Back~round of the Inv~ntio ..
DNA .S~q n"~i,.,~
Current knowledge I egal di~g gene structure, the control of gene activity and the function of cells on a molecular level all arose based on the determination of the 10 base sequence of millions of DNA molecules. DNA seq~len~in~ is still criticaJly important in research and for genetic the. apics and di~ostiçs, (e.g., to verify~ecol..b:nant clones and mutations).
DNA, a polyrner of deoxyribon~ eotides, is found in all living cells and 15 some viruses. DNA is the carrier of genetic info.-na~ion, which is passed from one gene.iltion to the next by homologous replication ofthe DNA molecule. Illru~ lion for the synthesis of all proteins is encoded in the seqUçnce of bases in the DNA.
To obtain the genetic information and therefore to reveal the base 20 sequence of a given DNA molecule, çh~m;c~l and el~malic seq~lçncin~ metho~s have been developed. DNA-sequ~n~.ing as proposed by Maxam-Gilbert (Maxam, A.M., W.
Gilbert, Proc. Natl. Acad. Sci. USA, 74:560-564 1977) is a chemical method of delel,nilf..lg base composition of a nucleic acid mole ~ e A single stranded DNAmolecule with ra~ioaGtive label at its 5' end is r.hçmi~lly modified in four base specific reactions and then cleaved at the modified positionC The cleavage products are sepa~ted on a polyacrylamide gel and typically are detected by autoradiography.
Currently favored is the el~yll,alic chain t~ ;oll reaction accolding to the Sanger-sequ~n~.ing method (Sanger, F. et. al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 1977). In the Sanger method, the four base specific sets of DNA fr~em~nts are - formed by starting with a p.;",el1lemrl~te system elong~ling the primer into the unknown DNA sequence area and thereby copying the t~mpl~te and s~ l.f.ci~ complçm~nt~ry strands using a DNA polymerase in the presence of chain-te....;.~ r~aef nlc The ~ . . ~ .
CA 022~279~ 1998-10-27 chain-tel,.,i.-~';,~g event is achieved by incorporating into the four separate reaction mixtures in addition to the four normal deoxynucleoside triphosphates, dATP, dGTP, dTTP and dCTP, only one ofthe chain-term~ tin~ dideoxyn.~cleosi~le triphosphates, ddATP, ddGTP, ddTTP or ddCTP, respectively, in a limiting small concentration. The incorporation of a ddNlP lacking the 3' hydroxyl function into the growing DNA strand by the enzyme DNA polymerase leads to chain termination through preventing the formation of a 3'-5'-phosphodiester bond by DNA polymerase. Due to the random incorporation ofthe ddNlPs, each reaction leads to a population of base specifictermin~ted fr~m~tlt~ of di~erent Iengths, which all together I epresenl the sequenced DNA-molecule.
A recent modification of the Sanger sequencing strategy involves the degradation of phosphorothioate-con~inine DNA fr~m~nts oblained by using alpha-thio dNTP instead of the normally used ddNTPs during the primer extension reaction medi~ted by DNA polymerase (Labeit et al., DNA 5, 173-177 (1986); Amel~llalll, PCT-Application GB86/00349; F.c~tein et al., Nucleic Acids Res. 16, 9947 (1988)).
Here, the four sets of base-specific sequencing ladders are obtained by limited digestion with exonuclease III or snake venom phosphodiesterase, subsequent separation on PAGE and vi.cll~li7~tion by radioisotopic labeling of either the primer or one of the dNTPs. In a further modification, the base-specific cleavage is achieved by alkylating the sulphur atom in the modified phosphodiester bond followed by a heat tre.~tment (Max-Planck-Gesçll~ch~, DE 3930312 Al).
DNA Amplification 2S DNA can be amplified by a variety of procedures insl~tlin~ cloning (Sambrook et al., Molecular Cloning: A ~aboratory Manual, Cold Spling Harbor Laboratory Press, 1989), polymerase chain reaction (PCR) (C.R. Newton and A.
Graham, PCR, BIOS Publishers, 1994), ligase chain reaction (LCR) (F. Barany Proc.
Na~l. Aca~ Sci USA 88, 189-93 (1991), strand ~i~pl;~cement amplification (SDA) (G.
Terrance Walker et al., Nucleic Acids Res. 22, 2670-77 (1994)) and variations such as RT-PCR, allele-specific amplification (ASA) etc.
CA 022~279~ 1998-10-27 The polymerase chain reaction (Mullis, K. et al., Methods Enzymol., 155:335-350 1987) permits the selective in vitro amplification of a particular DNA region by mimic~in~ the phenomena of in vivo DNA replicvtiQn Required reaction components S are single stranded DNA, primers (oligo~ cleotide sequences complemf nt~ry to the 5' and 3' ends of a defined sequence of the DNA template), deoxyn..c~eotidetriphosphates and a DNA polymerase enzyme. Typically, the single stranded DNA is generated by heat denaturation of provided double strand DNA. The reaction buffers contain m~gn~; .m ions and co-solvents for optimum enzyme stability and activity.
The amplification results from a repetition of such cycles in the following manner: The two di~rel~t primers, which bind selectively each to one of the comple .~ . y strands, are pytcn~led in the first cycle of amp!ificAti~ n. Each newly synth~i7ed DNA then co~ nc a binding site for the other primer. Thc.erore each new 15 DNA strand becol"f s a templAte for any further cycle of amplification e nl~ng the tçmplqte pool from cycle to cycle. ~peqted cycles theoretically lead to exponential synthesis of a DNA-fragment with a length defined by the 5' termini of the primer.
Initial PCR expe,i,-,ti--Ls used thermolabile DNA polymerase. However, 20 thermolabile DNA polymerase must be continually added to the reaction mixture after each denaturation cycle. Major advances in PCR practice were the development of a polymerase, which is stable at the near-boiling te.,.pe~ alLlre (Saiki, R. et al., Science 239:487-491 1988) and the development of a~ltomqted thermal cyclers.
The discovery of thermostable polymerases also allowed modifir.q~ion ofthe Sanger sequçnring reaction with si~nifirqnt advAntA~es The pol~",e.~aLion reaction could be carried out at high tc."pe, ~ re with the use of thermostable DNA
polymerase in a cyclic manner (cycle sequçncing). The con~itions of the cycles are similar to those of the PCR technique and COlllpl ;Sf della~ul a~ion~ ,~qnne~'ing~ and extension steps. Depf ~-~ing on the length of the primers only one anneAlin~ step at the beg;~ g of the reaction may be sufficient. Carrying out a sequenring reaction at ... ... . .
CA 022~279~ 1998-10-27 high temperature in a cyclic manner provides the advantage that each DNA strand can serve as template in every new cycle of extension which reduces the amount of DNA
necçs~ry for sequçn~ing~ thereby providing access to minim~l volumes of DNA, as well as res -lting in improved specificity of primer hybriAi~tion at higher te"~l)e~al~lle 5 and the reduction of secondary structures of the tçmpl~te strand.
However, amplification ofthe te,.~ ted fr~ onts is linear in conventional cycle seqllenrine approaches. A recently developed method, called semi-exponential cycle seq~l~ncing shortens the time ~ hed and incr~,ar,es the extent 10 of amplification obtained from conventional cycle sequçn~.in~e by using a second reverse primer in the sequçn~ing reaction. However, the reverse primer only generates additional temrl~te strands if it avoids being te, ...;.~ d prior to reaching the sequencing primer binding site. Needless to say, tel ...;..~led fr~mçnt~ generated by the reverse primer can not serve as a sufficient tçmp!~te. Therefore, in practice, 15 amplification by the semi-exponential approach is not entirely exponential. (Sarkat, G.
and Bolander Mark E., Semi Exponential Cycle Sequ~nt ing Nucleic Acids Research,1995, Vol. 23, No. 7, p. 1269-1270).
As pointed out above, current nucleic acid sequPn~i~ methods require 20 relatively large ~mo~lnt~ (typically about 1 g) of highly purified DNA template. Often, however, only a small amount of templ~te DNA is available. Although amplifications may be pe,ro.,ned, amplification procedures are typically time con~1ming can be limited in the amount of amplified template produced and the amplified DNA must be purified prior to seq~lçn~i~ A stre~mlined process for amplifying and sequerlcing DNA is 25 needed, particularly to f~.ilit~te highthroughput nucleic acid sequP.ncin~
Summary of the Invention In general, the instant invention provides a one-step process for 30 generating from a DNA template, base spec.ific?lly termin~ted fragmçnts of sllfficient quantity to enable DNA sequenci~
CA 022~279~ 1998-10-27 According to the process of the invention, a combined amplification and tel., inalion reaction is pelroll.lcd using at least two di~ren~ polymerase enzymes, each having a dillerenL affinity for the chain tel..~ -g nucleotide, so that polymerization by 5 an enzyme with relatively low affinity for the chain ttl~ ;llg nucleotide leads to exponential amplification whereas an enzyme with relatively high afflnity for the chain termin~ting nucleotide terminates the polymerization and yields sequencing products.
In another aspect, the invention features kits for directly amplifying 10 nucleic acid t~mp!~tes and generating base specifically termin~ted rlng...~..ls In one embodiment, the kit can comprise an approp.;ate amount of: i) a colllpl.,le set of chain-elongating nucleotides; ii) at least one chain-tel ~ nucleotide; (iii) a first DNA
polymerase, which has a relatively low affinity towards the chain tel...;~ nucleotide;
and (iv) a second DNA polymerase, which has a relatively high affinity towards the chain 15 te~ nucleotide. The kit can also optionally include an applol)liate primer or primers, appropliate buffers as well as instructions for use.
The instant invention allows DNA amplification and tel~l inalion to be pelrollned in one reaction vessel. Due to the use of two polyl,lel~ses with dif~re.ll 20 affinities for dideoxy nucleotide triphosphates, exponential amplification of the target sequence can be accompli~hed in col--bil-~lion with a tel.l..nalion reaction nucleotide. In addition, the process obviates the purification procedures, which are required when amplification is l~-ru--"ed separately f~om base termin?ted ~agment gen~;lalion. Further, the instant process requires less time to accQmrlish than separate amrlific~tion and base 25 specific te-., ina~ion reactions.
When combil-ed with a detection means, the process can be used to detect and/ or qu~ntit~te a particular nucleic acid sequence where only small amounts of template are available and fast and accurate sequence data acquisition is desirable. For 30 example, when comhined with a detection means, the process is useful for seq~lencing unknown genes or other nucleic acid sequences and for ~ grlosing or monitoring certain CA 022~279~ 1998-10-27 dice~es or contlition~, such as genetic ~ice~ces, chromosomal abno~ ;es, geneticpredispositions to certain ~ice~ces (e.g. cancer, obesity, artherosclerosis) and pathogenic (e.g. bacterial, viral, fungal, protistal) infections. Further, when double stranded DNA
molecllles are used as the starting material, the instant process provides an opportunity to 5 simlllt~neously sequence both strands, thereby providing greater cel ~aillLy of the sequ~nce data obtained or acquiring sequence i~ alion from both ends of a longer tP.mpl~te.
The above and further features and advantages of the instant invention will become clearer from the following Detailed Description and Claims.
Brief Description of the Fi~g ~reS
FIGURE 1 shows sequence data from an ABI-Prism ~utomqted sequencer (Model 373A) using a pOM8 derived ~t;co...binalll plasmid with a 400 base pair (BP) insert from the rrnB gene of E. coli as the tçmp!nte in the reaction described in the following Example l. The figure shows a reliable sequence readable to about 440 BP
which is the length of the PCR product.
FIGURE 2 shows sequence data from an ABI-Prism n~tQIn~ted sequçncer (Model 373A) again using the pOM8 derived recombinant plasmid with a 400 BP insert of from the rrnB gene of E. coli. However, because the sequencing reaction was carried out using standard sequencing protocols on a small amount of template (50ng), noreliable sequence was obtained.
FIGURE 3 is a srhPm~tic of a co-l~bined ~rnrlific~ti~n and sequçnring reaction using two polymerases and dye-labeled dideoxynucleotide triphosphate for detection and two reverse oriented primers.
FIGURE 4 is a fluol escenl chromotagram plot of a forward sequence ladder generated in a sim~1lt~neous amplification and sequencing reaction.
FIGllRE 5 is a fluorescent chromatogram plot of a reverse sequence ladder generated in a sirnlllt~neous amplification and seql~ncing reaction.
W O 97/42348 PCT~US97/07966 Detailed Description of the Invention In general, the invention features a process for directly amplifying and base specifically te, ...;l.~ a nucleic acid molecule. Accolding to the process of the 5 invention, a co,nbined amplification and tel" i"a~ion reaction is pe,ro.-..cd on a nucleic acid template using: i) a complete set of chain-elon~,qtine nucleotides; ii) at least one chain-termin~ting nucleotide; and (iii) a first DNA polymerase, which has a relatively low affinity towards the chain tel...;.~ g nucleotide; and (iv) a second DNA poly"~clase, which has a relatively high affinity towards the chain terminqtin~ nucleotide, so that 10 poly",eli~alion by the enzyme with relatively low affinity for the chain te~...;..~li.~g nucleotide leads to amplification of the tçrnrlqt~, whereas the enzyme with relatively high affinity for the chain t~....;.~1;.-g nucleotide terminates the polymerization and yields sequçnclng products.
The combined amplification and sequçncing can be based on any amplification procedure that employs an enzyme with polynucleotide synthetic ability (e.g. polyn.erase). One p.ere"ed process, based on the polymerase chain reaction(PCR), is comprised ofthe following three thermal steps: 1) denaturing a double stranded (ds) DNA molecule at an approp-iate telllpc~al~lre and for an approp,iale period 20 of time to obtain the two single stranded (ss) DNA molecules (the template: sense and ~q.nti~en~e strand); 2) contqctin~ the template with at least one primer that hybridizes to at least one ss DNA template at an app, op,iate temperature and for an approp, ;ate period of time to obtain a primer co.-l ~q,;~ g ss DNA tçmrl~te; 3) cont~ctin~ the primer co~ ni.~ template at an approp,iate temperature and for an appJop,iate period oftime 25 with: (i) a comrlete set of chain elongating nucleotides, (ii) at least one chain te. ".;nAI ;~g nucleotide, (iii) a first DNA polymerase, which has a relatively low affinity towards the chain terminqtin~ nucleotide; and (iv) a second DNA polymerase, which has a relatively high affinity towards the chain terminqting nucleotide.
Steps 1)- 3) can be sequentially pe.ru.. ed for an app.op.ia~e number of times (cycles) to obtain the desired amount of amplified seqUçnri~ Iadders. The CA 022~279~ 1998-10-27 quantity ofthe base spec.ific~lly tt,..~ led rla~ l desired dictates how many cycles are performed. Altho~lgh an increased number of cycles results in an increased level of amplification, it may also detract from the sensitivity of a subsequent detection. It is therero~ generally undesirable to perform more than about S0 cycles, and is morepreferable to pelrolll' less than about 40 cycles (e.g. about 20-30 cycles).
In a p-ere"ed embodiment, the first denaturation step is ~e.rullned at a temperature in the range of about 85~C to about 100~C (most prerel~bly about 92~C to about 96~C) for about 20 seconds (s) to about 2 mimltes (most preferably about 30s- 1 10 minute). The second hybridization step is prefelably pe-~,llled at a tel-lptlalure, which is in the range of about 40~C to about 80~C (most prererably about 45~C to about 72~C) for about 20s to about 2 mimltes (most p-erel~bly about 30s- 1 minute). The third, primer eYt~n.~ion step is preferably pe.~.lned at about 65~C to about 80~C (mostpreferably about 70~C to about 74~C) for about 30 s to about 3 minutes (most pl~relably 15 about 1 to about 2 minlltes) In order to obtain sequence ;l~l ..-alion on both the sense and antisense strands of a DNA molecule ~imlllt~neously, each of the single stranded sense and~ntic~n~e templates generated from the denaturing step can be cont~cted with app-op-iate 20 primers in step 2), so that amplified and chain termin~ted nucleic acid msleclllçs generated in step 3), are co...~ A~y to both strands.
Another pl~re--ed process for sim-~l~t~neously amplifying and chain tellllinA~ a nucleic acid sequ~nce is based on strand tlicrl~cemçnt a...pl;l'lcalion (SDA) (G. Terrance Walker et al., Nucleic Acids Res. 22, 2670-77 ( 1994); European Patent Publication Number 0 684 315 entitled Strand Disp.'~rement Amplif cation Using Therrnophilic Enzymes ). In essence, this process involves the following three steps, which altogether comprise a cycle: 1) denaturing a double stranded (ds) DNA m~lec~lle cont~ining the seqllence to be amplified at an applop.iale te.npel~ re and for an approp. iate period of time to obtain the two single stranded (ss) DNA molecules (the template: sense and ~ spl~ee strand); 2) cont~cting the tçmrl~te with at least one primer CA 022~279~ 1998-10-27 WO 97/4~348 PCT/US97/07966 _ 9 (P), that cQ~ ;...c a recognition/cleavage site for a restriction end~ n--c~e~e (RE) and that hybridizes to at least one ss DNA temr!~te at an app~up.;ate te...~ L.Ire and for an apploplid~e period oftime to obtain a primer CO..~ ss DNA templ~te; 3) cont~ctine the primer co.ll~;,.;ng template at an applop,iale telll~)e,~ re and for an app-opliale 5 period of time with: (i) a col..pl~te set of chain çlong~ti~ nucleotides; (ii) at least one chain te~ nucleotide, (iii) a first DNA polymerase, which has a relatively low affinity towards the chain te....;l-~ -g nucleotide; (iv) a second DNA polymerase~ which has a relatively high affinity towards the chain tc....;~ ;ne nucleotide; and v) an RE that nicks the primer recognition/cleavage site.
Steps 1) - 3) can be sequentially performed for an applopliale number of times (cycles~ to obtain the desired amount of arnplified sequçnrin~ ladders. As with the PCR based process, the quantity of the base specifically ttl ...;I-~ted fragment desired dictates how many cycles are pelrolllled. Plerelably, less than 50 cycles, more preferably less than about 40 cycles and most preferably about 20 to 30 cycles are pelrulllled.
The amplified sequçnr~ing ladders obtained as described above, can be separated and detected and/or ql~ntitated using well established methods, such as polyacrylamide gel ele~ opholèsis (PAGE), or capillary zone electrophoresis (CZE) (Jorgensonetal., J. Chrom~toyr~hy~, 337 (1986); Gestel~nd etal., Nucleic Acids Res. 18, 1415-1419 (1990)); or direct blotting electrophoresis (~BE) (Beck and Pohl, E~IBO J., vol. 3: Pp. 2905-2909 (1984)) in conjunction with, forexample, colorimetry, fluoli.,l.,lly, c~mil-lminescrnce and radioactivity.
Dye-terminator r~ try can be employed in the combined amplification and sequçncine reaction to enable the simultaneous generation of rul vvald and reverse sequence ladders, which can be separated based on the streptavidin-biotin system when one biotinylated primer is provided.
Figure 3 depicts a scheme for the co-nbined amplification and sequencing using two polymerases and dye-labeled chain tel ~ e nucleotide (ddNTP) for detection and two reverse oriented primers. A means of separation for the s~ t~neously generated forward and reverse sequence ladders is shown. Step A
represents the exponential amplification of a target sequence by the polymerase with a low affinity for ddNTPs. One ofthe sequence specific oligonl~cleotide primers isbiotinylated. Step B, ~prese,~ls the gcnc~ ~lion of a sequence ladder either from the S original template or the s;-nllh~neously generated arnrlific~tiQn product carried out by the polymerase with a high affinity for ddNTPs. After cc ~ on of the ~ tiQn, the products are incubated with a streptavidin coated solid support (Step C). Biotinylated forward sequenring products and reverse products hybridized to the r(" v~ralJ templâte are immobilized. In order to obtain readable sequ~.nce info"~-alion, the rolv~ald and 10 reverse sequence ladders are separated in Step D. The imm--bili7ed strands are washed and separated by denaturation with a",.,.ol-ium hydroxide at room temperature. The non-biotinylated reverse sequenrin~ products are removed from the beads with ammonium hydroxide sup~l ~,alanl during this procedure. The biotinylated forwardseq~lçnring products remain immobilized to the beads and are re-solubili7ed with15 ammonium hydroxide at 60~C. After ethanol prtc;y;lalion, both sequencing species can be resuspended in loading dye and run on an automated sequencer, for example.
When mass ~pe~ o",et~ y is used in conjunction with the direct ~mplific~tion and chain t~""-nalion processes, the sequPnci~ ladders can be directly 20 detected without first being separated using several mass s,~,e~;t, om~,ter formats.
Amenable formats for use in the invention include ionization te~hniq~les such as matrix-a~ ted laser desorption (MALDI), continuous or pulsed ele~,l.osp,~ (ESI) and related methods (e.g. Ionspray or Thermospray), and massive cluster impact (MSI); these ion sources can be m~tched with a detection format, such as linear or reflectron time-of-flight 25 (TOF), single or multiple quadrupole, single or multiple m~gn.o,tic sector, Fourier Transform ion cyclotron reSon~nre (FTICR), ion trap, or co,.lbin~l;on~ ofthese to give a hybrid detector (e.g. ion trap-TOF). For ionization, numerous matrix/wavelength combinalions (MALDI) or solvent co.,.bi~lalions (ESI) can be employed.
The above-described process can be pti,ru",.ed using virtually any nucleic acid molecule as the source of the DNA template. For example, the nucleic acid ...... . . . .. . .
CA 022~279~ 1998-10-27 molecule can be: a) single stranded or double stranded; b) linear or covalently closed circular in supe~ coiled or relaxed form; or c) RNA if combined with reverse transcription to generate a cDNA. For example, reverse transcription can be pc.ru~",cd using asuitable reverse transcriptase (e.g. Moloney murine leuk~mi~ virus reverse l,~nsc-i~lase) 5 using standard techniques (e.g. Kawasaki ( 1990) in PCR Protocols: A Gu~de to Methods and Applications, Ir~is et al., eds., ~c~dçmic Press, Berkeley, CA pp21-27).
Sources of nucleic acid t~ pl~es can include: a) pl~mi~is (naturally occurring or reco",bin~,l); b) RNA- or DNA- viruses and bacteriophages (naturally 10 occurring or recombil1a--~); c) chromosomal or episo-"al rep!ir~ting DNA (e.g. from tissue, a blood sample, or a biopsy); d) a nucleic acid fragment (e.g. derived by exonuclease, unspeeific endonuclease or restriction endonuclease digestion or by physical disruption (e.g. sonication or nebulization)); and e) RNA or RNA transcripts like mRNAs.
The nucleic acid to be ~mplified and sequenced can be oblained from virtually any biologic~l sample. As used herein, the term "biological sample" refers to any material obtained from any living source (e.g. human, animal, plant, bacteria, fungi, protist, virus). E~".~les of approp. ;ate biological samples for use in the instant 20 invention include: solid materials (e.g tissue, cell pellets, biopsies) and biological fluids (e.g. urine, blood, saliva, amniotic fluid, mouth wash, spinal fluid). The nucleic acid to be amplified and sequenced can be provided by unpurified whole cells, bacteria or virus.
Alternatively, the nucleic acid can first be purified from a sample using standard techniques, such as: a) cesium chloride gradient centrifi.~tiQn; b) alkaline Iysis with or 2S without RNAse lle<.~ c) ion exchange chlo.~ o~raphy; d) phenol/chlolo~ull.l extraction; e) isolation by hybridization to bound oligonucleotides; f) gel electrophoresis and elution; alcohol precipitation and h) co-..l,inaLions of the above.
As used herein, the phrases "chain-elongating nucleotides" and "chain-30 termin~ting nucleotides" are used in accordance with their art recognized m~ning Fort;~a"lple, for DNA, chain-elongating nucleotides include 2'-deoxyribonucleotides (e.g.
dATP, dCTP, dGTP and dTTP) and chain-te~ in~ g nucleotides include 2', 3'-dideoxyribonucleotides (e.g. ddATP, ddCTP, ddGTP, ddTTP). For RNA, chain-elongating nucleotides include ribonucleotides (e.g., ATP, CTP, GTP and UTP) andchain-te,...;~ nllcleotitles include 3'-deoxyribonucleotides (e.g. 3'dA, 3'dC, 3'dG and 5 3'dU). A complete set of chain elongating nucleotides refers to dATP, dCTP, dGl P and dTTP. The term "nucleotide" is also well known in the art. For the purposes of this invention, nucleotides include nucleoside mono-, di-, and triphosphates. Nucleotides also include modified nucleotides such as phosphorothioate nucleotides and deazapurine nucleotides. A complete set of chain-elongating nucleotides refers to four di~re"l 10 nucleotides that can hybridize to each of the four di~relll bases comprising the DNA
template.
If the ~mrlified seque1 çing ladders are to be detected by mass specllul~cLlic analysis, it may be useful to "condition" nucleic acid molçcllles, for 15 example to decrease the laser energy required for vol~ti7~tion and/or to ...;~
fragm~nl~l;on. Condiliûl~ing is preferably pc.r llllcd while the sequ~ncing ladders are immobilized. An e,.~.llple of conditioning is modification of the phos~horli~cter bac~hon~
ofthe nucleic acid molecule (e.g. cation eyrh~nee)7 which can be useful for çl;~il~ p peak brol~ening due to a heterogeneity in the cations bound per nucleotide unit.20 Cont~cting a nucleic acid molecule, which conlains an -thio-nucleoside-triphosph~te during pol~meli~lion with an alkylating agent such as akyliodide, iodoacet~m;de, -iodoethanol, or 2,3-epoxy-1-propanol, the monothio phosphodie~ter bonds of a nucleic acid molecule can be tran~Çull.,ed into a phosphotriester bond. Further condition;~
involves incorporating nucleotides which reduce sensitivity for depurination 25 (fr~g...~ ion during MS), e.g. a purine analog such as N7- or N9-dea_apurine nucleotides, and partial RNA con~ e oligodeoxynucleotide to be able to remove the unrnodified primer from the amplified and modified sequ~n~ g ladders by RNAse orine lle~ In DNA sequçnlin~ using fluoresce.lt cletection and ~ ~1 electrophoretic sepa.~lion, the N7 de~7.~rl-rine nucleotides reduce the formation of 30 secondary structure resulting in band coll.~)fess;on from which no sequ~ncing ;I~l..lation can be generated.
CA 022~279~ 1998-10-27 Critical to the novel process of the invention is the use of approp-iale amounts of two di~le"l polymerase c~ lles, each having a dil~lent affinity for the particular chain tel...;~ g nucleotide, so that pol~llllcl;,.alion by the enzyme with 5 relatively low affinity for the chain termin~ting nucleotide leads to amplification whereas the enzyme with relatively high affinity for the chain tel ..-in~ g nucleotide terminates the polymerization and yields sequen~ing products. PYererably about 0.5 to about 3 units of polymerase is used in the combined amplification and chain te,llfinalion re a~tion Most preferably about 1 to 2 units is used. Particularly pl~lled polylneld~es for use in 10 conjunction with PCR or other thermal amplification process are thermostable polymerases, such as Taq DNA polymerase (Boehringer ~vl~nnh~im), AmpliTaq FS DNApolymerase (Perkin-Elmer), Deep Vent (exo ), Vent, Vent (exo-) and Deep Vent DNApolymerases (New Fngl~nd Biolabs), Thermo Sequenase (Allle~l,an~) or exo(-) Pseudococcusfuriosus (Pfu) DNA poly"lc.asc (Stratagene, Heidelberg Germany).
15 AmpliTaq, Ultman, 9 degree Nm, Tth, Hot Tub, and Pyrococcus furiosus. In addition, preferably the polymerase does not have 5'-3' PYonllcle~se activity.
As sho~,vn in Example 1, the process of the invention can be carried out using AmpliTaq FS DNA polymerase (Perkin-Elmer), which has a relatively high affinity 20 and Taq DNA polymerase, which has a relatively low affinity for chain terrnin~ting nucleotides. Other appropriate polymerase pairs for use in the instant invention can be determined by one of skill in the art. (See e.g. S. Tabor and C.C. Richardsoll (1995) Proc. Na~. Acad. Sci. (USA), vol. 92: Pp. 6339-6343.) In addition to polymerases, which have a relatively high and a relatively low affinity to the chain t~ g nucleotide, a third polymerase, which has proofreading capacity (e.g. Pyrococcus woesei (Pwo)) DNA polymerase may also be added to the amplification mixture to enhance the fidelity of amplification.
Oligonucleotide primers, for use in the invention, can be desi~ed based on knowledge of the 5' and/or 3' regions of the nucleotide seq~lence to be CA 022~279~ 1998-10-27 WO 97/42348 PCT/US97107g66 amplified and sequenced, e.g., insert fl~r~ ng regions of cloning and sequenringvectors (such as M13, pUC, phagemid, cosmid). Optionally, at least one primer used in the chain extension and te,..~nalion reaction can be linked to a solid support to f~çilit~te purification of amplified product from primers and other react~nts, thereby S increasing yield or to separate the Sanger ladders from the sense and A~ n.~e template strand where ~imult~neous amplification-sequ~nring of both a sense and ~nti~e.n~e strand ofthe template DNA has been pelÇoll"cd.
Examples of appropliate solid supports include beads (silica gel, 10 controlled pore glass, m~gnetic beads, Seph~dçY/Sepharose beads, cçll-llose beads, etc.), capillaries, flat supports such as glass fiber filters, glass surfaces, metal surfaces (steel, gold, silver, ~ minllm, and copper), plastic materials or ,.le.llb.~.nes(polyethylene, polypropylene, polyamide, polyvinylide~-e~1ln.1oride) or beads in pits of flat surfaces such as wafers (e.g. silicon wafers), with or without filter plates.
Immobilization can be accomplished, for ~,,.a",plc, based on hybridization between a capture nucleic acid sequence, which has already been immobilized to the support and a co",pl~,l..e,~la.y nucleic acid sequence, which is also coll~ained within the nucleic acid molecule CGI-~ ~ the nucleic acid sequçnce to be 20 detecte~ So that hybrir~i7~tion between the col"ple .~F n~ r nucleic acid mnl e-nlles is not hindered by the support, the capture nucleic acid can include a spacer region of at least about five nucleotides in length between the solid support and the capture nucleic acid seqUçnce The duplex formed will be cleaved under the inflll~nee of the laser pulse and desorption can be initi~ted. The solid support-bound base sequence can be 25 presented through natural oligoribo- or oligodeoxylibo- nucleotide as well as analogs (e.g. thio-modified phosphndiester or phosphotriester backbone) or employing oligonucleotide mimetics such as PNA analogs (see e.g. Nielsen et al., Science, 254, 1497 (1991)) which render the base sequence less susceptible to enzymatic degradation and hence h~c".ases overall stability of the solid support-bound capture 30 base sequçnce CA 022~279~ 1998-10-27 WO 97/42348 PCTIUS97/0~966 Alternatively, a target detection site can be directly linked to a solid support via a reversible or irreversible bond between an appr~pliate functionality (L') on the target nucleic acid molecule and an applopliate filnctionqlity (L) on the capture molecule. A reversible linkage can be such that it is cleaved under the conditions of 5 mass spectrometry (i.e., a photocleavable bond such as a trityl ether bond or a charge transfer comrleY or a labile bond being formed between relatively stable organicradicals). Furthermore, the linkage can be formed with L' being a quaternary ammonium group, in which case, preferably, the surface of the solid support carries negative charges which repel the negatively charged nucleic acid backbone and thus 10 fq~ilitq~te the desorption required for analysis by a mass spe~ ,r.leler. Desorption can occur either by the heat created by the laser pulse and/or, depending on L,' by specific absorption of laser energy which is in resonance with the L' chromophore.
By way of ~ lllple, the L-L' çh~ni~try can be of a type of rliculfide 15 bond (chemically cleavable, for example, by lllercaploethanol or dithioerythrol), a biotin/streptavidin system, a heterobifunctional derivative of a trityl ether group (Koster et al., "A Versatile Acid-Labile Linker for Mo-lific~qtiQn of Synthetic Biomolecules," Tetrahedron Letters 31, 7095 (1990)) which can be cleaved under mild}y acidic conditions as well as under conriitiQnC of mass spec~ou~etry, a levulinyl 20 group cleavable under almost neutral con.~itions with a hydrq.7ini~lm/acetate buffer, an arginine-arginine or Iysine-lysine bond cleavable by an endopeptidase enzyme like trypsin or a pyrophosphate bond cleavable by a pyrophosphqtq~e or a ribonucleotide in between a deoxynucleotide sequence cleavable by an RNAse or alkali.
The functionalities, L and L,' can also form a charge llallsrer complex and thereby form the telllpol~ly L-L' linkage. Since in many cases the "charge-transfer band" can be determined by W/vis spectrometry (see e.g. Or~-q-ni~ Char~e Transfer Complexes by R. Foster, .Acq~de.mic Press, 1969), the laser energy can be tuned to the corresponding energy of the charge-lldnsrer wavelength and, thus, aspecific desorption offthe solid support can be initi-q~ted Those skilled in the art will recognize that several combinations can serve this purpose and that the donor .. .. ... .
CA 022~279~ 1998-10-27 functionality can be either on the solid support or coupled to the nucleic acid molecule to be detected or vice versa.
In yet another approach, a reversible L-L' linkage can be generated by 5 homolytically forming relatively stable radicals. Under the inflll~nce of the laser pulse, desorption (as ~ cussed above) as well as ionization will take place at the radical position. Those skilled in the art will recognize that other organic radicals can be selected and that, in relation to the dissociation energies needed to homolytically cleave the bond between them, a corresponding laser wavelength can be sPlected (see 10 e.g. Reactive Molecules by C. Wentrup, John Wiley & Sons, 1984).
An anchoring fimction L' can also be incorporated into a target capturing sequence by using appropliate primers during an amplification procedure, such as PCR, LCR or transcription amplification.
For certain applications, it may be useful to Qim~lt~neQusly amplify and chain terminate more than one (mutAted) loci on a particular captured nucleic acid fragment (on one spot of an array) or it may be useful to pelru---- parallel processi~ by using oli~on--cleotide or oligonucleotide mimetic arrays on various solid supports.
20 "Multiplexing" can be achieved either by the sequence itself (composition or length) or by the introduction of mass-modifying fi~nction~lities into the primer oligonucleotide. Such multiplexing is particularly useful in conjunction with mass spe-;l.on-cl,ic DNAsequrncing or mobility modified gel based fluolescence sequ~nr.in~
Without limiting the scope of the invention, the mass or mobility mo~ification can be introduced by using oligo-/polyethylene glycol derivatives. The oligo/polyethylene glycols can also be monoAlkylated by a lower alkyl such as methyl, ethyl, propyl, isopropyl, t-butyl and the like. Other cl-e~ ies can be used in the mass-modified compounds, as for example, those described recently in Olu~on..~leotides ~n-l 30 AnaloPues, A P~ .Al Approach, F. Fckctein, editor IRL Press, Oxford, 1991.
CA 022~279~ 1998-10-27 In yet another embodiment, various mass or mobility modifying fimctionalities, other than oligo/polyethylene glycolst can be selected and att~.h~d via apprupliate linking ch~mie~ries A simple modification can be achieved by using di~.enl alkyl, aryl or aralkyl moieties such as methyl, ethyl, propyl, isoplol.yl, t-butyl, hexyl, phenyl, substituted phenyl or benzyl. Yet another modification can be obtained by att~rhin~ homo- or heteropeptides to the nucleic acid molecule (e.g., primer) ornucleoside triphosphates. Simple olig~mides also can be used. Numerous other possibilities, in addition to those mentioned above, can be performed by one skilled in the art.
Different mass or mobility modified primers allow for multiplex sequçn~.in~ via sim~llt~neQus detection of primer-modified Sanger sequ~nrin~ ladders.
Mass or mobility modifications can be incorporated during the amplification process through nucleoside triphosphates or modified primers.
For use with certain detection means, such as polyacrylamide gel electrophoresis (PAGE), detect~hle labels must be used in either the primer (typically at the 5'-end) or in one of the chain e~rten-lin~ nucleotides, or chain termi~ting nucleotides.
Using radioisotopes such as 32p, 33p, or 35S iS still the most frequently used teçhniq~le.
After PAGE, the gels are exposed to X-ray films and silver grain exposure is analyzed.
Non-radioactive labeling techniques have been explored and, in recent years, integrated into partly ~ltom~ed DNA seqllen( ir~ procedures. All these improvements utilize the Sanger sequencing strategy. The label (e.g. fluorescenl dye) can be tagged to the primer (Smith et aL, Nature ~1, 674-679 (1986) and EPO
Patent No. 87300998.9; Du Pont De Nemours EPO Application No. 0359225;
Ansorge et al. J. Biochem. Biophys. Methods 13, 325-32 (1986)) or to the chain-tel..~ g dideoxynucloside triphosphates (Prober et aL Science ~, 336-41 (1987);
Applied Biosystems, PCT Application WO 91/05060). Based on either labeling the 30 primer or the ddNTP, systems have been developed by Applied Biosystems (Smith et al., Science ~, G89 (1987); U.S. Patent Nos. 570973 and 689013), Du Pont De , . .... , .. .. ,.. , . ~
Nemours (Proberetal. Science~, 336-341 (1987); U.S. PatentsNos. 881372 and 57566), Pharmacia-LKB (Ansorge et al. Nucleic Acids Res. 15, 4593-4602 (1987) and EMBL Patent Application DE P3724442 and P3805808.1) and Hitachi (JP 1-90844 and DE 4011991 A1). A somewhat similar approach was developed by S Brnmb~ugh e~ al. (Proc. Natl. Sci. USA 85, 5610-14 (1988) and U.S. Patent No.
4,729,947). An improved method for the Du Pont system using two clecl.ophol~lic lanes with two dif~lenL specific labels per lane is described (PCT Application W092/02635). A di~lenl approach uses fluorescelllly labeled avidin and biotin labeled primers. Here, the sequencing ladders ending with biotin are reacted during electrophoresis with the labeled avidin which results in the detection ofthe individual sequçnring bands (Br~.mb~lgh et al, U.s. Patent No. 594676).
More recently even more sensitive non-r~~ioac.tive labeling techniques for DNA using ~h~milllminescence triggerable and amplifyable by enzymes have been developed (Beck, O~eefe, Coull and Koster, Nucleic Acids Res. 17, 5115-5123 (1989) and Beck and Koster, An~l. Ch~ , 2258-2270 (1990)). These labeling methods were co---bined with multiplex DNA seq~lenring (Church et al. Scien~ 240, 185-188 (1988) and direct blotting electrophoresis (DBE) (Beck and Pohl, EMBO J
Vol. 3: p 2905-2909 (1984)) to provide for a strategy aimed at high throughput DNA
sequen~ing (Koster et al., Nucleic Acids Res. Symposium Ser. No. 24, 318-321 (1991), University of Utah, PCT Application No. WO 90/15883). However, this strategy still suffers from the disadvantage of being very laborious and difficult to automate.
In a.llon-aled sequ~on~ fluo. csce~ce labeled DNA fM~nent~ are cletected during migration through the seq-len~in~ gel by laser excitation.
Fluorescence label is incorporated during the sequ~nçing reaction via labeled primers or chain e~t~n-1ing nucleotides (Smith, L. et. al., Fluorescence detection in automated DNA seq-l~nce analysis, Nature 321:674-89 1986), (Knight, P., Automated DNA
sequencers, Biotechnology 6: 1095-96 1988).
.
CA 022~279~ 1998-10-27 Multiple ~ clly labeled primers can be used to dis~" s~e sequencing patterns. For example, four differently labeled sequenrin~ primers specific for the single termination reactions, e.g. with fluorescent dyes and online detection using laser excitation in an automated sequençin~ device. The use of eight Ji~. elllly labeled S primers allow the disc-il.l,nalion ofthe seq~lencin~ pattern from both strands. Instead of labeled primers, labeled ddNTP may be used for detection, if separation of the sequencing ~a~ment~ derived from both strand is provided. With one biotin labeled primer, sequçn~in~ fr~mPnts from one strand can be isolated for example via biotin-streptavidin coated magnetic beads. Possible is also the isolation via imm~lnoaffinity 10 chromatography in the case of a digoxigenin labeled primer or with affinity chromatography in case of complementary oligonucleotides bound to a solid support.
Another aspect of this invention concerns kits for directly generating from a nucleic acid template, amplified base specifically te..~ ed fragments. Such 15 kits include co-nl~ a~ions ofthe above-described re~ct~nts For in~ cç~ in oneembodiment, the kit can comprise: i) a set of chain-elongaliilg nucleotides; ii) a set of chain-tel...;.~ ;..g nucleotides; and (iii) a first DNA polymerase, which has a relatively low affinity towards the chain tt-~ n~ g nucleotide; and (iv) a second DNA
polymerase, which has a relatively high affinity towards the chain te~ p 20 nucleotide. The kit can also include appropriate solid supports for capture/purification and buffers as well as instructions for use.
The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited 25 references (in~.lu~iin~ literature references, issued patents, published patent applications (inclu~lin~ international patent application Publication Number WO
94/16101, entitled DNA Sequencing byMass Spectrometry by H. Koester; and international patent application Publication Number WO 94/21822 entitled "DNA
Sequ~ncing by Mass Spe.;l.ol.letry Via Exonuclease Degradation" by H. Koster), and - 30 co-pending patent applications, (incluclinp U.S Patent Application Serial No.
08/406,199, entitled DNA Diagnostics Based on Mass Spectromet7y by H. Koester), , CA 022~279~ 1998-10-27 and continuations-in-part of co-pending patent applications (published or unpublished) are hereby express}y incorporated by l ercrence.
Example 1: Direct Nucleic Acid Amp'ifi~ion and Sequencing s Materials and Metho~
Bacterial Strains and Plasmids E. coli K12 strains HB101 and XL1-Blue (Str~tag~n~, California) were grown in LB broth (Miller, J. (1972) Expe,imenls in Molecular G~ne~ics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.) suppl~mente~l with 100 g ~mpicillin/mL
(Binotal, Bayer, Germany). DNA-template used for sequçnr.ing was a pOM8 derived reco",binalll plasmid (Oberbaumer, I. (1986) Gene 49: 81-91 1986) with an 400 BPinsert from the rrnB gene of E. coli. The plasmid DNA was purified via Diagen plasmid purification tips-100 (Diagen, Hilden, Germany).
DNA Isolation The DNA template used in the sequencing reaction was isolated according to the method described by Birnboim and Doly (Birnboim, H.C., and Doly, J. Nucleic Acid Res. 7: 1513-1523 1979) with the improvements of Ish-Horowiczs and Burke (Ish-Horowicz, D., and Burke, J.F. Nucleic Acid Res. 9: 2989-2998 1981).
Sequencing reac~ions A control sequencinE reaction was pc.~-l.cd in accordance with the Perkin-Elmer, AmpliTaq FS kit protocol.
A combined nucleic acid amplification and sequencing reaction was performed as follows. The base specific reaction mixtures (A, C, G and T, as described below) each conlained a buffered solution of: i) DNA template; ii) two thermostrl le DNA polymerases, each with a di~rerc..l affinity to the ddNTP; iii) the ddNTP; iv) all 4 30 dNTPs (Pharmacia, Freiburg, Germany); a pair of sequ~nce specific oligonucleotide primers, one of them labeled with a fluorcsce--l dye. The reaction mixture was overlayed with wax.
.. ... . . . . . .
CA 022~279~ 1998-10-27 WO 97/42348 PCT/US97tO7966 The sequencin~ reaction was calTied out as follows:
A-reaction: 1 8 M ddATP, 80 M each dATP, dCTP, 7-deaza-dGTP, dTTP, 500 mM Tris-HCl (pH 9.0 at 25~C), 25 mM MgCI2 co.. ~ g lpmol JOE dye primer, thermal stable pyrophosphatase and 1.6 U AmpliTaq DNA polymerase, FS. Tothis 50 ng of the DNA template as pl epa. ed above (1 1), 1 U of Taq DNA polymerase (1 I) and 10 pmol reverse primer (0.5 1) were pipetted.
C-reaction: 4 1 8 M ddCTP, 80 M each dATP, dCTP, 7-deaza-dGTP, dTTP, 500 mM Tris-HC1 (pH 9.0 at 25~C), 25 mM MgCI2 co..~ g lpmol FAM dye primer, thermal stable pyrophosphatase and 1.6 U AmpliTaq DNA polymerase, FS. Tothis50ngoftheDNAtemplate(l 1), 1 UofTaqDNApolymerase(l I)and 10pmol reverse primer (0.5 1) were pipetted.
G-reactior~: 8 1 8 M ddGTP, 80 M each dATP, dCTP, 7-deaza-dGTP, dTTP, 500 mM Tris-HCI (pH 9.0 at 25~C) 25mM MgC12 C~ ,g 1 pmol TAMRA dye primer, thermal stable pyrophosphatase and 1.6 U AmpliTaq DNA polymerase, FS. Tothis 50 ng of the DNA template (1 1), 1 U of Taq DNA polymerase (1 1) and 10 pmol reverse primer (0.5 1) were pipetted.
T-reaction: 8 1 8 M ddTTP, 80 M each dATP, dCTP, 7-deaza-dGTP, dTTP, 500 mM Tris-HCl (pH 9.0 at 25~C), 25mM MgCI2 cont~ining 1 pmol ROX dye primer, thermal stable pyrophl-sph~t~se and 1.6 U AmpliTaq DNA polymerase, FS. To this 50 ng of the DNA t~n~pl?te (1 1), 1 U of Taq DNA polymerase (1 1) and 10 pmol reverse primer (0.5 1) were pipetted.
The incubation conditions inr.lllded an initial denaturation step of 4 min.
95~C, followed by 15 cycles of 30 sec. 95~C, 30 sec. 52~C, 60 sec. ?2~C. The reaction is completed by additional 15 cycles of 30 sec. 95~C, 30 sec. 52~C, 60 sec. 72~C.
The reaction mixture was separated from the wax by pipetting and ethanol precipitated. The samples were run on an ~--tom~tçd ABI prism sequencer model 377.
As can be seen from Figures 1 and 2, while the combined amplification and sequ~ncing reaction yielded a 440 BP readable sequence with 15 ng. of template DNA (Figure 1), no sequence data was obtained employing the same amount oftemplate 10 DNA and using the standard cycle sequencing protocol (Figure 2).
Example 2: Direct Nucleic Acid Amplification and Sequencing A combined nucleic acid amplification and sequencing reaction was performed as follows. The reaction contained a buffered solution of i) DNA template, 15 ii) two thermnst~hle DNA polylllel ~s~ s, each with a d~i enl affinity for the ddN~P; iii) dye-labeled ddNTP; iv) all 4 dNTP s (Pha~l.lacia, Freiburg, C~ .m~ly); a pair of sequence specific oligonucleotide primers, one of them labeled with a biotin group.
Combined amplifica~on and sequencing protocol using two DNA
pol~,,,.~l QS~S.
4ng (1 ~11) of p9G551D (pUC-derivative harbouring a 419 BP insert from human mutant CFTR gene) was subjected to seq~len~ by adding 4 ~,11 of 5 x TACS
buffer, 0.5 ~l of each dye ddNTP solution, 1 ~l dNTP mix (5mM dATP, dCTP, dTTP
and 10 mM N' deaza dGTP), I ,ul AmpliTaq FS, 0.75 U (0.3 ~11) (exo-) PJi( polymerase 25 and 40 pmol of each of the standard Ml 3 primers (for sequence see above) to a final volume of 20 ,LI. The cycling profile comprised one cycle of 95 ~C for 4 min., 20 cycles of 95 ~C for 45 sec, 52~C for 45 sec and 72~C for 45 sec. followed by 28 cycles of 96~C
for 50 sec, 55~C for 60 sec and 60~C for 4 min. The reaction was carried out using an EppendorfMastercycler 5330 with a heated lid.
Separation of the forward and reverse sequenc7ngJ~, ~.u nt~.
CA 022~279~ 1998-10-27 The reaction was mixed with an equal volume of 2x B/W-buffer (lOmM
Tris-HCl, pH 7.5, lmM EDTA, 2 M NaCl) cont~inin~ 300 ~lg of prewashed streptavidin M -280 Dynabeads (Dynal). After inc~lbation for 30 min. at room temperature the beads were washed twice with 50 ~,11 of lx B/W buffer in order to remove unwanted reaction 5 components and unincorporated dye terminators.
The reverse seqllencin~ products were recovered from immobilized biotinylated forward products by addition of 20 ~1 of a 20 % aqueouc ~m m onillmhydroxide solution (freshly prepared) and incub~tion at room telllpeldlllre for two 10 minutes. This step was repeated once the products were pooled.
The beads were then washed with 20 11l 25 % aqueous ammonium hydroxide solution for one minute at room te~")e, ~ re and the supernatant was discarded.
The forward seql~çnring reaction was removed from the beads using 30 ~11 of a 25% aqueous ammonium hydroxide solution and incubation at 60~C for 8 minlltçe This step was also repeated once and the products were pooled.
The separated forward and reverse sequrnçing reactions were finally precipiLaled each by addition of 200 ~ll ethanol to the a ....,o~ lm hydroxide solution, resuspended in 4 1ll loading dye (5 :1 ratio of deionized fo, .. .~. . .ide to 25mM EDTA (pH
8.0) /50 mg/ml Blue dextran) and analyzed with an ABI Prism sequencer (model 377).
RESULTS
Figure 4 and 5 depict the fluorescent chronlalogram plots of a forward (Fig. 4) and reverse (Fig. 5) sequence ladder generated in a siml~lt~neous amplification and seqUçnring reaction. In this experiment 1.5 units of Taq polymerase were added to a 30 dye terminator cycle seqllçnring mix cont~ininp AmpliTaq FS, and only 4 ng of plasmid DNA, a biotinylated fo, vvald primer and a reverse primer. A~er the cycling reaction, CA 022~279~ 1998-10-27 biotinylated fo- w~rd sequçncing products and reverse products hybridized to the forward tçmp!~te were immobilized on streptavidin coated m~netic beads. A separation of reverse and forward seql1çnl~.in~ ladders was achieved by incub~tion of the beads with ammonium hydroxide. Ammonium hydroxide acted as a denaturing agent for double 5 stranded DNA at room temperature, therefore r~le~cing the reverse sequçnr.ir.g products in the solution. The biotinylated products remained bound to the streptavidin, whereas the reverse products were removed with the supe,llata.,l. A recovery ofthe biotinylated products was carried out using arnmonium hydroxide at an incllb~tion temperature of 60~C. At elevated te"-pe~al-lre, ammonium hydroxide acted as a denaturing agent for 10 the streptavidin biotin interaction. The thus separated sequçn~ing ladders were then ethanol precipitated and separately analyzed on a ABI Prism sequencer.
CA 022~279~ 1998-10-27 F.quivalell~
Those skilled in the art will recognize or be able to ascertain using no more than routine e.~pe,;,~nlnl;on numerous equivalents to the specific procedures 5 described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.
Claims (48)
1. A kit for obtaining amplified and chain terminated nucleic acid molecules from a nucleic acid template comprising: i) an appropriate amount of a set of chain-elongating nucleotides; ii) an appropriate amount of at least one chain-terminating nucleotide; iii) an appropriate amount of a first polymerase, which has a relatively low affinity towards at least one chain terminating nucleotide; and iv) an appropriate amount of a second polymerase, which has a relatively high affinity towards at least one chain terminating nucleotide.
2. A kit of claim 1, which additionally comprises a restriction enzyme that can cleave the primer.
3. A kit of claim 1, wherein the first and second polymerases are thermostable DNA polymerases.
4. A kit of claim 3, wherein the thermostable DNA polymerases are selected from the group consisting of: Taq DNA polymerase, AmpliTaq FS DNA polymerase, Deep Vent (exo~) DNA polymerase, Vent DNA polymerase, Vent (exo~) DNA
polymerase and Deep Vent DNA polymerases, Thermo Sequenase, exo(-) Pseudococcus furiosus (Pfu) DNA polymerase, AmpliTaq, Ultman, 9 degree Nm, Tth, Hot Tub, Pyrococcus furiosus (Pfu), and Pyrococcus woesei (Pwo) DNA polymerase.
polymerase and Deep Vent DNA polymerases, Thermo Sequenase, exo(-) Pseudococcus furiosus (Pfu) DNA polymerase, AmpliTaq, Ultman, 9 degree Nm, Tth, Hot Tub, Pyrococcus furiosus (Pfu), and Pyrococcus woesei (Pwo) DNA polymerase.
5. A kit of claim 1, wherein the set of chain-elongating nucleotides areselected from the group consisting of: i) at least one deoxyadenosine triphosphate; ii) at least one deoxyguanosine triphosphate; iii) at least one deoxycytidine triphosphate; and iv) at least one thymidine triphosphate.
6. A kit of claim 5, wherein the deoxyadenosine and/or the deoxyguanosine is an N7- or N9- deazapurine nucleotide.
7. A kit of claim 1, wherein the chain terminating nucleotide is selected from the group consisting of: 2',3'-dideoxyadenosine triphosphate, 2',3'-dideoxyguanosine triphosphate, 2',3'-dideoxycytidine triphosphate, 2',3'-dideoxythymidine triphosphate.
8. A kit of claim 1, which additionally includes at least one primer.
9. A kit of claim 8, wherein the primer is linked to a solid support.
10. A kit of claim 9, wherein the solid support is selected from the group consisting of beads, capillaries, flat supports, membranes and wafers.
11. A kit of claim 9, wherein the primer contains a restriction site or aribonucleotide .
12. A kit of claim 8, wherein the primer is weight modified.
13. A kit of claim 8, wherein the primer is mobility modified.
14. A kit of claim 7, wherein at least one ddNTP is weight modified.
15. A kit of claim 1, which additionally comprises a proof reading DNA
polymerase.
polymerase.
16. A kit of claim 1, which additionally comprises a reverse transcriptase.
17. A process for simultaneously amplifying and sequencing a single stranded nucleic acid molecule, comprising the steps of:
a) contacting the single stranded nucleic acid molecule with: i) at least one primer that can hybridize to the template, (ii) a set of chain elongating nucleotides, (iii) at least one chain terminating nucleotide, (iv) a first DNA polymerase, which has a relatively low affinity towards the chain terminating nucleotide; and (v) a second DNA polymerase, which has a relatively high affinity towards the chain terminating nucleotide, so that polymerization by the first polymerase results in amplification and polymerization by the second polymerase results in the formation of chain terminated fragments;
b) detecting the chain terminated fragments by an appropriate detection means; and c) aligning the fragments by mobility or molecular weight to determine the sequence of the DNA template.
a) contacting the single stranded nucleic acid molecule with: i) at least one primer that can hybridize to the template, (ii) a set of chain elongating nucleotides, (iii) at least one chain terminating nucleotide, (iv) a first DNA polymerase, which has a relatively low affinity towards the chain terminating nucleotide; and (v) a second DNA polymerase, which has a relatively high affinity towards the chain terminating nucleotide, so that polymerization by the first polymerase results in amplification and polymerization by the second polymerase results in the formation of chain terminated fragments;
b) detecting the chain terminated fragments by an appropriate detection means; and c) aligning the fragments by mobility or molecular weight to determine the sequence of the DNA template.
18. A process of claim 17, wherein the first and second polymerases are thermostable DNA polymerases.
19. A process of claim 12, wherein the set of chain-elongating nucleotides are selected from the group consisting of: i) at least one deoxyadenosine triphosphate; ii) at least one deoxyguanosine triphosphate; iii) at least one deoxycytidine triphosphate; and iv) at least one thymidine triphosphate.
20. A process of claim 19, wherein the deoxyadenosine and/or the deoxyguanosine is an N7- or N9-deazapurine nucleotide.
21. A process of claim 17, wherein the chain terminating nucleotide is selected from the group consisting of: 2',3'-dideoxyadenosine triphosphate, 2',3'-dideoxyguanosine triphosphate, 2',3'-dideoxycytidine triphosphate, 2',3'-dideoxythymidine triphosphate.
22. A process of claim 17, wherein the detection means is a separation means selected from the group consisting of: polyacrylamide gel electrophoresis, capillary zone electrophoresis and mass spectrometry in conjunction with a visualization means selected from the group consisting of: colorimetry, fluorimetry, chemiluminescence, radioactivity and mass spectrometry.
23. A process of claim 17, wherein the detection means is mass spectrometry.
24. A process of claim 23, wherein the mass spectrometry is performed using an ion source selected from the group consisting of: matrix assisted laserdesorption ionization (MALDI), electrospray (ES), ionspray, thermospray and massive cluster impact; and the detection format is selected from the group consisting of: time-of-flight; quadrupole, magnetic sector, Fourier transform ion cyclotronresonance and ion trap.
25. A process of claim 17, wherein the double stranded DNA molecule has been synthesized from RNA using a reverse transcriptase.
26. A process of claim 17, wherein the primer is linked to a solid support.
27. A process of claim 26, wherein the solid support is selected from thegroup consisting of: beads, capillaries, flat supports, membranes and wafers.
28. A process of claim 26, wherein the primer contains a restriction site or a ribonucleotide.
29. A process of claim 26, wherein the primer is weight modified.
30. A process of claim 26, wherein the primer is mobility modified.
31. A process of claim 26, wherein at least one ddNTP is weight modified.
32. A process of claim 17, which additionally comprises a proofreading DNA
polymerase.
polymerase.
33. A process for simultaneously amplifying and sequencing a nucleic acidmolecule, comprising the steps of:
a) denaturing a double stranded DNA molecule at an appropriate temperature and for an appropriate period of time to obtain two complementary single stranded DNA molecules;
b) contacting at least one of the single stranded DNA molecules with a complementary primer at an appropriate temperature and for an appropriate period of time to obtain at least one primer containing single stranded DNA molecule;
c) contacting the at least one primer containing single stranded DNA
molecule at an appropriate temperature and for an appropriate period of time with: (i) a set of chain elongating nucleotides, (ii) at least one chain terminating nucleotide, (iii) a first DNA polymerase, which has a relatively low affinity towards the chain terminating nucleotide; and (iv) a second DNA polymerase, which has a relatively high affinity towards the chain terminating nucleotide, so that polymerization by the first polymerase results in amplification and polymerization by the second polymerase results in the formation of chain terminated fragments;
d) repeating steps a) through c) for an appropriate number of times to obtain a sufficient amount of chain terminated fragments for detection; and e) detecting the chain terminated fragments by an appropriate detection means and aligning the fragments by molecular weight to determine the sequence of the DNA template.
a) denaturing a double stranded DNA molecule at an appropriate temperature and for an appropriate period of time to obtain two complementary single stranded DNA molecules;
b) contacting at least one of the single stranded DNA molecules with a complementary primer at an appropriate temperature and for an appropriate period of time to obtain at least one primer containing single stranded DNA molecule;
c) contacting the at least one primer containing single stranded DNA
molecule at an appropriate temperature and for an appropriate period of time with: (i) a set of chain elongating nucleotides, (ii) at least one chain terminating nucleotide, (iii) a first DNA polymerase, which has a relatively low affinity towards the chain terminating nucleotide; and (iv) a second DNA polymerase, which has a relatively high affinity towards the chain terminating nucleotide, so that polymerization by the first polymerase results in amplification and polymerization by the second polymerase results in the formation of chain terminated fragments;
d) repeating steps a) through c) for an appropriate number of times to obtain a sufficient amount of chain terminated fragments for detection; and e) detecting the chain terminated fragments by an appropriate detection means and aligning the fragments by molecular weight to determine the sequence of the DNA template.
34. A process of claim 33, wherein the first and second polymerases are thermostable DNA polymerases.
35. A process of claim 33, wherein the set of chain-elongating nucleotides are selected from the group consisting of: i) at least one deoxyadenosine triphosphate; ii) at least one deoxyguanosine triphosphate; iii) at least one deoxycytidine triphosphate; and iv) at least one thymidine triphosphate.
36. A process of claim 35, wherein the deoxyadenosine and/or the deoxyguanosine is an N7- or N9- deazapurine nucleotide.
37. A process of claim 33, wherein the chain terminating nucleotide is selected from the group consisting of: 2',3'-dideoxyadenosine triphosphate, 2',3'-dideoxyguanosine triphosphate, 2',3'-dideoxycytidine triphosphate, 2',3'-dideoxythymidine triphosphate.
38. A process of claim 33, wherein the detection means is a separation means selected from the group consisting of: polyacrylamide gel electrophoresis, capillary zone electrophoresis and mass spectrometry, in conjunction with a visualization means selected from the group consisting of: colorimetry, fluorimetry, chemiluminescence, radioactivity, and mass spectrometry.
39. A process of claim 33, wherein the detection means is mass spectrometry.
40. A process of claim 39, wherein the mass spectrometry is performed using an ion source selected from the group consisting of: matrix assisted laser desorption ionization (MALDI), electrospray (ES), ionspray, thermospray and massive clusterimpact; and the detection format is selected from the group consisting of: time-of-flight;
quadrupole, magnetic sector, Fourier transform ion cyclotron resonance and ion trap.
quadrupole, magnetic sector, Fourier transform ion cyclotron resonance and ion trap.
41. A process of claim 33, wherein the double stranded DNA molecule has been synthesized from RNA using a reverse transcriptase.
42. A process of claim 33, wherein the primer is linked to a solid support.
43. A process of claim 42, wherein the solid support is selected from thegroup consisting of beads, capillaries, flat supports, membranes and wafers.
44. A process of claim 33, wherein the primer contains a restriction or aribonucleotide.
45. A process of claim 33, wherein the primer is weight modified.
46. A process of claim 33, wherein the primer is mobility modified.
47. A process of claim 33, wherein a ddNTP is weight modified.
48. A process of claim 33, which additionally comprises a proofreading DNA
polymerase.
polymerase.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/647,368 | 1996-05-09 | ||
| US08/647,368 US5928906A (en) | 1996-05-09 | 1996-05-09 | Process for direct sequencing during template amplification |
| PCT/US1997/007966 WO1997042348A1 (en) | 1996-05-09 | 1997-05-09 | Process for direct sequencing during template amplification |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2252795A1 true CA2252795A1 (en) | 1997-11-13 |
Family
ID=24596716
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002252795A Abandoned CA2252795A1 (en) | 1996-05-09 | 1997-05-09 | Process for direct sequencing during template amplification |
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| Country | Link |
|---|---|
| US (1) | US5928906A (en) |
| EP (1) | EP0901531B1 (en) |
| JP (1) | JP2000510341A (en) |
| AT (1) | ATE210192T1 (en) |
| AU (1) | AU718238B2 (en) |
| CA (1) | CA2252795A1 (en) |
| DE (1) | DE69708865T2 (en) |
| HK (1) | HK1018080A1 (en) |
| WO (1) | WO1997042348A1 (en) |
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|---|---|---|---|---|
| EP0679196B1 (en) | 1993-01-07 | 2004-05-26 | Sequenom, Inc. | Dna sequencing by mass spectrometry |
| US6194144B1 (en) | 1993-01-07 | 2001-02-27 | Sequenom, Inc. | DNA sequencing by mass spectrometry |
| US5605798A (en) * | 1993-01-07 | 1997-02-25 | Sequenom, Inc. | DNA diagnostic based on mass spectrometry |
| US6146854A (en) * | 1995-08-31 | 2000-11-14 | Sequenom, Inc. | Filtration processes, kits and devices for isolating plasmids |
| WO1997033000A1 (en) * | 1996-03-04 | 1997-09-12 | Genetrace Systems, Inc. | Methods of screening nucleic acids using mass spectrometry |
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| US5003059A (en) * | 1988-06-20 | 1991-03-26 | Genomyx, Inc. | Determining DNA sequences by mass spectrometry |
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| CA2114124A1 (en) * | 1991-07-24 | 1993-02-04 | Keith Gregg | Single step amplification and sequencing of nucleic acids |
| US5674679A (en) * | 1991-09-27 | 1997-10-07 | Amersham Life Science, Inc. | DNA cycle sequencing |
| EP0679196B1 (en) * | 1993-01-07 | 2004-05-26 | Sequenom, Inc. | Dna sequencing by mass spectrometry |
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