CA2114124A1 - Single step amplification and sequencing of nucleic acids - Google Patents

Abstract

Method for the amplification and sequencing of DNA or RNA. The method comprises the steps of (i) melting a double stranded nucleic acid to yield a pair of complementary nucleic acid strands, (ii) hybridising a primer to each of the strands, the primers being so chosen that the primer annealing to the sense strand is 3' to the position of the primer on the antisense strand, one of the primers being labelled so as to be capable of being visualized independently of the other primer, (iii) causing a polymerase enzyme to amplify the nucleic acid in the presence of a dideoxynucleotide analogue of one of the nucleotides present in the nucleic acid, the dideoxy analogue being present in such a concentration that a majority of the newly synthesised nucleic acid strands are terminated by incorporation of dideoxynucleotides without extending far enough to act as templates for synthesis of the opposite strand using the second primer, (iv) repeating steps (i) to (iii) sequentially a number of times, (v) repeating the steps (i) to (iv) using at least another two dideoxynucleotide analogues of the other three nucleotides present in the nucleic acid, and (vi) electrophoretically separating the reaction products of each of the repetitions of steps (i) to (iv) and visualizing the labelled strands.
The other of the nucleotides of at least a part of the strand of the nucleic acid to which the labelled primer annealed between the binding sites may be determined by comparing the separated and visualized gels for each of the nucleotide analogues used.

Description

2 ~ PCT/AU92/00372 Sinq~ Step Amplification and Sequencin~ of Nucleic Acids Field of the Invention The pre~ent invention relate~ to a method for the ~ingle-step amplification and sequencing of the nucleic 5 acids deoxyribonucleîc a~îd ~DNA) and rîbonuclQîc acid (RNA).
Back~round Art It is kno~n that DNA can be amplîfied by the polymerase chaîn reaction (PCR). Durîng PCR double-stranded DNA, such a~ genomic DNA, is melted to separate the two complementary DNA chains and two primers are ~dded one of which is complementary with a known seguence on each of ~he two DNA strandg. In the presence of 3uitable nucleotides the polymèras~ e~yme ~ill build up complementary strands of DN~ on the primed t~mplates until the r~action conditions are changed. The primer~ are 80 chosen that the primer annealing to ~h~ ~en~e strand i~ 3' : to the position of the primer on the anti~en~e ~trand.
Repetition of khis~cycle will caus~ the original genomic templates to be again reproduced. In addition the ~20 ~omplementary strands for~sd duri~g the first cycle will be reproduced but only to the extent of khe region of the original genomic DNA w~ich lies between and includes ~he ~ t~o primers. The amplified DN~ region between the primers : is then r~covered by electrophor~tic purification. ~his : ~5 PC~ reaction is described more fully in Saiki, R.K., Gelfa~d, D.H., Stoffel~ SOJ Scharft, S.J., ~iguchi~
R. Horn, G.T. Ilulli~, ~.B. and ~rlich, H.A. ( 1988) Science 239, 487-491.
It is also known to se~uence D~A using a process in 30 which a sinyle labelled primer is annealed to suitably melted DNA. The raaction is carried out in four sept~rate reacti.ons, each reaction vessel containing the dideoxyrluclec)tide analogue o~ one of the usual nucleotides. The presence of the dideoxy analos~ue in a 35 suitable concentration causes the chain reaction to stop . ~

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¦ SIJBSTI~UTE S~EE~J

~ I , L~ 2 -once out of every three or four hundred times that the respective complementary nucleotide occurs in the template. The extended DNA products from each of the four reactions are run side by side on a suitable electrophoretic gel and visualised. The relative positions of the bands on the gel allows the se~uence of the newly synthesized template DNA to b~ deduced. This dideoxynucleotide sequencing reaction is more fully described in Sanger, F., Nicklin, S and Coulsen, A.R.
(l977~ Proc. Natl. Acad. Soi. U.S.A. 74, 5483-5487.
It is also known to impro~e the ~ensitivity of sequencing reactions by ~hermocycling with a heat-stable DN~ polymerase (see ~e~, J.S. (1991) DNA and Cell Biol.
10, 87-73). In the existing proposals for using polymerase amplification of DNA together wi~h ~equencing, entirely separate steps have been provided or amplification o~ the ~emplate DNA, purification of products and ~e~uencing. The present invention pro~ides :for an alternati~e to the known processes for the : 20 amplification and sequencing of nucleic acids.

The present invention consists in a method for the : amplification and sequencing of a nucleic acid comprising th~ steps ofs~
: 25 i) melting a~double stranded nucleic acid to yield a pai.r of complementary strands of nucleic acid, ii) hybridising a~primer to each of the strands of nucleic acid, the primers being so chosen that the primer annealing to the sense strand is 3' to the position of the:primer on the antisense strand/ ~ne : : of~the prlmers~being:labelled so as to be capable of being ~isualised independently o~ the other primer;
iii) causing a polymerase enzyme to amplify the nucleic acid in the presence of a dideoxynucleotide analogue 39 of one of the nucleotidFs normally present in the :-~

W093/022~2 ~ J. !t i~ ~ PCT/AU92/00372 nucleic acid, the dideoxynucleo~ide analogue being present in such a concentration that a majori.ty of the newly synthesised nucleic acid strands are terminated by ineorporation o~ dideoxynucleotides without extending far enough to act as templates for synthesis of the opposite ~trand using khe second primer;
iv) repeating the steps i~ to iii) sequentially a number of times;
v) repeating the st~ps i) to iv) using di.deoxynucleo~ide a~alogues of at least al.~ but one of the other nucleotides present,in the nucl~ic acid; and vi) separa~ing the reaction products of each of the repetitîons of the steps i) to iv) and visualising the labelled strands to allow a determinakion of the sequence of at least a part of the strand of th~
nucleic acid to which the labelled primer annealed between the binding sit~s for the two primers.
While the method according to the present invention may be carried out on RNA it is preferable to form a double stranded DNR analogue of the RNA and tv seguence that. Similarly single stranded DNA may be converted to double stranded DNA and sequenced accordi~g to the present invention. The present method is particularly suitable ~or u~e with prokaxyotic DNA but may also be useful in sequencing eukaryotic DNA.
It is pr~ferred that ~he reaction products of the various repetitions of steps (i) to (iv~ be separated by electrophoretic separation in a suitable gel. This allows the various reaction products to be run side by side allowing a dixect reading of the nucleic acid sequence from the gel. If the various repeti~ions are run using pri~ers uni~uely labelle~, for instance by using a different fluorescent label ~or each repetition, it would be possible to run a single gel track and to use an ~J ~ ' J ~ -- 4 autosequencer to automatically read the nucleotide sequence from the gel.
The labelled primer may be labelled in any suitable manner known in the art. One suitable manner is by 5 incorporation of a radioactive iso~ope of an element in ::
the primer. Other suitable means include labelling the primer with a binding ligand such as biotin or labelling the primer with a fluorescent marker.
The distance between primers on the template can be varied to alter the ~ensit.ivity. The efficiency of : amplification depends upon th~ proportion of new DNA
: chains that extend far enough to act as templates for :~synthssis of a complementary strand using the second primer. It has been found that primer separation of about ~50 base pairs provides rea~onable levels of amplific~tion ~: with eukaryotic DNA and~allows rel7able sequence data to be obtained. In the case of prokaryotic DNA primer separation of 600 base:pairs is quite possible. The method according to this invention can be run using nanogram quantities of DNA however larger quantities are desirable with greater primer separation.
h The results obtained are sometimes confused adjacent to the originating primer~where one can get "cross ~: :banding". Closely adj~acent the other primer one may also find ~abnormalities such as ~Iblockage~ . The best results :` are obtained from the intermediate DNA betwe~n the two primers.
It would be normal that any nucleic acid studied would include four nucleotides. It would thus be normal to carry out the process of steps (i) to (i~) four times, once with the dideoxynucleotide analogue for each of the nucleotides occurring in the nucleic acid. It will be obvious t~at the method could ~e carried out for only three of the nucleotides with presence of the fourth nucleotide being implled by gaps in the separation. This W0~3/02212 ~ PCT/~U92/00372 s~rategy is deemed to be within the scope of this invention however it is not a preferred strategy.
Brief P~ tion of thç Dra~ings Fig 1 is a diagrammatic representation of the process according to the pres~nt invention, and Fig 2 is an electrophoreti~ gel showing the separatisn of strands of DNA for four duplicate reactions involving four different dideoxynucleotide analogues and showing the deduced nucleotide sequence of part of the DNA
being in~estigated.
The diagram of Fig 1 shows the procesC of the present invention. ~ double standed DNA 10 comprising a sense strand 11 and an anti~ense strand 12 is heated and anneal~d in the presence of two primers 13 and 1~. Primer 13 binds to the sense strand 11 3' to the position which primer 14 binds to the antisense strand 12. This means that the synthesis on the two strands is co~erging in direction. The primer 14 i6 labelled, such as with radioactive phosph~rou~. This allows the strands of DNA
derived from the antisense skxand to be visualised at the end of the process indep~ndently of the strands of DNA
derived from the sense strands. ~he DNA strands 11 and 12 to which the primers 13 and 14 have been annealed are : divided int~ four al:iquots 15, 16, 17 and 18.
Each of the aliquots lS, 16, 17 and 18 is amplified using a polymerase enzyme. A dideoxynucleotide analog of one of the nucleo~ides~in the DNA is added to each aliquot. Thus dideoxycytosine triphosphosphate is added to aliquot 15 and the corresponding dideoxynucleotides based on thy~ine, adenine and guanine are added to aliquots 16, 17 and 18 r~spectively. By cycling through a procass of heating and annealing each o~ the aliquots a I~rge number of DNA strands of different lengths will be buil~ up in each aliquot. In each case each of the strands ll ~nd 12 of the DNA will be copies in each WO93/022l2 . PCT/AU92/00372 ~ ~/1 6 -aliquot 15, 16, 17 and 18. The copying in each case will stop when a dideoxynucl~otide analogue is inserted into the developing copy of the s~rand in place o~ the corresponding deoxynucleotide. By adjusting the ratio of the dideoxynucleotide and the deoxynucleotide in each aliquot it is possible to produce a majority of DNA copy .
strands which stop short of reaching the point at which the opposin~ primer will anneal to ~he copy strand. As an example in ali~uot 15 strands 19, 21 and 22 have stopped short of reaching a length sufficient to allow binding of the pri~er 13. Thes~ strand~ will thus not be reproduced in further cycles of the heating and annealing. The ~strand 23~ by contrast, has extended to a point where the primer 13 will anneal to it and it served as a template ior the production of re~erse strands of DNA.
In this way is built up four populations of strands o~ varying lengthO Thesé populations are separated on an - elec~rophoretic gel 24 and visùalis~d on a suitable film.
:; ~ The film will only record the presence of the strands ~: 20 incorporatin~ the labelled primer. The four aliquots 15, 16, 17 and 18 are run side~by side on the gel 24 and ~he shorter strands will be carried furthest a~ong the gel 24. Reading the gel 24 in the direction of the arrow ~ shows that the strand sequenced reads CCTAGGGATCTA.
Figure 2:shows the visualisation of an actual gel : showing the sequencing of a section of DNA from the chromosome of a rumen bacterium ~ ~LL~L~L~la~
strain AR20. The gel is read in the direction of the arrow and the section identified reads 5' ~o 3', GGCTTTTACAGTT.
~est MQd~_o~ Carrying out the In~e~tion In order that the nature of the present invention may be more clearly understood a:prèferred embodiment thereof will now be described by reference to the followinq ;~
example.

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W093/02212 ~ i ;'J ~i PCT/AU92/00372 DNA PreparatiQn ~nd Se~ue~cLn~ Protocol Genomic DNA for amplification sequencing can be prepared from bacterial ~amples by any of ~he standard methods (Maniatis, T.l Fritsoh, E.F. and Sambrook, J.
~1982) Molecular Cloning- A Laboratory Manual. Cold Spring Harbor ~aboratory). A rapid and convenient method which may be used is as follows.
1.5 ml of bacterial suspension was centrifuged at 2000 g for S min, resuspended in 0.5 ml of TE buffer (Tris. HCl, pH 8.0, 10 mN; Ethylenediamine tetra acetic acid, EDTA, 1 mM) and recentrifuged at 2000 g. The bacterial pellet was suspended in 0.4 ml of TE buffer, containing 4 mg/ml Lysozyme, and incubated at 37C for 20 min. 10 yl of 10~ Sodium dodecyl sulfate (SDS) was mixed with the sample, followed by 10 ~l of ribonuclease solution (lO mg/ml), and it was incubated for 20 minutes at 37C. 10 ~l of Proteinase K ~10 mg/ml) w~s added, and the sample incubated at 55C for 30 minutes, or until clear. The cleared solution was ~20 extracted onre with an equal volume of phenol/chloroform (1:1), twice with 0.4 ml of chloroform, and the DNA was precipitated by mixing the aqueous phase with 1 ml of ethanol at room temperature for 4-5 min. and centrifuging at ~2000 g for 5 min. The ~NA was redissol~red in ;: 25 0.2 - 0.4 ml of dis~tiIled water and the purity and concentration estimated by u.v. spectrum of a 1:50 dilution.
For the four sequencing reactions, a total of approximately 5 - 10 pg of plasmid, or 5 - 10 ng of pure bacterial genomic DNA was used as template, although this .

: quantity could be increased without adverse effects.
: Mix~ures of bacterial DNAs were used at quantities sufficient to give at: least 5 ng of target DNA in the reactions. `
Four sequencing reactions were prepared, as for ~ 8 ~

con~entional DNA sequencing with the TaqTrack kit (Promega~. A single aliquot of template ~NA was mixed with 20 ng each of two PCR pximers, of which one had been labelled with 32p. Labelling was by polynucleotide kinase reaction ~Promega), with ~-32P-ATP, using the manufacturer's recommended conditions, at 37C for 80 - 90 min.
PCR reaction buffer ~Promega) was used in place of the normal DNA sequenci~g kit buffer, and was mixed with the template and prim~rs, and the ~olution adjusted to a final volume of 22 ~l with distillad water. The mixture was div~ded, 5 ~l into each of four tubes, each ~contalning 2 yl of a specific TaqTrack dNTP/ddNTP mixture.
Reaction mixtur~s were overlaid with 40 yl of mineral oil, to prevent evaporation, and subjected to 25 - 30 cycles in the:programmable incubator. Steps for denaturation of th~ double-s~randed template, and primer ~: : annealing/ prior to the sequencing reactions, were : 2Q unnecessary.
. Incubation conditions were as follows: denaturation 92~ for 90 s; annealing, 55C for 80 s;
: polymerization, 72C for 80 s. After completion of the :~ ~ reaction cycles,~5yl:of formamide stop-solution was ~ 25 added and the tubes centrifuged to pass it through the oil :~ layer (2000 g for 3~ s)~ R~action products were heat~d at 95C for 3 min. Samples could be withdrawn from bene~th the oil using a~glass capillary t:ube, or the oil could be -remo~ed by two gent~le~ether washes.
Products were separated by loading 2 ~l of each reaction mixture onto standard 6% (w/v) denaturing : poIyacrylamide gels containing 8 M urea in Tris-borate : EDTA buffer (Maniatis, T~ Fritsch, E.F. and Sambrook, J.
:~ (lg82) Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory)~. After electrophoresis, gels ~, WO93/02212 . 2~ c ~ .~ 1 PCT/AU92/0~372 were fixed with 12% (v/v) acetic acid for 10 minutes, washed with 1 litre of ~0% (v/v) ethanol, and dried at 95C, before being autoradiographed overnight with Fuji X-ray film.
During this process, a high proportion of nascent DNA
chains were terminated by incorporation of dideoxynucleotides. However, in each cycl~, a proportion were exten*ed far enough to allow annealing of ~he opposite primer and therefore to act as templates for ~y~thesis of the opposite strand. This resulted in ampli~ication of both strands at less than normal PCR
efficiency. Concurrently, the available template was re-used at each cyc~e, ~or the sequencing r*action.
The time taken, to obtain sequen~e from a sample of chromosomal DNA, is reduced to less than half that required in con~en~ional PCR/sequencing. Furthermore, amplification factors in this method are reduced considerably from those normally used in manufacturing template DNA. This would be expected to reduce the number : 20 of errors introduced during amplification. Concurrent duplication of the reactions provides immedia~e sequence :~ ~ confirmation.
; ~ This technique will clearly be advantageous in : ~ proces~es that require detailed gensti~ screening of numerous VNA samples: e.g. examination of specific genes for point mutations~, or in analyses that will benefit from specific~strain~identi~ication. Genes in which different s~rains o~an organis~possess different DNA sequences can be analysed rapidly, using small quantities of relatively impure DNA. A difference of one base-change, anywhere :~: : within the region: spanned by the primers, can be detected : by this method. In addition, application of the technique : to ~NA templates should allow rapid analysis of :: ~e.g.:16S ribosomal RNA for:bacterial taxonomy, or ~he detection and detail~ed identification of retroYiral : ~
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WO93/022l2 PCT/AU92~00372 strains.
Previous reports of thermocycling for DNA sequencing reactions have used a single, unlabelled primer, and the incorporation o radi~isotopes during the reaction, to label the sequencing products (Lee, J-S. (1991) DNA and Cell Biol. 10, 87-73). However, that approach is likely to result in problems through the attachment of radio-isotopes to non-target ~NA, in samples that are not entirely pure. Prelabelling the primer results in adequate sensitivity and avoids the complications that may arise in mix~ures of DNA.
It will be appreciated by pexsons skilled in the art .~that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments ::~
are~ therefore, to be considered in all respects as illustrative and not restrictive.

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Claims (8)

CLAIMS:-

1. A method for the amplification and sequencing of a nucleic acid comprising the steps of:-i) melting a double stranded nucleic acid to yield a pair of complementary strands of nucleic acid;
ii) hybridising a primer to each of the strands of nucleic acid, the primers being so chosen that the primer annealing to the sense strand is 3' to the position of the primer on the antisense strand, one of the primers being labelled so as to be capable of being visualised independently of the other primer;
iii) causing a polymerase enzyme to amplify the nucleic acid in the presence of a dideoxynucleotide analogue of one of the nucleotides normally present in the nucleic acid, the dideoxynucleotide analogue being present in such a concentration that a majority of the newly synthesised nucleic acid strands are terminated by incorporation of dideoxynucleotides without extending far enough to act as templates for synthesis of the opposite strand using the second primer;
iv) repeating the steps i) to iii) sequentially a number of times;
v) repeating the steps i) to iv) using dideoxynucleotide analogues of each of the other nucleotides present in the nucleic acid; and vi) separating the reaction products of each of the repetitions of the steps i) to ii) and visualising the labelled strands to allow a determination of the sequence of at least a part of the strand of the nucleic acid to which the labelled primer annealed between the binding sites for the two primers.

2. A method as claimed in claim l in which the double standed nucleic acid is a double stranded DNA derived from a prokaryotic organism.

3. A method as claimed in claim 1 in which double stranded nucleic acid is a double stranded DNA analogue of an RNA.

4. A method as claimed in claim 1 in which the one primer is labelled with a radioactive isotope.

5. A method as claimed in claim 1 in which the nucleic acid to be sequenced is derived from an eukaryotic organism and the binding sites of the primers are separated by about 250 base pairs.

6. A method as claimed in claim 1 in which the nucleic acid to be sequenced is derived from a prokaryotic organism and the binding sites of the primers are separated by at least 600 base pairs.

7. A method as claimed in claim 1 in which the reaction products are separated electrophoretically.

8. A method as claimed in claim 7 in which the reaction products of the replications of the steps (i) to (ii) are run side by side on a single eletrophoretic gel.