Note: Descriptions are shown in the official language in which they were submitted.
WO93/02212 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
WO93/02212 PCT/AU92/00372
~ 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
WO93/02212 PCT/AU92/00372
~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
WO93/02212 PCT/AU92/00372
~ 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
- 10 -
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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