U.S. patent application number 10/824465 was filed with the patent office on 2004-10-07 for method for the direct, exponential amplification and sequencing of dna molecules and its application.
This patent application is currently assigned to Roche Diagnostics GmbH. Invention is credited to Kilger, Christian, Paabo, Svante.
Application Number | 20040197811 10/824465 |
Document ID | / |
Family ID | 7815636 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040197811 |
Kind Code |
A1 |
Paabo, Svante ; et
al. |
October 7, 2004 |
Method for the direct, exponential amplification and sequencing of
DNA molecules and its application
Abstract
A method is described for the direct, exponential amplification
and sequencing ("DEXAS") of a DNA molecule from a complex mixture
of nucleic acids, wherein truncated DNA molecules as well as DNA
molecules of full length are synthesized simultaneously and
exponentially between two positions on the said DNA molecule, which
initially contains a DNA molecule in a thermocycling reaction, a
first primer, a second primer, a reaction buffer, a thermostable
DNA polymerase, a thermostable pyrophosphatase (optionally),
deoxynucleotides or derivatives thereof and a dideoxynucleotide or
derivatives thereof.
Inventors: |
Paabo, Svante; (Munchen,
DE) ; Kilger, Christian; (Munchen, DE) |
Correspondence
Address: |
ARENT FOX KINTNER PLOTKIN & KAHN
1050 CONNECTICUT AVENUE, N.W.
SUITE 400
WASHINGTON
DC
20036
US
|
Assignee: |
Roche Diagnostics GmbH
|
Family ID: |
7815636 |
Appl. No.: |
10/824465 |
Filed: |
April 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10824465 |
Apr 15, 2004 |
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09956342 |
Sep 20, 2001 |
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09956342 |
Sep 20, 2001 |
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09311723 |
May 14, 1999 |
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09311723 |
May 14, 1999 |
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08991347 |
Dec 16, 1997 |
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6107032 |
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Current U.S.
Class: |
435/6.11 ;
435/91.2 |
Current CPC
Class: |
C12Q 2527/143 20130101;
C12Q 1/6869 20130101; C12Q 1/6869 20130101; C12Q 2535/113 20130101;
C12Q 2521/101 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 1996 |
DE |
196 53 439.9 |
Claims
1-24. (canceled)
25. A method for sequencing of a selected region of a target
nucleic acid polymer in a sample containing the selected region in
substantially natural relative abundance, comprising the steps of:
(a) combining the sample containing the target region in
substantially natural relative abundance with first and second
primers, a nucleotide triphosphate feedstock mixture, a
chain-terminating nucleotide triphosphate and a thermally stable
polymerase enzyme which incorporates dideoxynucleotides into an
extending nucleic acid polymer at a rate which is no less than 0.4
times the rate of incorporation of deoxynucleotides in an
amplification mixture to form a reaction mixture, said first and
second primers binding to the sense and antisense strands,
respectively, of the target nucleic acid polymer at locations
flanking the selected region; (b) exposing the reaction mixture to
a plurality of temperature cycles each of which includes at least a
high temperature denaturation phase and a lower temperature
extension phase, thereby producing a plurality of terminated
fragments; and (c) evaluating terminated fragments produced during
the additional cycles to determine the sequence of the selected
region, wherein at least one of the first and second primers is
labelled with a-fluorescent label.
26. The method of claims 25, wherein the polymerase enzyme is
THERMO SEQUENASE.TM..
27. The method of claim 25, wherein the first and second primers
are each labeled with a different fluorescent label.
28. The method of claim 25, wherein the mole ratio of the
dideoxynucleotide triphosphate to the corresponding deoxynucleotide
triphosphate is from 1:100 to 1:300.
29. The method of claim 25, wherein one of the first and second
primers is labeled with a fluorescence label and the other of the
first and second primer is unlabelled.
Description
[0001] The present invention relates to a method for the direct,
exponential amplification and sequencing of DNA molecules as well
as the use of the method. The direct, exponential amplification and
sequencing of DNA molecules is referred to as "DEXAS" in the
following.
TECHNICAL FUNDAMENTALS
[0002] DNA sequence determination as developed by Sanger et al.
((1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467) is usually
carried out with a T7 DNA polymerase (Tabor S. and Richardson, C.
C. (1989) Proc. Natl. Acad. Sci. USA 86, 4076-4080). This method
requires relatively large amounts of a purified, single-stranded
DNA template. Recently cycle sequencing has been developed (Murray,
V. (1989) Nucleic Acids Res. 17, 8889). This method does not
require a single-stranded template and allows the sequence reaction
to be initiated with relatively small amounts of template. However,
the template DNA has to be purified to almost complete homogeneity
and is usually prepared by means of cloning in plasmids (Bolivar,
F. et al., (1977) Gene 2, 95-113) and subsequent plasmid
purification (Birnboim, H. C. and Doly, J. (1979) Nuicleic Acids
Res. 7, 1513-1523) or by means of PCR amplification (Mullis, K. B.
and Faloona, F. A. (1987) Methods Enzymol. 155, 335-350). Only one
primer is used in both of the methods described above.
[0003] In one embodiment of the cycle sequencing which is referred
to as "coupled amplification and sequencing" or "CAS" Ruano and
Kidd ((1991) Proc. Natl. Acad. Sci. USA 88, 2815-2819; U.S. Pat.
No. 5,427,91 1) have shown that one can use a two-step protocol to
generate sequences from DNA templates. In the first step 15 PCR
cycles are carried out with Taq DNA polymerase in the absence of
dideoxynucleotides in order to prepare an adequate amount of
sequencing template. In a second step in which dideoxynucleotides
and a labelled primer are added, CAS produces the sequence as well
as the additional amplification of the target sequence. Two primers
are used in both steps of the method.
[0004] Taq DNA polymerase, that is used in coupled DNA sequencing
reactions strongly discriminates against ddNTPs and preferably
incorporates dNTPs if it is furnished with a mixture of ddNTPs as
well as dNTPs. In addition it incorporates each ddNTP, i.e. ddATP,
ddCTP, ddGTP, ddTTP, with a strongly varying efficiency. Hence the
optimization of the CAS process requires careful titration of the
dideoxynucleotides.
[0005] Furthermore since coupled amplification and sequencing
depends on the amount of the initial DNA, the distance between the
two primers and the concentrations and the ratios of the ddNTPs and
dNTPs relative to one another and to each other, the optimization
of coupled amplification and sequencing reactions (CAS) requires
that the reaction conditions are individually optimized for a
particular DNA fragment.
[0006] All the methods described above require an interruption
between the first step of exponential amplification of the template
DNA and the second step for the synthesis of truncated DNA
molecules and also require the individual optimization of a given
DNA fragment which can be tedious and time-consuming and can lead
to errors especially when sequencing a large number of different
DNA molecules or when processing large amounts of samples in a
hospital or laboratory or when sequencing rare samples for forensic
or archaeological studies.
[0007] For this reason it would be advantageous to have available a
method for sequencing nucleic acids which simultaneously
potentiates the exponential amplification of molecules of full
length and of molecules of truncated length in the reaction which
leads to a reduction of the required amount of starting nucleic
acid molecules and does not require an interruption of the
exponential amplification step and of the sequencing step so that
the whole reaction can be carried out more rapidly and with fewer
manipulations.
SUMMARY OF THE INVENTION
[0008] The object of the present invention is to provide an
improved, rapid and reliable method for sequencing DNA molecules,
preferably genomic DNA.
[0009] A further object of the present invention is to provide a
direct method for nucleic acid sequencing which simultaneously
increases the exponential amplification of molecules of full length
as well as of molecules of truncated length in the reaction which
leads to a reduction of the initial amount of nucleic acid
molecules that are required for the cycling reaction.
[0010] A further object of the present invention is to provide an
improved, rapid and reliable method for sequencing DNA molecules,
preferably genomic DNA that can be carried out in a single step in
a single container.
[0011] A further object of the present invention is to provide an
application according to the invention for sequence determination
in medical diagnostics, forensics and population genetics.
[0012] Further objects of the invention are obvious to a person
skilled in the art from the description.
[0013] In contrast to the above-described "CAS" method a DNA
polymerase is used as the thermostable DNA polymerase which,
compared to wild-type Taq DNA polymerase, has a reduced
discrimination against the four ddNTPs in the buffer and under the
conditions that are used for the thermocycling. More preferably a
DNA polymerase is used which carries a "Tabor-Richardson" mutation
or a functional derivative thereof which also has no
5'-3'exonuclease activity such as e.g. AmplitaqFS.TM. (Taq DNA
polymerase (-exo5'- 3')(F667Y), Tabor and Richardson (1995), loc.
cit.), Taquenase.TM. (Taq DNA polymerase .DELTA.235 (-exo5'-3')
(F667Y), Tabor and Richardson (1995), loc. cit.) and Thermo
Sequenase.TM. (Taq DNA polymerase (-exo5'-3') (F667Y), Tabor and
Richardson (1995), loc. cit.) as well as mixtures thereof or other
DNA polymerases and mixtures thereof which are thermostable can
also be used in the method of the present invention.
[0014] Surprisingly the use of a DNA polymerase which, in
comparison to wild-type Taq DNA polymerase, has a reduced
discrimination against the four ddNTPs, enables the simultaneous
and exponential synthesis of truncated as well as of full fragments
from the start of the cycling reaction. Hence the present invention
concerns a method for the direct sequencing of a nucleic acid
molecule from a complex mixture of nucleic acids, such as e.g.
total genomic human DNA, containing a reaction buffer,
deoxynucleotides or derivatives thereof and a dideoxynucleotide or
another terminating nucleotide and a thermostable polymerase which
has a reduced discrimination against ddNTPs in comparison to
wild-type Taq DNA polymerase. Within the sense of the present
invention direct sequencing means that the nucleic acid fragment to
be sequenced is simultaneously amplified and sequenced in one step
without interrupting the reaction and without prior amplification
of the nucleic acid fragment to be sequenced by the known methods
and in such a manner that an unequivocal sequence ladder is
readable.
[0015] The principle of DEXAS is that the initial and subsequent
cycle sequencing reaction is carried out with two primers, a first
primer, and a second primer which lies on the strand complementary
to the first, which are preferably present in a non-equimolar ratio
and serve to simultaneously produce adequate template molecules of
full length as well as truncated molecules which contribute to the
sequencing of the DNA molecule. Four reactions are prepared, one
for the determination of each base, so that each reaction contains
two primers preferably in a non-equimolar ratio to one another of
which either one is labelled and the other is unlabelled or both
are differently labelled. The said non-equimolar ratio between the
first primer and the second primer enables the simultaneous and
exponential synthesis of the truncated as well as of the full
fragments from the start of the cycling reaction. Furthermore each
reaction contains from the start the DNA template to be sequenced
as well as a buffer solution, thermostable DNA polymerase,
thermostable pyrophosphatase (optionally), the four
deoxynucleotides or derivatives thereof and a dideoxynucleotide or
a terminating nucleotide e.g. 3-aminonucleotide or
3'-ester-derivatized nucleotides.
[0016] Thereafter cycles for denaturing and extension are carried
out so that in each of these cycles two types of extension products
are formed from each primer. Each primer functions such that it
initiates extension products which are long enough to reach the
other primer position. Simultaneously products are initiated by
each primer which, due to the incorporation of a dideoxynucleotide,
are terminated before the other primer position is reached. The
former said products (products of full length) serve in the
following cycles as a template for the production of further DNA
strands of full length and are also used as templates for
extensions that contribute to the sequence reaction, and the latter
products (truncated products) accumulate during the cycles and
contribute to the sequence ladder that is generated. Hence DEXAS
results in the simultaneous exponential production of a sequencing
template and a sequence ladder in a single tube without having to
interrupt the thermocycling reaction.
[0017] Therefore the use of the present invention enables the DNA
sequence of multicopy and single-copy regions of DNA to be
determined in a single step.
[0018] Hence the present invention for the first time provides a
method which enables the nucleic acid to be sequenced to be
simultaneously amplified and sequenced from a complex mixture of
nucleic acids, such as e.g. total genomic human DNA, without prior
amplification by the known methods, in one step i.e. without
interrupting the reaction and such that an unequivocal sequence
ladder is readable wherein at least one thermostable DNA
polymerase, a nucleic acid molecule, a first primer, a second
primer, a reaction buffer, deoxynucleotides or derivatives thereof
and at least one dideoxynucleotide or another terminating
nucleotide is present in the initial reaction mixture.
[0019] Furthermore the aforementioned object and goals of the
present invention are achieved by the provision of a method for
sequencing DNA molecules in which truncated DNA molecules as well
as DNA molecules of full length are simultaneously and
exponentially synthesized between two positions on the said DNA
molecule in a thermocycling reaction which initially contains a DNA
molecule, a first primer, a second primer, a reaction buffer, a
thermostable DNA polymerase, thermostable pyrophosphatase
(optionally), deoxynucleotides or derivatives thereof, and a
dideoxynucleotide or another terminating nucleotide thereof wherein
the initial ratio of the said primers in the said thermocycling
reaction is not equal to I.
[0020] In a preferred embodiment of the method of the invention the
ratio of the said primers to one another is about 2:1 to about 3:1,
most preferably 2:1.
[0021] In a further preferred embodiment of the method of the
invention the said primers have such a length that the
signal-to-noise ratio between the specific truncated DNA molecules
and the unspecific DNA molecules is large enough not to
substantially prevent the reading of the sequence. The said primers
preferably have a length of at least 25 nucleotides.
[0022] Primers can be synthesized by means of methods known in the
state of the art. For example primers can be synthesized using
known methods which do not significantly change the stability or
function of the said primers during the nucleic acid sequencing
method of the present invention.
[0023] Furthermore the PNA-DNA hybrid oligonucleotides (see Finn,
P. J. et al., N.A.R. 24, 3357-3363 (1996), Koch, T. et al.,
Tetrahedron Letters, 36, 6933-6936 (1995), Stetsenko, D. A, et al.,
Tetrahedron Letters 37, 3571-3574 (1996), Bergmann, F. et al.,
Tetrahedron Letters 36, 6823-6826 (1995) and Will, D. W. et al.,
Tetrahedron 51, 12069-12082 (1995)) are also regarded as primers
for the method according to the invention.
[0024] In a further preferred embodiment of the invention the said
first primer is labelled. Moreover it is preferable that the said
first primer and second primer are labelled differently. Any
suitable agents or methods known in the state of the art can be
used as single or differential labelling agents and methods,
provided that they do not significantly change the stability or
function of the said primer in the DNA sequencing method of the
present invention. For example single and differential labels can
be selected from the group which comprises those enzymes such as
.beta.-galactosidase, alkaline phosphatase and peroxidase, enzyme
substrates, coenzymes, dyes, chromophores, fluorescent,
chemiluminescent and bioluminescent labels such as FITC, Cy5,
Cy5.5, Cy7, Texas-red and IRD40 (Chen et al., (1993), J. Chromatog.
A 652: 355-360 and Kambara et al. (1992), Electrophoresis 13:
542-546) ligands or haptens such as e.g. biotin and radioactive
isotopes such as .sup.3H, .sup.35S, .sup.32P, .sup.125I and
.sup.14C.
[0025] The method according to the invention can also be carried
out as a "hot start" method. In this case it is ensured that the
activity of the polymerase or polymerases only starts at an
increased temperature in order to suppress a polymerization on
unspecifically hybridized primers at lower temperatures. One
possibility is that the thermocycling reaction additionally
contains a polymerase-inhibiting agent. Polymerase antibodies are
for example available commercially which only denature at higher
temperatures and thus release enzyme activity of the polymerase.
However, polymerases modified by genetic engineering that are
present in an inactive form at lower temperatures would also be
conceivable.
[0026] DEXAS is relatively insensitive to various buffers and
various deoxynucleotides and dideoxynucleotide concentrations and
can be carried out using various thermostable DNA polymerases.
[0027] The number of thermocycles can be from about 18 to about 50
cycles depending on the amount of template DNA and its purity.
[0028] Buffer components which can be used can include Tris-HCl at
a pH of about 9.0 to 9.5 and at a concentration of about 10 to 30
mM, ammonium sulfate at a concentration of about 10 to 20 mM
preferably 15 mM, MgCl.sub.2 at a concentration of about 3.5 to 5.5
mM optionally about 0.05 mM mercaptoethanol, about 0.28%
Tween20.RTM. and/or about 0.02% Nonidet 40.RTM. but, however, are
not limited to these.
[0029] Deoxynucleotides may be selected from dGTP, dATP, dTTP and
dCTP but are not limited to these. According to the invention it is
additionally also possible to use derivatives of deoxynucleotides
which are defined as those deoxynucleotides which are able to be
incorporated by a thermostable DNA polymerase into growing DNA
molecules that are synthesized in the thermocycling reaction. Such
derivatives can include thionucleotides, 7-deaza-2'-dGTP,
7-deaza-2'-dATP as well as deoxyinosine triphosphate that can also
be used as a substitute deoxynucleotide for dATP, dGTP, dTTP or
dCTP, but are not limited to these. The aforementioned
deoxynucleotides and derivatives thereof are preferably used at a
concentration between about 300 .mu.M and 2 mM.
[0030] Dideoxynucleotides can be selected from ddGTP, ddATP, ddTTP
and ddCTP but, however, are not limited to these. According to the
invention it is also additionally possible to use derivatives of
dideoxynucleotides which are defined as those dideoxynucleotides
that are able to be incorporated by a thermostable DNA polymerase
into growing DNA molecules that are synthesized in a thermo-cycling
reaction. In addition it is also possible to use other terminating
nucleotides e.g. 3'-amino nucleotide or 3'-ester-derivatized
nucleotides. Preferred concentrations of ddNTPs are between about 1
and 5 .mu.M.
[0031] In the method according to the invention the preferred ratio
of dNTPs to ddNTPs (dNTPs:ddNTPs) is between 100:1 and 1000:1
preferably between 300:1 and 600:1.
[0032] In a further preferred embodiment of the method of the
invention the said method is carried out at a temperature at which
the signal-to-noise ratio between the specific truncated DNA
molecules and the unspecific DNA molecules is large enough not to
substantially impede reading of the sequence. It is less important
to optimize the annealing temperature. In the case of human
single-copy DNA sequences the highest possible annealing
temperature drastically reduces the background. In this case the
annealing and synthesis steps of the thermocycling reaction are
preferably carried out at a minimum temperature of 62.degree. C.,
more preferably at 66.degree. C. and most preferably at at least
about 68.degree. C.
[0033] The template of the DNA molecule to be sequenced is
preferably present as a total genomic DNA molecule which does not
have to be cloned or purified, but this may be the case. In one
embodiment of the invention the genomic DNA has a length of more
than or equal to 2 kb. Other forms of DNA that can be used as
templates include cloned or uncloned mitochondrial DNA, partially
purified or unpurified DNA such as e.g. plasmid DNA of bacterial
colonies. DEXAS functions well with about 250 ng template DNA for
the determination of mitochondrial DNA sequences and about 1 .mu.g
template DNA for determining single-copy DNA sequences such as e.g.
total genomic DNA, but it also functions with smaller amounts of
mitochondrial or genomic DNA The method according to the invention
can also be used for the direct sequencing of unpurified
single-stranded or double-stranded DNA from bacteriophages. DEXAS
is in addition relatively independent of the base composition of
the template.
[0034] In a preferred embodiment the method according to the
invention is furthermore characterized in that each thermocycling
reaction to determine the position of A, G, C and T in the said DNA
molecule is carried out in a single step, in a single container,
vessel or tube.
[0035] Suitable sources of nucleic acid molecules in the method
according to the invention are body fluids such as sperm, urine,
blood or fractions of these, hairs, an individual cell, cells or
fractions thereof, hard tissue such as bones or soft tissue or
fractions thereof and cell cultures or fractions thereof.
[0036] The present invention also serves for the application of the
method according to the invention for the determination of a
nucleotide sequence of a given nucleic acid molecule e.g. for
sequencing Shotgun libraries with two labels for large-scale genome
projects and in medical diagnostics, forensics and population
genetics. The method of the present invention can be used to detect
genetic mutations or polymorphisms, to identify the origin of the
sequenced nucleic acid or to detect the presence of foreign or
infectious agents in a sample.
[0037] The present invention relates to all combinations of all
procedures of the above methods.
[0038] After preparation the sequencing reactions can be loaded
directly onto a sequencing gel such as e.g. after addition of a
commonly used application buffer (e.g. formamide which contains 20
mM EDTA (pH 7.4) and 6 mg/ml dextran blue) and denaturation (e.g.
for 4 minutes at 96.degree. C.). The sequence ladder can be read
according to known methods. The method of the invention is well
suited for automation. Since the two primers in the reaction are
provided with different labels which can for example be detected
with two different wavelengths, the method of the present invention
enables the simultaneous sequencing of both strands of a template
and the detection of both reactions in one or several gel lanes. In
general many DEXAS reactions that are carried out using different
dyes can be carried out simultaneously in the same tube and applied
to a sequencing instrument that is equipped with several lasers or
be detected by other methods such as e.g. autoradiography.
[0039] A further subject matter of the present invention is a kit
for the direct sequencing of a nucleic acid molecule from a complex
mixture of nucleic acids, such as e.g. total genomic human DNA,
containing a reaction buffer, deoxynucleotides or derivatives
thereof and a dideoxynucleotide or a further terminating nucleotide
and a thermostable polymerase which has a reduced discrimination
against ddNTPs compared to wild-type Taq DNA polymerase. Within the
sense of the present invention direct sequencing means that the
nucleic acid fragment to be sequenced is simultaneously amplified
and sequenced, without prior amplification of the nucleic acid
fragment to be sequenced by the known methods, in a single step
without interrupting the reaction and such that an unequivocal
sequence ladder can be read.
[0040] A further subject matter of the present invention is a kit
for the direct sequencing of a nucleic acid molecule of a complex
mixture of nucleic acids containing a reaction buffer,
deoxynucleotides or derivatives thereof and a dideoxynucleotide or
another terminating nucleotide, a thermostable polymerase and two
primers whose ratio is larger than 1. The kit particularly
preferably contains a thermostable polymerase which has a reduced
discrimination against ddNTPs in comparison to the wild-type Taq
DNA polymerase.
SHORT DESCRIPTION OF THE FIGURES
[0041] FIG. 1 Schematic representation of DEXAS. Two
oligonucleotides (27 mers), either a labelled and an unlabelled
oligonucleotide or an oligonucleotide labelled with FITC and an
oligonucleotide labelled with Cy5 (ratio 2:1) are mixed in four
tubes with human genomic DNA (250 ng to 3 .mu.g), a heat-resistant
DNA polymerase, the four deoxynucleotides and in each case one of
the dideoxynucleotides. Cycles for denaturing and subsequent
annealing and extension are carried out. During each extension the
primers are either extended up to the complementary primer position
or they are interrupted by the incorporation of a
dideoxynucleotide. In subsequent cycles these former products serve
as templates for the further generation of products of full length
as well as for the termination reactions whereas the latter
products accumulate during all of the cycles that are carried out
and contribute to the sequence signal. After the cycling the
reactions are denatured and, depending on their label, are either
analysed on an A.L.F. or an A.L.F. express.
[0042] FIG. 2. DEXAS reaction carried out on a 521 bp segment of
the human mitochondrial control region. Eight pmol of an
FITC-labelled (mtDNA1-L16026) and 4 pmol of an unlabelled primer
(mtDNA2-H16498) were used together with 250 ng of total genomic
human DNA (see text for details). A strong signal can be seen
before the first processed base and a strong stop can be seen at
about base number 440. The sequence was processed with the A.L.F.
software and was not edited manually. A total of 433 bases was
determined.
[0043] FIG. 3. DEXAS reaction carried out on single-copy genes.
FIG. 3A shows a sequence of the human p53 gene whereas FIG. 3B
shows a sequence of the human CCR-5 gene (see text for details).
The sequence was processed with the A.L.F. software and was not
edited manually. A total of 305 bases was determined in the case of
the p53 gene whereas 343 bases were determined for the CCR-5
gene.
[0044] FIG. 4. Two colour DEXAS reaction using different
oligonucleotide ratios. Each of the reactions was carried out using
250 ng genonic human DNA and a total amount of 12 pmol primer.
MtDNA1 was labelled with FITC (left panel) and MtDNA2 was labelled
with Cy5 (right panel). The ratios between FITC-MtDNA1 and
Cy5-MtDNA2 were varied between 2:1 (upper panel), 1:1 (middle
panel) and 1:2 (lower panel). The largest signal-to-noise ratio for
both primers is achieved when a ratio of 2:1 is used. The raw data
of the A.L.F. and of the A.L.F. express instruments are shown. The
assignment of the base signals from top to bottom is C, A, G, and T
respectively.
[0045] FIG. 5. DEXAS was carried out using simultaneously a
fluorescein-labelled `T3` primer and a Cy5 -labelled `universal`
primer. The figure shows the sequence that was obtained with the
Cy5-labelled primer. The two primers were used in a single reaction
using one bacterial colony. 4 .mu.l of each was analysed on an
A.L.F. or an A.L.F. express. The reaction with the `T3` primer
yielded 407 bases and the reaction with the `universal` primer
yielded 668 bases.
[0046] FIG. 6. The insert of a plasmid was sequenced from both
sides in a reaction using a FITC-labelled `T3` primer and an
opposite Cy5-labelled `universal` primer. The simultaneous use of
two differently labelled oligonucleotides in a DEXAS reaction
allowed the 548 base insert to be sequenced without leaving
ambiguous positions.
[0047] The primers were positioned at a distance of 670 bp to one
another.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The invention is described in more detail by the following
non-limiting examples.
EXAMPLE 1
Templata Preparation
[0049] Total genomic human DNA was prepared from 2 ml blood samples
using a rapid cleaning kit (Cambridge Molecular Technologies Ltd.,
Cambridge, UK). Purified DNA was diluted in ddH.sub.2O to a
concentration of 175 ng per .mu.l.
Sequencing Reagents and Conditions
[0050] Unlabelled and FITC-labelled oligonucleotides were
synthesized with an ABI DNA/RNA synthesizer model 392. Cy5-labelled
oligonucleotides were obtained from the Pharmacia Biotech Company
(Freiburg, Germany). The following oligonucleotides were used in
each case to sequence the mitochondrial control region (mtDNA), the
p53 gene (p53) and the CCR-5 gene (CCR-5):
1 SEQ ID NO. 1: (mtDNA1-L16026): 5'-GAT TCT AAT TTA AAG TAT TCT CTG
TTG-3'; SEQ ID NO.2: (mtDNA2-H16498): 5'-TTA TGA CCC TGA AGT AGG
AAC CAG ATG-3'; SEQ ID NO.3: (p53-1/exon-7): 5'-GGA GGC ACT TGC CAC
CCT GCA CAC TGG-3'; SEQ ID NO.4: (53-2/intron-8): 5'-CTC CTC CAC
CGC TTG TTG TTC TGC TTG-3' SEQ ID NO. 5: (CCR5-1): 5'-GGC TGG TCC
TGC CGC TGC TTG TCA T-3'; SEQ ID NO. 6: (CCR5-2): 5'-CTG CTC CCC
AGT GGA TCG GGT GTA AAC-3'.
[0051] The numbering of the mtDNA primers refers to the 3'end
according to Anderson et al. ((1981) Nature 290, 457-465) and L and
H refer to the L strand and the H strand respectively. The DEXAS
reaction was carried either using ThermoSequenase.TM. (Tabor, S.
and Richardson, C. C. (1995) Proc, Natl. Acad. Sci. USA 92,
6339-6343) (Amersham, UK) reagents or using the following
10.times.buffer: 500 mM Tris-HCl (pH 9.2), 160 mM
(NH.sub.4).sub.2SO.sub.4, 35 mM MgCl.sub.2 (ScienTech Corp., St.
Louis, Mo.). Three different nucleotide mixtures were evaluated for
the termination: (i) 1:333, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM
dTTP, in which the A, C, G and T reaction each contained 3 .mu.M of
the corresponding dideoxy-nucleotide. (ii) 1:666 also containing in
each case 1 mM of each deoxynucleotide but 1.5 .mu.M of the
corresponding dideoxynucleotide. (iii) 1:1000 also containing in
each case 1 mM of each deoxynucleotide but 1.0 .mu.M of the
corresponding dideoxynucleotide. All termination mixtures were
prepared using 50 mM Tris-HCl (pH 9.2), 16 mM
(NH.sub.4).sub.2SO.sub.4, 5 mM MgCl.sub.2.
[0052] A premix of 1 .mu.l (units not defined) Taquenase.TM.
(ScienTech Corp., St. Louis, Mo.) and 1 unit thermostable
pyrophosphatase (NEB, Beverly, Mass.) was prepared for each
sequencing reaction. In the case of the ThermoSequenase reactions,
the reactions were prepared as recommended by the manufacturer. In
the other cases a 20 .mu.l mixture of primer (2 pmol to 12 pmol),
DNA (15 ng to 1.5 .mu.g), sequencing buffer (2 .mu.l of the
10.times.buffer, see above) and enzyme was prepared and a 5 .mu.l
aliquot of this was added to 2 .mu.l termination mix. The
sequencing reactions were carried out in a thermocycler with a
heatable cover (MJ-Research, Watertown, Mass.). The reactions were
stopped by adding 5 .mu.l formamide (20 mM EDTA (pH 7.4) and 6
mg/ml dextran blue) which was followed by a 4 minute denaturation
at 95.degree. C.
[0053] The sequencing reactions were analysed on an A.L.F. when
FITC-labelled primers were used and on an A.L.F. express when
Cy5-labelled primers were used (both Pharmacia Biotech, Uppsala,
Sweden). HydroLink Long Ranger.TM. (FMC, Rockland, Me.) gels and 30
cm glass plates were used in all cases. The gel conditions were in
accordance with the manufacturer's recommendations.
EXAMPLE 2
DEXAS of Mitochondrial DNA Sequences
[0054] Two oligonucleotides were synthesized both having a length
of 27 nucleotides which span a 521 base pair region of the human
mitochondrial control region. 27-mers were used to minimize an
unspecific annealing of the primers to incorrect priming positions
and to enable the reaction temperatures to remain above 62.degree.
C. during all synthesis steps. One of the two oligonucleotides was
labelled at the 5'end with fluorescein (mtDNA1) whereas the other
(mtDNA2) was unlabelled. 4 pmol of each of the primers was mixed
with ThermoSequenase.TM. (Amersham, UK) reagent which contained
enzyme (DNA polymerase and thermostable pyrophosphatase), reaction
buffer and a mixture of deoxynucleotides and dideoxynucleotide.
Different amounts of human DNA (500 ng, 250 ng, 125 ng, 62 ng, 0
ng) were added to individual aliquots of this mixture. 500 ng
template DNA but no unlabelled primer was added to one tube. The
reactions were incubated for 3 minutes at 95.degree. C. in order to
enable a complete denaturation of the template DNA. Subsequently 35
cycles each of 30 sec. at 62.degree. C. and 40 sec. at 95.degree.
C. were carried out. The reactions were stopped and denatured by
the addition of formamide and heating to 95.degree. C. for 4 min
before they were loaded onto an A.L.F. sequencing gel.
[0055] In the case were no DNA had been added no sequence was
detectable. No sequence was detectable when only the labelled
primer and no unlabelled primer had been added. However, sequence
curves were obtained in cases in which 62 ng or more template had
been used. In the reactions in which 250 ng and 500 ng had been
used the A.L.F. software was able to determine more than 400
bases.
[0056] Using a constant amount of template DNA of 500 ng and a
total amount of 12 pmol of the two primers, the ratios between the
labelled primer and the unlabelled primer were varied in each case
between 3:1, 2:1, 1:1, 1:2 and 1:3. The reaction in which the
primers were present in equimolar amounts yielded poor signals
whereas all other ratios, independently of whether the labelled or
the unlabelled primer was present in excess, yielded better
results. The ratios 2:1 and 1:2 yielded the best results. It was
surprising and unexpected that both non-equimolar ratios are
advantageous. Using 8 pmol of the primer mtDNA1 and 4 pmol of the
primer mtDNA2 we presently routinely determine 450 base pairs of
the mitochondrial control region.
[0057] The ratio of the deoxynucleotides (dNTPs) to
dideoxy-nucleotides (ddNTPs) can be varied in the DEXAS reaction. A
higher proportion of dNTPs will probably allow an increased
template production in each cycle whereas a higher proportion of
ddNTPs would lead to an increased termination of the extension
products before the priming position of the second unlabelled
primer is reached. The latter products will contribute to the
sequence reaction but not to the further template amplification. In
order to determine to what extent the ratio of ddNTPs to dNTPs
influences the reaction, ddNTPs were mixed with dNTPs in ratios of
1:333, 1:666 and 1:1000 and used in a DEXAS reaction containing 8
pmol of an FITC-labelled primer (mtDNA1), 4 pmol of an unlabelled
primer (mtDNA2) and 300 ng human DNA. The reaction conditions were
as described above. The results showed that the ratio 1:666
(ddNTPs:dNTPs) yielded stronger signals.
EXAMPLE 3
Dexas of Single Copies of Human Dna Sequences
[0058] In order to evaluate the applicability of DEXAS to
single-copy DNA sequences, primers were synthesized which flank a
507 base pair segment of the intron 7 and the exon 8 of the human
p53 gene. DEXAS reactions were prepared which each contained 8 pmol
of an FITC-labelled sequencing primer (p53-1), 4 pmol of an
unlabelled (p53-2) primers and 3.5 .mu.g, 1.75 .mu.g, 875 ng and
430 ng human DNA. These reactions were denatured for 3 minutes at
95.degree. C. and 40 cycles comprising 30 seconds at 62.degree. C.
and 40 seconds at 95.degree. C. were carried out. The results
showed a clearly readable sequence. In order to improve the results
various modifications of the protocol were evaluated. The annealing
temperature was increased and the amount of the sequencing primer
was reduced. Additionally the number of cycles was increased to 47
and various primer ratios and template concentrations were
evaluated. The best results were obtained using 8 pmol of the
FITC-labelled primer and 4 pmol of the unlabelled primer and
cycling temperatures of 30 seconds at 68.degree. C. and 30 seconds
at 95.degree. C. These conditions yielded between 260 and 320 bases
of the sequence with 1 to 5 ambiguities per reaction in five
experiments (FIG. 3A). If about 1 .mu.g or more template was used,
the sequence signals were read with the A.L.F. software using
automated processing.
[0059] In order to further evaluate the general applicability of
DEXAS to single copy genes, primers were synthesized which flank a
382 base pair segment of the CCR-5 gene. 3 pmol of the CCR5-1, 6
pmol of the FITC-labelled primer CCR5-2, 0.5-1.0 .mu.g template DNA
and 45 cycles DEXAS were used. In the sequence reactions carried
out on 40 samples the reading lengths varied between 230 bp and 351
bp (average 294 bp). A typical reaction is shown in FIG. 3B.
EXAMPLE 4
Simultaneous Sequencing of Both Dna Strands
[0060] It was shown that it is possible to sequence both
complementary DNA strands of plasmid DNA in a single reaction using
two different fluorescently-labelled primers (Wiemann, S., et al.,
(1995) Analytical Biochemistry 224, 117-121). The applicability of
this approach to DEXAS was analysed using an FITC-labelled primer
(mtDNA1), a Cy5-labelled primer (mtDNA2) and 500 ng human DNA as
the template. While retaining the above reaction conditions the
primer ratios were varied (FITC-mtDNA1 :Cy5-mtDNA2) (3:1, 2:1, 1:1,
1:2 and 1:3). After the cycling reaction and denaturation 5 .mu.l
of the reaction was applied to an A.L.F. or an A.L.F. express
instrument. As in previous experiments considerably poorer results
were obtained if equimolar amounts of primer were used compared to
reactions in which non-equimolar amounts were used (FIG. 4). A
ratio of 2:1 at a total amount of 12 pmol gave the best
signal-to-noise ratio. In such reactions 450 bases were read on
both strands without yielding ambiguous positions. The observation
that a larger amount of FITC-labelled primer than Cy5-labelled
primer is advantageous is probably due to experiments it was
possible to show that the two colour approach can also be applied
to single-copy the better signal-to-noise ratio of Cy5 compared to
FITC. In further genes.
Sequence CWU 1
1
6 1 27 DNA Artificial Sequence Description of Artificial Sequence
synthetic primer 1 gattctaatt taaactattc tctgttc 27 2 27 DNA
Artificial Sequence Description of Artificial Sequence synthetic
primer 2 ttatgaccct gaagtaggaa ccagatg 27 3 27 DNA Artificial
Sequence Description of Artificial Sequence synthetic primer 3
ggaggcactt gccaccctgc acactgg 27 4 27 DNA Artificial Sequence
Description of Artificial Sequence synthetic primer 4 ctcctccacc
gcttcttgtt ctgcttg 27 5 25 DNA Artificial Sequence Description of
Artificial Sequence synthetic primer 5 ggctggtcct gccgctgctt gtcat
25 6 27 DNA Artificial Sequence Description of Artificial Sequence
synthetic primer 6 ctgctcccca gtggatcggg tgtaaac 27
* * * * *