U.S. patent application number 09/755088 was filed with the patent office on 2002-11-28 for method for the uncoupled, direct, exponential amplification and sequencing of dna molecules with the addition of a second thermostable dna polymerase and its application.
This patent application is currently assigned to Roche Diagnostics GmbH. Invention is credited to Kilger, Christian, Paabo, Svante.
Application Number | 20020177129 09/755088 |
Document ID | / |
Family ID | 7815667 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177129 |
Kind Code |
A1 |
Paabo, Svante ; et
al. |
November 28, 2002 |
Method for the uncoupled, direct, exponential amplification and
sequencing of DNA molecules with the addition of a second
thermostable DNA polymerase and its application
Abstract
Method for sequencing a nucleic acid molecule in a thermocycling
reaction which initially comprises a nucleic acid molecule, a first
primer, a second primer, a reaction buffer, a first thermostable
DNA polymerase, (optionally) a thermostable pyrophosphatase,
deoxynucleotides or derivatives thereof and a dideoxynucleotide or
a derivative thereof and which is characterized in that the
thermocycling reaction additionally contains a second thermostable
DNA polymerase which, in comparison to the said first thermostable
DNA polymerase, has a reduced ability to incorporate
dideoxynucleotides as well as the use of the said method.
Inventors: |
Paabo, Svante; (Munchen,
DE) ; Kilger, Christian; (Munchen, DE) |
Correspondence
Address: |
ARENT FOX KINTNER PLOTKIN & KAHN, PLLC
Suite 600
1050 Connecticut Avenue, N.W.
Washington
DC
20036-5339
US
|
Assignee: |
Roche Diagnostics GmbH
|
Family ID: |
7815667 |
Appl. No.: |
09/755088 |
Filed: |
January 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09755088 |
Jan 8, 2001 |
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09357166 |
Jul 19, 1999 |
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09357166 |
Jul 19, 1999 |
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08991184 |
Dec 16, 1997 |
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Current U.S.
Class: |
435/6.11 ;
435/6.1; 435/91.2 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6869 20130101; C12Q 1/6869 20130101; C12Q 1/6869 20130101;
C12Q 2521/101 20130101; C12Q 2531/113 20130101; C12Q 2521/101
20130101; C12Q 2521/101 20130101; C12Q 2535/113 20130101; C12Q
2527/101 20130101; C12Q 2527/101 20130101; C12Q 2535/101 20130101;
C12Q 2535/113 20130101; C12Q 2535/113 20130101; C12Q 2527/101
20130101; C12Q 2549/101 20130101; C12Q 2549/101 20130101; C12Q
2535/113 20130101; C12Q 2521/101 20130101; C12Q 1/6869 20130101;
C12Q 1/6869 20130101; Y10S 435/81 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 1996 |
DE |
196 53 494.1 |
Claims
1.) Method for sequencing a nucleic acid molecule in a
thermocycling reaction which initially comprises 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, wherein the
thermocycling reaction contains at least one thermostable DNA
polymerase with different enzyme activities for incorporating
dideoxynucleotides.
2.) Method for sequencing a nucleic acid molecule as claimed in
claim 1, wherein the thermocycling reaction contains a first
thermostable DNA polymerase and additionally a second thermostable
DNA polymerase which has a reduced ability to incorporate
dideoxynucleotides in comparison to the said first thermostable DNA
polymerase.
3.) Method as claimed in claim 1 or 2, wherein the said first
thermostable polymerase has a reduced discrimination against ddNTPs
compared to wild-type Taq DNA polymerase in the buffer or under the
conditions that are used for the thermocycling.
4.) Method as claimed in one of the claims 1 to 3, wherein the said
first thermostable DNA polymerase has a higher processivity than
ThermoSequenase and the said second thermostable DNA polymerase has
a higher processivity than wild-type Taq DNA polymerase.
5.) Method as claimed in one of the claims 1 to 4, wherein the said
first thermostable polymerase is a DNA Taq polymerase with a
"Tabor-Richardson" mutation, which also lacks the 5'-3' exonuclease
activity, or a functional derivative thereof.
6.) Method as claimed in one of the claims 1 to 5, wherein the said
first thermostable polymerase is Taq DNA polymerase
(-exo5'-3')(F667Y) or a functional derivative thereof.
7.) Method as claimed in one of the claims 1 to 6, wherein the said
second thermostable DNA polymerase is Taq polymerase or a
functional derivative thereof.
8.) Method as claimed in one of the claims 1 to 7, wherein the said
method is carried out in one step, in a single container, vessel or
tube.
9.) Method as claimed in one of the claims 1 to 8, wherein the
ratio of the said primers is not equal to 1.
10.) Method as claimed in one of the claims 1 to 9, wherein the
ratio of the said primers is about 2:1.
11.) Method as claimed in one of the claims 1 to 10, wherein the
said first primer is labelled.
12.) Method as claimed in one of the claims 1 to 11, wherein the
said first primer and said second primer are differently
labelled.
13.) Method as claimed in one of the claims 1 to 12, wherein the
annealing steps of the thermocycling reaction are carried out at a
temperature of at least 55.degree. C.
14.) Method as claimed in one of the claims 1 to 13, wherein the
thermocycling reaction additionally contains a thermostable
pyrophosphatase.
15.) Method as claimed in one of the claims 1 to 14, wherein the
said primers have a length of at least 18 nucleotides.
16.) Method as claimed in one of the claims 1 to 15, wherein the
said nucleic acid molecule is genomic DNA.
17.) Method as claimed in one of the claims 1 to 16, wherein the
said nucleic acid molecule is RNA, the said second polymerase is a
thermostable DNA polymerase with reverse transcriptase
activity.
18.) Method as claimed in claim 17, wherein the said second
polymerase is Tth DNA polymerase or a functional derivative thereof
and the reaction is carried out in the presence of MnCl.sub.2 or Mn
acetate.
19.) Method as claimed in one of the claims 1 to 18, wherein the
source of the nucleic acid molecules to be sequenced is body fluids
such as sperm, urine, blood or blood samples, hairs, single cells
or fractions thereof, tissue or fractions thereof, cell cultures,
bacteria, viruses or bacteriophages.
20.) Method as claimed in one of the claims 1-19, wherein the
thermocycling reaction additionally contains a
polymerase-inhibiting agent so that the enzyme activity only occurs
at an increased temperature.
21. Use of the method as claimed in one of the claims 1 to 20 for
the determination of a sequence of a nucleic acid.
22. Use of the method as claimed in one of the claims 1 to 20 for
the direct sequencing of eukaryotic genomic DNA.
23. Use of the method as claimed in one of the claims 1 to 20 for
the direct sequencing of human chromosomal or mitochondrial
DNA.
24. Use of the method as claimed in one of the claims 1 to 20 for
the direct sequencing of human RNA.
25. Use of the method as claimed in one of the claims 1 to 20 for
the direct sequencing of unpurified plasmid DNA from bacterial
colonies.
26. Use of the method as claimed in one of the claims 1 to 20 for
the direct sequencing of unpurified single-stranded or
double-stranded DNA from bacteriophages.
27. Use of the method as claimed in one of the claims 1 to 20 for
the detection of genetic mutations or polymorphisms.
28. Use of the method as claimed in one of the claims 1 to 20 for
identifying the origin of the sequenced nucleic.
29. Use of the method as claimed in one of the claims 1 to 20 for
the detection of the presence of foreign or infectious agents in a
sample.
30. Use of the method as claimed in one of the claims 1 to 20 for
sequencing a nucleic acid molecule from body fluids such as sperm,
urine, blood or blood samples, hairs, single cells or fractions
thereof, tissues or fractions thereof, cell cultures, bacteria,
viruses or bacteriophages.
31. Kit for sequencing a nucleic acid molecule containing a
reaction buffer, deoxynucleotides or derivatives thereof and at
least one dideoxynucleotide or another terminating nucleotide and
at least one thermostable DNA polymerase with different abilities
to incorporate dideoxynucleotides.
32. Kit for sequencing a nucleic acid molecule as claimed in claim
31, wherein it contains a first thermostable DNA polymerase and
additionally a second thermostable DNA polymerase which, in
comparison to the said first thermostable DNA polymerase, has a
reduced ability to incorporate dideoxynucleotides.
33. Kit for sequencing a nucleic acid molecule as claimed in one of
the claims 31 or 32, wherein the said first thermostable polymerase
is Taq DNA polymerase (-exo5'-3')(F667Y) or a functional derivative
thereof and the said second thermostable DNA polymerase is Taq
polymerase or a functional derivative thereof.
Description
[0001] The present invention relates to a method for the uncoupled,
direct, exponential amplification and sequencing of DNA molecules
by the addition of a second thermostable DNA polymerase and it also
relates to the application of the said method. The uncoupled,
direct, exponential amplification and sequencing of DNA molecules
by the addition of a second thermostable DNA polymerase is referred
to as "DEXTAQ" in the following.
TECHNICAL FUNDAMENTALS
[0002] The 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) Nucleic 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] Known thermostable polymerases that are used for cycle
sequencing e.g. ThermoSequenase and Taquenase carry a mutation
which is known as the "Tabor Richardson" mutation (Tabor, S. &
Richardson, C. C. (1995) Proc. Natl. Acad. Sci. USA 92, 6339-6343)
in which a tyrosine is present instead of a phenylalanine in the
crevice of the enzyme which, during polymerization of the DNA
molecule being formed, is responsible for discriminating between
the incorporation of either deoxynucleotides or dideoxynucleotides.
Such enzymes or functional derivatives thereof have an increased
ability to incorporate dideoxynucleotides into DNA fragments that
are being formed and can be used to improve the signal uniformity
in sequencing reactions. The increased ability of the
aforementioned DNA polymerases with a Tabor-Richardson mutation to
incorporate dideoxy-nucleotides increases the statistical
probability that a chain termination occurs due to incorporation of
a dideoxynucleotide into a DNA molecule being formed.
[0004] 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. 5,427,911) 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.
[0005] 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,
ddGTP, ddCTP, ddTTP, with a strongly varying efficiency. Hence the
optimization of the CAS process requires careful titration of the
dideoxynucleotides.
[0006] 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.
[0007] All methods described above require a separate step for
template production, CAS accomplishes this with an interruption
between the first step for the exponential amplification of the
template DNA and the second step for the synthesis of truncated DNA
molecules. Also, all methods 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.
[0008] 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.
[0009] Furthermore it would also be advantageous to have available
a method for sequencing nucleic acid molecules which allows an
increase in the distance between the positions of the two primers
on the nucleic acid molecule to be sequenced, is relatively
independent of the distance between the said primers and in general
does not require an optimization of the reaction conditions for
each DNA fragment to be sequenced.
SUMMARY OF THE INVENTION
[0010] An object of the present invention is to provide an
improved, rapid and reliable method for sequencing nucleic acid
molecules.
[0011] A further object of the present invention is to provide a
method for sequencing nucleic acid molecules that can be carried
out in an uninterrupted manner, in a single step and in a single
container.
[0012] A further object of the present invention is to provide a
nucleic acid sequencing which simultaneously increases the
exponential amplification of molecules of full length as well as of
molecules of truncated length which leads to a reduction of the
initial amount of nucleic acid molecules that are required for the
cycling reaction.
[0013] A further object of the present invention is to provide a
method for sequencing nucleic acid molecules which leads to an
increase in the distance at which both primers can be positioned on
the nucleic acid molecule to be sequenced.
[0014] A further object of the present invention is to provide a
method for sequencing a nucleic acid which increases the
signal-to-noise ratio of specific, correctly terminated molecules
to unspecifically terminated molecules.
[0015] A further object of the present invention is to provide an
application of the method according to the invention for sequence
determination in medical diagnostics, forensics and population
genetics.
[0016] Further objects of the invention can be deduced by a person
skilled in the art from the description.
[0017] The thermocycling reaction of the present invention
comprises a first primer and a second primer which serve to
simultaneously produce sufficient template molecules of full length
as well as molecules of truncated length which contribute to the
sequencing of the nucleic acid molecule. Either one primer is
labelled and the other is not or both are differently labelled. In
addition each reaction initially contains the nucleic acid template
to be sequenced as well as a buffer solution and the four
deoxynucleotides or derivatives thereof and one dideoxynucleotide
or another terminating nucleotide e.g. 3'-aminonucleotides or
3'-ester-derivatized nucleotides. A thermostable pyrophosphatase
can be optionally added. Four reaction mixtures are prepared one
for the determination of each base.
[0018] However, in contrast to the methods known in the state of
the art, it was surprisingly found that direct, exponential
amplification and sequencing can be carried out by adding two
different types of DNA polymerases to the initial cycle sequencing
reaction: a first thermostable DNA polymerase and a second
thermostable DNA polymerase with a reduced ability to incorporate
dideoxynucleotides compared to the said first thermostable DNA
polymerase. The first DNA polymerase mainly produces truncated
products that accumulate exponentially during the cycles and
contribute to the sequence ladder that is generated whereas the
second DNA polymerase, which has a reduced ability to incorporate
dideoxynucleotides compared to the first said thermostable DNA
polymerase, primarily produces products of full length which
accumulate exponentially and serve in subsequent cycles as a
template for the production of further DNA strands of fill length
as well as templates for extensions which contribute to the
sequencing reaction. Hence the combination of the different
properties of the two polymerases, i.e. the ability of the first
DNA polymerase to efficiently incorporate dideoxynucleotides and
the ability of the second DNA polymerase to efficiently incorporate
deoxynucleotides, leads to a considerably increased efficiency of
the uncoupled, direct, exponential amplification and sequencing
reaction.
[0019] Therefore the present invention provides a method for
nucleic acid sequencing DEXTAQ) which simultaneously enables the
exponential amplification of molecules of full length as well as of
truncated length in a thermocycling reaction and leads to a
reduction of the amount of initial nucleic acid molecules that are
necessary for the reaction. This enables the sequencing of
single-copy DNA fragments in amounts as small as ca. 60 ng genomic
DNA.
[0020] Since, in addition, all reagents that are necessary for the
exponential amplification of fragments of full length as well as of
truncated fragments are present in the initial reaction mixture,
the method of the present invention (DEXTAQ) achieves the
simultaneous, exponential production of a sequencing template and
of a sequence ladder in a single tube without the necessity of
interrupting the thermocycling reaction. This means that using the
method of the present invention it is possible to determine the
nucleic acid sequence in a single step.
[0021] Furthermore the method of the present invention (DEXTAQ)
allows the distance at which the two primers can be positioned on
the template nucleic acid molecule to be enlarged. Thus the method
according to the invention for example enables the 3'-ends of the
first and of the second primer to be positioned on the DNA template
at a distance that is larger than or equal to 500 bases.
[0022] Hence the aforementioned object of the present invention is
achieved by providing a method for sequencing nucleic acid
molecules in a thermocycling reaction which initially contains a
nucleic acid molecule, a first primer, a second primer, a reaction
buffer, a first thermostable DNA polymerase, deoxynucleotides or
derivatives thereof, and a dideoxynucleotide or another terminating
nucleotide and is characterized in that the thermocycling reaction
additionally contains a second thermostable DNA polymerase which,
in comparison to the said first thermostable DNA polymerase, has a
reduced ability to incorporate dideoxynucleotides.
[0023] A single enzyme would also be suitable for use in the method
according to the invention that has different enzyme activities
e.g. by using a chimeric polymerase or by the fact that a fraction
of the polymerase has a modified ability to incorporate
dideoxynucleotides by the continuous or partial action of agents.
If this enzyme is composed of several subunits then these subunits
can be covalently or non-covalently linked together.
[0024] The present invention also enables three or more DNA
polymerases to be used in this method.
[0025] In a preferred embodiment the method according to the
invention is furthermore characterized in that each thermocycling
reaction for the determination of 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.
[0026] The use of a DNA polymerase is preferred as the thermostable
first DNA polymerase which, in contrast to wild-type Taq DNA
polymerase, has a reduced discrimination against 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 ThermoSequenase.TM. (Taq DNA polymerase .DELTA.272
(-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. The use of Thermosequenase or some other DNA
polymerase which has a better ability to incorporate ddNTPs is
particularly preferred for the method of the present invention.
[0027] A DNA polymerase which carries no "Tabor-Richardson"
mutation such as e.g. Taq DNA polymerase, Tth DNA polymerase,
Klentaq (Taq DNA polymerase) (-exo5'-3'), (Korolev et al. (1995)
Proc. Natl. Acad. Sci. USA 92, 9246-9268, W. Barnes in Proc. Natl.,
Acad. Sci. USA 91 (1994), 2216-2220 and U.S. Pat. No. 5,436,149 is
preferably used as the thermostable second DNA polymerase which has
a reduced ability to incorporate dideoxynucleotides compared to the
first thermostable DNA polymerase. The use of Taq DNA polymerase in
the method of the present invention is particularly preferred.
[0028] Processive polymerases are preferably used for the method
according to the invention i.e. the polymerase with a reduced
discrimination against ddNTPs preferably has a higher processivity
than ThermoSequenase and the polymerase which discriminates against
ddNTPs preferably has a higher processivity than the wild-type Taq
DNA polymerase. Polymerases according to the invention are most
preferably used for the present method whose processivity is higher
than that of the wild-type Taq DNA polymerase. Hence it would for
example be advantageous to use two polymerases whose processivity
is the same as that of T7 polymerase.
[0029] The method according to the invention can also be carried
out as a "hot start" method. This ensures 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
commercially available 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.
[0030] In a further preferred embodiment of the method of the
invention the ratio of the said primers is preferably higher than
1:1, more preferably between about 2:1 and about 3:1 and most
preferably 2:1.
[0031] In a further preferred embodiment of the method of the
invention the said primers have a length that can prevent annealing
to unspecific DNA fragments by a high temperature during the
cycling. This leads to a good signal-to-noise ratio. The said
primers preferably have a length of at least 18 nucleotides.
[0032] 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.
[0033] Furthermore 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.
[0034] In a further preferred embodiment of the method 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 agents or methods known in the state of
the art can be used as single or differential labelling agents and
methods, provided that the stability or function of the said primer
in the DNA sequencing method of the present invention is not
significantly changed. For example single and differential labels
can be selected from the group which comprises those enzymes such
as .beta.-galactosidase, alkaline phosphatase, peroxidase and
enzyme substrates, coenzymes, dyes, chromophores, fluorescent,
chemiluminescent and bioluminescent labels such as FITC, Cy5,
Cy5.5, Cy7, Texas-red and IRD40 (Chen et al., (1 993), 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.
[0035] DEXTAQ is relatively insensitive to various buffers and
various deoxynucleotides and dideoxynucleotide concentrations.
[0036] The number of thermocycles can be from about 18 to about 50
cycles depending on the amount of template DNA and its purity.
[0037] 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.
[0038] 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 a thermocycling reaction. Such
derivatives 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 of about 300 .mu.M to 2 mM.
[0039] 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. Preferred concentrations of ddNTPs are between about 1
and 5 .mu.M.
[0040] The preferred ratio of dNTPs to ddNTPs (dNTPs:ddNTPs) that
is used in the method according to the invention is between 100:1
and 1000:1 more preferably between 300:1 and 600:1.
[0041] 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 of the specific, truncated DNA molecules
compared to the unspecific DNA molecules is large enough not to
substantially impede reading of the sequence. In the case of human
single-copy DNA sequences the highest possible annealing
temperature drastically reduces the background. For this the
annealing and synthesis steps of the thermocycling reaction are
carried out at a temperature of at least 55.degree. C., preferably
at 66.degree. C. and most preferably at at least about 68.degree.
C.
[0042] In a further preferred embodiment of the method of the
invention the nucleic acid molecule to be sequenced can be present
as total genomic DNA which is in an uncloned or unpurified state.
The genomic DNA can have a length of more than or equal to 2 kb
DEXTAQ functions with about 60 ng total genomic DNA, but also
functions with smaller amounts of DNA if multicopy fragments are
analysed. Other forms of DNA that can be used as templates include
purified, partially purified or unpurified cloned DNA such as e.g.
unpurified plasmid DNA from bacterial colonies or cloned or
uncloned mitochondrial DNA etc. Furthermore the method of the
present invention is relatively independent of the base composition
of the template.
[0043] In a further preferred embodiment of the method of the
invention the nucleic acid molecule to be sequenced can be present
as RNA. A mixture of two polymerases is used: a first DNA
polymerase according to the invention e.g. one which contains a
"Tabor-Richardson" mutation or a functional derivative thereof,
such as ThermoSequenase and a second DNA polymerase that is able to
reversely transcribe RNA into DNA and has the ability to act as a
PCR enzyme. Any thermostable DNA polymerase which has reverse
transcriptase activity can be used as a second DNA polymerase for
the method of the invention in which RNA is used as the template.
Taq DNA polymerase (Jones et al., Nucl. Acids Res. 17:8387-8388
(1989)) or Tth DNA polymerase (Myers et al., Biochemistry
30:7666-7672 (1991)) is preferably used and more preferably Tth DNA
polymerase. Tth polymerase reversely transcribes the RNA template
into DNA which can then be used by both enzymes: Tth polymerase
will primarily generate products of full length which can serve as
templates and ThermoSequenase will produce truncated products
(ddNTP incorporation) and thus a sequence ladder.
[0044] Suitable buffers include those that are described in Myers
et al. (1991) Biochemistry 30: 7666-7672. The following buffer can
be used for both polymerases and guarantees the function of both
polymerases: 10 mM Tris-HCl (pH 8.3), 40 mM KCl, 1 mM MnCl.sub.2. A
reverse transcription using a reaction buffer and optionally
MgCl.sub.2 at a concentration of about 1 mM to 5 mM and which also
includes both polymerases, both primers and nucleotides, is
subjected to an incubation step (15 minutes at 70.degree. C.).
Afterwards the MgCl.sub.2 concentration is adjusted to between 1 mM
and 5 mM and a DEXTAQ reaction is carried out.
[0045] 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 and soft tissue or
fractions thereof and cell cultures or fractions thereof as well as
bacteria, viruses or bacteriophages.
[0046] The present invention also provides an 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 using 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.
[0047] The present invention provides for the first time a method
which enables the simultaneous amplification and sequencing of a
nucleic acid fragment to be sequenced from a complex mixture of
nucleic acids such as total genomic human DNA without prior
amplification of the nucleic acid to be sequenced by means of known
methods, in a single step i.e. without interrupting the reaction
and indeed such that an unequivocal sequence ladder is
readable.
[0048] A particular advantage of the method according to the
invention is therefore the ability to directly sequence nucleic
acids. Thus the method according to the invention can be used for
the direct sequencing of e.g.
[0049] eukaryotic genomic DNA such as e.g. of human chromosomal DNA
or mitochondrial DNA,
[0050] human RNA
[0051] unpurified plasmid DNA from bacterial colonies as well
as
[0052] unpurified single-stranded or double-stranded DNA from
bacteriophages.
[0053] The present invention relates to all combinations of all
procedures of the above methods.
[0054] After preparation the sequencing reactions can be applied
directly to a sequencing gel such as e.g. after addition of a
commonly used loading 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 in
correspondence with 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 DEXTAQ reactions 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 can
be detected by other methods such as e.g. autoradiography.
[0055] Furthermore a kit for sequencing a nucleic acid molecule is
also a subject matter of the present invention, wherein this kit
contains a reaction buffer, deoxynucleotides or derivatives thereof
and at least one dideoxynucleotide or a derivative thereof and at
least one thermostable DNA polymerase with different abilities to
incorporate dideoxynucleotides. A suitable first primer and the
second primer are then added individually depending on the
application and the nucleic acid molecule to be sequenced.
[0056] In order to sequence a nucleic acid molecule the kit
contains, in a preferred embodiment, a first thermostable DNA
polymerase and additionally a second thermostable DNA polymerase
which, compared to the said first thermostable DNA polymerase, has
a reduced ability to incorporate dideoxynucleotides.
[0057] Most preferably the kit for sequencing a nucleic acid
molecule contains a first thermostable polymerase, Taq DNA
polymerase (-exo5'-3') (F667Y) or a functional derivative thereof
and a second thermostable DNA polymerase, Taq polymerase or a
functional derivative thereof.
SHORT DESCRIPTION OF THE FIGURES
[0058] FIG. 1. Schematic representation of the method of the
present invention (DEXTAQ). A first polymerase of the present
invention (A) which carries a "Tabor-Richardson" mutation for
discriminating towards ddNTPs preferentially incorporates ddNTPs
and produces the sequence ladder. A second polymerase of the
present invention which, compared to the said first thermostable
DNA polymerase (B), has a reduced ability to incorporate
dideoxynucleotides, preferably incorporates dNTPs and mainly
produces products of full length and provides the uncoupled,
direct, exponential amplification and sequence reaction with
additional sequencing templates.
[0059] FIG. 2. 60 ng of total genomic DNA was subjected to a
direct, uncoupled sequencing reaction using 6 pmol of an
FITC-labelled primer (CCR5-2) and 3 pmol of an unlabelled primer
(CCR5-1). The section shown in all windows is only at a distance of
20 base pairs from the end of the template and the last bases are
part of the primer that generates the second template. No
additional Taq DNA polymerase was added to the reaction that is
shown in window A. Increasing amounts of Taq DNA polymerase were
added to the reactions that are shown in window B (0.25 units), C
(0.5 units), D (1.0 units) and E (2.0 units). In cases where no Taq
DNA polymerase had been added, the A.L.F. software was not able to
process a sequence. A better ratio between signal and noise is seen
in the cases in which Taq DNA polymerase had been added.
[0060] FIG. 2A. 60 ng of total genomic DNA was subjected to an
uncoupled, direct amplification and sequencing reaction using
equimolar amounts i.e. 3 pmol each of an FITC-labelled primer
(CCR5-2) and of an unlabelled primer (CCR5-1). The A.L.F. software
was able to process 290 bases. The reactions were carried out using
0.25 units Taq DNA polymerase and standard ThermoSequenase
reagents.
[0061] FIG. 3. An uncoupled, direct, exponential amplification and
sequencing reaction was carried out in combination with various
thermostable polymerases which do not carry the "Tabor-Richardson"
mutation. Window A shows a reaction in which 2.5 units Klentaq
polymerase were added to a direct, uncoupled amplification and
sequencing reaction which was carried out with 60 ng total genomic
DNA Window B shows a direct, uncoupled, exponential amplification
and sequencing reaction which was carried out with standard Taq DNA
polymerase and window C shows a reaction in which 0.25 units Tth
polymerase was added.
[0062] FIG. 4. 300 ng of total genomic human DNA was subjected to a
direct, uncoupled, amplification and sequencing reaction using
non-equimolar amount of primers i.e. 6 pmol of an FITC-labelled
primer (CCR5-2) and 3 pmol of an unlabelled primer (CCR5-3). The
primers span a region of 560 base pairs of the human single-copy
gene CCR5. The A.L.F. software was able to process 260 bases. The
reactions were carried out using 0.25 units Taq DNA polymerase and
standard ThermoSequenase reagents.
[0063] FIG. 5. 4 .mu.l of a bacterial colony lysate was subjected
to a direct, uncoupled amplification and sequencing reaction using
non-equimolar amounts of differentially labelled primers i.e. 6
pmol of an FITC-labelled primer (universal) and 3 pmol of a
Cy5-labelled primer (reverse). The primers span a region of 650
base pairs of the plasmid insert. The A.L.F. software was able to
process 502 bases for the FITC labelled primer. The figure shows
the curve results in the case of the FITC labelled universal
primer. The reactions were carried out using 0.25 units Taq DNA
polymerase and standard ThermoSequenase reagents.
[0064] The invention is described more exactly and in more detail
by the following non-limiting examples.
EXAMPLE 1
TEMPLATE PREPARATION
[0065] Total genomic 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
[0066] 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:
1 SEQ ID NO. 1: (CCR5-1): 5'-GGC TGG TCC TGC CGC TGC TTG TCA T-3';
SEQ ID NO. 2: (CCR5-2): 5'-CTG CTC CCC AGT GGA TCG GGT GTA AAC-3';
SEQ ID NO. 3: (CCR5-3)5'-CAC CTT TGG GGT GGT GAC AAG TGT GAT-3'
(Samson, M. et al., Biochemistry 35 (11), 3362-3367 (1996)), SEQ ID
NO. 4: (universal primer) 5'-CGA CGT TGT AAA ACG ACG GCC AGT-3'and
SEQ ID NO. 5: (reverse) 5'-CAG GAA ACA GCT ATG AC-3' (Pharmacia
Biotech).
[0067] Direct, exponential amplification and sequencing reactions
were carried out using Thermosequenase reagents (2). A 24 .mu.l
mixture composed of 6 pmol of an FITC-labelled and 3 pmol of an
unlabeled primer, 6 pmol of an FITC-labelled and 3 pmol of a
Cy5-labelled primer, or 3 pmol of an FITC-labelled and 3 pmol of an
unlabelled primer total genomic DNA (0.5 to 8 .mu.l at a
concentration of 120 ng/.mu.l) and additional polymerase if
necessary (0.1 to 2 .mu.l depending on the unit definition) was
prepared and 6 .mu.l aliquots were added to 2 .mu.l Thermosequenase
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 mg EDTA
(pH 7.4) and 6 mg/ml dextran blue) which was followed by a 4 minute
denaturation at 95.degree. C. The sequencing reactions were
analysed on an A.L.F. (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 corresponded to
the manufacturer's recommendations.
EXAMPLE 2
[0068] Two oligonucleotides with a length of 25 and 27 nucleotides
which span 382 base pairs of the CCR5 gene were synthesized. One of
the two oligonucleotides was labelled at the 5'-end with
fluorescein (CCR5-2) whereas the other (CCR5-1) was unlabelled. Two
reactions were prepared each containing 6 pmol of the labelled
primer and 3 pmol of the unlabelled primer, 500 ng total genomic
DNA and ThermoSequenase reagent which was composed of enzyme,
reaction buffer and deoxy and dideoxy mixtures. 2.5 units of
standard Taq polymerase was added to one of the two reactions. The
reactions were incubated for 3 min. at 95.degree. C. in order to
enable a complete denaturation of the template DNA. Afterwards 45
cycles were carried out each consisting of 30 sec. at 68.degree. C.
and 40 sec. at 95.degree. C. The reactions were stopped and
denatured by the addition of formamide and heating to 95.degree. C.
for 4 minutes before they were applied to an A.L.F. sequencing
apparatus.
[0069] If no additional Taq DNA polymerase had been added, the
A.L.F. was able to process 344 bases. If, in contrast, 0.25 units
Taq polymerase was added to the direct exponential amplification
and sequencing reaction, 351 bases were determined. A visual
analysis of the A.L.F. curves showed that the signal intensity
improved where Taq DNA polymerase had been added. Furthermore the
stops at the start of the run which can occur in direct exponential
amplification and sequencing reactions were substantially reduced.
In contrast the signal strength remained continuously high towards
the amplifying primer. In contrast in the case in which no Taq DNA
polymerase was present, the signal was weaker after 200 base pairs.
The signal at the end of the run which corresponds to the product
of full length was substantially stronger in the reaction in which
Taq DNA polymerase was present.
EXAMPLE 3
[0070] In order to confirm the fact that additional Taq DNA
polymerase is able to amplify the exponential factor and to reduce
the background and also to determine the amount of Taq DNA
polymerase required for an optimal reaction, a reduced amount of
template DNA was used in combination with varying amounts of Taq
DNA polymerase. Five reactions were prepared each containing 120 ng
total genomic DNA, 6 pmol of the labelled primer and 3 pmol of
unlabelled primer and ThermoSequenase reagent. In one reaction no
additional Taq DNA polymerase was added. In the other 4 reactions
0.25, 0.5, 1.0 and 2.0 units Taq polymerase was added respectively.
In order to exclude a possible effect of the Taq polymerase storage
buffer, the storage buffer was added to that reaction to which no
polymerase had been added. The reactions were subjected to the
cycles described above.
[0071] In the case where no Taq DNA polymerase had been added the
A.L.F. Manager was only able to process a few bases and the signal
intensities were too low to be considered useful for routine
sequencing. In the reactions in which 0.25 units and 0.5 units had
been added the A.L.F. Manager was able to process 374 bases and 364
bases respectively. 226 bases were determined by the A.L.F. Manager
in the reaction in which one unit Taq polymerase had been
added.
[0072] The reactions were repeated under identical conditions but
using an even smaller amount of genornic DNA. 60 ng template DNA
was sequenced using varying amounts of Taq DNA polymerase. No
signal was detectable where no Taq polymerase had been added but
the A.L.F. Manager was again able to process the sequence in cases
where between 0.1 and 0.4 units Taq DNA polymerase had been added
(FIG. 2).
EXAMPLE 4
[0073] Of the thermostable polymerases without a "Tabor-Richardson"
mutation, a number of different polymerases were tested with regard
to their effect on the direct, exponential amplification and
sequencing reaction. Tth polymerase, Klentaq (U.S. Pat. No.
5,436,149, Korolev. S., et al., (1995) Proc. Natl. Acad. Sci. USA
92, 9264-9268), Sequitherm.RTM. and standard Taq DNA polymerase
were cycled with 60 ng total genomic DNA. Different unit amounts of
0.25 to 25 units (in the case of Klentaq) were tested for all
polymerases (FIG. 3). The best results were achieved with Taq DNA
polymerase.
EXAMPLE 5
[0074] In order to test whether DEXTAQ can be applied to
single-copy DNA sequences if the primers are positioned at a
distance of over 500 base pairs to one another, 300 ng total
genomic human DNA was subjected to an uncoupled, direct,
exponential amplification and sequencing reaction using
non-equimolar amounts of an FITC-labelled and of an unlabelled
primer (6 pmol:3 pmol) which span a region of 560 base pairs of the
human single-copy gene. Good sequence curves were obtained for a
length of about 300 base pairs (FIG. 4).
EXAMPLE 6
[0075] In order to test whether DEXTAQ can also be applied to
plasmid sequencing of crude, bacterial lysates and in order to
check whether DEXTAQ can be carried out using two differentially
labelled primers, 4 .mu.l of a bacterial colony lysate was
subjected to an uncoupled, direct, exponential amplification and
sequencing reaction using non-equimolar amounts of differentially
labelled primers i.e. 6 pmol of an FITC-labelled primer (universal)
and 3 pmol of a Cy5-labelled primer (reverse). The primers span a
length of 650 base pairs of the plasmid insert. The A.L.F. software
was able in the case of the FITC-labelled primer to process 502
bases (FIG. 5).
Sequence CWU 1
1
* * * * *