U.S. patent application number 09/741664 was filed with the patent office on 2001-11-15 for stable compositions for nucleic acid amplification and sequencing.
Invention is credited to Rashtchian, Ayoub, Solus, Joseph.
Application Number | 20010041334 09/741664 |
Document ID | / |
Family ID | 27104492 |
Filed Date | 2001-11-15 |
United States Patent
Application |
20010041334 |
Kind Code |
A1 |
Rashtchian, Ayoub ; et
al. |
November 15, 2001 |
Stable compositions for nucleic acid amplification and
sequencing
Abstract
The present invention is directed to compositions comprising
mixtures of reagents, including thermostable enzymes (e.g.,
thermostable DNA polymerases), buffers, cofactors and other
components, suitable for immediate use in nucleic acid
amplification or sequencing techniques without dilution or addition
of further components. The compositions contain no stablizing
agents (e.g., glycerol or serum albumin) and unexpectedly maintain
activity for extended periods of time upon storage at temperatures
above freezing. These compositions are useful, alone or in the form
of kits, for nucleic acid amplification (e.g., by the Polymerase
Chain Reaction) and sequencing (e.g., by dideoxy or "Sanger"
sequencing), or for any procedure utilizing thermostable DNA
polymerases in a variety of medical, forensic and agricultural
applications. In particular, the compositions and methods are
useful for amplifying and sequencing nucleic acid molecules that
are larger than about 7 kilobases in size.
Inventors: |
Rashtchian, Ayoub;
(Gaithersburg, MD) ; Solus, Joseph; (Gaithersburg,
MD) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W., SUITE 600
WASHINGTON
DC
20005-3934
US
|
Family ID: |
27104492 |
Appl. No.: |
09/741664 |
Filed: |
December 21, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09741664 |
Dec 21, 2000 |
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09049021 |
Mar 27, 1998 |
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09049021 |
Mar 27, 1998 |
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08801720 |
Feb 14, 1997 |
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08801720 |
Feb 14, 1997 |
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08689815 |
Aug 14, 1996 |
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Current U.S.
Class: |
435/6.11 ;
435/194; 435/6.12; 435/91.2; 530/388.26; 536/23.1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/686 20130101; C12Q 2527/125 20130101; C12Q 2527/149
20130101; C12Q 2527/137 20130101; C12Q 2527/149 20130101; C12Q
2527/149 20130101; C12Q 2527/137 20130101; C12Q 2527/149 20130101;
C12N 9/96 20130101; C12Q 1/686 20130101; C12Q 2527/125 20130101;
C12Q 1/686 20130101; C12Q 1/6869 20130101; C12N 9/1252 20130101;
C12P 19/34 20130101; C12Q 1/6869 20130101 |
Class at
Publication: |
435/6 ; 435/91.2;
435/194; 536/23.1; 530/388.26 |
International
Class: |
C12Q 001/68; C07H
021/02; C12P 021/08; C07H 021/04; C12P 019/34; C12N 009/12; C07K
016/00 |
Claims
What is claimed is:
1. A stable composition comprising a mixture of reagents at working
concentrations, wherein said reagents are at least one thermostable
enzyme and at least one buffer salt.
2. A stable composition for nucleic acid amplification comprising a
mixture of reagents, wherein said reagents are at least one
thermostable DNA polymerase, at least one buffer salt and at least
one deoxynucleoside triphosphate.
3. A stable composition for nucleic acid sequencing comprising a
mixture of reagents, wherein said reagents are at least one
thermostable DNA polymerase, at least one deoxynucleoside
triphosphate, at least one dideoxynucleoside triphosphate and at
least one buffer salt.
4. The stable composition of claim 2 or claim 3, wherein said
reagents are present at working concentrations.
5. The composition of claim 2 or claim 3, wherein said thermostable
DNA polymerase is selected from the group of thermostable DNA
polymerases consisting of a Taq DNA polymerase, a Tne DNA
polymerase, a Tma DNA polymerase, and mutants thereof.
6. The composition of claim 2 or claim 3, wherein said thermostable
DNA polymerase is selected from the group of thermostable DNA
polymerases consisting of a Pfu DNA polymerase, a Pwo DNA
polymerase, VENT.TM. DNA polymerase, DEEPVENT.TM. DNA polymerase,
and mutants thereof.
7. The composition of claim 5, wherein said mixture further
comprises DEEPVENT.TM. DNA polymerase or VENT.TM. DNA
polymerase.
8. The composition of claim 5, wherein the concentration of Taq DNA
polymerase or mutant thereof is about 0.1 to 200 units per
milliliter.
9. The composition of claim 8, wherein the concentration is about
20 units per milliliter.
10. The composition of claim 5, wherein the concentration of Tne
DNA polymerase or mutant thereof is about 0.1 to 200 units per
milliliter.
11. The composition of claim 10, wherein the concentration is about
20 units per milliliter.
12. The composition of claim 5, wherein the concentration of Tma
DNA polymerase or mutant thereof is about 0.1 to 200 units per
milliliter.
13. The composition of claim 12, wherein the concentration is about
20 units per milliliter.
14. The composition of claim 6, wherein the concentration of
VENT.TM. DNA polymerase or mutant thereof is about 0.1 to 200 units
per milliliter.
15. The composition of claim 14, wherein the concentration is about
20 units per milliliter.
16. The composition of claim 6, wherein the concentration of
DEEPVENT.TM. DNA polymerase or mutant thereof is about 0.1 to 200
units per milliliter.
17. The composition of claim 16 wherein the concentration is about
20 units per milliliter.
18. The composition of claim 6, wherein the concentration of Pfu
DNA polymerase or mutant thereof is about 0.1 to 200 units per
milliliter.
19. The composition of claim 18 wherein the concentration is about
20 units per milliliter.
20. The composition of claim 6, wherein the concentration of Pwo
DNA polymerase or mutant thereof is about 0.1 to 200 units per
milliliter.
21. The composition of claim 20 wherein the concentration is about
20 units per milliliter.
22. The composition of claim 7, wherein the concentration of
DEEPVENT.TM. DNA polymerase or VENT DNA polymerase is about 0.002
to 200 units per milliliter.
23. The composition of claim 22, wherein the concentration is about
0.40 units per milliliter.
24. The composition of claim 2 or claim 3, wherein said DNA
polymerase retains at least 90% of the enzymatic activity for at
least four weeks when stored at about 20.degree. C. to 25.degree.
C.
25. The composition of claim 5, wherein said DNA polymerase retains
at least 90% of the enzymatic activity for at least one year when
stored at about 4.degree. C.
26. The composition of claim 2 or claim 3, further comprising a
magnesium salt.
27. The composition of claim 2 or claim 3, further comprising at
least one nonionic detergent.
28. The composition of claim 2 or claim 3, wherein the
concentration of said deoxynucleoside triphosphate is about 200 to
about 300 micromolar.
29. The composition of claim 3, wherein the concentration of said
dideoxynucleoside triphosphate is about 0.08 to about 5
micromolar.
30. A nucleic acid amplification kit comprising one or more
containers, wherein a first container contains a stable composition
comprising a mixture of reagents, wherein said reagents are at
least one thermostable DNA polymerase, at least one buffer salt,
and at least one deoxynucleoside triphosphate.
31. A nucleic acid sequencing kit comprising one or more
containers, wherein a first container contains a stable composition
comprising a mixture of reagents, wherein said reagents are at
least one thermostable DNA polymerase, at least one buffer salt, at
least one deoxynucleoside triphosphate and at least one
dideoxynucleoside triphosphate.
32. The kit of claim 30 or 31, wherein said reagents are present at
working concentrations.
33. A method of amplifying a nucleic acid molecule comprising
contacting said nucleic acid molecule with the composition of claim
2.
34. A method of amplifying a nucleic acid molecule comprising
contacting said nucleic acid molecule with a composition selected
from the group consisting of: a composition comprising a
thermostable 3' exo+ DNA polymerase and a thermostable 3' exo- DNA
polymerase wherein the concentrations of said 3' exo+ DNA
polymerase and of said 3' exo- DNA polymerase are equal, and a
composition comprising a thermostable 3' exo+ DNA polymerase and a
thermostable 3' exo- DNA polymerase wherein the concentration of
said 3' exo+ DNA polymerase is higher than the concentration of
said 3' exo- DNA polymerase.
35. A method of sequencing a nucleic acid molecule comprising
contacting said nucleic acid molecule with the composition of claim
3.
36. A method of sequencing a nucleic acid molecule comprising
contacting said nucleic acid molecule with a composition selected
from the group consisting of: a composition comprising a
thermostable 3' exo+ DNA polymerase and a thermostable 3' exo- DNA
polymerase wherein the concentrations of said 3' exo+ DNA
polymerase and of said 3' exo- DNA polymerase are equal, and a
composition comprising a thermostable 3' exo+ DNA polymerase and a
thermostable 3' exo- DNA polymerase wherein the concentration of
said 3' exo+ DNA polymerase is higher than the concentration of
said 3' exo- DNA polymerase.
37. The method of any one of claims 33-36, wherein said nucleic
acid molecule is larger than about 4 kilobases in size.
38. The method of claim 37, wherein said nucleic acid molecule is
larger than about 7 kilobases in size.
39. The method of claim 38, wherein said nucleic acid molecule is
larger than about 8 kilobases in size.
40. A nucleic acid molecule amplified by the method of claim 33 or
claim 34.
41. The nucleic acid molecule of claim 40, wherein said nucleic
acid molecule is larger than about 4 kilobases in size.
42. The nucleic acid molecule of claim 41, wherein said nucleic
acid molecule is larger than about 7 kilobases in size.
43. The nucleic acid molecule of claim 41, wherein said nucleic
acid molecule is larger than about 8 kilobases in size.
44. The composition of claim 1, further comprising at least one
antibody that specifically binds to said thermostable enzyme.
45. The composition of claim 2 or claim 3, further comprising at
least one antibody that specifically binds to said thermostable
enzyme.
46. The kit of claim 30 or claim 31, wherein said mixture of
reagents further comprises at least one antibody that specifically
binds to said thermostable DNA polymerase.
47. The kit of claim 30 or claim 31, further comprising one or more
additional containers containing at least one antibody that
specifically binds to said thermostable DNA polymerase.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/801,720, filed Feb. 14, 1997, which is a
continuation-in-part of U.S. application Ser. No. 08/689,815, filed
Aug. 14, 1996 (now abandoned), the contents of both of which are
fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention is in the fields of molecular and cellular
biology. The invention is particularly directed to reagent
compositions for use in techniques whereby nucleic acids (DNA or
RNA) are amplified or sequenced, and to methods of amplifying and
sequencing long nucleic acid molecules, especially via the
polymerase chain reaction (PCR).
BACKGROUND OF THE INVENTION
[0003] DNA Polymerases
[0004] During growth and reproduction of viruses and cellular
organisms, the DNA containing the parental genetic information must
be faithfully copied and passed on to the progeny. This highly
regulated process of DNA replication is carried out in vivo by a
complex of enzymes and associated proteins and cofactors (Komberg,
A., Science 131:1503-1508, 1959; Hibner, U., and Alberts, B. M.,
Nature 285:300-305, 1980). The primary enzymes taking part in this
process are the DNA polymerases, which catalyze the addition of
deoxynucleoside triphosphate (dNTP) bases into the newly forming
DNA strands. Together with other enzymes (e.g., helicases, ligases
and ATPases), the DNA polymerases thus ensure rapid and relatively
faithful replication of DNA in preparation for proliferation in
prokaryotes, eukaryotes and viruses.
[0005] DNA polymerases are also used to manipulate DNA in vitro in
a variety of molecular genetic techniques. These enzymes have
proven useful not only for in vitro DNA synthesis, but also for
determining the nucleotide sequence (i.e., "sequencing") of DNA
fragments or genes. This latter application relies on the fact
that, in addition to an activity which adds dNTPs to DNA in the 5'
to 3' direction (i.e., "polymerase" activity), many DNA polymerases
also possess activities which remove dNTPs in the 5' to 3' and/or
the 3' to 5' direction (i.e., "exonuclease" activity). DNA
polymerases can also be used for sequencing via their incorporation
of labeled chain-terminating agents such as dideoxynucleoside
triphosphates (See U.S. Pat. Nos. 4,962,020; 5,173,411; and
5,498,523). Thus the same enzyme, e.g., DNA polymerases I and III
from the bacterium Escherichia coli, DNA polymerase .gamma. from
animal cells or DNA polymerase from bacteriophage T7 (See U.S. Pat.
No. 4,795,699), may be used in vitro both for DNA synthesis,
involving the elongation of the DNA strand, and for DNA sequencing,
involving either the synthesis or the digestion of the DNA
strand.
[0006] The dual activity of certain DNA polymerases is, however, a
drawback for some in vitro applications. For example, the in vitro
synthesis of an intact copy of a DNA fragment by the polymerase
activity, an elongation process which proceeds in a 5' to 3'
direction along the template DNA strand, is jeopardized by the
exonuclease activities which may simultaneously or subsequently
degrade the newly formed DNA. To overcome this technical problem, a
fragment of E. coli DNA polymerase I lacking the 5' to 3'
exonuclease activity (named the "Klenow fragment" after its
discoverer) is often employed for in vitro DNA synthesis. The
Klenow fragment provides for in vitro DNA synthesis at
approximately the same rate as intact E. coil DNA polymerase I, but
the newly synthesized DNA molecules are less subject to enzymatic
degradation and are correspondingly more stable.
[0007] Unfortunately, the error rate (i e., the rate at which
incorrect dNTPs are incorporated into the new DNA strand by the
enzyme) is somewhat higher for the Klenow fragment than for many
other commonly used DNA polymerases, including E. coli DNA
polymerases I and II and the polymerases from bacteriophages T4 and
T7 (Kunkel, T. A., et al., J. Biol. Chem. 259:1539-1545, 1984;
Tindall, K. R., and Kunkel, T. A., Biochemistry 27:6008-6013, 1988;
Mattila, P., et al., Nucl. Acids Res. 19:4967-4973, 1991). Thus,
until recently the rates of synthesis, degradation and error had to
be weighed together when choosing which DNA polymerase to use for
in vitro DNA synthesis.
[0008] DNA Sequencing
[0009] In general, two techniques have been traditionally used to
sequence nucleic acids. In the first method, termed "Maxam and
Gilbert sequencing" after its co-developers (Maxam, A. M. and
Gilbert, W., Proc. Natl. Acad Sci. USA 74:560-564, 1977), DNA is
radiolabeled, divided into four samples and treated with chemicals
that selectively destroy specific nucleotide bases in the DNA and
cleave the molecule at the sites of damage. By separating the
resultant fragments into discrete bands by gel electrophoresis and
exposing the gel to X-ray film, the sequence of the original DNA
molecule can be read from the film. This technique has been used to
determine the sequences of certain complex DNA molecules, including
the primate virus SV40 (Fiers, W., et al., Nature 273:113-120,
1978; Reddy, V. B., et al., Science 200:494-502, 1978) and the
bacterial plasmid pBR322 (Sutcliffe, G., Cold Spring Harbor Symp.
Quant. Biol. 43:77-90,1979).
[0010] An alternative technique for sequencing, named "Sanger
sequencing" after its developer (Sanger, F., and Coulson, A. R., J.
Mol. Biol. 94:444-448, 1975), is more commonly employed. This
method uses the DNA-synthesizing activity of DNA polymerases which,
when combined with mixtures of reaction-terminating
dideoxynucleoside triphosphates (Sanger, F., et al., Proc. Natl.
Acad. Sci. USA 74:5463-5467, 1977) and a short primer (either of
which may be detectably labeled), gives rise to a series of newly
synthesized DNA fragments specifically terminated at one of the
four dideoxy bases. These fragments are then resolved by gel
electrophoresis and the sequence determined as described for Maxam
and Gilbert sequencing above. By carrying out four separate
reactions (once with each ddNTP), the sequences of even fairly
complex DNA molecules may rapidly be determined (Sanger, F., et
al., Nature 265:678-695, 1977; Barnes, W., Meth. Enzymol.
152:538-556, 1987). While Sanger sequencing usually employs E. coli
or T7 DNA polymerase (U.S. Pat. No. 4,795,699), recent
modifications of this technique using T7 polymerase mutants allow
sequencing to be accomplished using a single sequencing reaction
containing all four chain-terminating ddNTPs at different
concentrations (U.S. Pat. Nos. 4,962,020 and 5,173,411). Further
modifications to the technique, to reduce or eliminate the buildup
of reaction-poisoning pyrophosphate in the reaction mixtures, have
also been described (U.S. Pat. No. 5,498,523).
[0011] The Polymerase Chain Reaction
[0012] Soon after their identification and characterization, it was
recognized that the activities of the various enzymes and cofactors
involved in DNA synthesis could be exploited in vitro to
dramatically increase the concentration of, or "amplify," one or
more selected DNA sequences. For many medical, diagnostic and
forensic applications, amplification of a particular DNA sequence
is essential to allow its detection in, or isolation from, a sample
in which it is present in very low amounts. More recently, in vitro
amplification of specific genes has provided powerful and less
costly means to facilitate the production of therapeutic proteins
by molecular biological techniques, and may have applications in
genetic therapy as well.
[0013] While a variety of nucleic acid amplification processes has
been described, the most commonly employed is the Polymerase Chain
Reaction (PCR) technique disclosed in U.S. Pat. Nos. 4,683,195 and
4,683,202. In this process, a sample containing the nucleic acid
sequence to be amplified (the "target sequence") is first heated to
denature or separate the two strands of the nucleic acid. The
sample is then cooled and mixed with specific oligonucleotide
primers which hybridize to the target sequence. Following this
hybridization, DNA polymerase in a buffered aqueous solution is
added to the sample, along with a mixture of the dNTPs that are
linked by the polymerase to the replicating nucleic acid strand.
After allowing polymerization to proceed to completion, the
products are again heat-denatured, subjected to another round of
primer hybridization and polymerase replication, and this process
repeated any number of times. Since each nucleic acid product of a
given cycle of this process serves as a template for production of
two new nucleic acid molecules (one from each parent strand), the
PCR process results in an exponential increase in the concentration
of the target sequence. Thus, in a well-controlled, high-fidelity
PCR process, as few as 20 cycles can result in an over one
million-fold amplification of the target nucleic acid sequence (See
U.S. Pat. Nos. 4,683,195 and 4,683,202).
[0014] Thermostable DNA Polymierases
[0015] Overview
[0016] Initially, the DNA polymerases of choice for use in DNA
sequencing or PCR were E. coli DNA polymerase I, the Klenow
fragment, or T4 or T7 polymerases owing to their ease of isolation
and well-characterized activities. However, the use of these
enzymes necessitated their addition prior to the start of each
sequencing or PCR cycle, due to their thermolability at the
temperatures used to denature the DNA strands in the initial steps
of the processes (typically 70.degree. to 95.degree. C.) (Saiki, R.
K., et al., Science 230:1350-1354, 1985; Mullis, K. B., and
Faloona, F. A., Meth. Enzymol. 155:335-350, 1987; U.S. Pat. No.
4,795,699). This need for the addition of fresh enzyme at the
beginning of each cycle increased the amount of time required for
these processes, and increased the risk of operator error and
contamination of the samples during reagent introduction, often
leading to undesirable results.
[0017] These difficulties were partially overcome by the use of
thermostable DNA polymerases in the PCR process (Saiki, R. K., et
al., Science 239:487-491, 1988). The thermostable DNA polymerase
most commonly used in PCR is Taq polymerase, isolated from the
thermophilic bacterium Thennus aquaticus (Saiki et al., 1988, Id.;
U.S. Pat. Nos. 4,889,818 and 4,965,188). Taq polymerase functions
optimally at temperatures of 70-80.degree. C., and is able to
maintain substantial activity upon repeated exposure to
temperatures of 92.degree.-95.degree. C. as are often used in the
initial steps of PCR (Gelfand, D. H., and White, T. J., in: PCR
Protocols: A Guide toMethodsandApplications, Innis, M. A., et al.,
eds., Academic Press, pp. 129-141, 1989; Bej, A. K., and Mahbubani,
M. H., in: PCR Technology: Current Innovations, Griffin, H. G., and
Griffin, A. M., eds., CRC Press, pp. 219-237, 1994).
[0018] The use of Taq polymerase in PCR eliminated the need to add
fresh enzyme to the reaction mix prior to each PCR cycle' Instead,
a quantity of Taq polymerase sufficient to catalyze DNA
polymerization over the desired number of cycles can be mixed with
the other components prior to the initiation of the first PCR
cycle, and the enzyme continues to function throughout the
repetitive cycles of increased and decreased temperatures. The use
of Taq polymerase has also facilitated the automation of the PCR
process (Gelfand and White, Id.), thereby at once dramatically
reducing time constraints and the risks of operator error and
sample contamination that are problematic with thermolabile
polymerases. Currently, most PCR amplification of nucleic acids for
industrial and academic applications is performed using Taq
polymerase and automated thermal cycling instrumentation.
[0019] In addition to Taq polymerase, other thermostable
polymerases have found similar application in PCR (Bej and
Mahbubani, Id.). Particularly useful as substitutes for Taq
polymerase in PCR are polymerases isolated from the thermophilic
bacteria Thermus thermophilus (Tth polymerase), Thermococcus
litoralis (Tli or VEN.TM. polymerase), Pyrococcus furiosus (Pfu or
DEEPVENT polymerase), Pyrococcus woosii (Pwo polymerase) and other
Pyrococcus species, Bacillus sterothennophilus (Bst polymerase),
Sulfolobus acidocaldarius (Sac polymerase), Thennoplasma
acidophilum (Tac polymerase), Thermus flavus (Tfl/Tub polymerase),
Thermus ruber (Tru polymerase), Thennus brockianus (DYNAZYME.TM.
polymerase), Thermotoga neapolitana (Tne polymerase; See WO
96/10640), Thermotoga maritima (Tma polymerase; See U.S. Pat. No.
5,374,553) and other species of the Thermotoga genus (Tsp
polymerase) and Methanobacterium thermoautotrophicum (Mth
polymerase). While each of these polymerases is useful for
particular applications (See Bej and Mahbubani, Id., p. 222), Taq
polymerase is still by far the most commonly used polymerase in
PCR.
[0020] Thermostable polymerases have also found application in DNA
sequencing techniques, particularly in automated methods of dideoxy
sequencing such as "cycle sequencing." These approaches resemble
PCR in most respects except that, in place of dNTPs, automated DNA
sequencing uses ddNTPs which allow determination of the sequence of
the template DNA as described above. Use of higher denaturation
temperatures in automated sequencing also improves sequencing
efficiency (i.e., fewer misincorporations occur) and allows the
sequencing of templates that are GC-rich or contain significant
secondary structure (such as supercoiling).
[0021] The use of thermolabile DNA polymerases such as E. coli or
T7 DNA polymerases in these approaches, however, is subject to the
same limitations described above for their use in PCR. Accordingly,
automated methods of DNA sequencing utilizing higher temperatures
have increasingly employed thermostable DNA polymerases, the most
commonly used of which is, as for PCR, Taq polymerase.
[0022] Technical Limitations
[0023] The use of Taq and other thermostable polymerases in
sequencing and PCR is not, however, without drawback. For example,
the error rate for Taq polymerase is substantially higher (i.e.,
the final product is of "lower fidelity") than that for most of the
thermolabile DNA polymerases, including the Klenow fragment of E.
coli DNA polymerase I (Tindall and Kunkel, Id.), averaging about
10.sup.-4 misincorporations per base pair per cycle. In addition,
Taq polymerase is only useful for amplifying relatively short
stretches of DNA (maximum length on the order of 5-6 kilobases;
Barnes, W. M., Proc. Natl. Acad Sci. USA 91:2216-2220, 1994), thus
precluding its use in PCR amplification of large genes and whole
genomes as is necessary in many current applications.
[0024] These technical limitations are apparently related: it has
been theorized that the Taq polymerase PCR length limitation is due
to the low efficiency of elongation of the newly synthesized DNA
strands at the sites of incorporation of mismatched bases in the
parent strands (Bames, Id.). Further contributing to this
difficulty is the absence in Taq polymerase of a 3' to 5'
exonuclease activity, which in other polymerases acts in a
"proofreading" capacity to correct these mismatches and reduce the
error rate (Bej and Mahbubani, Id.). The 5' to 3' exonuclease
activity present in most thermostable DNA polymerases can also
degrade the 5' ends of the oligonucleotide primers (also a
complication with the 3' to 5' exonuclease activity which can
degrade the 3' ends of the primers), yielding undesirable results
due to an early termination of the PCR process (See WO 92/06200;
Barnes, Id.).
[0025] In sequencing reactions, Taq polymerase is subject to a
limitation shared by E. coli polymerase I and the Klenow fragment.
These enzymes each are "discriminatory," meaning that they
preferentially incorporate dNTPs over ddNTPs into newly synthesized
DNA. Thus, to use Taq polymerase in automated sequencing reactions,
relatively high concentrations of ddNTPs must be maintained in the
reaction mixtures, to kinetically favor ddNTP incorporation by the
enzyme. This need for high levels of ddNTPs can be prohibitively
expensive, particularly when large DNA fragments are being
sequenced.
[0026] As another technical limitation, the DNA polymerases have
heretofore been maintained in highly concentrated stock solutions
in storage buffers containing glycerol, bovine serum albumin and/or
other stabilizing agents and stored at -20.degree. C. or lower
(See, e.g., WO 92/06188; U.S. Pat. No. 5,436,149). The conventional
understanding in the field has been that the enzymes would rapidly
lose activity at more dilute working concentrations (as do many
bioactive proteins) and in solutions without glycerol or other
stabilizing agents. Moreover, the solutions of enzymes had to be
mixed with dNTPs or ddNTPs, cofactors (such as Mg.sup.++) and one
or more detergents immediately prior to use in the sequencing or
PCR processes, as it was believed that premixture and storage of
these solutions would also deleteriously affect their stability. In
addition, dNTPs have traditionally been stored at temperatures
below -20.degree. C. and have also been thought to be unstable if
stored otherwise (Maniatis, T., et al., Molecular Cloning, A
Laboratory Manual, CRC Press, 1992). Together, these limitations
have made the use of compositions containing thermostable DNA
polymerases in DNA sequencing and PCR more costly and
time-consuming than would be desired.
[0027] Overcoming Technical Limitations
[0028] Several approaches have been undertaken to attempt to
surmount these technical difficulties. The results of DNA
sequencing methodologies, for example, have been improved by the
use of mutant Taq enzymes such as .DELTA.Taq (WO 92/06188) lacking
the 5' to 3' exonuclease activity. Improved sequencing results have
also been obtained by including in the reaction mixture an agent
such as pyrophosphatase which breaks down the pyrophosphate that
can be formed during dideoxy sequencing reactions (See U.S. Pat.
No. 5,498,523). To overcome the discrimination between ddNTPs and
dNTPs, some investigators have used T7 DNA polymerase in
sequencing, as this enzyme is "nondiscriminatory," meaning that it
incorporates ddNTPs at approximately the same rate as dNTPs (See
U.S. Pat. No. 4,795,699). Alternatively, mutants of DNA polymerase
from a variety of organisms (e.g., E. coli) which are
nondiscriminatory have also been described; see copending U.S.
patent application Ser. No. 08/525,057 of Deb K. Chattetjee, filed
Sep. 8, 1995, entitled "Mutant DNA Polymerases and Use Thereof,"
the disclosure of which is expressly incorporated herein by
reference. However, as described above, both E. coli and T7 DNA
polymerases are thermolabile, so their use in automated sequencing
requires addition of fresh enzyme at the beginning of each
cycle.
[0029] More recently, mutations in Tne polymerase from
Thermotoganeapolitana have been described, which overcome these
limitations (WO 96/10640). One of these mutations, in which a
phenylalanine residue at amino acid position number 730 in the
wildtype protein (SEQ ID NO:1) is replaced with a tyrosine residue,
results in a mutant Tne polymerase (SEQ ID NO:2) which is both
thermostable and nondiscriminatory. This mutant Tne polymerase thus
provides a solution to both the problems of thermolability and
ddNTP discrimination found in other enzymes used in automated DNA
sequencing. See also the co-pending U.S. patent application of A.
John Hughes and Deb K. Chatteree, entitled "Cloned DNA Polymerases
from Thermotoga and Mutants Thereof," filed on even day herewith,
which is incorporated by reference herein in its entirety.
[0030] In PCR applications, the low fidelity of Taq-produced PCR
products has been alleviated to some extent by the use of Pfu DNA
polymerase which contains the proofreading 3' to 5' exonuclease
activity lacking in Taq polymerase (Lundberg, K. S., et al., Gene
108:1-6, 1991). Other thermostable DNA polymerases, including
Tli/VENT.TM. (Bej and Mahbubani, Id.) and DEEPVENT.TM. (Flaman, J.
-M., et al., Nucl. Acids Res. 22(15):3259-3260, 1994) have also
been shown to improve the fidelity of PCR products.
[0031] As a means of overcoming this length limitation, mutant
enzymes lacking the 5' to 3' exonuclease activity have been
prepared, including N-terminal deletion mutants of Taq polymerase
that are analogous to the Klenow fragment of E. coli DNA polymerase
I. Several of these mutant enzymes, including Klentaq-1 and
Klentaq-278 (Barnes, W. M., Gene 112:29-35,1992; U.S. Pat. No.
5,436,149), the Taq Stoffel fragment (Lawyer, F. C., et al., PCR
Meth. Appl. 2:275-287, 1993), and mutants of other thermostable DNA
polymerases lacking the 5' to 3' exonuclease activity (e.g., those
disclosed in WO 92/06200), have been shown to provide increasingly
stable PCR products and primers. However, as these enzymes also
lack the 3' to 5' proofreading activity, their use subjects the PCR
process to the increased error rates described above. Thus, even
with these mutant enzymes, high-fidelity PCR amplification of DNA
fragments larger than 5-6 kilobases has proven exceedingly
difficult.
[0032] By combining specific quantities of several enzymes,
however, high-fidelity PCR amplification of large DNA sequences has
been achieved. For example, use of a combination of a high
concentration of a thermostable DNA polymerase lacking the 3' to 5'
exonuclease activity (e.g., Klentaq278) and a low concentration of
a thermostable DNA polymerase exhibiting the 3' to 5' exonuclease
activity (e.g., Pfu/DEEPVENT or Tli/VENT.TM.) provides for
amplification to high concentrations of DNA sequences of at least
35 kilobases in length with significantly improved fidelity
(Barnes, Id; U.S. Pat. No. 5,436,149). Similar results have been
obtained with mixtures of Tth polymerase and low levels of
thermostable polymerases from Pyrococcus, Tli or Tma (U.S. Pat. No.
5,512,462). Apparently, the low level of 3' to 5' exonuclease
activity is sufficient to remove any mismatched bases incorporated
by the majority polynerase, but is insufficient to significantly
degrade the primers. While this approach has heretofore been
applied to simple DNA sequences such as those from bacteriophage
.lambda., it may prove applicable to larger and more complex
sequences as well, including those of the genomes of bacteria,
yeast, plants and animals.
[0033] Despite its widespread use, however, conventional PCR can
produce non-specific amplification fragments which range from small
primer-dimer products to target fragments of various yields and of
heterogeneous size. These non-specific products not only obscure
PCR results, but can also limit the sensitivity of PCR product
detection and can also interfere with downstream processes such as
DNA sequencing and cloning of PCR fragments. These artifact
amplification products are often due to non-specific annealing and
extension of primers at low temperatures, and to the presence of a
low level of polymerase activity in the reaction mixtures during
setup and start of PCR (Li, H., et al., Proc. Natl. Acad. Sci. USA
87:4580 (1990); Frohman, M. A., et al., Proc. Natl. Acad. Sci. USA
85:8998 (1988); Chou, Q., et al., Nucl. Acids Res. 20:1717 (1992)).
Consequently, a number of physical manipulation methods have been
developed to circumvent the non-specific priming during the PCR
setup and start of the reaction. These manual methods are often
referred to as "Hot Start" and involve the addition of Taq DNA
polymerase to preheated (typically to about 80.degree. C.) PCR
reactions (Chou, Q., et al., Nucl. Acids Res. 20:1717 (1992);
D'Aquila, R. T., et al. Nucl. Acids Res. 19:3749 (1991). These
methods, however, are often cumbersome and are not used for routine
or high throughput applications.
[0034] It has recently been shown that specific monoclonal
antibodies to thermostable DNA polymerases can be used to improve
specificity of PCR amplification (see U.S. Pat. No. 5,338,671, the
disclosure of which is incorporated herein by reference in its
entirety; Sharkey, D. J., et al., BioTechnology 12:506 (1994);
Daiss, J. L. et al., .J Immunol. Meth. 183:15 (1995)). These
monoclonal antibodies prevent the polymerization activity of the
enzyme and result in inactivity of Taq DNA polymerase during the
PCR setup and start of reactions. However, during the initial
denaturation step of PCR, antibodies are denatured and active Taq
DNA polymerase is released into the reaction. This approach
provides an effective and automatic method for control of
non-specific PCR products in all PCR reactions.
[0035] Despite these successes in overcoming the ddNTP
discrimination, length, fidelity, and artifact limitations,
however, compositions comprising the reagents necessary for DNA
sequencing or PCR capable of extended storage above freezing at
working concentrations, without stabilizing agents, have not
heretofore been reported. Thus, the time and economic constraints
to the use of solutions of thermostable enzymes in most
applications have yet to be overcome.
BRIEF SUMMARY OF THE INVENTION
[0036] The present invention overcomes these temporal and economic
limitations of previously available reagent compositions used in
nucleic acid amplification and sequencing methods. Specifically,
the invention is directed to compositions comprising mixtures of
reagents at working concentrations suitable for use with or without
dilution and maintaining activity upon storage for an extended
time, said mixtures consisting essentially of at least one
thermostable enzyme and at least one buffer salt. The invention
further provides such compositions for use in nucleic acid
amplification further comprising at least one deoxynucleoside
triphosphate, magnesium salts and at least one nonionic detergent,
wherein the thermostable enzyme is at least one thermostable DNA
polymerase. In another embodiment, the invention provides such
compositions for use in nucleic acid sequencing further comprising
at least one deoxynucleoside triphosphate, at least one
dideoxynucleoside triphosphate, magnesium salts and at least one
nonionic detergent, wherein the thermostable enzyme is at least one
thermostable DNA polymerase. More specifically, the invention is
directed to such compositions wherein the thermostable DNA
polymerase is Taq, Tne, Tma, Pfu, Pwo or Tth DNA polymerase, or
mutants thereof, preferably at concentrations of about 0.1-200
units per milliliter, about 0.1-50 units per milliliter, about
0.1-40 units per milliliter, about 0.1-36 units per milliliter,
about 0.1-34 units per milliliter, about 0.1-32 units per
milliliter, about 0.1-30 units per milliliter, or about 0.1-20
units per milliliter, and most preferably at concentrations of
about 20 units per milliliter. The invention is also directed to
such compositions that further comprise VENT or DEEPVENT.TM. DNA
polymerase, preferably at concentrations of about 0.0002-200 units
permilliliter, about 0.002-100 units permilliliter, about 0.002-20
units per milliliter, about 0.002-2.0 units per milliliter, about
0.002-1.6 units per milliliter, about 0.002-0.8 units per
milliliter, about 0.002-0.4 units per milliliter, or about
0.002-0.2 units per milliliter, most preferably at concentrations
of about 0.40 units per milliliter.
[0037] In another embodiment, the invention is directed to such
compositions which optionally further comprise at least one
antibody which specifically binds to the one or more thermostable
enzymes (such as the one or more DNA polymerases) in the
compositions. The antibodies used in this aspect of the invention
may be polyclonal or monoclonal, and are preferably monoclonal, and
may include (but are not limited to) anti-DNA polymerase
antibodies, particularly antibodies which bind specifically to one
or more thermostable DNA polymerases, such as anti-Taq antibodies,
anti-Tne antibodies, anti-Tma antibodies, anti-Pfu antibodies,
anti-Pwo antibodies, anti-Tth antibodies, and the like. Preferably,
the antibodies are used in the compositions at an antibody to
polymerase concentration ratio of up to about 100:1, up to about
50:1, up to about 25:1, up to about 20:1, up to about 15:1, up to
about 10:1, up to about 9:1, up to about 8:1, up to about 7.5:1, up
to about 7:1, up to about 6:1, up to about 5:1, up to about 4:1, up
to about 3:1, up to about 2.5:1, up to about 2:1, or up to about
1:1. Most preferably, the antibodies are used in the compositions
at an antibody to polymerase concentration ratio of about 1:1 to
about 10:1, or about 1:1 to about 5:1.
[0038] The invention is further directed to kits for DNA
amplification or sequencing, said kits comprising a carrier means,
such as a box, carton, tube or the like, having in close
confinement therein one or more container means, such as vials,
tubes, ampules, bottles or the like, wherein a first container
means contains a composition comprising a mixture of reagents at
working concentrations suitable for use without dilution and
maintaining activity upon storage for extended time, said mixture
consisting essentially of at least one thermostable DNA polymerase,
buffer salts, magnesium salts and at least one nonionic detergent.
In additional embodiments, the kits may optionally comprise one or
more antibodies, in the first container means or in a separate
container means, which specifically bind to one or more of the DNA
polymerases present in the compositions of the kits, such as those
antibodies described above. The first container means may also
contain a mixture of dNTPs (for PCR applications) or ddNT's (for
sequencing applications). Alternatively, the dNTPs or ddNTPs may be
included in a second container means also closely confined within
the carrier means of the kit.
[0039] The invention is also directed to methods of amplifying or
sequencing a nucleic acid molecule, comprising contacting the
nucleic acid molecule to be amplified or sequenced, which is
preferably larger than about 4-8 kilobases in size, more preferably
larger than about 5-7 kilobases in size and most preferably larger
than about 7 kilobases in size, with the compositions of the
invention. The invention also provides nucleic acid molecules
amplified by these methods.
[0040] The present invention is also directed more generally to
compositions containing thermostable proteins or enzymes useful in
molecular biology. These compositions comprise mixtures of reagents
at working concentrations suitable for use with or without dilution
which maintain the enzyme or protein activity upon storage for an
extended time. The compositions of this aspect of the invention
comprise at least one thermostable enzyme and at least one buffer
salt. The thermostable enzymes in these compositions include, but
are not limited to, polymerases, restriction enzymes, alkaline
phosphatases, reverse transcriptases, ligases, nucleases,
pyrophosphatases, DNAses, RNAses, exonucleases, RNAse inhibitors,
kinases, topoisomerases, guanylyltransferases and glycosylases
(e.g., uracil DNA glycosylase).
[0041] The invention is further directed to kits for conducting a
procedure associated with the thermostable enzymes or proteins
(restriction enzymes, phosphatases, etc.), the kits comprising a
container means such as a box, carton, tube and the like, having in
close confinement therein one or more container means, such as a
vial, tube, ampule, bottle or the like, wherein a first container
means contains a composition comprising a mixture of reagents at
working concentrations suitable for use with or without dilution.
The reagents in the first container means include at least one
thermostable enzyme or protein and at least one buffer salt.
[0042] The compositions of the invention have unexpectedly been
found to maintain enzyme activity for extended periods of time
compared to conventional compositions. For example, the present
compositions maintain enzyme activity for at least four weeks when
stored at ambient temperature (about 20.degree.-25.degree. C.), for
at least one year at about 4.degree. C. and for at least two years
at about -20.degree. C.
[0043] These stable, ready-to-use reagent compositions are useful
for nucleic acid amplification (including the Polymerase Chain
Reaction or PCR) and sequencing (including dideoxy sequencing), or
for any procedure using thermostable DNA polymerases or other
enzymes (restriction enzymes, phosphatases, kinases, etc.) in
fields such as medical therapeutics and diagnostics, forensics and
agricultural science.
[0044] Other features, advantages and applications of the present
invention will be apparent to those skilled in the art from the
following description of the preferred embodiments thereof, and
from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 is a photograph of an agarose gel (visualized by
ethidium bromide fluorescence under ultraviolet illumination) of
PCR amplification of a 1.3 kilobase human genomic DNA fragment from
10 nanograms of template using 15 different reagent compositions
(corresponding to samples 10-24 in Table 1) stored as indicated in
Table 1, and a freshly made control sample. Lane marked "M"
contains markers indicating amount of DNA loaded; bands correspond
to (from top to bottom) 100 nanograms, 60 nanograms, 40 nanograms
and 20 nanograms of DNA mass markers.
[0046] FIG. 2 is a photograph of an agarose gel (visualized by
ethidium bromide fluorescence under ultraviolet illumination) of
PCR amplification of a 4.1 kilobase human genomic DNA fragment
using the amounts indicated (in nanograms) of template for each
amplification reaction. The samples on the upper portion of the gel
were amplified with a nucleic acid amplification composition stored
for 10 weeks at 4.degree. C., while those on the lower portion of
the gel were amplified with a composition stored for 10 weeks at
ambient temperature (about 20-25.degree. C.). Leftmost lanes in
each portion are markers as indicated in FIG. 1.
DETAILED DESCRIPTION OF THE INVENTION
[0047] Definitions
[0048] Throughout this disclosure, various terms that are generally
understood by those of routine skill in the art are used. Certain
terms as used herein, however, have specific meanings for the
purposes of the present invention. The term "dNTP" (plural "dNTPs")
generically refers to the deoxynucleoside triphosphates (e.g.,
dATP, dCTP, dGTP, dTTP, dUTP, dITP, 7-deaza-dGTP, adATP, adTTP,
adGTP and adCTP), and the term "ddNTP" (plural "ddNT~s") to their
dideoxy counterparts, that are incorporated by polymerase enzymes
into newly synthesized nucleic acids. The term "unit" as used
herein refers to the activity of an enzyme. When referring to a
thermostable DNA polymerase, one unit of activity is the amount of
enzyme that will incorporate 10 nanomoles of dNTPs into
acid-insoluble material (i.e., DNA or RNA) in 30 minutes under
standard primed DNA synthesis conditions. "Working concentration"
is used herein to mean the concentration of a reagent that is at or
near the optimal concentration used in a solution to perform a
particular function (such as amplification, sequencing or digestion
of nucleic acids). The term "detergent" as used herein refers to a
nonionic surfactant such as TRITON X-1000, Nonidet P-40 (NP-40),
Tween 20 or Brij 35. The terms "stable" and "stability" as used
herein generally mean the retention by an enzyme of at least 70%,
preferably at least 80%, and most preferably at least 90%, of the
original enzymatic activity (in units) after the enzyme or
composition containing the enzyme has been stored for at least four
weeks at a temperature of about 20-25.degree. C., at least one year
at a temperature of about 4.degree. C. or at least 2 years at a
temperature of -20.degree. C. Overview The present invention
provides, in a first preferred embodiment, compositions comprising
mixtures of at least one thermostable enzyme (e.g., a thermostable
DNA polymerase, restriction enzyme, etc.), at least one buffer
salt, and other reagents necessary for carrying out the procedure
associated with the enzyme(s) (e.g., deoxynucleoside triphosphates
(dNTPs) for amplification of nucleic acids, dNTPs and
dideoxynucleoside triphosphates (ddNTPs) for sequencing of nucleic
acids, etc.). In additional preferred embodiments, the invention
provides such compositions which may further comprise one or more
antibodies which specifically bind to the one or more thermostable
enzymes (such as the one or more DNA polymerases) in the
compositions. The compositions of the invention contain no
stabilizing compounds (e.g., glycerol, serum albumin or gelatin)
that have been traditionally included in stock reagent solutions,
and exhibit increased stability (measured as maintenance of enzyme
activity) even upon storage at temperatures above freezing.
Furthermore, the invention provides these reagent compositions in
ready-to-use concentrations, obviating the time-consuming dilution
and pre-mixing steps necessary with previously available solutions.
Unexpectedly, even at these diluted concentrations the reagent
compositions are stable for extended periods of time at
temperatures ranging from ambient (about 20-25.degree. C.) to about
-70.degree. C.
[0049] In additional preferred embodiments, the present invention
provides these ready-to-use compositions in the form of kits that
are suitable for immediate use to carry out the procedure
associated with the enzyme(s) (e.g. nucleic acid amplification or
sequencing in the case of DNA polymerases). These kits are also
stable for extended periods of time at temperatures ranging from
ambient (about 20-25.degree. C.) to -70.degree. C.
[0050] In additional preferred embodiments, the invention provides
ready-to-use compositions for PCR amplification. The ready-to-use
reagents will contain all necessary components for PCR
amplification such as one or more DNA polymerase(s), one or more
deoxynucleoside triphosphates (dNTPs) and buffers, and optionally
one or more other components contributing to efficient
amplification of nucleic acid templates by automatic "hot start."
Automatic Hot Start PCR can be accomplished by reaction of specific
antibodies, e.g., monoclonal antibodies, that bind to and
inactivate one or more DNA polymerases, such as thermostable DNA
polymerases (e.g., Taq DNA polymerase), that are present in the
ready-to-use compositions of the invention. In additional
embodiments, the invention provides formulation of ready-to-use PCR
reagents which contain one or more thermostable DNA polymerases
(e.g., Taq DNA polymerase), one or more dNTPs, one or more buffers,
and one or more antibodies that bind to a DNA polymerase.
[0051] Sources of Reagents
[0052] The compositions of the present invention may be formed by
mixing the component reagents at the concentrations described
below. The components for making the ready-to-use compositions can
be obtained from, for example, Life Technologies, Inc. (Rockville,
Md.).
[0053] Thermostable Enzymes
[0054] The thermostable enzymes (e.g., DNA polymerases, restriction
enzymes, phosphatases, etc.) used in the present invention may be
isolated from natural or recombinant sources, by techniques that
are well-known in the art (See Bej and Mahbubani, Id.; WO 92/06200;
WO 96/10640), from a variety of thermophilic bacteria that are
available commercially (for example, from American Type Culture
Collection, Rockville, Md.) or may be obtained by recombinant DNA
techniques (WO 96/10640). Suitable for use as sources of
thermostable enzymes or the genes thereof for expression in
recombinant systems are the thermophilic bacteria Thermus
thermophilus, Thermococcus litoralis, Pyrococcus furiosus,
Pyrococcus woosii and other species of the Pyrococcus genus,
Bacillus sterothennophilus, Sulfolobus acidocaldarius, Thermoplasma
acidophilum, Thermus flavus, Thermus ruber, Thermus brockianus,
Thermotoga neapolitana, Thermotoga maritima and other species of
the Thennotoga genus, and Methanobacterium thermoautotrophicum, and
mutants thereof. It is to be understood, however, that thermostable
enzymes from other organisms may also be used in the present
invention without departing from the scope or preferred embodiments
thereof. As an alternative to isolation, thermostable enzymes
(e.g., DNA polymerases) are available commercially from, for
example, Life Technologies, Inc. (Rockville, Md.), New England
Biolabs (Beverly, Mass.), Finnzymes Oy (Espoo, Finland) and Perkin
Elmer Cetus (Norwalk, Conn.). Once obtained, the purified enzymes
may be placed into solution at working concentrations and stored
according to the methods of the present invention.
[0055] dNTPs
[0056] The dNTP components of the present compositions serve as the
"building blocks" for newly synthesized nucleic acids, being
incorporated therein by the action of the polymerases. These
dNTPs--deoxyadenosine triphosphate (dATP), deoxycytosine
triphosphate (dCTP), deoxyguanosine triphosphate (dGTP),
deoxythymidine triphosphate (dTTP), and for some applications
deoxyuridine triphosphate (dUTP) and deoxyinosine triphosphate
(dITm), a-thio-dATP and 7-deaza-dGTP--are available commercially
from sources including Life Technologies, Inc. (Rockville, Md.),
New England Biolabs (Beverly, Mass.) and Sigma Chemical Company
(Saint Louis, Mo.). The dNTPs may be unlabeled, or they may be
detectably labeled by coupling them by methods known in the art
with radioisotopes (e.g., .sup.3H, .sup.14C, .sup.32P or .sup.35S),
vitamins (e.g., biotin), fluorescent moieties (e.g., fluorescein,
rhodamine, Texas Red, or phycoerythrin) or other detection agents.
Labeled dNTPs may also be obtained commercially, for example from
Life Technologies, Inc. (Rockville, Md.) or Sigma Chemical Company
(Saint Louis, Mo.). Once obtained, the dNTPs may be placed into
solution at working concentrations and stored according to the
methods of the present invention.
[0057] ddNTPs
[0058] The ddNTP components of the present compositions serve as
the "terminating agents" in the dideoxy nucleic acid sequencing
methodologies, being incorporated into newly synthesized nucleic
acids by the action of the polymerases. These
ddNTPs--dideoxyadenosine triphosphate (ddATP), dideoxycytosine
triphosphate (ddCTP), dideoxyguanosine triphosphate (ddGTP),
dideoxythymidinetriphosphate (ddTTP), and for some applications
dideoxyuridine triphosphate (ddUTP) and dideoxyinosine triphosphate
(ddITP)--are available commercially from sources including Life
Technologies, Inc. (Rockville, Md.), New England Biolabs (Beverly,
Mass.) and Sigma Chemical Company (Saint Louis, Mo.). The ddNTPs
may be unlabeled, or they may be detectably labeled by coupling
them by methods known in the art with radioisotopes (e.g., .sup.3H,
.sup.14C, .sup.32P or .sup.35S), vitamins (e.g., biotin),
fluorescent moieties (e.g., fluorescein, rhodamine, Texas Red, or
phycoerythrin) or other detection agents. Labeled ddNTPs may also
be obtained commercially, for example from Life Technologies, Inc.
(Rockville, Md.) or Sigma Chemical Company (Saint Louis, Mo.). Once
obtained, the ddNTPs may be placed into solution at working
concentrations and stored according to the methods of the present
invention.
[0059] Buffers/Salts
[0060] All buffers and cofactor salts comprising the compositions
of the present invention, and concentrated stock solutions thereof
are available from a variety of commercial sources including Life
Technologies, Inc. (Rockville, Md.) and Sigma Chemical Company
(Saint Louis, Mo.). Particularly preferred buffers for use in
forming the present compositions are the sulfate, hydrochloride,
phosphate or free acid forms of tris-(hydroxymethyl)aminomethane
(TRIS.RTM.), although alternative buffers of the same approximate
ionic strength and pKa as TRIS.RTM. may be used with equivalent
results. In addition to the buffer salts, cofactor salts such as
those of potassium (preferably potassium chloride) and magnesium
(preferably magnesium chloride or sulfate) are included in the
compositions. Once obtained, the buffers and cofactor salts may be
placed into solution at working concentrations and stored according
to the methods of the present invention.
[0061] Detergents
[0062] At least one detergent may be included as a component of the
present compositions, to provide for both increased stability and
activity of the component enzymes. Nonionic detergents are
preferred, to maintain a balanced ionic strength and prevent
chelation of cofactors and aggregation or inactivation of proteins.
Particularly preferred as detergents are TRITONX-100.RTM., Brij 35,
Tween20 and Nonidet P-40 (NP-40), although other nonionic
surfactants and mixtures thereof may also be used in the present
compositions. These detergents are available commercially from
sources such as Sigma Chemical Company (Saint Louis, Mo.), usually
as concentrated aqueous solutions or in powder form. Once obtained,
the detergents may be placed into solution at working
concentrations and stored according to the methods of the present
invention.
[0063] Antibodies
[0064] In additional embodiments of the invention, the compositions
may optionally comprise one or more antibodies which specifically
bind to the one or more thermostable enzymes, such as the one or
more DNA polymerases, present in the compositions of the invention.
According to this aspect of the invention, the one or more
antibodies will specifically bind to the one or more thermostable
enzymes (such as the one or more DNA polymerases) at temperatures
below about 45.degree. C.; as a result of this binding, the
enzymatic activity of the enzyme will be completely or
substantially completely inhibited. However, once the composition
or reaction mixture containing the composition is raised to a
temperature above about 60-65.degree. C. (e.g., the temperatures at
which standard PCR methods are conducted), the antibody is
denatured and the activity of the enzyme is restored. Thus, such
compositions will have utility in such applications as "Hot Start"
PCR amplification protocols. Antibodies for use in this aspect of
the invention include polyclonal antibodies, monoclonal antibodies,
and enzyme-binding fragments (such as F(ab') or F(ab').sub.2
fragments) thereof According to the invention, any antibody or
fragment thereof which specifically binds to one or more of the
thermostable enzymes in the present compositions, such as the DNA
polymerases, may be used, including but not limited to anti-Taq
antibodies, anti-Tne antibodies, anti-Tma antibodies, anti-Pfu
antibodies, anti-Pwo antibodies, anti-Tth antibodies, and the like.
These and other antibodies suitable for use in this aspect of the
invention may be obtained commercially, e.g., from Life
Technologies, Inc. (Rockvflle, Md.). Alternatively, antibodies may
be produced in animals by routine methods of production of
polyclonal antibodies (see, e.g., Harlow, E., and Lane, D.,
Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.: Cold
Spring Harbor Laboratory Press (1988); Kaufinan, P. B., et al., In:
Handbook of Molecular and Cellular Methods in Biology and Medicine,
BocaRaton, Fla.: CRC Press, pp. 468-469 (1995) or monoclonal
antibodies (see, e.g., Kohler et al., Nature 256:495 (1975); Kohler
et al., Eur. J Immunol. 6:511 (1976); Kohler et al., Eur. J.
Immunol. 6:292 (1976); Hammerling et al., In: Monoclonal Antibodies
and T-Cell Hybridomas, New York: Elsevier, pp. 563-681 (1981);
Kaufinan, P. B., et al., In: Handbook of Molecular and Cellular
Methods in Biology and Medicine, Boca Raton, Fla.: CRC Press, pp.
444-467 (1995)), using the corresponding thermostable enzyme (such
as the corresponding DNA polymerase) as an immunogen.
[0065] Formulating the Reagent Compositions
[0066] Once the reagent components have been obtained, they are
mixed at working concentrations to form a solution suitable for
immediate use with or without dilution or addition of further
reagents. The water used in the formulations of the present
invention is preferably distilled, deionized and sterile filtered
(through a 0.1-0.2 micrometer filter), and is free of contamination
by DNase and RNase enzymes. Such water is available commercially,
for example from Sigma Chemical Company (Saint Louis, Mo.), or may
be made as needed according to methods well known to those skilled
in the art.
[0067] Although the components of the present compositions may be
admixed in any sequence, it is often preferable to first dissolve
the buffer(s) and cofactor salts in water and to adjust the pH of
the solution prior to addition of the remaining components. In this
way, the pH-sensitive components (particularly the enzymes, ddNTPs
and dNTPs) will be less subject to acid- or alkaline-hydrolysis
during formulation.
[0068] To formulate the buffered salts solution, a buffer salt
which is preferably a salt of tris(hydroxymethyl)aminomethane
(TRIS.RTM.), and most preferably the hydrochloride salt thereof, is
combined with a sufficient quantity of water to yield a solution
having a TRIS.RTM. concentration of 5-150 millimolar, preferably
10-60 .millimolar, and most preferably about 20-60 millimolar. To
this solution, a salt of magnesium (preferably either the chloride
or sulfate salt thereof) may be added to provide a working
concentration thereof of 1-10 milimolar, preferably 1.5-5
millimolar, and most preferably about 1.5-2 millimolar. A salt of
potassium (most preferably potassium chloride) may also be added to
the solution, at a working concentration of 10-100 millimolar and
most preferably about 50 millimolar. An ammonium salt, for example
ammonium sulfate, may also be added to the mixture, at a working
concentration of 2-50 millimolar, preferably 10-30 millimolar and
most preferably 18 millimolar. Combinations of ammonium sulfate and
potassium chloride (or other salts) may also be used in formulating
the compositions of the present invention. A small amount of a salt
of ethylenediaminetetraacetat- e (EDTA) may also be added
(preferably about 0.1 millimolar), although inclusion of EDTA does
not appear to be essential to the function or stability of the
compositions of the present invention. After addition of all
buffers and salts, this buffered salt solution is mixed well until
all salts are dissolved, and the pH is adjusted using methods known
in the art to a pH value of 7.4 to 9.2, preferably 8.0 to 9.0, and
most preferably about 8.3 for compositions to be used in
amplification or sequencing of nucleotide fragments up to about 5-6
kilobases in size (hereinafter referred to as "standard
compositons"), and about 8.9 for compositions to be used for
amplification or sequencing of nucleotide fragments larger than
about 5-6 kilobases in size (hereinafter referred to as "large
sequence compositions").
[0069] To the buffered salt solution, the remaining components of
the present composition are added. It is well known in the field
that the addition of one or more detergents to an aqueous buffer
will aid in the subsequent solubilization of added proteins.
Accordingly, at least one nonionic detergent such as TRITON
X-100.RTM. (preferably at a working concentration of 0.1-1%), Brij
35 (preferably at a concentration of 0.01-1% and most preferably of
about 0.1%) or Nonidet P-40 (NP-40, preferably as an admixture with
a concentration of 0.004-1%, and most -preferably in admixture with
Tween 20 at a working concentration of 0.1% for standard
compositions and 0.02% for large sequence compositions) maybe added
to the buffer solution. This detergent is preferably added prior to
the introduction of the remaining components into the solution,
although the detergent may equivalently be added at any step of
formulation. Following formulation, the buffered salt solutions may
be filtered through a low protein-binding filter unit that is
available commercially (for example from Millipore Corporation,
Bedford, Mass.) and stored until use.
[0070] The remaining components are then added to the solution to
formulate the compositions of the present invention. At least one
thermostable enzyme (e.g., DNA polymerase) is added and the
solution is gently mixed (to minimize protein denaturation). For
standard DNA amplification (including via PCR) or sequencing of DNA
segments up to about 5-6 kilobases in length, any thermostable DNA
polymerase (hereinafter the "primary polymerase") may be used in
the standard compositions, although Taq, Tne, Tma, VENT.TM.,
DEEPVEN.TM., Pfu or Pwo polymerases are preferable at a working
concentration in the solution of about 0.1-200 units per
milliliter, about 0.1-50 units per milliliter, about 0.1-40 units
per milliliter, about 0.1-36 units per milliliter, about 0.1-34
units per milliliter, about 0.1-32 units per milliliter, about
0.1-30 units per milliliter, or about 0.1-20 units per milliliter,
and most preferably at a working concentration of about 20 units
per milliliter. For amplification of DNA segments larger than 5-6
kilobases in length, large sequence compositions should be
formulated by adding to the standard compositions a low
concentration of one or more additional thermostable DNA
polymerases (hereinafter the "secondary polymerase") containing a
3'-5' exonuclease activity. Particularly suited for this
application are VENT.TM., Pfu, Pwo or Tne, and most preferably
DEEPVENT.TM., DNA polymerases. The additional polymerase(s) should
be added to the solution in sufficient quantity to give a final
working concentration of about 0.0002-200 units per milliliter,
about 0.002-100 units per milliliter, about 0.002-20 units per
milliliter, about 0.002-2.0 units per milliliter, about 0.002-1.6
units per milliliter, about 0.002-0.8 units per milliliter, about
0.002-0.4units per milliliter, or about 0.002-0.2 units per
milliliter, most preferably at concentrations of about 0.40 units
per milliliter.
[0071] It has heretofore been thought that the activity ratios of
the primary to secondary polymerases should be maintaied at
4:1-2000:1 for large sequence amplification (see U.S. Pat. No.
5,436,149). It has now been discovered, however, that in the
compositions of the present invention that activity ratios of the
primary to secondary polymerases of 1:1, 1:2, 1:4, 1:5, 1:8, 1:10,
1:25, 1:50, 1:100, 1:250, 1:500, 1:1000 and 1:2000 maybe suitable
for amplification of large nucleotide sequences.
[0072] For nucleic acid sequencing, the reagent compositions may be
used as formulated above. For nucleic acid sequencing by the
dideoxy method (See U.S. Pat. Nos. 4,962,020, 5,173,411 and
5,498,523), however, preferably the mutant Tne DNA polymerase shown
in SEQ ID NO:2 is added to the reagent compositions. Tne polymerase
is added to the solution to give a working concentration of about
0.1-10,000 units per milliliter, about 0.1-5000 units per
milliliter, about 0.1-2500 units per milliliter, about 0.1-2000
units per milliliter, about 0.1-1500 units per milliliter, about
0.1-1000 units per milliliter, about 0.1-500 units per milliliter,
about 0.1-300 units per milliliter, about 0.1-200 units per
milliliter, about 0.1-100 units per milliliter, or about 0.1-50
units per milliliter, and most preferably of about 300 units per
milliliter.
[0073] For dideoxy sequencing, a solution of each ddNTP is also
prepared. The base of each solution contains dATP, dCTP, dTTP,
7-deaza-GTP and/or other dNTPs, each at a working concentration of
about 10-1000 micromolar, about 10-500 micromolar, about 10-250
micromolar, or about 10-100 micromolar, most preferably at a
concentration of about 100 micromolar, in a solution of buffer and
chelating salts, for example TRIS.RTM.-HCl most preferably at a
working concentration of about 10 millimolar (pH about 7.5) and
disodium-EDTA most preferably at a concentration of about 0.1
millimolar. To this base, one of the ddNT's is added to make each
of four solutions. Preferably, the sodium or lithium salt of ddATP,
ddCTP, ddGTP or ddTTP is added to the solution to give a working
concentration of the ddNTP of about 0.5-10 micromolar, about 0.5-8
micromolar, about 0.5-5 micromolar, about 0.5-3 micromolar, about
0.5-2.5 micromolar, or about 0.5-2 micromolar, and most preferably
about 2 micromolar. For cycle sequencing applications, the pH of
the ddNTP solutions will preferably be about 9.0, and the
concentrations of ddNTPs may be lower, preferably about 0.05 to 1.0
micromolar or about 0.05 to 0.8 micromolar, and most preferably
about 0.08 to 0.8 micromolar. For some applications, it may be
desirable to also incorporate or substitute ddITP, ddUTP, and/or
.alpha.-thio-dATP into the compositions at approximately the same
working concentrations. Thus, four solutions are prepared, each
containing one of the four ddNTPs, which are combined with the
polymerase compositions of the present invention to carry out the
four separate reactions used in dideoxy sequencing. Alternatively,
for single-solution sequencing as disclosed in U.S. Pat. Nos.
4,962,020 and 5,173,411, the four ddNTPs may be combined into a
single solution which is added to the polymerase compositions of
the present invention to perform the sequencing reaction.
[0074] For nucleic acid amplification, including PCR, dNTP salts
are added to the reagent compositions. Preferably, the sodium or
lithium salts of dATP, dCTP, dGTP and dTTP are added to the
solution to give a working concentration of each dNTP of 10-1000
micromolar, preferably 200-300 micromolar, and most preferably
about 200 micromolar. For some applications, it may be desirable to
also incorporate or substitute dITP or dUTP into the compositions
at the same working concentrations.
[0075] In certain embodiments as noted above, one or more
antibodies that specifically bind to the one or more thermostable
enzymes in the compositions, such as the one or more DNA
polymerases, may optionally be added to the compositions.
Preferably, the antibodies are used in these compositions at an
antibody to polymerase concentration ratio of up to about 100:1, up
to about 50:1, up to about 25:1, up to about 20:1, up to about
15:1, up to about 10:1, up to about 9:1, up to about 8:1, up to
about 7.5:1, up to about 7:1, up to about 6:1, up to about 5:1, up
to about 4:1, up to about 3:1, up to about 2.5:1, up to about 2:1,
or up to about 1:1. Most preferably, the antibodies are used in the
compositions at an antibody to polymerase concentration ratio of
about 1:1 to about 10:1, or about 1:1 to about 5:1.
[0076] To reduce component denaturation, storage of the reagent
compositions is preferably in conditions of diminished light, e.g.,
in amber or otherwise opaque containers or in storage areas with
controlled low lighting. The ready-to-use reagent compositions of
the present invention are unexpectedly stable at ambient
temperature (about 20.degree.-25.degree. C.) for about 4-10 weeks,
are stable for at least one year upon storage at 4.degree. C., and
for at least two years upon storage at -20.degree. C. Surprisingly,
storage of the compositions at temperatures below freezing (e.g.,
-20.degree. C. to -70.degree. C.), as is traditional with stock
solutions of bioactive components, is not necessary to maintain the
stability of the compositions of the present invention.
[0077] In other preferred embodiments, the compositions of the
present invention may be assembled into kits for use in nucleic
acid amplification or sequencing. Sequencing kits according to the
present invention comprise a carrier means, such as a box, carton,
tube or the like, having in close confinement therein one or more
container means, such as vials, tubes, ampules, bottles and the
like, wherein a first container means contains a stable composition
comprising a mixture of reagents, at working concentrations, which
are at least one thermostable DNA polymerase, at least one buffer
salt, at least one deoxynucleoside triphosphate, at least one
dideoxynucleoside triphosphate, and optionally at least one
antibody which specifically binds to at least one thermostable DNA
polymerase present in the compositions. The sequencing kits may
further comprise additional reagents and compounds necessary for
carrying out standard nucleic sequencing protocols, such as
pyrophosphatase, agarose or polyacrylamide media for formulating
sequencing gels, and other components necessary for detection of
sequenced nucleic acids (See U.S. Pat. Nos. 4,962,020 and
5,498,523, which are directed to methods of DNA sequencing).
[0078] Similarly, amplification kits according to the present
invention comprise carrier means, such as a box, carton, tube or
the like, having in close confinement therein one or more container
means, such as vials, tubes, ampules, bottles and the like, wherein
a first container means contains a stable composition comprising a
mixture of reagents, at working concentrations, which are at least
one thermostable DNA polymerase, at least one buffer salt, at least
one deoxynucleoside triphosphate, and optionally at least one
antibody which binds specifically to at least one thermostable DNA
polymerase present in the composition. The amplification kits
encompassed by this aspect of the present invention may further
comprise additional reagents and compounds necessary for carrying
out standard nucleic amplification protocols (See U.S. Pat. Nos.
4,683,195 and 4,683,202, which are directed to methods of DNA
amplification by PCR).
[0079] Use of the Reagent Compositions
[0080] The compositions and kits embodied in the present invention
will have general utility in any application utilizing nucleic acid
sequencing or amplification methodologies. Amplification methods in
which the present compositions may be used include PCR (U.S. Pat.
Nos. 4,683,195 and 4,683,202), Strand Displacement Amplification
(SDA; U.S. Pat. No. 5,455,166; EP 0 684 315), and Nucleic Acid
Sequence-Based Amplification (NASBA; U.S. Pat. No. 5,409,818; EP 0
329 822). Nucleic acid sequencing techniques which may employ the
present compositions include dideoxy sequencing methods such as
those disclosed in U.S. Pat. Nos. 4,962,022 and 5,498,523, as well
as more complex PCR-based nucleic acid fingerprinting techniques
such as Random Amplified Polymorphic DNA (RAPD) analysis (Williams,
J. G. K., et al., Nucl. Acids Res. 18(22):6531-6535, 1990),
Arbitrarily Primed PCR (AP-PCR; Welsh, J., and McClelland, M.,
Nucl. Acids Res. 18(24):7213-7218, 1990), DNA Amplification
Fingerprinting (DAF; Caetano-Anolles et al, Bio/Technology
9:553-557, 1991), microsateffite PCR or Directed Amplification of
Minisatellite-region DNA (DAMD; Heath, D. D., et al., Nucl. Acids
Res. 21(24): 5782-5785, 1993), and Amplification Fragment Length
Polymorphism (AFLP) analysis (EP 0 534 858; Vos, P., et al., Nucl.
Acids Res. 23(21):4407-4414,1995; Lin, J. J., and Kuo, J., FOCUS
17(2):66-70, 1995). In particular, the compositions and kits of the
present invention will be useful in the fields of medical
therapeutics and diagnostics, forensics, and agricultural and other
biological sciences, in any procedure utilizing thermostable DNA
polymerases. Furthermore, the methods by which the compositions of
the present invention are formulated may be extendable to all
thermostable enzymes or mixtures thereof, and may allow the
formulation of ready-to-use compositions of a variety of bioactive
enzymes or other proteins that demonstrate increased stability upon
extended storage at temperatures above freezing.
[0081] The compositions and kits of the invention are particularly
useful in methods for amplifying and sequencing nucleic acid
molecules. Nucleic acid amplification methods according to this
aspect of the invention comprise contacting a nucleic acid molecule
to be amplified with one or more of the compositions of the
invention, thus providing a population of amplified copies of the
nucleic acid molecule. Nucleic acid sequencing methods according to
this aspect of the invention comprise contacting the nucleic acid
molecule to be sequenced with one or more of the compositions of
the invention. According to these methods, amplification and
sequencing of the nucleic acid molecule may be accomplished by any
of the above-described amplification and sequencing techniques,
most preferably by PCR. The present amplification and sequencing
methods are particularly useful for amplification and sequencing of
large nucleic acid molecules (e.g., by "long PCR"), preferably
nucleic acid molecules that are larger than about 4-8 kilobases in
size, more preferably larger than about 5-7 kilobases in size, and
most preferably nucleic acid molecules that are larger than about 7
kilobases in size.
[0082] It will be readily apparent to those of ordinary skill in
the relevant arts that other suitable modifications and adaptations
to the methods and applications described herein are obvious and
may be made without departing from the scope of the invention or
any embodiment thereof. Having now described the present invention
in detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
EXAMPLE 1
Formulation of Standard Compositions
[0083] As an initial step in formulating stable, ready-to-use
reagent compositions for nucleic acid amplification and sequencing,
components were mixed in varying amounts as shown in Table 1 to
provide 24 different formulations. The pH on all formulations was
adjusted to about 8.3. After filtration through a low
protein-binding filter, all compositions were formulated with 20
units/ml of Taq polymerase and were suitable for use as standard
compositions for amplification or sequencing of nucleic acid
fragments up to about 5-6 kilobases in size.
1TABLE 1 FORMULATIONS OF STANDARD COMPOSITIONS Formulation Number 1
2 3 4 5 6 7 8 9 10 11 Tris-HCl, 20 mM 20 mM 20 mM 20 mM 20 mM 20 mM
20 mM 20 mM 20 mM 20 mM 20 mM pH 8.4 KCl 50 mM 50 mM 50 mM 50 mM 50
mM 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM (NH.sub.4).sub.2SO.sub.4 0 0
0 0 0 0 0 0 0 0 0 dNTP 200 .mu.M 200 .mu.M 200 .mu.M 200 .mu.M 200
.mu.M 200 .mu.M 200 .mu.M 200 .mu.M 200 .mu.M 200 .mu.M 200 .mu.M
Na Na Li Li MgCl.sub.2 1.5 mM 1.5 mM 1.5 mM 1.5 mM 1.5 mM 1.5 mM
1.5 mM 1.5 mM 1.5 mM 3 mM 3 mM Detergents 0.004% 0.1% 0.1% 0.1%
0.1% 0.004% 0.1% 0.004% 0.1% 0.1% 0.1% Tween20/ Tween20/ Tween20/
Tween2O/ Tween20/ Tween20/ Tween20/ Tween20/ Tween20/ Tween20/
Brij35 NP40 NP40 NP40 NP40 NP40 NP40 NP40 NP40 Taq, U/ml 20 20 20
20 20 20 20 20 20 20 20 Formulation Number 12 13 14 15 16 17 18 19
20 21 22 23 24 Tris-HCl, 20 20 mM 20 20 mM 20 mM 20 20 20 20 mM 20
mM 20 mM 50 mM 20 mM pH 8.4 mM mM mM mM mM (pH 9.0) (pH 8.8) KCl 50
50 mM 50 50 Mm 50 mM 50 50 50 50 mM 50 mM 50 mM 0 10 mM mM Mm Mm mM
mM (NH.sub.4).sub.2SO.sub.4 0 0 0 0 0 0 0 0 0 0 0 mM 10 mM dNTP 200
200 .mu.M 200 .mu.M 200 .mu.M 200 .mu.M 200 .mu.M 200 200 200 .mu.M
200 .mu.M 200 .mu.M 200 .mu.M 200 .mu.M .mu.M .mu.M .mu.M
MgCl.sub.2 3 3 mM 3 3 mM 3 mM 3 3 3 3 mM 3 mM 3 mM 1.5 mM 2 mM mM
mM mM mM mM Detergents 0.1% 0.01% 0.01% 0.01% 1% 1% 1% none 0.1%
0.1% 0.1% 0.1% 0.1% Tri- Tween20/ Brij35 TritonX- Tween20/ Brij35
Tri- Tween20/ Tween20/ Tween20/ Tween20/ Tri- tonX- NP40 100 NP40
tonX- NP40 NP40 NP40 NP40 tonX100 100 100 Taq,U/ml 20 20 20 20 20
20 20 20 20 20 20 20 20
EXAMPLE 2
Stability of Standard Compositions
[0084] To examine the stability of the standard compositions
formulated in Example 1, samples of each formulation were aliquoted
and stored, under diminshed light, at ambient temperature (about
20.degree.-25.degree. C.), 4.degree. C., -20.degree. C. and
-70.degree. C. Samples of each formulation from each temperature
were taken daily for the first week, and weekly thereafter, and
used in stability assays. These stability assays were performed by
amplifying, via standard PCR, suboptimal amounts of human genomic
DNA as a template using a template titration (if few samples were
to be compared in a time point) or a fixed amount of template (if a
larger number of samples were to be compared). To the desired
amount of template DNA in a given formulation, 10 picomoles of
primer was added, and the reaction mixtures were subjected to 35
cycles of PCR of 30 seconds at 94.degree. C., 30 seconds at
55.degree. C. and 1 minute/kilobase at 72.degree. C. A portion of
each reaction was then subjected to agarose gel electrophoresis and
visualized by ethidium bromide fluorescence under ultraviolet
illumination.
[0085] The results of these assays indicated that certain of the
formulations demonstrated enhanced stability upon storage. As shown
in FIG. 1, after about three months of storage at 4.degree. C.,
formulations 15 and 19 had completely lost enzymatic activity, as
evidenced by an absence of bands in the lanes corresponding to
these samples. At this same time point, however, formulations
10-14, 16-18 and 20-24 demonstrated about the same levels of
enzymatic activity as a freshly made ("control") formulation.
[0086] Storage temperature was also found to have a significant
effect upon the stability of the formulations, even within a given
formulation. As shown in FIG. 2, when samples of formulation 4 were
examined after about 15 weeks of storage at either 4.degree. C. or
about 20-25.degree. C., the samples stored at 4.degree. C. retained
full enzymatic activity when compared to a control sample. Those
stored at about 20-25.degree. C., however, had lost some activity,
as indicated by the lower yields of the target fragment obtained at
all template concentrations.
[0087] The results for all of the formulations at the various
storage temperatures are summarized in Table 2.
2TABLE 2 STABILITY OF STANDARD COMPOSITIONS Formulation
Number.sup.1 1 2 3 4 5 6 7 8 9 10 11 12 Storage Temp- erature,
.degree. C. 20-25 <12 weeks <12 weeks <15 weeks <12
weeks nd.sup.2 nd nd nd nd <10 weeks <6 weeks <6 weeks 4
>26 weeks >26 weeks >56 weeks >27 weeks >8 >8
>8 >8 >40 weeks >26 weeks >26 weeks >26 weeks
weeks weeks weeks weeks -20 >13 weeks >13 weeks nd >15
weeks nd nd nd nd >10 weeks >26 weeks >26 weeks >26
weeks -70 nd nd nd >15 weeks nd nd nd nd nd nd nd nd Formulation
Number.sup.1 13 14 15 16 17 18 19 20 21 22 23 24 Storage Temp-
erature, .degree. C. 20-25 <12 weeks <10 weeks <7 days
<12 weeks <6 weeks <6 0 days <10 weeks <10 weeks
<12 <6 weeks <12 weeks weeks weeks 4 >26 weeks >26
weeks <1 day >26 weeks >26 weeks >26 0 days >26
weeks >26 weeks >13 >26 weeks >26 weeks weeks weeks -20
>26 weeks >26 weeks <7 days >26 weeks >26 weeks
>26 0 days >26 weeks >26 weeks >26 >26 weeks >26
weeks weeks weeks -70 nd nd nd nd nd nd nd nd nd nd nd nd
.sup.1Formulation numbers correspond to those in Table 1; .sup.2nd
= not done.
[0088] These results indicate that several of the compositions
unexpectedly maintained enzymatic activity for 6-12 weeks upon
storage at 20-25.degree. C., and for over one year at 4.degree. C.
Formulation 19, however, had completely lost activity within 24
hours of formulation. Formulation 15 also exhibited a rapid loss of
activity.
[0089] For further analysis of the stability of these ready-to-use
compositions, several formulations were stored at 20-25.degree. C.
or at 4.degree. C. for up to six months, with samples taken monthly
for stability assays performed by a determination of polymerase
unit activity. The results of these assays are summarized in Tables
3 and 4.
3TABLE 3 STABILITY OF STANDARD COMPOSITIONS (PERCENTAGE OF ENZYME
ACTIVITY REMAINING) UPON STORAGE AT 20-25.degree. C. Formulation
No..sup.1 1 Month 2 Months 3 Months 4 Months 10 89 65 nd.sup.2 nd
11 106 3 nd nd 12 106 91 nd ud 13 93 72 nd nd 14 91 76 nd nd 15 78
63 nd nd 16 94 85 nd nd 17 89 90 nd nd 18 90 84 nd nd 19 0 0 nd nd
20 88 81 72 77 21 nd 103 nd nd 22 97 84 nd nd 23 83 77 nd nd 24 81
106 nd nd .sup.1Formulation numbers correspond to those in Table 1.
.sup.2nd = not done
[0090]
4TABLE 4 STABILITY OF STANDARD COMPOSITIONS (PERCENTAGE OF ENZYME
ACTIVITY REMAINING) UPON STORAGE AT 4.degree. C. Formulation
No..sup.1 1 Month 2 Months 4 Months 5 Months 6 Months 10 84 86 98
nd.sup.2 105 11 94 97 98 nd nd 12 94 97 106 nd nd 13 86 93 93 nd nd
14 85 91 105 nd nd 15 89 89 98 nd nd 16 88 103 104 nd nd 17 83 94
91 nd nd 18 90 97 99 nd nd 19 95 59 nd nd 38 20 97 100 94 94 103 21
93 97 100 nd nd 22 100 109 35 nd nd 23 94 97 89 nd 107 24 93 94 97
nd nd .sup.1Formulation numbers correspond to those in Table 1.
.sup.2nd--not done
[0091] Several of the formulations were stable upon storage at
20-25.degree. C., most notable formulation 20 which retained
>70% of its enzymatic activity even after storage for four
months at this temperature. As described earlier, formulation 19
had completely lost activity within the first month, as determined
by the PCR assay. Interestingly, however, as determined by the
polymerase unit activity assay, formulation 19 was stable for one
month when stored at 4.degree. C. but had lost substantial activity
by the second month of storage at this temperature (Table 4).
Formulation 20, shown previously to be stable upon extended storage
at 20-25.degree. C., was stable upon storage at 4.degree. C. for at
least six months.
[0092] Taken together, these results indicate that the compositions
of the present invention are readily suitable for use in nucleic
acid amplification reactions, and demonstrate extended stability
upon storage at 20-25.degree. C. or 4.degree. C.
EXAMPLE 3
Formulation and Stability of Large Sequence Compositions
[0093] For use in amplification and sequencing of nucleic acid
fragments larger than 5-6 kilobases, it has been suggested as
described above that a mixture of Taq and VENT.TM. or DEEPVENT.TM.
polymerases (U.S. Pat. No. 5,436,149; Barnes, Id.), or of Tth and
either Tli, Pyrococcus or Tma (U.S. Pat. No. 5,512,462), be used.
Accordingly, Taq and DEEPVENT.TM. DNA polymerases were formulated,
at activity ratios of 100:1 (for samples 1-3) or 50:1 (for sample
4) into solutions containing the buffer salts, cofactors and
detergents shown in Table 5. Each of these formulations was
adjusted to about pH 9.1, which is optimal for the activity of
DEEPVENT.TM. DNA polymerase (Bej and Mahbubani, Id.). Samples were
then stored at about 20-25.degree. C. or at 4.degree. C. and
assayed weekly for 12 weeks, and monthly thereafter, in stability
assays in which a 20 kilobase target in 100 nanograms of human
genomic template was amplified by PCR. Reaction mixtures included
10 picomoles of primer and were subjected to 35 PCR cycles of 30
seconds at 94.degree. C., 30 seconds at 62.degree. C. and 1
minute/kilobase at 68.degree. C. Portions of the reaction were
subjected to agarose gel electrophoresis and were visualized by
ethidium bromide fluorescence under ultraviolet illumination, as
shown above for FIGS. 1 and 2. Results are summarized in Table
5.
5TABLE 5 STABILITY OF LARGE SEQUENCE COMPOSITIONS Formula-
Stability at: tion No. Formulation 20-25.degree. C. 4.degree. C. 1
66 mM Tris-SO.sub.4 (pH 9.1) <12 weeks 11 months 19.8 mM
(NH.sub.4).sub.2 SO.sub.4 2.2 mM MgSO.sub.4 22 units/ml Taq DNA
Polymerase 0.22 units/ml DEEPVENT DNA Polymerase 0.11% Tween-20
0.011% NP-40 2 66 mM Tris-SO.sub.4 (pH 9.1) <12 weeks >11
months 19.8 mM (NH.sub.4).sub.2 SO.sub.4 2.2 mM MgSO.sub.4 24.42
units/ml Taq DNA Polymerase 0.242 units/ml DEEPVENT DNA Polymerase
0.066% Tween-20 0.066% NP-40 3 66 mM Tris-SO.sub.4 (pH 9.1)
nd.sup.1 11 months 19.8 mM (NH.sub.4).sub.2 SO.sub.4 2.2 mM
MgSO.sub.4 22 units/ml Taq DNA Polymerase 0.22 units/ml DEEPVENT
DNA Polymerase 0.01% Tween-20 0.01% NP-40 4 66 mM Tris-SO.sub.4 (pH
9.1) nd.sup.1 11 months 19.8 mM (NH.sub.4).sub.2 SO.sub.4 2.2 mM
MgSO.sub.4 22 units/ml Taq DNA Polymerase 0.44 units/ml DEEPVENT
DNA Polymerase 0.01% Tween-20 0.01% NP-40 .sup.1nd = not done
[0094] Upon storage at ambient temperature (20.degree.-25.degree.
C.), all of the formulations were stable for 6-12 weeks. Storage of
these formulations at 4.degree. C. provided enhanced stability of
over 11 months. Similar results may be obtained with formulations
in which Taq and Tne DNA polymerases were used in an activity ratio
of 1:1, 1:2, 1:4, 1:5, 1:8, 1:10, 1:25, 1:50, 1:100, 1:250, 1:500,
1:1000 or 1:2000. These results indicate that the large sequence
compositions of the present invention are readily suitable for use
in amplification of nucleic acid sequences larger than 5-6
kilobases and demonstrate extended stability upon storage at
20.degree. to 25.degree. C., or at 4.degree. C.
EXAMPLE 4
Combinations of Thermus flavis (Tfl) DNA Polymerase and Thermotoga
neapolitana (Tne) DNA Polymerase
[0095] To examine other DNA polymerase compositions for their
utility in amplification of nucleic acid molecules, a mixture of
Tfl and Tne DNA polymerases, at a 1:1 ratio, was used to amplify
the 2.7 kilobase Puc19 plasmid. Amplification reactions were in a
50 .mu.l final volume in buffer containing 1 mM magnesium acetate.
80 pg of Puc19 linearized by treatment with AdtII was used as the
template, and was contacted with 1 .mu.l of enzyme mixture. PCR
conditions were 1 min at 94.degree. C., followed by 35 cycles of
94.degree. C. for 30 seconds/60.degree. C. for 30
seconds/68.degree. C. for 5 minutes.
[0096] Upon analysis of the amplification products by gel
electrophoresis, this composition comprising Tfl and Tne DNA
polymerases was found to efficiently amplify the 2.7 kilobase Puc19
plasmid. The efficiency of amplification was comparable to
amplification of Puc19 using 1 .mu.l of Taq DNA polymerase.
EXAMPLE 5
Amplification of Genomic DNA Using Tfl/Tne Compositions
[0097] Having demonstrated that compositions comprising Tfl and Tne
DNA polymerases efficiently amplify plasmid-sized nucleic acid
molecules, these compositions were examined for their ability to
amplify nucleic acid molecules from genomic DNA templates. Six
different primer sets were constructed (ranging in size from 0.25
to 4.1 kilobases) and used to amplify the human .beta.-globin gene
from a genomic DNA template from the K562 human leukemia cell line.
Each reaction was performed in a volume of 50 .mu.l comprising
template at 40 ng/reaction, and Tfl/Tne mixture at either 0.5
unit/reaction or 1 unit/reaction (Tfl and Tne at a 1:1 ratio in
both mixtures). PCR conditions were 1 min at 94.degree. C.,
followed by 35 cycles of 94.degree. C. for 30 seconds/55.degree. C.
for 30 second/68.degree. C. for 5 minutes.
[0098] Upon analysis of the amplification products by gel
electrophoresis, efficient amplification was observed for all
primers. The sizes of the amplification products produced using the
different primers were 0.25 kilobase, 0.7 kilobase, 1.1 kilobases,
2.0 kilobases and 4.1 kilobases. These results demonstrate that the
Tfl/Tne compositions efficiently amplify nucleic acid molecules
derived from genomic DNA templates.
EXAMPLE 6
Use of Various Enzyme Ratios in DNA Polymerase Compositions
[0099] To determine the efficacy of different enzyme ratios in
nucleic acid amplification, a composition comprising a higher
amount of Tne (3' exo+) DNA polymerase than Tfl (3' exo-) DNA
polymerase was made and tested for its ability to amplify Puc19 and
the .beta.-globin gene. A 1:3 mixture of Tfl/Tne was made and used
to amplify Puc19 under the conditions described in Example 4, and
.beta.-globin under the conditions described in Example 5.
[0100] Upon analysis of the amplification products by gel
electrophoresis, efficient amplification was observed for both
Puc19 and for .beta.-globin (all template sizes). These results
demonstrate that compositions in which a 3' exo+ DNA polymerase
(Tne) is present in higher quantity than a 3' exo- DNA polymerase
(Tfl) efficiently amplify nucleic acid molecules derived from
plasmid and genomic DNA templates.
EXAMPLE 7
Amplification of Large Nucleic Acid Molecules Using DNA Polymerase
Mixtures
[0101] Having demonstrated that compositions comprising mixtures of
3' exo+ and 3' exo- DNA polymerases efficiently amplify
plasmid-sized and small genomic nucleic acid molecules, such
compositions were examined for their ability to amplify larger
nucleic acid molecules. For these studies, mixtures of Taq (3'
exo+) DNA polymerase and Tma (3' exo-) DNA polymerase (ULTma.TM.;
LTI, Rockville, Md.) were prepared. Two sets of mixtures were
prepared: one set contained 1 unit of Taq and varying amounts of
ULTma (0.3 unit, 0.6 unit, 0.8 unit or 1 unit), and the other set
contained no Taq and only varying amounts of ULTma (0.3 unit, 0.6
unit, 0.8 unit or 1 unit). These compositions were used to amplify
80 mg of human genomic DNA using primers specific for a 7.5
kilobase region of the human .beta.-globin gene. PCR conditions
were 1 min at 94.degree. C., followed by 35 cycles of 94.degree. C.
for 30 seconds/60.degree. C. for 30 seconds/68.degree. C. for 5
minutes.
[0102] Upon analysis of the amplification products by gel
electrophoresis, the 7.5 kilobase .beta.-globin fragment was found
to be efficiently amplified by all compositions comprising both Taq
and Tma DNA polymerase. Compositions comprising only Tma DNA
polymerase, however, were unable to amplify this large fragment. In
control experiments, compositions comprising only Taq DNA
polymerase were also unable to amplify this large fragment. These
results demonstrate that compositions comprising a mixture of 3'
exo+ and 3' exo- DNA polymerases are useful in efficiently
amplifying large nucleic acid molecules, particularly in amplifying
nucleic acid molecules larger than about 7 kilobases in size.
EXAMPLE 8
Amplification of Large Nucleic Acid Molecules Using Various Ratios
of Taq and Tma DNA Polymerases
[0103] Having demonstrated that compositions comprising mixtures of
3' exo+ (Taq) and 3' exo- (Tma) DNA polymerases efficiently amplify
large nucleic acid molecules, compositions comprising these enzymes
in various ratios were made and tested for their abilities to
amplify the 7.5 kilobase .beta.-globin gene fragment from Example
7. Two sets of mixtures were prepared: one set contained 1 unit of
ULTma DNA polymerase and different amounts (0.25, 0.5, 0.6, 0.7,
0.8, 0.9 or 1.0 unit) of Taq DNA polymerase, and the other set
contained 1 unit of Taq DNA polymerase and different amounts (1.0,
1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 3.0, 4.0 or 6.0 units) of ULTma DNA
polymerase. Amplification templates, primers and cycling conditions
were as described for Example 7.
[0104] Upon analysis of the amplification products by gel
electrophoresis, efficient amplification of the 7.5 kilobase
.beta.-globin gene was observed for the first set of mixtures only
in those compositions containing 0.5 to 1.0 unit of Taq DNA
polymerase and 1.0 unit of ULTma. Compositions in this set
containing less than 0.5 unit of Taq DNA polymerase did not amplify
this fragment. However, all enzyme mixtures in the other set, i.e.,
compositions comprising 1.0 unit of Taq DNA polymerase and 1.0 to
6.0 units of ULTma DNA polymerase, demonstrated efficient
amplification of the 7.5 kilobase fragment. In separate
experiments, a 13.5 kilobase fragment of the .beta.-globin gene was
efficiently amplified using mixtures containing a 1:1 or a 1:2
ratio of Taq to ULTma. Together, these results indicate that
compositions in which Tma DNA polymerase is present in equal or
higher quantity than Taq DNA polymerase efficiently amplify large
nucleic acid molecules, particularly those that are larger than
about 7-13 kilobases in size.
EXAMPLE 9
Use of Taq and Tne DNA Polymerase Mixtures for Long PCR
[0105] To determine if other DNA polymerases could be used in
compositions also comprising Taq DNA polymerase in amplification of
large nucleic acid molecules, various mixtures of Taq and Tne DNA
polymerases were made. For these experiments, a Tne DNA polymerase
deletion mutant (5' exo-; 3' exo+) was mixed in amounts ranging
from 0.05 to 2.0 units with 1 unit of Taq DNA polymerase and used
to amplify the 7.5 kilobase .beta.-globin fragment under conditions
described for Example 7.
[0106] Upon analysis of the amplification products by gel
electrophoresis, all of the combinations of Tne and Taq DNA
polymerases were found to efficiently amplify the 7.5 kilobase DNA
fragment. These results indicate that compositions comprising
combinations of Taq and Tne DNA polymerases are useful in
amplifying large nucleic acid molecules, particularly those larger
than about 7 kilobases in size.
EXAMPLE 10
Preparation and Use of Compositions Comprising Anti-Taq
Antibodies
[0107] To examine the stability of ready-to-use PCR reagents
containing anti-Taq antibodies, Taq DNA polymerase was reacted with
monoclonal antibody TP4-3 at ratios of 5:1, 2:1, 1:1 and 0:1 of
antibody to Taq DNA polymerase. Binding of the antibody to Taq DNA
polymerase inhibited polymerase activity of Taq almost completely
at 5:1 and 2:1 ratios. The 1:1 ratio of antibody to Taq resulted in
partial inhibition of polymerase activity ranging from 54% to 83%
of the control Taq DNA polymerase with no antibody.
[0108] The ready-to-use reaction mixtures were stored at 4.degree.
C. or -20.degree. C. for determination of stability of Taq DNA
polymerase as well as stability of anti-Taq antibodies. Long-term
storage of these mixtures showed no reduction in activity of Taq
DNA polymerase or anti-Taq antibody after 17 months at 4.degree. C.
or -20.degree. C. (Table 6).
6TABLE 6 Stability of Ready-to-Use PCR Reagents Ready-to-Use
Reagents Ready-to-Use Reagents Control Fresh Mixtures (4.degree. C.
Storage) (-20.degree. C. Storage) Antibody: Taq Antibody
Activity.sup.1 Antibody Activity Antibody Activity Ratio A B C D 14
months 17 months 14 months 17 months 5:1 97% 98% 97% 97.9% 94.8%
97.5% 94.8% 98.5% 2:1 N.D..sup.2 96.9% 96.4% N.D. N.D. N.D. 94.8%
98% 1:1 54% 79% 82.6% 68% 71% 65% 73% 67% 0:1 (control) 0% 0% 0% 0%
0% 0% 0% 0% .sup.1Antibody activity is expressed as % inhibition of
DNA polymerization activity compared to no antibody (0:1 ratio)
control. .sup.2N.D. = not determined.
[0109] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
[0110] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1
1
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