U.S. patent application number 14/108863 was filed with the patent office on 2014-06-05 for thermostable chimeric nucleic acid polymerases and uses thereof.
This patent application is currently assigned to QIAGEN GMBH. The applicant listed for this patent is QIAGEN GMBH. Invention is credited to Jie Kang, Dirk Loffert, Andreas Missel.
Application Number | 20140154748 14/108863 |
Document ID | / |
Family ID | 24013413 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140154748 |
Kind Code |
A1 |
Loffert; Dirk ; et
al. |
June 5, 2014 |
THERMOSTABLE CHIMERIC NUCLEIC ACID POLYMERASES AND USES THEREOF
Abstract
Novel thermostable chimeric nucleic acid polymerases and methods
for their generation and use are disclosed. It is shown that these
chimeric nucleic acid polymerases, such as DNA polymerases, can be
constructed using enzymatically active domains, isolated from
different proteins or chemically synthesized. It is demonstrated
that chimeric nucleic acid polymerases of the present invention
possess the chemical and physical properties of their component
domains (e.g., exonuclease activity, thermostability) and that the
chimeric polymerases are thermostable.
Inventors: |
Loffert; Dirk; (Dusseldorf,
DE) ; Missel; Andreas; (Dusseldorf, DE) ;
Kang; Jie; (Mettmann, DE) |
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Applicant: |
Name |
City |
State |
Country |
Type |
QIAGEN GMBH |
Hilden |
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DE |
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Assignee: |
QIAGEN GMBH
Hilden
DE
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Family ID: |
24013413 |
Appl. No.: |
14/108863 |
Filed: |
December 17, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10216682 |
Aug 8, 2002 |
8637288 |
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14108863 |
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PCT/EP01/01790 |
Feb 16, 2001 |
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10216682 |
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09506153 |
Feb 17, 2000 |
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PCT/EP01/01790 |
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Current U.S.
Class: |
435/91.2 |
Current CPC
Class: |
C12N 9/1252 20130101;
C12P 19/34 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/91.2 |
International
Class: |
C12P 19/34 20060101
C12P019/34 |
Claims
1. A method of synthesizing a recombinant nucleic acid encoding a
thermostable chimeric nucleic acid polymerase having two,
non-naturally associated, enzymatically active domains, the method
comprising: (a) isolating a first nucleic acid encoding a first
enzymatically active domain; (b) isolating a second nucleic acid
encoding a second enzymatically active domain, wherein said second
enzymatically active domain is non-naturally associated with said
first enzymatically active domain; and (c) combining said first
nucleic acid and said second nucleic acid to form said recombinant
nucleic acid encoding said thermostable chimeric nucleic acid
polymerase.
2. The method of claim 1, wherein said first enzymatically active
domain is a 3'-5' exonuclease domain.
3. The method of claim 1, wherein said second enzymatically active
domain is a 5'-3' polymerase domain.
4. The method of claim 1, wherein said isolating in (a) and (b)
comprises amplifying the first nucleic acid and the second nucleic
acid by polymerase chain reaction (PCR) with a PCR primer
comprising a first nucleotide sequence complementary to a terminal
region of a 3'-5' exonuclease domain of said first nucleic acid and
a second nucleotide sequence complementary to a terminal region of
a 5'-3' polymerase domain of said second nucleic acid.
5. The method of claim 1, wherein said combining comprises
hybridizing said first nucleic acid to said second nucleic acid to
form a composite polynucleotide template, and amplifying said
composite polynucleotide template to form said recombinant nucleic
acid encoding said thermostable chimeric nucleic acid
polymerase.
6. The method of claim 2, wherein said 3'-5' exonuclease domain
comprises a 3'-5' exonuclease domain of Pho DNA polymerase.
7. The method of claim 6, wherein said 3'-5' exonuclease domain
comprises amino acid residues 1 to 396 of Pho DNA polymerase (SEQ
ID NO:3).
8. The method of claim 2, wherein said 3'-5' exonuclease domain
comprises a 3'-5' exonuclease domain of Pwo DNA polymerase.
9. The method of claim 8, wherein said 3'-5' exonuclease domain
comprises amino acid residues 1 to 396 of Pwo DNA polymerase (SEQ
ID NO:4).
10. The method of claim 8, wherein said 3'-5' exonuclease domain
comprises amino acid residues 1 to 421 of Pwo DNA polymerase (SEQ
ID NO:5).
11. The method of claim 2, wherein said 3'-5' exonuclease domain
comprises a 3'-5' exonuclease domain of Sso DNA polymerase.
12. The method of claim 11, wherein said 3'-5' exonuclease domain
comprises amino acid residues 1 to 508 of Sso DNA polymerase (SEQ
ID NO:6).
13. The method of claim 2, wherein said 3'-5' exonuclease domain
comprises a 3'-5' exonuclease domain of Tpac DNA polymerase.
14. The method of claim 13, wherein said 3'-5' exonuclease domain
comprises amino acid residues 1 to 395 of Tpac DNA polymerase (SEQ
ID NO:16).
15. The method of claim 3, wherein said 5'-3' polymerase domain is
a 5'-3' polymerase domain of Taq DNA polymerase.
16. The method of claim 3, wherein said 5'-3' polymerase domain is
a 5'-3' polymerase domain of Tth DNA polymerase.
17. The method of claim 15, wherein said 5'-3' polymerase domain
comprises amino acid residues 281 to 832 of Taq DNA polymerase (SEQ
ID NO:1).
18. The method of claim 15, wherein said 5'-3' polymerase domain
comprises amino acid residues 271 to 832 of Taq DNA polymerase (SEQ
ID NO:7).
19. The method of claim 16, wherein said 5'-3' polymerase domain
comprises amino acid residues 273 to 834 of Tth DNA polymerase (SEQ
ID NO:2).
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 10/216,682, filed Aug. 8, 2002 (currently pending), which is a
continuation of International Application No. PCT/EP01/01790, filed
Feb. 16, 2001 (now abandoned), which is a continuation-in-part of
U.S. Ser. No. 09/506,153, filed Feb. 17, 2000 (now abandoned), the
disclosures of which are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing which has been
submitted in ASCII format via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created Dec. 16, 2013,
is named 0051.sub.--0003US2--Sequence_Listing.txt and is 84489
bytes in size.
FIELD OF THE INVENTION
[0003] The present invention is in the field of molecular biology.
The present invention is directed to novel thermostable chimeric
enzymes useful for the generation of nucleic acids, methods for
making thermostable chimeric nucleic acid polymerases, and methods
useful for polymerizing nucleic acids using a thermostable chimeric
nucleic acid polymerase. Specifically, the invention is directed to
chimeric thermostable DNA polymerases and their uses.
BACKGROUND OF THE INVENTION
[0004] Nucleic acid polymerases are an important class of compounds
that enzymatically link (polymerize) nucleotides to form larger
polynucleotide chains (e.g., DNA or RNA strands). Nucleic acid
polymerases typically utilize a template polynucleotide (in either
a single-strand or double-strand form) for nucleic acid synthesis,
as in conventional nucleic acid replication, transcription, or
reverse transcription. Other nucleic acid polymerases, e.g.,
terminal transferase (TdT), are capable of de novo polymerization,
that is, template independent nucleic acid synthesis.
[0005] All known nucleic acid polymerases possess an enzymatic
domain that catalyzes the formation of a phosphodiester bond
between two nucleotides, utilizing the 5' carbon triphosphate of
one nucleotide and the 3' carbon hydroxyl group of another
nucleotide. Nucleic acid polymerases synthesize nascent
polynucleotides by linking the 5' phosphate of one nucleotide to
the 3' OH group of the growing polynucleotide strand. This process
is known and commonly referred to by persons skilled in the art as
5'-3' polymerization.
[0006] In addition, nucleic acid polymerases possess a wide range
of ancillary chemical properties useful for nucleic acid synthesis.
These properties include, but are not limited to: [0007] product
and/or template specificity (e.g., RNA or DNA); [0008]
single-strand or double-strand template specificity; [0009]
processivity--a measure of the ability of a nucleic acid polymerase
to generate a nascent polynucleotide from a template polynucleotide
without dissociating from the template; [0010] extension rate--a
measure of the rate at which nucleotides are added to a growing
polynucleotide strand; [0011] fidelity--a measure of the accuracy
(or conversely the error rate) with which a nucleic acid polymerase
synthesizes a polynucleotide complementary to a template
polynucleotide; [0012] nick translation--the ability of a nucleic
acid polymerase to degrade the preceding nucleotide strand of a
double strand molecule simultaneous to polymerizing a nascent
strand; [0013] proofreading--the ability of a nucleic acid
polymerase to remove an incorrectly linked nucleotide from a
polynucleotide before further polymerization occurs; and [0014]
thermostability--the ability of a nucleic acid polymerase to retain
activity after exposure to elevated temperatures.
[0015] Many of these properties are the result of one or more
discrete functional domains within a polymerase holoenzyme. Three
extensively studied enzymatically active domains of nucleic acid
polymerase include: a 5'-3' polymerase domain, responsible for
polynucleotide synthesis; a 5'-3' exonuclease domain, responsible
for polynucleotide digestion of the 5' end of a polynucleotide,
useful for nick translation; and a 3'-5' exonuclease domain,
responsible for polynucleotide digestion of the 3' end of a
polynucleotide, allowing for proofreading, and thus improving the
fidelity of the polymerase. Some studies indicate that selection,
incorporation, and extension of the correct nucleotide, versus an
incorrect nucleotide, is a variable property of the 5'-3'
polymerase domain, thus affecting polymerase fidelity in concert
with proofreading activity (Mendelman et al., 1990; Petruska et
al., 1988).
[0016] DNA polymerases can be categorized into six families based
on amino acid homology. These families consist of pol I, pol
.alpha., SONDZEICHEN pol .beta., SONDZEICHEN DNA-dependent RNA
polymerase, (Joyce and Steitz, 1994). Table 1 summarizes the
enzymatic features of a few representative DNA polymerases.
TABLE-US-00001 TABLE 1 DNA polymerase enzymatic activity (N
terminus --------- C terminus) 5'-3' 3'-5' 5'-3' de novo DNA exonu-
exonu- poly- Thermo- poly- polymerase clease clease merase
stability merase E. coli pol I (+) (+) (+) (-) (-) Klenow fragment
(-) (+) (+) (-) (-) E. coli pol II (-) (+) (+) (-) (-) E. coli pol
III (+) (+) (+) (-) (-) T4 pol (-) (+) (+) (-) (-) T7 pol (-) (+)
(+) (-) (-) M-MuLV RT (-) (-) (+) (-) (-) TdT (-) (-) (+) (-) (+)
Taq pol (+) (-) (+) (+) (-) Stoffel fragment (-) (-) (+) (+) (-)
Tbr pol (+) (-) (+) (+) (-) Tli pol (-) (+) (+) (+) (-) Tma pol (-)
(+) (+) (+) (-) Tth pol (+) (-) (+) (+) (-) Pfu pol (-) (+) (+) (+)
(-) Psp pol (-) (+) (+) (+) (-) Pwo pol (-) (+) (+) (+) (-)
[0017] Because of the diversity of properties and characteristics
potentially exhibited by nucleic acid polymerases generally,
practitioners in the art have sought to modify, to alter, or to
recombine various features of nucleic acid polymerases in an effort
to develop new and useful variants of the enzyme. Initially,
polymerase truncations and deletions were developed. The Klenow
fragment, for example, was the first nucleic acid polymerase
variant developed. Klenow fragments exist as a large C-terminal
truncation of DNA polymerase I (pol I), possessing an enzymatically
active 3'-5' exonuclease and 5'-3' polymerase domains, but lacking
altogether the 5'-3' exonuclease domain of native pol I (Klenow and
Henningsen, 1970; Jacobson et al., 1974; and Joyce and Grindley,
1983).
[0018] Since the advent of the polymerase chain reaction (PCR)
methodology (including derivative methodologies such as reverse
transcription PCR, or RT-PCR), resilient nucleic acid polymerases,
capable of withstanding temperature spikes as high as 95.degree. C.
without a subsequent significant loss in enzymatic activity (i.e.,
thermostable) have become vital tools in modern molecular biology.
The use of thermostable enzymes to amplify nucleic acid sequences
is described in U.S. Pat. Nos. 4,683,195 and 4,683,202. A
thermostable DNA polymerase from Thermus aquaticus (Taq) has been
cloned, expressed and purified from recombinant cells (Lawyer et
al., 1989; U.S. Pat. Nos. 4,889,818 and 5,079,352. PCR is also
described in many U.S. patents, including U.S. Pat. Nos. 4,965,188,
4,683,195, 4,683,202, 4,800,159, 4,965,188, 4,889,818, 5,075,216,
5,079,352, 5,104,792, 5,023,171, 5,091,310, and 5,066,584.).
[0019] As depicted in Table I, Taq DNA polymerase possesses
enzymatically active 5'-3' polymerase and 5'-3' exonuclease
domains, but it exhibits only background levels of 3'-5'
exonuclease activity (Lawyer et al., 1989; Bernard et al., 1989;
Longley et al., 1990). Crystallographic data revealed that Taq
polymerase contains a 3'-5' exonuclease domain (Eom et al., 1996);
comparisons of the crystal structure of the Klenow fragment from
Bacillus DNA polymerase I, Taq DNA polymerase, and E. coli DNA
polymerase I have shown, however, that critical residues required
to carry out a 3'-5' exonuclease activity are missing in the 3'-5'
exonuclease domain of Taq DNA polymerase (Kiefer et al., 1997).
Park et al. (1997), have determined that Taq DNA polymerase
possesses none of three sequence motifs (Exo I, II, and III) within
the 3'-5' exonuclease domain and necessary for 3'-5' exonuclease
activity. Because Taq polymerase exhibits essentially no 3'-5'
exonuclease activity (i.e., proofreading capability), the error
rate of Taq DNA polymerase is high compared to other DNA
polymerases that possess an enzymatically active 3'-5' exonuclease
domain (Flaman et al., 1994). The Taq DNA polymerase structure thus
comprises a 5'-3' exonuclease domain occurring at the N-terminal
region of the polypeptide (residues 1-291), followed by an
enzymatically inactive 3'-5' exonuclease domain (residues 292-423),
and a C-terminal 5'-3' polymerase domain (Park et al., 1997).
[0020] Since Taq DNA polymerase does not possess an enzymatically
active 3'-5' exonuclease domain, providing a proofreading feature
to the polymerase, the use of Taq DNA polymerase becomes less
desirable for most nucleic acid amplification applications, e.g.,
for PCR sequencing protocols or amplification for protein
expression, which require complete identity of replication products
to the template nucleic acid. Depending on the phase of PCR during
which an error becomes incorporated into the PCR product (e.g., in
an early replication cycle), the entire population of amplified DNA
could contain one or more sequence errors, giving rise to a
nonfunctional and/or mutant gene product. Nucleic acid polymerases
that possess an enzymatically active 3'-5' exonuclease domain
(i.e., proofreading activity), therefore, are especially preferred
for replication procedures requiring high fidelity.
[0021] Due to the scientific and commercial importance of PCR in
modern molecular biology, the reliance of PCR protocols on nucleic
acid polymerases of particular characteristics, and in view of the
enzymatic deficiencies of Taq polymerase, an enormous amount of
research and development has focussed on developing new and useful
thermostable DNA polymerase variants and/or assemblages.
[0022] One approach has been directed to the discovery and
isolation of new thermophilic nucleic acid polymerases, which may
possess a unique and/or improved collection of catalytic
properties. As a result, thermostable nucleic acid polymerases have
been isolated from a variety of biological sources, including, but
not limited to, species of the taxonomic genera, Thermus,
Thermococcus, Thermotoga, Pyrococcus, and Sulfolobus. These
polymerases possess a variety of chemical characteristics, as
illustrated in Table 1. Some of these naturally occurring
thermostable DNA polymerases possess enzymatically active 3'-5'
exonuclease domains, providing a natural proofreading capability
and, thus, exhibiting higher fidelity than Taq DNA polymerase.
Naturally occurring proofreading thermostable polymerases include:
Pfu polymerase (isolated from Pyrococcus furiosus), Pwo polymerase
(isolated from Pyrococcus woesei), Tli polymerase (isolated from
Thermococcus litoralis), and Psp polymerase (isolated from
Pyrococcus sp. GB-D). All of these naturally occurring thermostable
polymerases are commercially available (Tli polymerase and Psp
polymerase are marketed as Vent.RTM. and Deep Vent SONDZEICHEN.RTM.
DNA polymerase, respectively, by New England Biolabs, Beverly,
Mass.). These DNA polymerases show slower DNA extension rates and
an overall lower processivity when compared to Taq DNA polymerase,
however, thus rendering these naturally occurring thermostable DNA
polymerases less desirable for PCR, despite their higher
fidelity.
[0023] In an effort to compensate for the deficiencies of
individual thermostable polymerases, a second approach has been to
develop multiple enzyme assemblages, combining, for example, Taq
polymerase and a proofreading enzyme, such as Pfu polymerase or
Vent SONDZEICHEN.RTM. polymerase. These multiple-enzyme mixtures
exhibit higher PCR efficiency and reduced error rates when compared
to Taq polymerase alone (Barnes, 1994). Mixtures of multiple
thermostable enzymes are commercially available (e.g., the
Failsafe.TM. PCR system from Epicentre, Madison, Wis.). PCR
protocols utilizing multiple polymerase mixtures are still prone to
error, however, and require the practitioner to perform preliminary
experimental trials, to determine special optimized solution
conditions necessary for multiple-enzyme reaction mixtures.
[0024] A third approach has been to develop new and useful variants
of Taq polymerase through deletion/truncation techniques. The
Stoffel fragment, for example, is a 544 amino acid C-terminal
truncation of Taq DNA polymerase, possessing an enzymatically
active 5'-3' polymerase domain but lacking 3'-5' exonuclease and
5'-3' exonuclease activity. Other commercially available
thermostable polymerase deletions include Vent SONDZEICHEN.RTM.
(exo.sup.-) and Deep Vent SONDZEICHEN.RTM. (exo.sup.-) (New England
Biolabs, Beverly, Mass.). Deletion mutations serve only to remove
functional domains of a nucleic acid polymerase, however, and do
not add any novel features or enzymatic properties.
[0025] Polymerase mutagenesis is yet another approach that has been
attempted to develop new and useful nucleic acid polymerase
variants. Park et al. (1997) performed site-directed mutagenesis of
4 amino acids in the enzymatically inactive 3'-5' exonuclease
domain of Taq polymerase in an effort to activate the proofreading
ability of this domain. The resultant mutant exhibited an increase
of exonuclease activity over that of naturally occurring Taq
polymerase. The reported increase was a mere two-fold increase
above background exonuclease activity, however; an insignificant
rise in exonuclease activity that is unlikely to increase PCR
fidelity.
[0026] Bedford et al. (1997) developed a recombinant mesophilic DNA
pol I from E. coli. They succeeded to insert a thioredoxin binding
domain from T7 DNA polymerase into E. coli pol I. The inserted 76
amino acid binding domain improved polymerase binding to a template
polynucleotide, thus increasing the processivity of the recombinant
E. coli pol I but did not improve or provide any novel enzymatic
activity to the polymerase.
[0027] Recently Gelfand et al. (1999) combined fusion protein
technology with mutagenesis to eliminate or substantially reduce
5'-3' exonuclease activity and 3'-5' exonuclease activity in
recombinant polymerases. Once again, no improved or additional
enzymatic activity was provided by the fusion polymerase.
[0028] Frey et al. (1999) attempted to engineer chimeric
polymerases utilizing enzymatically active domains from Taq, Tne,
and E. coli DNA polymerases. Although they successfully substituted
the non-functional 3'-5' exonuclease domain of Taq DNA polymerase
with a functional 3'-5' exonuclease domain from another DNA
polymerase, their resultant chimeric polymerase lost significant,
if not all, enzymatic activity after only one minute at 80.degree.
C. or 95.degree. C. (i.e., they are not thermostable), and thus are
not useful for performing PCR protocols without the successive
addition of fresh polymerase for each cycle.
[0029] Despite these intense research efforts, there remains a need
in the art for thermostable nucleic acid polymerases that possess
improved or novel assemblages of enzymatically active domains.
Despite its enzymatic deficiencies, Taq DNA polymerase remains the
most widely used enzyme for processing in vitro amplification of
nucleic acids. In particular, there has been long felt need for a
nucleic acid polymerase possessing the 5'-3' polymerization
qualities of Taq polymerase, but which also possesses 3'-5'
exonuclease (proofreading) activity.
SUMMARY OF THE INVENTION
[0030] In response to the long felt need for new and useful nucleic
acid polymerases, a novel approach for producing thermostable
nucleic acid polymerases was invented. The present invention
represents the first thermostable chimeric nucleic acid polymerase,
useful for continuous PCR protocols, obtained by combining at least
two enzymatically active domains from different proteins by means
of recombinant DNA techniques.
[0031] The present invention is directed to novel thermostable
chimeric enzymes useful for the generation of nucleic acids,
methods for making thermostable chimeric nucleic acid polymerases,
and methods useful for polymerizing nucleic acids using a
thermostable chimeric nucleic acid polymerase. The thermostable
chimeric nucleic acid polymerase of the present invention comprises
at least two enzymatically active domains, which are non-naturally
associated. The recombinant association of the enzymatically active
domains results in a composite enzyme not found in nature. The
thermostable chimeric nucleic acid polymerase of the present
invention possesses new or improved catalytic properties compared
to nucleic acid polymerases known in the art.
[0032] The thermostable chimeric nucleic acid polymerase of the
present invention offers several advantages over previous
approaches to develop novel nucleic acid polymerases. The present
invention provides a single enzyme that possesses a suite of
chemical properties, the combination of which may not necessarily
exist in nature, but nonetheless is useful in molecular biology.
The chimeric nucleic acid polymerase of the present invention
eliminates the need to specifically develop multiple-enzyme
reaction mixtures, which are often difficult to optimize and
expensive to use, and the necessity to add successive amounts of
fresh enzyme during each cycle of a PCR program. The invention thus
facilitates the rapid, efficient, and accurate generation of
nucleic acid molecules, particularly in regard to PCR
protocols.
DEFINITIONS
[0033] As used herein, an "enzymatically active domain" refers to
any polypeptide, naturally occurring or synthetically produced,
capable of mediating, facilitating, or otherwise regulating a
chemical reaction, without, itself, being permanently modified,
altered, or destroyed. Binding sites (or domains), in which a
polypeptide does not catalyze a chemical reaction, but merely forms
noncovalent bonds with another molecule, are not enzymatically
active domains as defined herein. In addition, catalytically active
domains, in which the protein possessing the catalytic domain is
modified, altered, or destroyed, are not enzymatically active
domains as defined herein. Enzymatically active domains, therefore,
are distinguishable from other (nonenzymatic) catalytic domains
known in the art (e.g., detectable tags, signal peptides, alosteric
domains, etc.).
[0034] As defined herein, a 3'-5' exonuclease domain refers to any
polypeptide capable of enzymatically cleaving a nucleotide from the
3' end of a di- or polynucleotide, a 5'-3' exonuclease domain
refers to any polypeptide capable of enzymatically cleaving a
nucleotide from the 5' end of a di- or polynucleotide, and a 5'-3'
polymerase domain refers to any polypeptide capable of
enzymatically linking the 5' phosphate of one nucleotide to the 3'
OH group of another nucleotide.
[0035] Polypeptide domains that are "non-naturally associated",
refer to specific polypeptides that are not naturally produced
within a single polypeptide; that is, the polypeptide domains are
not naturally translated from a common nucleic acid transcript in a
naturally occurring organism. Non-naturally associated polypeptide
domains include domains isolated from functionally distinct
proteins, separately produced by an organism of one or more
species, or synthetically generated, as well as polypeptide domains
isolated from functionally similar proteins, but naturally produced
by organisms of different species, or synthetically generated. The
term "non-naturally associated polypeptide domains" refers to
domains that are associated or fused only through human
intervention; the term expressly excludes naturally occurring
enzymes or fragments thereof.
[0036] As used herein, the term "chimeric protein" encompasses all
proteins that contain two or more polypeptide domains that are
non-naturally associated (regardless of whether the domains are
naturally produced by organisms of the same species, different
species, or synthetically generated). A chimeric nucleic acid
polymerase of the present invention must necessarily possess two or
more non-naturally associated domains, as defined herein.
[0037] The term "thermostable" generally refers to the resilience
of a substance to relatively high temperature treatment. A
thermostable enzyme is an enzyme that retains its definitive
enzymatic activity despite exposure to relatively high temperature.
A thermostable nucleic acid polymerase, as generally understood by
practitioners in the art and as defined herein, refers to a
polymerase that is useful for PCR protocols; i.e., not requiring
successive or supplemental addition of enzyme after each high
temperature step of the PCR program cycle. The chimeric nucleic
acid polymerase of the present invention is thermostable, in that
it is useful for PCR protocols, because it does not require
successive or supplemental addition of polymerase after each high
temperature step of the PCR program cycle.
[0038] A preferred thermostable chimeric polymerase of the present
invention is one that allows a thermal polymerase chain reaction to
proceed with only an initial supply of polymerase at the start of
the PCR program. Preferably, a thermostable chimeric nucleic acid
polymerase retains some measurable enzymatic activity at its normal
operating temperature (typically about 72.degree. C.) after
exposure to 95.degree. C. for three minutes. More preferably, a
thermostable chimeric nucleic acid polymerase is able to withstand
one minute at 95.degree. C. without significant loss (>5% loss)
in enzymatic activity. In other words, a preferred thermostable
chimeric nucleic acid polymerase retains at least about 95% of its
polymerase activity at its normal operating temperature (typically
about 72.degree. C.) after one minute at 95.degree. C. Even more
preferably, a thermostable chimeric nucleic acid polymerase is able
to withstand three minutes at 95.degree. C. without significant
loss in enzymatic activity. A most preferred thermostable chimeric
nucleic acid polymerase is able to withstand ten minutes at
90.degree. C. and still retain at least about 50% of its enzymatic
activity at its normal operating temperature. In other words, the
polymerase displays a "half life" (the length of time it takes for
a substance to lose one half of its initial activity) of ten
minutes at 90.degree. C. Ideally, a thermostable chimeric nucleic
acid polymerase displays a half-life comparable to the half-life
measurement of naturally occurring thermostable nucleic acid
polymerases. For example a most desirable thermostable chimeric
nucleic acid polymerase displays a half-life at 90.degree. C.
comparable to that of Taq polymerase, approximately 90 minutes.
[0039] The present invention is directed generally to all
thermostable chimeric nucleic acid polymerases comprising at least
two non-naturally associated enzymatically active domains. As
defined herein, a nucleic acid polymerase is any enzyme that
catalyzes the formation of chemical bonds between (chemically
bonds) nucleotides to form polynucleotide chains, that is, any
enzyme that promotes nucleic acid polymerization. The thermostable
chimeric nucleic acid polymerases of the present invention include
all types of nucleic acid polymerases, without limitation to
product or template specificity, molecular requirements, or
chemical properties (e.g., RNA vs. DNA, single strand vs. double
strand, high fidelity, etc.).
[0040] One embodiment of the present invention is directed to a
thermostable chimeric DNA polymerase, preferably a chimeric DNA
polymerase wherein the enzymatically active domains are isolated
from naturally occurring proteins from two or more species, or any
mutants, variants, or derivatives thereof.
[0041] As used herein, mutant, variant, and derivative polypeptides
refer to all chemical permutations of a given polypeptide, which
may exist or be produced, that still retain the characteristic
molecular activity that is definitive of that polypeptide.
[0042] The thermostable chimeric nucleic acid polymerase of the
present invention is unexpected in view of the fact that
enzymatically active domains may be isolated from a wide variety of
sources, yet still retain their enzymatic activities (e.g.,
polymerase, exonuclease) and chemical properties (e.g.,
thermostability, processivity). Enzymatically active domains
isolated from organisms of different taxonomic kingdoms and from
completely different families of proteins may be fused to produce
an entirely novel, yet functional, nucleic acid polymerase. For
example, enzymatically active domains from a eubacterium polymerase
of e.g., Taq polymerase may be chimerically joined with
enzymatically active domains from an archaeon polymerase (e.g.,
Pwo, Sso, and Pho polymerases).
[0043] Retention of thermal stability in a fusion protein
engineered from different thermophilic proteins is highly
unexpected. Attempts to construct chimeric polymerases have failed
to produce thermostable chimeric polymerases (see Frey et al.,
1999). The underlying principles of thermal stability of proteins
derived from thermophilic organisms are not known. Even small
changes in the amino acid sequence of thermoresistant proteins
result in a significant decrease in thermal stability and an
associated reduction in enzymatic activity of the protein.
Maintenance of, or an increase in, thermal stability of
thermostable DNA polymerase has only been accomplished by
truncation of a DNA polymerase (e.g., Barnes, 1995). The present
invention represents the first chimeric nucleic acid polymerase,
containing enzymatically active domains from different thermostable
proteins, that possess thermostable properties.
[0044] In a preferred embodiment, at least one of the enzymatically
active domains of the chimeric nucleic acid polymerase is isolated
from a DNA polymerase produced by a thermophilic organism,
preferably an organism of a genus selected from the group of genera
consisting of: Thermus, Thermococcus, Thermotoga, Pyrococcus,
Pyrodictium, Bacillus, Sulfolobus, and Methanobacterium. Most
preferably, at least one of the enzymatically active domains of the
chimeric nucleic acid polymerase is isolated from a DNA polymerase
selected from the group consisting of: Thermoplasma acidophilum
(Tac) polymerase; Thermus aquaticus (Taq) polymerase; Thermococcus
barossii (Tba) polymerase; Thermus brockianus (Tbr) polymerase; Tfi
polymerase; Thermus flavus (Tfl) polymerase; Thermococcus litoralis
(Tli) polymerase; Thermus ruber (Tru) polymerase; Thermus
thermophilus (Tth) polymerase; Pyrodictium abyssi (Pab) polymerase;
Pyrococcus furiosus (Pfu) polymerase; Pyrococcus hellenicus (Phe)
polymerase; Pyrococcus horikoshii (Pho) polymerase; Pyrococcus
kodakarensis (Pko) polymerase; Pyrococcus sp. strain KOD1 (KOD)
polymerase; Pyrococcus sp. strain ES4 (ES4) polymerase; Pyrodictium
occultum (Poc) polymerase; Pyrococcus sp. GB-D (Psp) polymerase;
Pyrococcus woesei (Pwo) polymerase; Thermotoga maritima (Tma)
polymerase; Thermotoga neapolitana (Tne) polymerase; Bacillus
sterothermophilus (Bst) polymerase; Sulfolobus acidocaldarius (Sac)
polymerase; Sulfolobus solfataricus (Sso) polymerase;
Methanobacterium thermoautotrophicum (Mth) polymerase; and mutants,
variants, and derivatives thereof.
[0045] In another embodiment of the invention, the enzymatically
active domains are selected from the group consisting of: 5'-3'
exonuclease domain, 3'-5' exonuclease domain, and 5'-3' polymerase
domain. Preferably the enzymatically active domains are naturally
occurring domains, isolated from two or more species, most
preferably the enzymatically active domains are isolated from
naturally occurring thermostable proteins, mutants, variants, or
derivatives thereof.
[0046] Another aspect of the present invention relates to an
isolated polynucleotide encoding a thermostable chimeric nucleic
acid polymerase comprising at least two non-naturally associated
enzymatically active domains. Preferably the enzymatically active
domains are isolated from different species.
[0047] A related aspect of the invention is directed to a method
for synthesizing a recombinant nucleic acid that encodes a
thermostable chimeric nucleic acid polymerase comprising at least
two non-naturally associated enzymatically active domains.
[0048] A further aspect of the invention relates to a vector
comprising a polynucleotide that encodes a thermostable chimeric
nucleic acid polymerase having at least two non-naturally
associated enzymatically active domains. Preferred vectors are
expression vectors, which will be suitable for production of the
encoded chimeric nucleic acid polymerase in transformed host
cells.
[0049] Another aspect of the invention includes a recombinant host
cell transformed with a vector comprising a polynucleotide that
encodes a thermostable chimeric nucleic acid polymerase possessing
at least two non-naturally associated enzymatically active
domains.
[0050] A related aspect of the invention is directed to a method
for producing a thermostable chimeric nucleic acid polymerase
comprising at least two non-naturally associated enzymatically
active domains.
[0051] Another aspect of the invention is directed to a process of
nucleic acid polymerization, which necessarily utilizes a
thermostable chimeric nucleic acid polymerase having at least two
non-naturally associated enzymatically active domains.
[0052] A related aspect of the invention is directed to a kit
useful for polymerization of nucleic acid, comprising a
thermostable chimeric nucleic acid polymerase having at least two
non-naturally associated enzymatically active domains. Preferably,
the kit further comprises at least one reagent suitable for nucleic
acid polymerization. Most preferably, the kit further comprises at
least one reagent selected from the group consisting of one or more
additional enzymes, one or more oligonucleotide primers, a nucleic
acid template, any one or more nucleotide bases, an appropriate
buffering agent, a salt, or other additives useful in nucleic acid
polymerization.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1 is a photograph of an ethidium bromide
(EtdBr)-stained agarose gel, which depicts the polymerase activity
of thermostable chimeric DNA polymerases using a primer extension
reaction. Lane 1 shows a nucleic acid ladder, used as a gel
reference marker. Lanes 2, 6 and 10 show negative controls (without
addition of polymerase). Lane 3, 4, 5 show the activity of 0.05,
0.03 and 0.01 units Taq DNA polymerase, respectively. Lanes 7-9
illustrate polymerase activity of undiluted cleared lysate, a 1:1,
and 1:5 diluted cleared lysate, of a Pho/Taq chimeric polymerase,
respectively.
[0054] FIG. 2 is a photograph of an ethidium bromide
(EtdBr)-stained agarose gel, which depicts the thermostability of a
thermostable chimeric DNA polymerase compared to Taq DNA
polymerase, using a primer extension reaction. DNA polymerases were
incubated for various time spans at 90.degree. C. and assayed for
remaining polymerase activity. Lanes 1 and 11 show a nucleic acid
ladder, used as a gel reference marker. Lanes 2, 10, 12, and 20
represent negative control reactions (without addition of
polymerase). Lanes 3-9 and lanes 13-19 illustrate DNA polymerase
activity after incubation of Taq DNA polymerase and a Pho/Taq
chimeric DNA polymerase at 90.degree. C. for 0, 10, 15, 30, 60, 90,
and 120 min, respectively.
[0055] FIG. 3 is a photograph of an ethidium bromide
(EtdBr)-stained agarose gel, which depicts 3'-5' exonuclease
activity of three different thermostable DNA polymerases. (A)
illustrates PCR product using a wild type primer combination. (B)
illustrates PCR product using a mutant primer pair. Lane 1 is a
nucleic acid ladder, used as a gel reference marker. The PCR
amplification product of Taq DNA polymerase is shown in lanes 2-5;
Pfu DNA polymerase I PCR product is shown in lanes 6-9; and a
Pho/Taq thermostable chimeric DNA polymerase PCR product is shown
in lanes 10-13. Duplicate side-by-side reactions are shown
representing undigested (the first and third lane for each enzyme
used), and digested (the second and fourth lane for each enzyme
used) PCR product.
[0056] FIG. 4 is a photograph of an ethidium bromide
(EtdBr)-stained agarose gel, which illustrates the combined effect
of primer extension efficiency and polymerase processivity on PCR
efficiency of three different thermostable DNA polymerases. The
photograph illustrates PCR products obtained in duplicate reactions
using different primer extension times. (A) indicates PCR products
obtained with Taq DNA polymerase. (B) illustrates PCR products
obtained with a Pho/Taq thermostable chimeric DNA polymerase. (C)
shows PCR products generated with Pfu DNA polymerase I. Lane 1 is a
nucleic acid ladder, used as a gel reference marker. Lanes 2-3 show
PCR products amplified after primer extension for 1 min. Lanes 4-5
show PCR products amplified after primer extension for 30 sec.
Lanes 6-7 show PCR products amplified after primer extension for 10
sec. Lanes 8-9 show PCR products amplified after primer extension
for 5 sec.
DETAILED DESCRIPTION OF THE INVENTION
[0057] Genetic engineering techniques were successfully employed to
generate the first thermostable chimeric nucleic acid polymerase,
containing enzymatically active domains, not naturally found within
a single protein. The chimeric nucleic acid polymerase and methods
described herein encompass all thermostable nucleic acid
polymerases, without limitation to product or template specificity,
molecular requirements, or chemical properties. For example,
chimeric nucleic acid polymerases of the present invention include
single or double strand DNA polymerases, RNA polymerases, and
reverse transcriptases. Thermostable chimeric nucleic acid
polymerases of the present invention may possess any number and/or
combination of properties and features including, but not limited
to, template dependence or independence, high processivity, high
fidelity, proofreading, nick translation, and high extension rates.
Persons skilled in the art will understand and appreciate that
these features are due, in large part, to the presence and
characteristics of discrete polypeptide domains within the
holoenzyme. Essential to the chimeric nucleic acid polymerase of
the present invention is that it possess at least two enzymatically
active domains that are not naturally associated, and the chimeric
nucleic acid is thermostable.
[0058] Enzymatically active domains may be isolated from any
natural polypeptide, or may be synthetically produced. Natural
polypeptides include any polypeptide found in nature, and from any
organism of any taxonomic group. Enzymatically active domains
useful in the present invention also include variant, mutant, or
derivative forms of domains found in nature. Enzymatically active
domains further include domains that may not be found in nature,
e.g., polypeptides randomly generated or engineered in the
laboratory or selected from a non-naturally generated library of
polypeptides. For the purposes of this invention, enzymatically
active domains need only necessarily possess an enzymatic activity
that is functional within the chimeric nucleic acid polymerase of
the invention. The thermostable chimeric nucleic acid polymerases
of the present invention specifically contemplates incorporation
into a nucleic acid polymerase, enzymatically active domains that
are absent, inactive, or weakly active in the naturally occurring
protein.
[0059] Persons skilled in the art will know and appreciate that a
wide variety of enzymatic domains exist that perform the same or
similar enzymatic functions. For example, DNA polymerases possess
3'-5' exonuclease domains of a wide range of enzymatic
functionality; from little or no 3'-5' exonuclease activity (as
seen in Taq polymerase), to fully functional 3'-5' exonuclease
activity (as seen in E. coli pol I), to thermostable 3'-5'
exonuclease activity (as seen in Pwo polymerase). It is understood
by practitioners in the art that enzymatically active domains of
individual polymerases are considered separate and distinct
enzymatically active domains, as defined herein. Thus, the
incorporation of an enzymatically active domain from one polymerase
into a second polymerase produces, by definition, a chimeric
polymerase, regardless of whether the second polymerase naturally
possesses its own enzymatically active domain of similar
functionality.
[0060] Preferably, genetic engineering techniques may be used to
generate novel thermostable DNA polymerases possessing either 5'-3'
polymerase activity and 3'-5' exonuclease activity; or 5'-3'
polymerase activity, 3'-5' exonuclease activity and 5'-3'
exonuclease activity derived from different thermostable DNA
polymerases, e.g. Taq polymerase, Pho polymerase, Pwo polymerase,
and Sso polymerase.
[0061] Preferred thermostable chimeric nucleic acid polymerases of
the present invention include a 5'-3' polymerase domain of Taq
polymerase. For example, the Stoffel fragment is a 544 residue
N-terminal deletion of Taq polymerase possessing an enzymatically
active 5'-3' polymerase domain and an enzymatically inactive 3'-5'
exonuclease domain. Generally, a Taq 5'-3' polymerase domain is at
least about 544 residues in length, and includes any mutant,
variant, or derivative of the Stoffel fragment of Taq polymerase,
as defined herein. A 552 amino acid polypeptide, residue numbers
281-832 of Taq polymerase (SEQ ID NO:1), is an especially preferred
enzymatically active Taq 5'-3' polymerase domain useful in the
present invention.
[0062] Alternatively, the thermostable chimeric nucleic acid
polymerases of the present invention may include a 5'-3' polymerase
domain of Tth polymerase. Tth polymerase is capable of reverse
transcription. Thermostable chimeric nucleic acid polymerases,
which include the Tth 5'-3' polymerase domain, therefore, may be
used for reverse transcription reactions (e.g., RT-PCR).
Preferably, the 5'-3' polymerase domain of Tth polymerase is about
562 residues in length, including residue numbers 273-834 of Tth
polymerase (SEQ ID NO:2), and includes any mutant, variant, or
derivative thereof.
[0063] Preferred thermostable chimeric nucleic acid polymerases of
the present invention also include an enzymatically active 3'-5'
exonuclease domain of a thermostable polymerase. Preferred 3'-5'
exonuclease domains include the enzymatically active 3'-5'
exonuclease domains of Pho polymerase, Pwo polymerase, and Sso
polymerase. Most preferred are residues 1-396 of Pho polymerase
(SEQ ID NO:3), residues 1-396 of Pwo polymerase (SEQ ID NO:4),
residues 1-421 of Pwo polymerase (SEQ ID NO:5), residues 1-508 of
Sso polymerase (SEQ ID NO:6), and any mutants, variants, or
derivatives of any one of these 3'-5' exonuclease domains, as
defined herein.
[0064] A process for synthesizing a recombinant nucleic acid
encoding a thermostable chimeric nucleic acid polymerase of the
invention necessarily comprises isolating at least two nucleic acid
fragments each encoding at least one enzymatically active domain,
which is not naturally associated with the other enzymatically
active domain (i.e., derived from separate polypeptides), and
genetically combining the nucleic acids of the enzymatically active
domains to form a chimeric nucleic acid.
[0065] For production of thermostable chimeric nucleic acid
polymerases according to the invention, the nucleic acid encoding a
chimeric nucleic acid polymerase may be stably inserted into a
genetic vector, preferably the nucleic acid is operably inserted
into an expression vector, and most preferably the vector construct
is capable of replication within a host organism, such that the
nucleic acid encoding a thermostable chimeric nucleic acid
polymerase is capable of being transcribed and translated into a
polypeptide. A preferred mode of making the chimeric nucleic acid
polymerase of the present invention includes culturing a host cell
containing a nucleic acid encoding a thermostable chimeric nucleic
acid polymerase under conditions suitable for expression of the
chimeric nucleic acid polymerase by the host cell, and isolating
the chimeric nucleic acid polymerase expressed from said cell
culture.
[0066] Methods for generating recombinant nucleic acids, vector
construction, host cell transformation, and polypeptide expression
systems useful in the practice of this invention can involve a wide
variety of modern genetic engineering techniques, tools, and
biological sources that are well known in the art and routinely
practiced by those skilled in the art. Exemplary techniques and
methods are described in detail herein by way of preferred example,
but are not limiting to the practice of the invention. The present
invention incorporates by reference in their entirety techniques
and supplies well known in the field of molecular biology,
including, but not limited to, techniques and supplies described in
the following publications: [0067] Ausubel, F. M. et al. eds.,
Short Protocols In Molecular Biology (4th Ed. 1999) John Wiley
& Sons, NY. (ISBN 0-471-32938-X). [0068] Freshney, R. I.
Culture of Animal Cells (1987) Alan R. Liss, Inc. [0069] Old, R. W.
& S. B. Primrose, Principles of Gene Manipulation: An
Introduction To Genetic Engineering (3d Ed. 1985) Blackwell
Scientific Publications, Boston. Studies in Microbiology; V.2:409
pp. (ISBN 0-632-01318-4). [0070] Sambrook, J. et al. eds.,
Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring
Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6).
[0071] Winnacker, E. L. From Genes To Clones: Introduction To Gene
Technology (1987) VCH Publishers, NY (translated by Horst
Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).
[0072] The thermostable chimeric nucleic acid polymerases described
herein are especially useful for generating a desired target
nucleic acid. Thermostable chimeric nucleic acid polymerases of the
invention, having at least two enzymatically active domains that
are not naturally associated may be utilized under conditions
sufficient to allow polymerization of a nascent nucleic acid.
Generally, this method includes any method of nucleic acid
generation, replication, amplification, transcription, or reverse
transcription known in the art that utilizes a conventional nucleic
acid polymerase, wherein the nucleic acid polymerase is substituted
or combined with a chimeric nucleic acid polymerase of the present
invention. Preferably the method of amplification is polymerase
chain reaction, utilizing a thermostable chimeric nucleic acid
polymerase. PCR is described herein as an exemplary protocol
capable of utilizing the compositions and methods of the present
invention without limitation. Persons skilled in the art will
understand that the present invention has utility in other
processes requiring the polymerization of nucleic acid (e.g.,
RT-PCR).
[0073] PCR is a technique well known in the art. PCR is used to
amplify nucleic acids by subjecting a reaction mixture to cycles
of: (i) nucleic acid denaturation, (ii) oligonucleotide primer
annealization, and (iii) nucleic acid polymerization. Preferred
reaction conditions for amplification comprise thermocycling, i.e.,
alternating the temperature of the reaction mixture to facilitate
each of the steps of the PCR cycle. PCR is typically extended
through multiple cycles of denaturation, annealization and
replication, augmented (optionally and preferably) with an initial
prolonged denaturation step and a final prolonged extension
(polymerization) step. To perform the repetitive steps of
thermocycling, it is preferable to employ an enzyme that is capable
of tolerating exposure to relatively high temperature without a
subsequent significant loss in enzyme activity; i.e., a
thermostable enzyme. The use of a thermostable enzyme for PCR
protocols permits the repetitive steps of increasing and decreasing
reaction temperatures without the need to supplement, or otherwise
add, enzyme after each successive high temperature step of the PCR
program cycle.
[0074] Also included in the invention is a kit that includes a
thermostable chimeric nucleic acid polymerase and one or more
additional reagents suitable for nucleic acid polymerization
reactions. Such components may include, but are not limited to: one
or more additional enzymes, one or more oligonucleotide primers, a
nucleic acid template, any one or more nucleotide bases, an
appropriate buffering agent, a salt, or other additives useful in
nucleic acid polymerization.
[0075] Additional enzymes of the kit include any enzyme that may be
used in combination with the thermostable chimeric nucleic acid
polymerase of the invention. For example, multiple-polymerase kits
are known in the art. Numerous polymerases are known and
commercially available to persons skilled in the art, and include
DNA polymerases, RNA polymerases, and reverse transcriptases
(commercial suppliers include: Roche Diagnostics., Indianapolis,
Ind.; Life Technologies, Inc., Rockville, Md.; New England Biolabs,
Inc., Beverly, Mass.; Perkin Elmer Corp., Norwalk, Conn.; Pharmacia
LKB Biotechnology, Inc., Piscataway, N.J.; Qiagen, Inc., Valencia,
Calif.; Stratagene, La Jolla, Calif.).
[0076] Oligonucleotide primers useful in the present invention may
be any oligonucleotide of two or more nucleotides in length.
Preferably, PCR primers are about 15 to about 30 bases in length
and are not palindromic (self-complementary) or complementary to
other primers that may be used in the reaction mixture. Primers may
be, but are not limited to, random primers, homopolymers, or
primers specific to a target oligonucleotide template (e.g., a
sequence specific primer). Oligonucleotide primers are
oligonucleotides used to hybridize to a region of a target nucleic
acid to facilitate the polymerization of a complementary nucleic
acid. In PCR protocols, primers serve to facilitate polymerization
of a first nucleic acid molecule complementary to a portion of an
oligonucleotide template, and also to facilitate replication of the
oligonucleotide. Any primer may be synthesized by a practitioner of
ordinary skill in the art or may be ordered and purchased from any
of a number of commercial venders (e.g., from Roche Diagnostics,
Indianapolis, Ind.; Life Technologies, Inc., Rockville, Md.; New
England Biolabs, Inc., Beverly, Mass.; Pharmacia LKB Biotechnology,
Inc., Piscataway, N.J.). It will be understood that a vast array of
primers may be useful in the present invention, including those not
specifically disclosed herein, without departing from the scope or
preferred embodiments thereof.
[0077] A nucleic acid template is defined as any polynucleotide
molecule used to provide a nucleic acid sequence from which a
polynucleotide complementary to the template may be generated. The
synthesis of DNA from a DNA template may be accomplished according
to the invention by utilizing a thermostable chimeric DNA
polymerase. The synthesis of RNA from a DNA template may be
accomplished according to the invention by utilizing a thermostable
chimeric RNA polymerase. The synthesis of DNA from an RNA template
may be accomplished according to the invention by utilizing a
thermostable chimeric nucleic acid polymerase that exhibits reverse
transcriptase activity.
[0078] Nucleotide bases useful in the present invention may be any
nucleotide useful in the polymerization of a nucleic acid.
Nucleotides may be naturally occurring, unusual, modified,
derivative, or artificial. Nucleotides may be unlabeled, or
detectably labeled by methods known in the art (e.g., using
radioisotopes, vitamins, fluorescent or chemiluminescent moieties,
digoxigenin). Preferably the nucleotides are deoxynucleoside
triphosphates, dNTPs (e.g., dATP, dCTP, dGTP, dTTP, dITP, dUTP,
.alpha.-thioSONDZEICHEN-dNTPs, biotin-dUTP, fluorescein-dUTP,
digoxigenin-dUTP, 7-deaza-dGTP). dNTPs are also well known in the
art and are commercially available (e.g., from Roche Diagnostics,
Indianapolis, Ind.; New England Biolabs, Inc., Beverly, Mass.;
Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.).
[0079] Buffering agents and salts useful in the present invention
provide appropriate stable pH and ionic conditions for nucleic acid
synthesis. A wide variety of buffers and salt solutions and
modified buffers are known in the art that may be useful in the
present invention, including agents not specifically disclosed
herein. Preferred buffering agents include, but are not limited to,
TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, CAPS.
Preferred salt solutions include, but are not limited to solutions
of; potassium chloride, potassium acetate, potassium sulfate,
ammonium sulfate, ammonium chloride, ammonium acetate, magnesium
chloride, magnesium acetate, magnesium sulfate, manganese acetate,
sodium chloride, sodium acetate, lithium chloride, and lithium
acetate.
[0080] Other additives capable of facilitating nucleic acid
generation and amplification, other than those disclosed for the
first time by this invention, are known in the art. In accordance
with the compositions and methods of this invention, one or more of
these additives may be incorporated in a DNA/RNA polymerization kit
according to the present invention to optimize the generation and
replication of polynucleotides. Additives may be organic or
inorganic compounds. Agents useful in the present invention
include, but are not limited to, polypeptides such as phosphatase,
human serum albumin, bovine serum albumin (BSA), ovalbumin,
albumax, casein, gelatin, collagen, globulin, lysozyme,
transferrin, .alpha.-lactalbumin, .beta.-lactoglobulin,
phosphorylase b, myosin, actin, .beta.-galactosidase, lectins, E.
coli single-stranded binding (SSB) protein, phage T4 gene 32
protein, and the like, or fragments or derivatives thereof.
Examples of nonpolypeptide additives include, but are not limited
to; homopolymeric nucleic acid, heteropolymeric nucleic acid, tRNA,
rRNA, sulfur-containing compounds, acetate-containing compounds,
dimethylsulfoxide (DMSO), glycerol, formamide, betain,
tetramethylammonium chloride (TMAC), polyethylene glycol (PEG),
Tween 20, NP 40, ectoine, and polyoles.
[0081] It will be readily apparent to those skilled in the art that
other suitable modifications and adaptations of the compositions
and methods of the invention described herein are obvious and may
be made without departing from the scope of the invention or the
embodiments disclosed herein. Having now described the present
invention in detail, the same will be more clearly understood by
reference to the following examples, which are included for
purposes of illustration only and are not intended to be limiting
of the invention.
Example 1
Construction of a Thermostable Chimeric DNA Polymerase Gene
[0082] Chimeric thermostable DNA polymerase constructs containing
enzymatically active domains from different (source) thermostable
DNA polymerases were generated using recombinant DNA techniques.
The 3'-5' exonuclease domain of various thermostable polymerases
were recombinantly linked to the 5'-3' polymerase domain of Taq
polymerase or Tth polymerase. The particularly preferred enzymatic
domains and domain borders, described herein in detail, were
selected and tested as preferred embodiments, and are not to be
considered limiting in scope of the thermostable chimeric nucleic
acid polymerase of the invention, or the enzymatically active
domains useful therein.
[0083] Appropriate microbial strains or genomic DNA preparations,
from which the enzymatically active domains used in the
construction of chimeric nucleic acid polymerase were isolated,
were purchased from commercial suppliers, e.g., from DSMZ GmbH
(Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH),
Braunschweig, Germany. Specifically chosen strains included Thermus
aquaticus (order # DSM 625), Thermus thermophilus (order # DSM
579), Pyrococcus furiosus (order # DSM 3638). Pyrococcus woesei
(order # DSM 3773). Pyrococcus horikoshii (DSM 3638), and
Sulfolobus solfataricus (order # DSM 5833). A multiplicity of
genomic DNA extraction, purification, and isolation techniques
useful to obtain the desired enzymatically active domains are well
known in the art.
[0084] Modified PCR amplification techniques and/or cloning
procedures such as restriction digestion and ligation using
appropriate enzymes were used to obtain the chimeric DNA polymerase
constructs. Primers appropriate to amplify polynucleotides encoding
particular enzymatic domains from the source thermostable DNA
polymerases were synthesized according to the nucleotide sequences
of the source thermostable DNA polymerase. DNA sequences of the
source thermostable DNA polymerases are published in GenBank. The
synthesis of oligonucleotide primers is well known to practitioners
in the art, and may also be ordered from commercial oligonucleotide
suppliers (e.g., Life Technologies, Gaithersburg, Md.).
[0085] PCR primers were of special design. The primers contained a
nucleotide sequence complementary to the terminal region of a
particular enzymatic domain of interest within a source DNA
polymerase. The primers also contained a noncomplementary
nucleotide sequence region as well to provide; i) an appropriate
restriction enzyme site, to facilitate genetic manipulation (e.g.,
vector insertion), or ii) sequence information (e.g.,
complementarity), to facilitate fusion to a second, non-naturally
associated enzymatic domain. For example, primers designed to
facilitate fusion of a 3'-5' exonuclease domain to a 5'-3'
polymerase domain contained a sequence, one half of which was
complementary to a terminal region of the 3'-5' exonuclease domain
of interest (e.g., residues 388-396 of Pho polymerase) and one half
of which was complementary to a terminal region of the 5'-3'
polymerase domain (e.g., residues 281-288 of Taq polymerase).
[0086] As an initial step, various enzymatic domains were amplified
by PCR. The PCR reaction mixture contained: 2.5 units of Taq
polymerase (Qiagen, Valencia, Calif.) and 0.1 to 0.2 units of Pfu
polymerase (Stratagene, La Jolla, Calif.); an appropriate amount of
the specially designed primers, as described above (0.2 to 1.0
.mu.M); genomic DNA isolated from the appropriate microorganism
containing the source thermostable polymerase; and 200 .mu.M of
each dNTP in a 1.times.PCR buffer (Qiagen, Valencia, Calif.). A
3-step PCR cycling program was run, consisting of an initial
denaturation step at 94.degree. C., an annealing step and an
extension step. The PCR ran for 25-35 cycles, depending upon the
desired amount of product. The size of the PCR product was checked
by agarose gel electrophoresis against an appropriate DNA size
marker. The correctly sized PCR product was gel-purified using the
QIAquick.TM. Gel Extraction Kit (Qiagen, Valencia, Calif.).
[0087] Once isolation and amplification of the polynucleotides
encoding the enzymatic domains chosen for chimeric polymerase
construction were obtained, the component enzymatic domains were
combined, in equivalent concentrations, in a composite PCR
reaction, together with 2-5 units of Pfu polymerase (Stratagene, La
Jolla, Calif.), and 200 .mu.M of each dNTP in 1.times.PCR buffer
(Qiagen, Valencia, Calif.). This PCR mixture did not contain any
primer oligonucleotides. This reaction mixture was subjected to 10
to 15 PCR cycles.
[0088] During the composite PCR, the single strand polynucleotides
encoding each of the enzymatically active domains hybridize at
their respective terminal regions of complementarity (due to the
specially designed primers as described above). The hybridized
single strand polynucleotides encoding each of the enzymatically
active domains form a single composite polynucleotide template,
thus serving as primers for each other. Pfu polymerase extends the
3' terminal end of each of the enzymatically active domains,
creating a single polynucleotide containing the chimeric DNA
polymerase gene construct.
[0089] After the initial 10 to 15 cycles of chimeric DNA polymerase
gene construction, oligonucleotide primers, appropriate to amplify
the full-length chimeric DNA polymerase gene, were added to the PCR
mixture. The PCR ran for 20-30 additional cycles, depending upon
the desired amount of chimeric DNA polymerase PCR product. The size
of the PCR product was checked by agarose gel electrophoresis and
the correctly sized PCR product was gel-purified as described
above.
[0090] The purified chimeric DNA polymerase gene was then subjected
to restriction digestion with the appropriate restriction enzyme to
cut the polynucleotide at restriction sites located at the terminal
ends of the chimeric DNA polymerase gene. These sites were
originally generated by the specially designed primers described
above.
Example 1.1
Construction of a Pho/Taq Thermostable Chimeric DNA Polymerase
Gene
[0091] A polynucleotide encoding the enzymatically active 3'-5'
exonuclease domain of Pho DNA polymerase was linked to a
polynucleotide encoding the enzymatically active 5'-3' polymerase
domain and the nonfunctional 3'-5' exonuclease domain of Taq DNA
polymerase. A polynucleotide encoding amino acids 271-832 (SEQ ID
NO:7) of Taq DNA polymerase was recombinantly linked to the 3' end
of a polynucleotide encoding amino acids 1-396 (SEQ ID NO:3) of Pho
DNA polymerase following the procedures detailed in Example 1
above, producing a polynucleotide that encodes a novel Pho/Taq
thermostable chimeric DNA polymerase (SEQ ID NO:8).
Example 1.2
Construction of a Pwo/Taq Thermostable Chimeric DNA Polymerase
Gene
[0092] A polynucleotide encoding the enzymatically active 3'-5'
exonuclease domain of Pwo DNA polymerase was linked to a
polynucleotide encoding the enzymatically active 5'-3' polymerase
domain of Taq DNA polymerase. A polynucleotide encoding amino acids
271-832 (SEQ ID NO:7) of Taq DNA polymerase was recombinantly
linked to the 3' end of a polynucleotide encoding amino acids 1-396
(SEQ ID NO:4) of Pwo DNA polymerase following the procedures
detailed in Example 1 above, producing a polynucleotide that
encodes a novel Pwo/Taq thermostable chimeric DNA polymerase (SEQ
ID NO:9).
Example 1.3
Construction of a Sso/Taq Thermostable Chimeric DNA Polymerase
Gene
[0093] A polynucleotide encoding the enzymatically active 3'-5'
exonuclease domain of Sso DNA polymerase was linked to a
polynucleotide encoding the enzymatically active 5'-3' polymerase
domain of Taq DNA polymerase. A polynucleotide encoding amino acids
281-832 (SEQ ID NO:1) of Taq DNA polymerase was recombinantly
linked to the 3' end of a polynucleotide encoding amino acids 1-508
(SEQ ID NO:6) of Sso DNA polymerase following the procedures
detailed in Example 1 above, producing a polynucleotide that
encodes a novel Sso/Taq thermostable chimeric DNA polymerase (SEQ
ID NO:10).
[0094] This chimeric construct, possessing a smaller Taq 5'-3'
polymerase domain than that used in Examples 1.1 and 1.2, also
demonstrates that specifically determined domain borders of an
enzymatic domain are not essential to the invention. What is
essential for the domain is that it retain its definitive enzymatic
activity.
Example 1.4
Construction of Variant Thermostable Chimeric DNA Polymerase
Genes
[0095] To further demonstrate that a thermostable chimeric nucleic
acid polymerase may be generated using an enzymatically active
domain of varying domain borders (provided the enzymatic activity
of the domain is retained), a Pwo/Taq chimeric DNA polymerase
variant of the thermostable chimeric polymerase generated in
Example 1.2 was constructed. This variant construct comprised a
polynucleotide encoding amino acids 271-832 (SEQ ID NO:7) of Taq
DNA polymerase recombinantly linked to the 3' end of a
polynucleotide encoding amino acids 1-421 (SEQ ID NO:5) of Pwo DNA
polymerase following the procedures detailed in Example 1 above,
producing a polynucleotide that encodes a second novel Pwo/Taq
thermostable chimeric DNA polymerase (SEQ ID NO:11).
Example 1.5
Construction of a Pho/Tth Thermostable Chimeric DNA Polymerase
Gene
[0096] To demonstrate that a thermostable chimeric nucleic acid
polymerase may be generated using an enzymatically active
polymerase domain other than that of Taq polymerase, a
polynucleotide encoding the enzymatically active 3'-5' exonuclease
domain of Pho DNA polymerase was linked to a polynucleotide
encoding the enzymatically active 5'-3' polymerase domain of Tth
DNA polymerase. A polynucleotide encoding amino acids 273-834 (SEQ
ID NO:2) of Tth DNA polymerase was recombinantly linked to the 3'
end of a polynucleotide encoding amino acids 1-396 (SEQ ID NO:3) of
Pho DNA polymerase following the procedures detailed in Example 1
above, producing a polynucleotide that encodes a novel Pho/Tth
thermostable chimeric DNA polymerase (SEQ ID NO:12).
[0097] This chimeric construct, possessing a Tth 5'-3' polymerase
domain that is also capable of reverse transcription activity, also
demonstrates a thermostable chimeric nucleic acid polymerase of the
present invention useful for RT-PCR protocols.
Example 1.6
Construction of a Thermostable Chimeric DNA Polymerase Gene
Encoding More than Two Enzymatically Active Domains
[0098] The chimeric nucleic acid polymerase gene of the invention
may encode two or more enzymatically active domains, of which two
more domains are non-naturally occurring. In addition the
enzymatically active domains may be derived from any polypeptide
source naturally occurring or synthetically produced.
[0099] For example, the practitioner may wish to construct a
thermostable chimeric nucleic acid polymerase possessing both the
5'-3' polymerase domain and the 5'-3' exonuclease domain of Taq
polymerase, as well as the 3'-5' exonuclease domain of another
polymerase (e.g., Pho polymerase). In this instance, a
polynucleotide encoding the 5'-3' exonuclease domain of Taq
polymerase (known to be contained within amino acids 1-291 of Taq
polymerase) would be recombinantly linked to 5' end of a
polynucleotide encoding the 3'-5' exonuclease domain of Pho
polymerase (e.g., SEQ ID NO: 3) and the 5'-3' polymerase domain of
Taq DNA polymerase (e.g., SEQ ID NOs: 1 or 7), which was earlier
demonstrated in Examples 1.1 and 1.4.
Example 2
Construction of a Thermostable Chimeric DNA Polymerase Vector
[0100] The isolated chimeric DNA polymerase genes of Examples 1.1
through 1.5 were each ligated into a vector, linearized using the
appropriate restriction enzyme. Ligation was performed overnight at
16.degree. C. using T4 DNA ligase and an appropriate buffer (Life
Technologies, Gaithersburg, Md.) in a final volume of 20 .mu.l.
Example 3
Construction of a Thermostable Chimeric DNA Polymerase Host
Cell
[0101] The ligated recombinant vectors of Example 2 were used to
transform calcium-competent M15[pRep4] cells (Qiagen, Valencia,
Calif.) or DH5SONDZEICHEN.alpha. competent cells. Aliquots of the
transformation mixture were spread onto agar plates containing
ampicillin and kanamycin (for M15[pRep4] cells), or ampicillin only
(for DH5.alpha. competent cells), and incubated overnight at
37.degree. C.
[0102] Colonies of successfully transformed cells were transferred
to LB media containing the appropriate antibiotic selection, and
incubated overnight. Plasmid isolation preparations were performed
using QIAprep.TM. Spin Kit or Plasmid Midi Kit (both from Qiagen,
Valencia, Calif.). Presence of the chimeric DNA polymerase gene was
verified by restriction digest analysis and the chimeric DNA
polymerase gene sequenced by techniques well known in the art.
[0103] The chimeric DNA polymerase genes were cloned into either
pQE-30 or pQE-31 expression vectors (Qiagen, Valencia, Calif.)
containing a six-histidine tag sequence preceding the respective
DNA polymerase sequence.
Example 4
Expression and Purification of a Thermostable Chimeric DNA
Polymerase
[0104] Thermostable chimeric DNA polymerase gene expression of the
successfully transformed host cells from Example 3, was induced by
IPTG. Harvested cells were lysed by sonification and lysozyme
treatment or a simple heat treatment. Chimeric His-tagged protein
was purified in batch format using Ni-NTA agarose (Qiagen,
Valencia, Calif.) following standard protocol procedures.
[0105] Eluates were ultrafiltrated using NanosepSONDZEICHEN.RTM.
ultrafiltration units (Pall Deutschland GmbH Holding, Dreieich,
Germany). Alternatively, heat treated cleared lysate was
centrifuged through Ultrafree filterunits 300,000 (Sigma,
Deisenhofen, Germany), to remove contaminating nucleic acids, and
was subsequently concentrated using NanosepSONDZEICHEN.RTM. or
MicrosepSONDZEICHEN.RTM. ultrafiltration units (Pall Deutschland
GmbH Holding, Dreieich, Germany).
[0106] Concentrated samples were mixed with a storage buffer
containing 20 mM TrisHCl (pH 8.0 at 20.degree. C.), 100 mM KCl, 1
mM EDTA, 0.5% (v/v) Nonidet P-40 substitute, 0.5% (v/v) Tween 20
and 50% (v/v) glycerol. Chimeric polymerase preparations were
stored at -20.degree. C. In some cases, the cleared lysate of the
polymerase preparation was directly used for subsequent analysis;
chimeric polymerase preparations were then stored at +4.degree.
C.
Example 5
5'-3' Polymerase Activity of Thermostable Chimeric DNA
Polymerases
[0107] To demonstrate the polymerase activity of thermostable
chimeric DNA polymerases produced from Example 4, an assay for
measuring primer extension activity was performed. This assay is
based on the difference in mobility of single-versus double-strand
DNA molecules on an agarose gel in the presence of a DNA
intercalating dye. Annealing of a primer to a single-stranded DNA
molecule creates a priming site for a DNA polymerase. The primer is
then extended by the polymerase, converting the single-strand DNA
into double-strand molecules. The extension rate is dependent upon
the polymerase used. The final amount of DNA extension (i.e.,
polymerization) is dependent on the amount of polymerase provided,
the extension rate of the polymerase, and the length of time the
reaction is allowed to proceed.
[0108] All polymerization reaction mixtures contained 50 ng M13 mp
18 DNA (20 fmol; 7250 nt), 0.1 .mu.M 30-mer oligonucleotide primer
5'-TTTCCCAGTCACGACGTTGTAAAACGACGG-3' (SEQ ID NO: 13), and 50 .mu.M
of each dNTP in 10 .mu.l of 10 mM Tris HCl.
[0109] Polymerization reactions containing Taq DNA polymerase and a
thermostable chimeric DNA polymerase were performed in 1.times.PCR
buffer (Qiagen, Valencia, Calif.).
[0110] Taq DNA polymerase was used for external standard reactions
(0.05, 0.03, 0.01 units) in order to determine polymerase activity
of the thermostable chimeric DNA polymerases. DNA polymerases were
diluted in the reaction buffer containing 1 .mu.g/ml bovine serum
albumin (BSA) to compensate for possible protein interactions with
the surface of the polypropylene tube.
[0111] The assay was performed in a MJ Research PTC-200
Thermocycler (Biozym, Hess. Oldendorf, Germany) or a Biometra UnoII
Thermocycler (Biometra, Gottingen, Germany). The thermal program
consisted of a 10 sec. denaturation step 94.degree. C.; a 30 sec.
annealing step at 55.degree. C.; and a 3 min. polymerization step
at 72.degree. C. Heating of the reaction mixture to 94.degree. C.
was done to destroy possible secondary structures of the
single-stranded M13 DNA and to facilitate specific primer annealing
during the lowering of reaction temperature to 55.degree. C.
[0112] Results of primer extension reactions at 72.degree. C. were
reproducible. After completing the reaction, reaction products were
mixed with 1 .mu.l gel loading solution (50% Glycerol, 1.times.TAE
buffer, 0.02 mg/ml Bromphenol blue) and loaded on a 1% agarose gel
containing 0.5 .mu.g/ml ethidium bromide. The gel was run at 80 mA
for 15 min in 1.times.TAE buffer. These conditions facilitated
discrimination between extended-(ds) and non-extended (ss) M13 DNA
fragments. The results, as represented in FIG. 1, illustrate the
polymerase activity of the thermostable chimeric polymerase is
comparable to that of wild type Taq polymerase.
Example 6
Thermostability of Chimeric DNA Polymerases
[0113] The primer extension assay described in Example 5 was also
used to measure the resilience of chimeric DNA polymerases to
thermal degradation (i.e., thermostability). Heat-treatment of
chimeric DNA polymerases (0.2 units) consisted of incubation of the
enzyme for 0, 10, 15, 30, 60, 90 and 120 min at 90.degree. C.,
followed by primer extension at 72.degree. C. Polymerase activity
of heat-treated chimeric polymerase was compared to untreated
chimeric DNA polymerase based on the amount of polymerized (i.e.,
double strand) M13 DNA. The same assay was performed, under
identical reaction conditions, on identical amounts of Taq DNA
polymerase, as a standard. A control consisted of a polymerase
reaction mixture, without any DNA polymerase. After completing the
reaction, reaction products were mixed with 1 .mu.l gel loading
solution (50% Glycerol, 1.times.TAE buffer, 0.02 mg/ml Bromphenol
blue), and loaded on a 1% agarose gel containing 0.5 .mu.g/ml
ethidium bromide. The gel was run at 80 mA for 15 min in
1.times.TAE buffer. The results, presented in FIG. 2 and quantified
in Table 2 below, are representative of the thermostability
assay.
TABLE-US-00002 TABLE 2 Thermostability of chimeric polymerase
compared to Taq polymerase Incubation Pho/Taq Chimeric Taq
Polymerase at 90.degree. C. (min.) Polymerase % Activity % Activity
0 100 100 10 84 99 15 84 89 30 82 74 60 66 69 90 53 31* 120 45 43
*single non-reproducible data; value expected to be higher
[0114] FIG. 2 and Table 2 confirm the thermostability of the
chimeric polymerase of the present invention. Table 2 illustrates
that although the activity of the chimeric DNA polymerase shows an
initial drop in activity (within the first 10 min at 90.degree. C.)
greater than that of Taq DNA polymerase, the overall
thermostability is comparable to Taq DNA polymerase. Chimeric DNA
polymerase of the invention displays the same half life at
90.degree. C. as Taq DNA polymerase (approximately 90 min).
[0115] The thermostability assay was also performed under extreme
temperature conditions. The primer extension assay was run after
heat-treatment at 95.degree. C. for 0, 3, 5, and 10 min. The
results, quantified in Table 3 below, are representative of the
95.degree. C. thermostability assay, and further confirm that the
chimeric DNA polymerase of the present invention is highly
thermostable.
TABLE-US-00003 TABLE 3 Thermostability of chimeric polymerase
Incubation Pho/Taq Chimeric at 95.degree. C. (min.) Polymerase %
Activity 0 100 3 100 5 86 10 86
[0116] These results confirm the thermostability of the chimeric
DNA polymerase of the present invention, making it useful for in
vitro reactions under heat denaturing conditions such as PCR,
without requiring successive addition of enzyme at each cycle of
the PCR program.
Example 7
3'-5' Exonuclease Activity of Thermostable Chimeric DNA
Polymerases
[0117] Fidelity of DNA replication is based on a two step process:
misinsertion and misextension. In PCR, if the DNA polymerase
inserts an incorrect nucleotide, and the resulting 3'-mismatched
terminus of the growing DNA chain is not extended, the truncated
primer extension product cannot be amplified during subsequent PCR
cycles since the downstream primer binding site is missing.
Additionally, mismatched termini are less efficiently extended than
DNA ends harboring the complementary base. DNA polymerases
possessing an enzymatically active 3'-5' exonuclease domain are
capable of removing a misincorporated nucleotide, thus increasing
fidelity of the PCR product and increasing primer extension
efficiency.
[0118] A PCR and restriction endonuclease digestion assay,
developed to assess the ability of thermostable DNA polymerases to
remove mismatched primer termini by 3'-5' exonuclease activity, was
performed using the protocol disclosed in U.S. Pat. No. 5,491,086
(incorporated by reference). Wild type primers, perfectly matching
the BamHI restriction enzyme recognition sequence in the Taq
polymerase gene, and mutant primers, possessing a 3'-mismatch
(employing every possible combination) to the first nucleotide of
the BamHI restriction enzyme recognition sequence, were used in
side-by-side PCR trials.
[0119] Wild type primers to 5'-GCACCCCGCTTGGGCAGAG-3' (SEQ ID
NO:14) and 5'-TCCCGCCCCTCCTGGAAGAC-3' (SEQ ID NO:15) yield a 151 bp
PCR product that becomes digested upon incubation with BamHI
restriction enzyme, generating a 132 bp and 19 bp fragment.
[0120] Three forward primers containing a single 3'-mismatched
nucleotide representing a C:A, C:T, and C:C mismatch to SEQ ID
NO:14 were used as mutant primers. Any extension product from these
mutant primers would corrupt the BamHI restriction site, rendering
the resulting PCR products unaffected by BamHI digestion, thus
leaving the 151 bp PCR product intact. The presence of an
enzymatically active 3'-5' exonuclease domain, would correct the
3'-mismatched nucleotide of the mutant primer, however, thus
restoring the BamHI restriction site, rendering the PCR product
susceptible to BamHI digestion, thus producing the 132 bp and 19 bp
digestion fragments.
[0121] Using this PCR fidelity assay, the chimeric thermostable DNA
polymerase was tested for the ability to correct a 3'-primer
mismatch during PCR. Chimeric polymerase trials were run in
parallel with wild type Taq DNA polymerase and Pfu DNA polymerase
I. The Taq DNA polymerase trials served as a negative control,
representing a DNA polymerase possessing an enzymatically inactive
3'-5' exonuclease domain (i.e., proofreading capability). The Pfu
DNA polymerase I trials served as a positive control, representing
a thermostable DNA polymerase possessing an enzymatically active
3'-5' exonuclease domain.
[0122] PCR mixtures comprised 20 ng plasmid pQE-31 containing the
(target) Taq polymerase gene sequence; 0.5 units of the test DNA
polymerase; 0.4 .mu.M of the appropriate trial primers (wild type
vs. mutant primers); 200 .mu.M of each dNTP; 1.times. Qiagen PCR
buffer (Qiagen, Valencia, Calif.) or 1.times.Pfu reaction buffer
(Stratagene, La Jolla, Calif.) and 1.5 mM MgCl.sub.2 in a final
reaction volume of 50 .mu.l.
[0123] PCR was performed using a MJ Research PTC-200 Thermocycler
(Biozym, Hess. Oldendorf, Germany) or a Biometra UnoII Thermocycler
(Biometra, Gottingen, Germany). The PCR program consisted of an
initial 1 min template denaturation step at 94.degree. C. followed
by 40 cycles of a 30 sec. denaturation step 94.degree. C.; a 30
sec. annealing step at 62.degree. C.; and a 1 min. polymerization
step at 72.degree. C. for 1 min. The PCR concluded with a final
prolonged extension step for 2 min. at 72.degree. C.
[0124] PCR products were analyzed on a 2% agarose gel by gel
electrophoresis (approximately 35 min. at 85 volts) in 1.times.TAE
electrophoresis buffer and Ethidium bromide. PCR products were
visualized using UV irradiation, and quantified using the 200 bp
DNA fragment of the Low DNA MassSONDZEICHEN.TM. Ladder (Life
Technologies, Gaithersburg, Md., USA) as standard by gel
densitometry. PCR products were purified using QIAquick.TM. PCR
Purification Kit (Qiagen, Valencia, Calif.).
[0125] Identical amounts of PCR product were digested in the same
final reaction volume using 1 unit BamHI (Life Technologies,
Gaithersburg, Md., USA) per 100 ng PCR product and corresponding
reaction buffer. Restriction digest was performed for 90 min. at
37.degree. C. Digestion products were analyzed on a 4%
MetaphorSONDZEICHEN.RTM. agarose gel (Biozym, Hess. Oldendorf,
Germany) FIG. 3. is representative of the results of the 3'-5'
exonuclease activity assay.
[0126] FIG. 3(A) illustrates the PCR product of the three nucleic
acid polymerases (Taq polymerase, Pfu polymerase, and the
thermostable chimeric polymerase) using wild type primers.
Alternating lanes represent undigested PCR product and PCR product
subjected to BamHI digestion. Undigested product shows the intact
151 bp PCR product. Digestion treated product shows the 132 bp
digestion fragment.
[0127] FIG. 3(B) illustrates the PCR product of the three
polymerases (Taq polymerase, Pfu polymerase, and the thermostable
chimeric polymerase) using mutant primers. Once again, alternating
lanes represent undigested PCR product and PCR product subjected to
BamHI digestion. Taq polymerase PCR product was unaffected by BamHI
digestion (lanes 3 and 5), due to the lack of a BamHI site
resulting for normal extension of the mutant primer. Pfu polymerase
PCR product was effectively digested by BamHI (lanes 7 and 9),
producing the expected 132 bp digestion fragment. These results are
indicative of the proofreading ability (i.e., 3'-5' exonuclease
activity) of Pfu polymerase, which corrected the nucleotide
mismatch of the mutant primer, thus restoring the BamHI site of the
template DNA.
[0128] The thermostable chimeric polymerase PCR product displayed
results similar to the Pfu polymerase PCR product. The chimeric
polymerase PCR product was also effectively digested by BamHI
(lanes 11 and 13), producing the expected 132 bp digestion fragment
and indicative of polymerase proofreading ability. These results
confirm that the thermostable chimeric polymerase, which possesses
the 5'-3' polymerase domain of Taq polymerase, also possesses an
enzymatically active 3'-5' exonuclease domain not naturally
occurring in Taq polymerase.
Example 8
PCR efficiency of Thermostable Chimeric DNA Polymerases
[0129] PCR efficiency of a DNA polymerase can be described as the
combined effect of primer extension activity and processivity of
the enzyme. PCR efficiency of the thermostable chimeric DNA
polymerase was tested in comparison with Taq DNA polymerase, known
to possess a higher PCR efficiency than common proofreading
polymerases, and Pfu DNA polymerase (both serving as controls).
[0130] One unit of the respective polymerase was used to amplify a
750 bp large product from human genomic DNA using a thermocycling
profile with varying primer extension times at 72.degree. C.
Limiting primer extension time was used to measure polymerase
efficiency in PCR, using the same amount of enzyme activity in the
assay. Taq DNA polymerase was assayed in its optimized PCR buffer
(Qiagen, Valencia, Calif.), a Pho/Taq thermostable chimeric DNA
polymerase was used in a 1.times. buffer consisting of 50 mM
TrisHCl (pH 8.9 at room temperature), 10 mM
(NH.sub.4).sub.2SO.sub.4, and Pfu DNA polymerase was used in the
reaction buffer supplied with the enzyme (Stratagene, La Jolla,
Calif.). All reactions contained 1 unit of enzyme, 0.4 .mu.M of
each primer, 200 .mu.M of each dNTP, and a final MgCl.sub.2
concentration of 1.5 mM (Taq polymerase, chimeric DNA polymerase)
or 2.0 mM (Pfu polymerase).
[0131] Thermocycling was performed in a Biometra Uno thermocycler
using the following cycling conditions: initial denaturation at
94.degree. C. for 3 min followed by a denaturation step at
94.degree. C. for 30 sec, an annealing step at 60.degree. C. for 30
sec, and a primer extension step at 72.degree. C. for 1 min, 30
sec, 10 sec or 5 sec. The reaction proceeded for 34 cycles, and
concluded with a final extension step at 72.degree. C. for 10
min.
[0132] The results are depicted in FIG. 4. Taq DNA polymerase (A)
shows a high PCR efficiency even when primer extension time is as
low as 5 sec. The thermostable chimeric DNA polymerase (B) shows a
higher PCR efficiency than Taq polymerase at extension times of 1
min and 30 sec, but a slightly lower efficiency than Taq polymerase
at 5 sec extension time. Pfu DNA polymerase I (C) generates a
visible PCR product only when using the 1 min extension time.
[0133] These results indicate that the overall processivity of the
chimeric polymerase is comparable to that of Taq DNA polymerase,
and is dramatically better than Pfu DNA polymerase I. The
thermostable chimeric polymerase of the present invention performs
as well as Taq DNA polymerase (the standard enzyme of PCR
protocols), and outperforms Pfu DNA polymerase I (the standard
enzyme for high fidelity PCR protocols). In addition, the
thermostable chimeric polymerase of the present invention combines
the beneficial features of each of the standard enzymes for PCR
protocols formerly not obtained with either Taq DNA polymerase or
proofreading polymerases: removal of misincorporated nucleotides
required for high fidelity PCR, and high PCR efficiency.
REFERENCES
[0134] Barnes, PNAS USA 91:2216-2220 (1994). [0135] Barnes. U.S.
Pat. No. 5,436,149 (1995). [0136] Bedford et al., WO 97/29209 (14
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et al., Nature 382: 278-281 (1996) [0139] Flaman et al. N.A.R.
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(1992).
[0164] Each of the publications mentioned herein is incorporated by
reference.
Sequence CWU 1
1
171552PRTThermus aquaticus 1Leu Leu His Glu Phe Gly Leu Leu Glu Ser
Pro Lys Ala Leu Glu Glu 1 5 10 15 Ala Pro Trp Pro Pro Pro Glu Gly
Ala Phe Val Gly Phe Val Leu Ser 20 25 30 Arg Lys Glu Pro Met Trp
Ala Asp Leu Leu Ala Leu Ala Ala Ala Arg 35 40 45 Gly Gly Arg Val
His Arg Ala Pro Glu Pro Tyr Lys Ala Leu Arg Asp 50 55 60 Leu Lys
Glu Ala Arg Gly Leu Leu Ala Lys Asp Leu Ser Val Leu Ala 65 70 75 80
Leu Arg Glu Gly Leu Gly Leu Pro Pro Gly Asp Asp Pro Met Leu Leu 85
90 95 Ala Tyr Leu Leu Asp Pro Ser Asn Thr Thr Pro Glu Gly Val Ala
Arg 100 105 110 Arg Tyr Gly Gly Glu Trp Thr Glu Glu Ala Gly Glu Arg
Ala Ala Leu 115 120 125 Ser Glu Arg Leu Phe Ala Asn Leu Trp Gly Arg
Leu Glu Gly Glu Glu 130 135 140 Arg Leu Leu Trp Leu Tyr Arg Glu Val
Glu Arg Pro Leu Ser Ala Val 145 150 155 160 Leu Ala His Met Glu Ala
Thr Gly Val Arg Leu Asp Val Ala Tyr Leu 165 170 175 Arg Ala Leu Ser
Leu Glu Val Ala Glu Glu Ile Ala Arg Leu Glu Ala 180 185 190 Glu Val
Phe Arg Leu Ala Gly His Pro Phe Asn Leu Asn Ser Arg Asp 195 200 205
Gln Leu Glu Arg Val Leu Phe Asp Glu Leu Gly Leu Pro Ala Ile Gly 210
215 220 Lys Thr Glu Lys Thr Gly Lys Arg Ser Thr Ser Ala Ala Val Leu
Glu 225 230 235 240 Ala Leu Arg Glu Ala His Pro Ile Val Glu Lys Ile
Leu Gln Tyr Arg 245 250 255 Glu Leu Thr Lys Leu Lys Ser Thr Tyr Ile
Asp Pro Leu Pro Asp Leu 260 265 270 Ile His Pro Arg Thr Gly Arg Leu
His Thr Arg Phe Asn Gln Thr Ala 275 280 285 Thr Ala Thr Gly Arg Leu
Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile 290 295 300 Pro Val Arg Thr
Pro Leu Gly Gln Arg Ile Arg Arg Ala Phe Ile Ala 305 310 315 320 Glu
Glu Gly Trp Leu Leu Val Ala Leu Asp Tyr Ser Gln Ile Glu Leu 325 330
335 Arg Val Leu Ala His Leu Ser Gly Asp Glu Asn Leu Ile Arg Val Phe
340 345 350 Gln Glu Gly Arg Asp Ile His Thr Glu Thr Ala Ser Trp Met
Phe Gly 355 360 365 Val Pro Arg Glu Ala Val Asp Pro Leu Met Arg Arg
Ala Ala Lys Thr 370 375 380 Ile Asn Phe Gly Val Leu Tyr Gly Met Ser
Ala His Arg Leu Ser Gln 385 390 395 400 Glu Leu Ala Ile Pro Tyr Glu
Glu Ala Gln Ala Phe Ile Glu Arg Tyr 405 410 415 Phe Gln Ser Phe Pro
Lys Val Arg Ala Trp Ile Glu Lys Thr Leu Glu 420 425 430 Glu Gly Arg
Arg Arg Gly Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg 435 440 445 Tyr
Val Pro Asp Leu Glu Ala Arg Val Lys Ser Val Arg Glu Ala Ala 450 455
460 Glu Arg Met Ala Phe Asn Met Pro Val Gln Gly Thr Ala Ala Asp Leu
465 470 475 480 Met Lys Leu Ala Met Val Lys Leu Phe Pro Arg Leu Glu
Glu Met Gly 485 490 495 Ala Arg Met Leu Leu Gln Val His Asp Glu Leu
Val Leu Glu Ala Pro 500 505 510 Lys Glu Arg Ala Glu Ala Val Ala Arg
Leu Ala Lys Glu Val Met Glu 515 520 525 Gly Val Tyr Pro Leu Ala Val
Pro Leu Glu Val Glu Val Gly Ile Gly 530 535 540 Glu Asp Trp Leu Ser
Ala Lys Glu 545 550 2562PRTThermus thermophilus 2Ala Phe Leu Glu
Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly 1 5 10 15 Leu Leu
Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro 20 25 30
Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp 35
40 45 Ala Glu Leu Lys Ala Leu Ala Ala Cys Arg Asp Gly Arg Val His
Arg 50 55 60 Ala Ala Asp Pro Leu Ala Gly Leu Lys Asp Leu Lys Glu
Val Arg Gly 65 70 75 80 Leu Leu Ala Lys Asp Leu Ala Val Leu Ala Ser
Arg Glu Gly Leu Asp 85 90 95 Leu Val Pro Gly Asp Asp Pro Met Leu
Leu Ala Tyr Leu Leu Asp Pro 100 105 110 Ser Asn Thr Thr Pro Glu Gly
Val Ala Arg Arg Tyr Gly Gly Glu Trp 115 120 125 Thr Glu Asp Ala Ala
His Arg Ala Leu Leu Ser Glu Arg Leu His Arg 130 135 140 Asn Leu Leu
Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr 145 150 155 160
His Glu Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala 165
170 175 Thr Gly Val Arg Arg Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu
Glu 180 185 190 Leu Ala Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe
Arg Leu Ala 195 200 205 Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gln
Leu Glu Arg Val Leu 210 215 220 Phe Asp Glu Leu Arg Leu Pro Ala Leu
Gly Lys Thr Gln Lys Thr Gly 225 230 235 240 Lys Arg Ser Thr Ser Ala
Ala Val Leu Glu Ala Leu Arg Glu Ala His 245 250 255 Pro Ile Val Glu
Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys 260 265 270 Asn Thr
Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly 275 280 285
Arg Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu 290
295 300 Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro
Leu 305 310 315 320 Gly Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala
Gly Trp Ala Leu 325 330 335 Val Ala Leu Asp Tyr Ser Gln Ile Glu Leu
Arg Val Leu Ala His Leu 340 345 350 Ser Gly Asp Glu Asn Leu Ile Arg
Val Phe Gln Glu Gly Lys Asp Ile 355 360 365 His Thr Gln Thr Ala Ser
Trp Met Phe Gly Val Pro Pro Glu Ala Val 370 375 380 Asp Pro Leu Met
Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu 385 390 395 400 Tyr
Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr 405 410
415 Glu Glu Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys
420 425 430 Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys
Arg Gly 435 440 445 Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val
Pro Asp Leu Asn 450 455 460 Ala Arg Val Lys Ser Val Arg Glu Ala Ala
Glu Arg Met Ala Phe Asn 465 470 475 480 Met Pro Val Gln Gly Thr Ala
Ala Asp Leu Met Lys Leu Ala Met Val 485 490 495 Lys Leu Phe Pro Arg
Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gln 500 505 510 Val His Asp
Glu Leu Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu Glu 515 520 525 Val
Ala Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala 530 535
540 Val Pro Leu Glu Val Glu Val Gly Met Gly Glu Asp Trp Leu Ser Ala
545 550 555 560 Lys Gly 3396PRTPyrococcus horikoshii 3Met Ile Leu
Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile 1 5 10 15 Arg
Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Val Glu Tyr Asp Arg 20 25
30 Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Ala Ile
35 40 45 Asp Glu Ile Lys Lys Ile Thr Ala Gln Arg His Gly Lys Val
Val Arg 50 55 60 Ile Val Glu Thr Glu Lys Ile Gln Arg Lys Phe Leu
Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Leu Glu His Pro
Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Glu His Pro
Ala Val Val Asp Ile Phe Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys
Arg Tyr Leu Ile Asp Lys Gly Leu Thr Pro 115 120 125 Met Glu Gly Asn
Glu Lys Leu Thr Phe Leu Ala Val Asp Ile Glu Thr 130 135 140 Leu Tyr
His Glu Gly Glu Glu Phe Gly Lys Gly Pro Val Ile Met Ile 145 150 155
160 Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Ile
165 170 175 Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met
Ile Lys 180 185 190 Arg Leu Ile Arg Val Ile Lys Glu Lys Asp Pro Asp
Val Ile Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr
Leu Leu Lys Arg Ala Glu 210 215 220 Lys Leu Gly Ile Lys Leu Leu Leu
Gly Arg Asp Asn Ser Glu Pro Lys 225 230 235 240 Met Gln Lys Met Gly
Asp Ser Leu Ala Val Glu Ile Lys Gly Arg Ile 245 250 255 His Phe Asp
Leu Phe Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr
Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu 275 280
285 Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu Thr Gly Glu Gly
290 295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val
Thr Tyr 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala
Gln Leu Ala Arg Leu 325 330 335 Val Gly Gln Pro Val Trp Asp Val Ser
Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg
Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp
Glu Lys Glu Tyr Glu Arg Arg Leu Arg Glu Ser 370 375 380 Tyr Glu Gly
Gly Tyr Val Lys Glu Pro Glu Lys Gly 385 390 395 4396PRTPyrococcus
woesei 4Met Ile Leu Asp Val Asp Tyr Ile Thr Glu Glu Gly Lys Pro Val
Ile 1 5 10 15 Arg Leu Phe Lys Lys Glu Asn Gly Lys Phe Lys Ile Glu
His Asp Arg 20 25 30 Thr Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg
Asp Asp Ser Lys Ile 35 40 45 Glu Glu Val Lys Lys Ile Thr Gly Glu
Arg His Gly Lys Ile Val Arg 50 55 60 Ile Val Asp Val Glu Lys Val
Glu Lys Lys Phe Leu Gly Lys Pro Ile 65 70 75 80 Thr Val Trp Lys Leu
Tyr Leu Glu His Pro Gln Asp Val Pro Thr Ile 85 90 95 Arg Glu Lys
Val Arg Glu His Pro Ala Val Val Asp Ile Phe Glu Tyr 100 105 110 Asp
Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120
125 Met Glu Gly Glu Glu Glu Leu Lys Ile Leu Ala Phe Asp Ile Glu Thr
130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile
Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Asn Glu Ala Lys Val Ile
Thr Trp Lys Asn Ile 165 170 175 Asp Leu Pro Tyr Val Glu Val Val Ser
Ser Glu Arg Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Ile Ile Arg
Glu Lys Asp Pro Asp Ile Ile Val Thr 195 200 205 Tyr Asn Gly Asp Ser
Phe Asp Phe Pro Tyr Leu Ala Lys Arg Ala Glu 210 215 220 Lys Leu Gly
Ile Lys Leu Thr Ile Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240
Met Gln Arg Ile Gly Asp Met Thr Ala Val Glu Val Lys Gly Arg Ile 245
250 255 His Phe Asp Leu Tyr His Val Ile Thr Arg Thr Ile Asn Leu Pro
Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys
Pro Lys Glu 275 280 285 Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp
Glu Ser Gly Glu Asn 290 295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met
Glu Asp Ala Lys Ala Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe
Leu Pro Met Glu Ile Gln Leu Ser Arg Leu 325 330 335 Val Gly Gln Pro
Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu
Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Val Ala 355 360 365
Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gln Arg Arg Leu Arg Glu Ser 370
375 380 Tyr Thr Gly Gly Phe Val Lys Glu Pro Glu Lys Gly 385 390 395
5421PRTPyrococcus woesei 5Met Ile Leu Asp Val Asp Tyr Ile Thr Glu
Glu Gly Lys Pro Val Ile 1 5 10 15 Arg Leu Phe Lys Lys Glu Asn Gly
Lys Phe Lys Ile Glu His Asp Arg 20 25 30 Thr Phe Arg Pro Tyr Ile
Tyr Ala Leu Leu Arg Asp Asp Ser Lys Ile 35 40 45 Glu Glu Val Lys
Lys Ile Thr Gly Glu Arg His Gly Lys Ile Val Arg 50 55 60 Ile Val
Asp Val Glu Lys Val Glu Lys Lys Phe Leu Gly Lys Pro Ile 65 70 75 80
Thr Val Trp Lys Leu Tyr Leu Glu His Pro Gln Asp Val Pro Thr Ile 85
90 95 Arg Glu Lys Val Arg Glu His Pro Ala Val Val Asp Ile Phe Glu
Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly
Leu Ile Pro 115 120 125 Met Glu Gly Glu Glu Glu Leu Lys Ile Leu Ala
Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly
Lys Gly Pro Ile Ile Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Asn
Glu Ala Lys Val Ile Thr Trp Lys Asn Ile 165 170 175 Asp Leu Pro Tyr
Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys 180 185 190 Arg Phe
Leu Arg Ile Ile Arg Glu Lys Asp Pro Asp Ile Ile Val Thr 195 200 205
Tyr Asn Gly Asp Ser Phe Asp Phe Pro Tyr Leu Ala Lys Arg Ala Glu 210
215 220 Lys Leu Gly Ile Lys Leu Thr Ile Gly Arg Asp Gly Ser Glu Pro
Lys 225 230 235 240 Met Gln Arg Ile Gly Asp Met Thr Ala Val Glu Val
Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr His Val Ile Thr Arg
Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu
Ala Ile Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Ala Asp Glu
Ile Ala Lys Ala Trp Glu Ser Gly Glu Asn 290 295 300 Leu Glu Arg Val
Ala Lys Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr 305 310 315 320 Glu
Leu Gly Lys Glu Phe Leu Pro Met Glu Ile Gln Leu Ser Arg Leu 325 330
335 Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu
Val Ala 355 360 365 Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gln Arg Arg
Leu Arg Glu Ser 370 375 380 Tyr Thr Gly Gly
Phe Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn 385 390 395 400 Ile
Val Tyr Leu Asp Phe Arg Ala Leu Tyr Pro Ser Ile Ile Ile Thr 405 410
415 His Asn Val Ser Pro 420 6508PRTSulfolobus solfataricus 6Met Thr
Lys Gln Leu Thr Leu Phe Asp Ile Pro Ser Ser Lys Pro Ala 1 5 10 15
Lys Ser Glu Gln Asn Thr Gln Gln Ser Gln Gln Ser Ala Pro Val Glu 20
25 30 Glu Lys Lys Val Val Arg Arg Glu Trp Leu Glu Glu Ala Gln Glu
Asn 35 40 45 Lys Ile Tyr Phe Leu Leu Gln Val Asp Tyr Asp Gly Lys
Lys Gly Lys 50 55 60 Ala Val Cys Lys Leu Phe Asp Lys Glu Thr Gln
Lys Ile Tyr Ala Leu 65 70 75 80 Tyr Asp Asn Thr Gly His Lys Pro Tyr
Phe Leu Val Asp Leu Glu Pro 85 90 95 Asp Lys Val Gly Lys Ile Pro
Lys Ile Val Arg Asp Pro Ser Phe Asp 100 105 110 His Ile Glu Thr Val
Ser Lys Ile Asp Pro Tyr Thr Trp Asn Lys Phe 115 120 125 Lys Leu Thr
Lys Ile Val Val Arg Asp Pro His Ala Val Arg Arg Leu 130 135 140 Arg
Asn Asp Val Pro Lys Ala Tyr Glu Ala His Ile Lys Tyr Phe Asn 145 150
155 160 Asn Tyr Met Tyr Asp Ile Gly Leu Ile Pro Gly Met Pro Tyr Val
Val 165 170 175 Lys Asn Gly Lys Leu Glu Ser Val Tyr Leu Ser Leu Asp
Glu Lys Asp 180 185 190 Val Glu Glu Ile Lys Lys Ala Phe Ala Asp Ser
Asp Glu Met Thr Arg 195 200 205 Gln Met Ala Val Asp Trp Leu Pro Ile
Phe Glu Thr Glu Ile Pro Lys 210 215 220 Ile Lys Arg Val Ala Ile Asp
Ile Glu Val Tyr Thr Pro Val Lys Gly 225 230 235 240 Arg Ile Pro Asp
Ser Gln Lys Ala Glu Phe Pro Ile Ile Ser Ile Ala 245 250 255 Leu Ala
Gly Ser Asp Gly Leu Lys Lys Val Leu Val Leu Asn Arg Asn 260 265 270
Asp Val Asn Glu Gly Ser Val Lys Leu Asp Gly Ile Ser Val Glu Arg 275
280 285 Phe Asn Thr Glu Tyr Glu Leu Leu Gly Arg Phe Phe Asp Ile Leu
Leu 290 295 300 Glu Tyr Pro Ile Val Leu Thr Phe Asn Gly Asp Asp Phe
Asp Leu Pro 305 310 315 320 Tyr Ile Tyr Phe Arg Ala Leu Lys Leu Gly
Tyr Phe Pro Glu Glu Ile 325 330 335 Pro Ile Asp Val Ala Gly Lys Asp
Glu Ala Lys Tyr Leu Ala Gly Leu 340 345 350 His Ile Asp Leu Tyr Lys
Phe Phe Phe Asn Lys Ala Val Arg Asn Tyr 355 360 365 Ala Phe Glu Gly
Lys Tyr Asn Glu Tyr Asn Leu Asp Ala Val Ala Lys 370 375 380 Ala Leu
Leu Gly Thr Ser Lys Val Lys Val Asp Thr Leu Ile Ser Phe 385 390 395
400 Leu Asp Val Glu Lys Leu Ile Glu Tyr Asn Phe Arg Asp Ala Glu Ile
405 410 415 Thr Leu Gln Leu Thr Thr Phe Asn Asn Asp Leu Thr Met Lys
Leu Ile 420 425 430 Val Leu Phe Ser Arg Ile Ser Arg Leu Gly Ile Glu
Glu Leu Thr Arg 435 440 445 Thr Glu Ile Ser Thr Trp Val Lys Asn Leu
Tyr Tyr Trp Glu His Arg 450 455 460 Lys Arg Asn Trp Leu Ile Pro Leu
Lys Glu Glu Ile Leu Ala Lys Ser 465 470 475 480 Ser Asn Ile Arg Thr
Ser Ala Leu Ile Lys Gly Lys Gly Tyr Lys Gly 485 490 495 Ala Val Val
Ile Asp Pro Pro Ala Gly Ile Phe Phe 500 505 7562PRTThermus
aquaticus 7Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu
Phe Gly 1 5 10 15 Leu Leu Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro
Trp Pro Pro Pro 20 25 30 Glu Gly Ala Phe Val Gly Phe Val Leu Ser
Arg Lys Glu Pro Met Trp 35 40 45 Ala Asp Leu Leu Ala Leu Ala Ala
Ala Arg Gly Gly Arg Val His Arg 50 55 60 Ala Pro Glu Pro Tyr Lys
Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly 65 70 75 80 Leu Leu Ala Lys
Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly 85 90 95 Leu Pro
Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro 100 105 110
Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp 115
120 125 Thr Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe
Ala 130 135 140 Asn Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu
Trp Leu Tyr 145 150 155 160 Arg Glu Val Glu Arg Pro Leu Ser Ala Val
Leu Ala His Met Glu Ala 165 170 175 Thr Gly Val Arg Leu Asp Val Ala
Tyr Leu Arg Ala Leu Ser Leu Glu 180 185 190 Val Ala Glu Glu Ile Ala
Arg Leu Glu Ala Glu Val Phe Arg Leu Ala 195 200 205 Gly His Pro Phe
Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu 210 215 220 Phe Asp
Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly 225 230 235
240 Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His
245 250 255 Pro Ile Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys
Leu Lys 260 265 270 Ser Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile His
Pro Arg Thr Gly 275 280 285 Arg Leu His Thr Arg Phe Asn Gln Thr Ala
Thr Ala Thr Gly Arg Leu 290 295 300 Ser Ser Ser Asp Pro Asn Leu Gln
Asn Ile Pro Val Arg Thr Pro Leu 305 310 315 320 Gly Gln Arg Ile Arg
Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu 325 330 335 Val Ala Leu
Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu 340 345 350 Ser
Gly Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile 355 360
365 His Thr Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val
370 375 380 Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly
Val Leu 385 390 395 400 Tyr Gly Met Ser Ala His Arg Leu Ser Gln Glu
Leu Ala Ile Pro Tyr 405 410 415 Glu Glu Ala Gln Ala Phe Ile Glu Arg
Tyr Phe Gln Ser Phe Pro Lys 420 425 430 Val Arg Ala Trp Ile Glu Lys
Thr Leu Glu Glu Gly Arg Arg Arg Gly 435 440 445 Tyr Val Glu Thr Leu
Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu 450 455 460 Ala Arg Val
Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn 465 470 475 480
Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val 485
490 495 Lys Leu Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu
Gln 500 505 510 Val His Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg
Ala Glu Ala 515 520 525 Val Ala Arg Leu Ala Lys Glu Val Met Glu Gly
Val Tyr Pro Leu Ala 530 535 540 Val Pro Leu Glu Val Glu Val Gly Ile
Gly Glu Asp Trp Leu Ser Ala 545 550 555 560 Lys Glu
8958PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Pho/Taq Chimeric polymerase polypeptide 8Met Ile Leu Asp
Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile 1 5 10 15 Arg Ile
Phe Lys Lys Glu Asn Gly Glu Phe Lys Val Glu Tyr Asp Arg 20 25 30
Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Ala Ile 35
40 45 Asp Glu Ile Lys Lys Ile Thr Ala Gln Arg His Gly Lys Val Val
Arg 50 55 60 Ile Val Glu Thr Glu Lys Ile Gln Arg Lys Phe Leu Gly
Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Leu Glu His Pro Gln
Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Glu His Pro Ala
Val Val Asp Ile Phe Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg
Tyr Leu Ile Asp Lys Gly Leu Thr Pro 115 120 125 Met Glu Gly Asn Glu
Lys Leu Thr Phe Leu Ala Val Asp Ile Glu Thr 130 135 140 Leu Tyr His
Glu Gly Glu Glu Phe Gly Lys Gly Pro Val Ile Met Ile 145 150 155 160
Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Ile 165
170 175 Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile
Lys 180 185 190 Arg Leu Ile Arg Val Ile Lys Glu Lys Asp Pro Asp Val
Ile Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu
Leu Lys Arg Ala Glu 210 215 220 Lys Leu Gly Ile Lys Leu Leu Leu Gly
Arg Asp Asn Ser Glu Pro Lys 225 230 235 240 Met Gln Lys Met Gly Asp
Ser Leu Ala Val Glu Ile Lys Gly Arg Ile 245 250 255 His Phe Asp Leu
Phe Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr
Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu 275 280 285
Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu Thr Gly Glu Gly 290
295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr
Tyr 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln
Leu Ala Arg Leu 325 330 335 Val Gly Gln Pro Val Trp Asp Val Ser Arg
Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys
Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu
Lys Glu Tyr Glu Arg Arg Leu Arg Glu Ser 370 375 380 Tyr Glu Gly Gly
Tyr Val Lys Glu Pro Glu Lys Gly Ala Phe Leu Glu 385 390 395 400 Arg
Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu Glu Ser 405 410
415 Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly Ala Phe
420 425 430 Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp
Leu Leu 435 440 445 Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg
Ala Pro Glu Pro 450 455 460 Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala
Arg Gly Leu Leu Ala Lys 465 470 475 480 Asp Leu Ser Val Leu Ala Leu
Arg Glu Gly Leu Gly Leu Pro Pro Gly 485 490 495 Asp Asp Pro Met Leu
Leu Ala Tyr Leu Leu Asp Pro Ser Asn Thr Thr 500 505 510 Pro Glu Gly
Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu Glu Ala 515 520 525 Gly
Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu Trp Gly 530 535
540 Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu Val Glu
545 550 555 560 Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr
Gly Val Arg 565 570 575 Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu
Glu Val Ala Glu Glu 580 585 590 Ile Ala Arg Leu Glu Ala Glu Val Phe
Arg Leu Ala Gly His Pro Phe 595 600 605 Asn Leu Asn Ser Arg Asp Gln
Leu Glu Arg Val Leu Phe Asp Glu Leu 610 615 620 Gly Leu Pro Ala Ile
Gly Lys Thr Glu Lys Thr Gly Lys Arg Ser Thr 625 630 635 640 Ser Ala
Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile Val Glu 645 650 655
Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr Tyr Ile 660
665 670 Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu His
Thr 675 680 685 Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
Ser Ser Asp 690 695 700 Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro
Leu Gly Gln Arg Ile 705 710 715 720 Arg Arg Ala Phe Ile Ala Glu Glu
Gly Trp Leu Leu Val Ala Leu Asp 725 730 735 Tyr Ser Gln Ile Glu Leu
Arg Val Leu Ala His Leu Ser Gly Asp Glu 740 745 750 Asn Leu Ile Arg
Val Phe Gln Glu Gly Arg Asp Ile His Thr Glu Thr 755 760 765 Ala Ser
Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met 770 775 780
Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly Met Ser 785
790 795 800 Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
Ala Gln 805 810 815 Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys
Val Arg Ala Trp 820 825 830 Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg
Arg Gly Tyr Val Glu Thr 835 840 845 Leu Phe Gly Arg Arg Arg Tyr Val
Pro Asp Leu Glu Ala Arg Val Lys 850 855 860 Ser Val Arg Glu Ala Ala
Glu Arg Met Ala Phe Asn Met Pro Val Gln 865 870 875 880 Gly Thr Ala
Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu Phe Pro 885 890 895 Arg
Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His Asp Glu 900 905
910 Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala Arg Leu
915 920 925 Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro
Leu Glu 930 935 940 Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala
Lys Glu 945 950 955 9958PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Pwo/Taq Chimeric polymerase
polypeptide 9Met Ile Leu Asp Val Asp Tyr Ile Thr Glu Glu Gly Lys
Pro Val Ile 1 5 10 15 Arg Leu Phe Lys Lys Glu Asn Gly Lys Phe Lys
Ile Glu His Asp Arg 20 25 30 Thr Phe Arg Pro Tyr Ile Tyr Ala Leu
Leu Arg Asp Asp Ser Lys Ile 35 40 45 Glu Glu Val Lys Lys Ile Thr
Gly Glu Arg His Gly Lys Ile Val Arg 50 55 60 Ile Val Asp Val Glu
Lys Val Glu Lys Lys Phe Leu Gly Lys Pro Ile 65 70 75 80 Thr Val Trp
Lys Leu Tyr Leu Glu His Pro Gln Asp Val Pro Thr Ile 85 90 95 Arg
Glu Lys Val Arg Glu His Pro Ala Val Val Asp Ile Phe Glu Tyr 100 105
110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125 Met Glu Gly Glu Glu Glu Leu Lys Ile Leu Ala Phe Asp Ile
Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro
Ile Ile Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Asn Glu Ala Lys
Val Ile Thr Trp Lys Asn Ile 165 170 175 Asp Leu Pro Tyr Val Glu Val
Val Ser Ser Glu Arg Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Ile
Ile Arg Glu Lys Asp Pro Asp Ile Ile Val Thr 195
200 205 Tyr Asn Gly Asp Ser Phe Asp Phe Pro Tyr Leu Ala Lys Arg Ala
Glu 210 215 220 Lys Leu Gly Ile Lys Leu Thr Ile Gly Arg Asp Gly Ser
Glu Pro Lys 225 230 235 240 Met Gln Arg Ile Gly Asp Met Thr Ala Val
Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr His Val Ile
Thr Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val
Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Ala
Asp Glu Ile Ala Lys Ala Trp Glu Ser Gly Glu Asn 290 295 300 Leu Glu
Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr 305 310 315
320 Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ile Gln Leu Ser Arg Leu
325 330 335 Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly
Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg
Asn Glu Val Ala 355 360 365 Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gln
Arg Arg Leu Arg Glu Ser 370 375 380 Tyr Thr Gly Gly Phe Val Lys Glu
Pro Glu Lys Gly Ala Phe Leu Glu 385 390 395 400 Arg Leu Glu Phe Gly
Ser Leu Leu His Glu Phe Gly Leu Leu Glu Ser 405 410 415 Pro Lys Ala
Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly Ala Phe 420 425 430 Val
Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp Leu Leu 435 440
445 Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro Glu Pro
450 455 460 Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu
Ala Lys 465 470 475 480 Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu
Gly Leu Pro Pro Gly 485 490 495 Asp Asp Pro Met Leu Leu Ala Tyr Leu
Leu Asp Pro Ser Asn Thr Thr 500 505 510 Pro Glu Gly Val Ala Arg Arg
Tyr Gly Gly Glu Trp Thr Glu Glu Ala 515 520 525 Gly Glu Arg Ala Ala
Leu Ser Glu Arg Leu Phe Ala Asn Leu Trp Gly 530 535 540 Arg Leu Glu
Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu Val Glu 545 550 555 560
Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly Val Arg 565
570 575 Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala Glu
Glu 580 585 590 Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly
His Pro Phe 595 600 605 Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val
Leu Phe Asp Glu Leu 610 615 620 Gly Leu Pro Ala Ile Gly Lys Thr Glu
Lys Thr Gly Lys Arg Ser Thr 625 630 635 640 Ser Ala Ala Val Leu Glu
Ala Leu Arg Glu Ala His Pro Ile Val Glu 645 650 655 Lys Ile Leu Gln
Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr Tyr Ile 660 665 670 Asp Pro
Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu His Thr 675 680 685
Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp 690
695 700 Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln Arg
Ile 705 710 715 720 Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu
Val Ala Leu Asp 725 730 735 Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala
His Leu Ser Gly Asp Glu 740 745 750 Asn Leu Ile Arg Val Phe Gln Glu
Gly Arg Asp Ile His Thr Glu Thr 755 760 765 Ala Ser Trp Met Phe Gly
Val Pro Arg Glu Ala Val Asp Pro Leu Met 770 775 780 Arg Arg Ala Ala
Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly Met Ser 785 790 795 800 Ala
His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu Ala Gln 805 810
815 Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg Ala Trp
820 825 830 Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val
Glu Thr 835 840 845 Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu
Ala Arg Val Lys 850 855 860 Ser Val Arg Glu Ala Ala Glu Arg Met Ala
Phe Asn Met Pro Val Gln 865 870 875 880 Gly Thr Ala Ala Asp Leu Met
Lys Leu Ala Met Val Lys Leu Phe Pro 885 890 895 Arg Leu Glu Glu Met
Gly Ala Arg Met Leu Leu Gln Val His Asp Glu 900 905 910 Leu Val Leu
Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala Arg Leu 915 920 925 Ala
Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro Leu Glu 930 935
940 Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 945 950
955 101060PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Sso/Taq Chimeric polymerase polypeptide 10Met Thr Lys Gln
Leu Thr Leu Phe Asp Ile Pro Ser Ser Lys Pro Ala 1 5 10 15 Lys Ser
Glu Gln Asn Thr Gln Gln Ser Gln Gln Ser Ala Pro Val Glu 20 25 30
Glu Lys Lys Val Val Arg Arg Glu Trp Leu Glu Glu Ala Gln Glu Asn 35
40 45 Lys Ile Tyr Phe Leu Leu Gln Val Asp Tyr Asp Gly Lys Lys Gly
Lys 50 55 60 Ala Val Cys Lys Leu Phe Asp Lys Glu Thr Gln Lys Ile
Tyr Ala Leu 65 70 75 80 Tyr Asp Asn Thr Gly His Lys Pro Tyr Phe Leu
Val Asp Leu Glu Pro 85 90 95 Asp Lys Val Gly Lys Ile Pro Lys Ile
Val Arg Asp Pro Ser Phe Asp 100 105 110 His Ile Glu Thr Val Ser Lys
Ile Asp Pro Tyr Thr Trp Asn Lys Phe 115 120 125 Lys Leu Thr Lys Ile
Val Val Arg Asp Pro His Ala Val Arg Arg Leu 130 135 140 Arg Asn Asp
Val Pro Lys Ala Tyr Glu Ala His Ile Lys Tyr Phe Asn 145 150 155 160
Asn Tyr Met Tyr Asp Ile Gly Leu Ile Pro Gly Met Pro Tyr Val Val 165
170 175 Lys Asn Gly Lys Leu Glu Ser Val Tyr Leu Ser Leu Asp Glu Lys
Asp 180 185 190 Val Glu Glu Ile Lys Lys Ala Phe Ala Asp Ser Asp Glu
Met Thr Arg 195 200 205 Gln Met Ala Val Asp Trp Leu Pro Ile Phe Glu
Thr Glu Ile Pro Lys 210 215 220 Ile Lys Arg Val Ala Ile Asp Ile Glu
Val Tyr Thr Pro Val Lys Gly 225 230 235 240 Arg Ile Pro Asp Ser Gln
Lys Ala Glu Phe Pro Ile Ile Ser Ile Ala 245 250 255 Leu Ala Gly Ser
Asp Gly Leu Lys Lys Val Leu Val Leu Asn Arg Asn 260 265 270 Asp Val
Asn Glu Gly Ser Val Lys Leu Asp Gly Ile Ser Val Glu Arg 275 280 285
Phe Asn Thr Glu Tyr Glu Leu Leu Gly Arg Phe Phe Asp Ile Leu Leu 290
295 300 Glu Tyr Pro Ile Val Leu Thr Phe Asn Gly Asp Asp Phe Asp Leu
Pro 305 310 315 320 Tyr Ile Tyr Phe Arg Ala Leu Lys Leu Gly Tyr Phe
Pro Glu Glu Ile 325 330 335 Pro Ile Asp Val Ala Gly Lys Asp Glu Ala
Lys Tyr Leu Ala Gly Leu 340 345 350 His Ile Asp Leu Tyr Lys Phe Phe
Phe Asn Lys Ala Val Arg Asn Tyr 355 360 365 Ala Phe Glu Gly Lys Tyr
Asn Glu Tyr Asn Leu Asp Ala Val Ala Lys 370 375 380 Ala Leu Leu Gly
Thr Ser Lys Val Lys Val Asp Thr Leu Ile Ser Phe 385 390 395 400 Leu
Asp Val Glu Lys Leu Ile Glu Tyr Asn Phe Arg Asp Ala Glu Ile 405 410
415 Thr Leu Gln Leu Thr Thr Phe Asn Asn Asp Leu Thr Met Lys Leu Ile
420 425 430 Val Leu Phe Ser Arg Ile Ser Arg Leu Gly Ile Glu Glu Leu
Thr Arg 435 440 445 Thr Glu Ile Ser Thr Trp Val Lys Asn Leu Tyr Tyr
Trp Glu His Arg 450 455 460 Lys Arg Asn Trp Leu Ile Pro Leu Lys Glu
Glu Ile Leu Ala Lys Ser 465 470 475 480 Ser Asn Ile Arg Thr Ser Ala
Leu Ile Lys Gly Lys Gly Tyr Lys Gly 485 490 495 Ala Val Val Ile Asp
Pro Pro Ala Gly Ile Phe Phe Leu Leu His Glu 500 505 510 Phe Gly Leu
Leu Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro 515 520 525 Pro
Pro Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro 530 535
540 Met Trp Ala Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val
545 550 555 560 His Arg Ala Pro Glu Pro Tyr Lys Ala Leu Arg Asp Leu
Lys Glu Ala 565 570 575 Arg Gly Leu Leu Ala Lys Asp Leu Ser Val Leu
Ala Leu Arg Glu Gly 580 585 590 Leu Gly Leu Pro Pro Gly Asp Asp Pro
Met Leu Leu Ala Tyr Leu Leu 595 600 605 Asp Pro Ser Asn Thr Thr Pro
Glu Gly Val Ala Arg Arg Tyr Gly Gly 610 615 620 Glu Trp Thr Glu Glu
Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu 625 630 635 640 Phe Ala
Asn Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp 645 650 655
Leu Tyr Arg Glu Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met 660
665 670 Glu Ala Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu
Ser 675 680 685 Leu Glu Val Ala Glu Glu Ile Ala Arg Leu Glu Ala Glu
Val Phe Arg 690 695 700 Leu Ala Gly His Pro Phe Asn Leu Asn Ser Arg
Asp Gln Leu Glu Arg 705 710 715 720 Val Leu Phe Asp Glu Leu Gly Leu
Pro Ala Ile Gly Lys Thr Glu Lys 725 730 735 Thr Gly Lys Arg Ser Thr
Ser Ala Ala Val Leu Glu Ala Leu Arg Glu 740 745 750 Ala His Pro Ile
Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys 755 760 765 Leu Lys
Ser Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg 770 775 780
Thr Gly Arg Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly 785
790 795 800 Arg Leu Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val
Arg Thr 805 810 815 Pro Leu Gly Gln Arg Ile Arg Arg Ala Phe Ile Ala
Glu Glu Gly Trp 820 825 830 Leu Leu Val Ala Leu Asp Tyr Ser Gln Ile
Glu Leu Arg Val Leu Ala 835 840 845 His Leu Ser Gly Asp Glu Asn Leu
Ile Arg Val Phe Gln Glu Gly Arg 850 855 860 Asp Ile His Thr Glu Thr
Ala Ser Trp Met Phe Gly Val Pro Arg Glu 865 870 875 880 Ala Val Asp
Pro Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly 885 890 895 Val
Leu Tyr Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile 900 905
910 Pro Tyr Glu Glu Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe
915 920 925 Pro Lys Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly
Arg Arg 930 935 940 Arg Gly Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg
Tyr Val Pro Asp 945 950 955 960 Leu Glu Ala Arg Val Lys Ser Val Arg
Glu Ala Ala Glu Arg Met Ala 965 970 975 Phe Asn Met Pro Val Gln Gly
Thr Ala Ala Asp Leu Met Lys Leu Ala 980 985 990 Met Val Lys Leu Phe
Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu 995 1000 1005 Leu Gln
Val His Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg 1010 1015 1020
Ala Glu Ala Val Ala Arg Leu Ala Lys Glu Val Met Glu Gly Val 1025
1030 1035 Tyr Pro Leu Ala Val Pro Leu Glu Val Glu Val Gly Ile Gly
Glu 1040 1045 1050 Asp Trp Leu Ser Ala Lys Glu 1055 1060
11983PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Pwo/Taq Chimeric polymerase polypeptide 11Met Ile Leu Asp
Val Asp Tyr Ile Thr Glu Glu Gly Lys Pro Val Ile 1 5 10 15 Arg Leu
Phe Lys Lys Glu Asn Gly Lys Phe Lys Ile Glu His Asp Arg 20 25 30
Thr Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Lys Ile 35
40 45 Glu Glu Val Lys Lys Ile Thr Gly Glu Arg His Gly Lys Ile Val
Arg 50 55 60 Ile Val Asp Val Glu Lys Val Glu Lys Lys Phe Leu Gly
Lys Pro Ile 65 70 75 80 Thr Val Trp Lys Leu Tyr Leu Glu His Pro Gln
Asp Val Pro Thr Ile 85 90 95 Arg Glu Lys Val Arg Glu His Pro Ala
Val Val Asp Ile Phe Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg
Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Glu Glu
Glu Leu Lys Ile Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His
Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile 145 150 155 160
Ser Tyr Ala Asp Glu Asn Glu Ala Lys Val Ile Thr Trp Lys Asn Ile 165
170 175 Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile
Lys 180 185 190 Arg Phe Leu Arg Ile Ile Arg Glu Lys Asp Pro Asp Ile
Ile Val Thr 195 200 205 Tyr Asn Gly Asp Ser Phe Asp Phe Pro Tyr Leu
Ala Lys Arg Ala Glu 210 215 220 Lys Leu Gly Ile Lys Leu Thr Ile Gly
Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Met Gln Arg Ile Gly Asp
Met Thr Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu
Tyr His Val Ile Thr Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr
Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu 275 280 285
Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu Ser Gly Glu Asn 290
295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Ala Thr
Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ile Gln
Leu Ser Arg Leu 325 330 335 Val Gly Gln Pro Leu Trp Asp Val Ser Arg
Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys
Ala Tyr Glu Arg Asn Glu Val Ala 355 360 365 Pro Asn Lys Pro Ser Glu
Glu Glu Tyr Gln Arg Arg Leu Arg Glu Ser 370 375 380 Tyr Thr Gly Gly
Phe Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn 385 390 395 400 Ile
Val Tyr Leu Asp Phe Arg Ala Leu Tyr Pro Ser Ile Ile Ile Thr 405 410
415 His Asn Val Ser Pro Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu
420 425 430 Leu His Glu Phe Gly Leu Leu Glu Ser Pro Lys Ala Leu Glu
Glu Ala 435 440 445 Pro Trp Pro Pro Pro Glu Gly Ala Phe Val Gly Phe
Val Leu
Ser Arg 450 455 460 Lys Glu Pro Met Trp Ala Asp Leu Leu Ala Leu Ala
Ala Ala Arg Gly 465 470 475 480 Gly Arg Val His Arg Ala Pro Glu Pro
Tyr Lys Ala Leu Arg Asp Leu 485 490 495 Lys Glu Ala Arg Gly Leu Leu
Ala Lys Asp Leu Ser Val Leu Ala Leu 500 505 510 Arg Glu Gly Leu Gly
Leu Pro Pro Gly Asp Asp Pro Met Leu Leu Ala 515 520 525 Tyr Leu Leu
Asp Pro Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg 530 535 540 Tyr
Gly Gly Glu Trp Thr Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser 545 550
555 560 Glu Arg Leu Phe Ala Asn Leu Trp Gly Arg Leu Glu Gly Glu Glu
Arg 565 570 575 Leu Leu Trp Leu Tyr Arg Glu Val Glu Arg Pro Leu Ser
Ala Val Leu 580 585 590 Ala His Met Glu Ala Thr Gly Val Arg Leu Asp
Val Ala Tyr Leu Arg 595 600 605 Ala Leu Ser Leu Glu Val Ala Glu Glu
Ile Ala Arg Leu Glu Ala Glu 610 615 620 Val Phe Arg Leu Ala Gly His
Pro Phe Asn Leu Asn Ser Arg Asp Gln 625 630 635 640 Leu Glu Arg Val
Leu Phe Asp Glu Leu Gly Leu Pro Ala Ile Gly Lys 645 650 655 Thr Glu
Lys Thr Gly Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala 660 665 670
Leu Arg Glu Ala His Pro Ile Val Glu Lys Ile Leu Gln Tyr Arg Glu 675
680 685 Leu Thr Lys Leu Lys Ser Thr Tyr Ile Asp Pro Leu Pro Asp Leu
Ile 690 695 700 His Pro Arg Thr Gly Arg Leu His Thr Arg Phe Asn Gln
Thr Ala Thr 705 710 715 720 Ala Thr Gly Arg Leu Ser Ser Ser Asp Pro
Asn Leu Gln Asn Ile Pro 725 730 735 Val Arg Thr Pro Leu Gly Gln Arg
Ile Arg Arg Ala Phe Ile Ala Glu 740 745 750 Glu Gly Trp Leu Leu Val
Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg 755 760 765 Val Leu Ala His
Leu Ser Gly Asp Glu Asn Leu Ile Arg Val Phe Gln 770 775 780 Glu Gly
Arg Asp Ile His Thr Glu Thr Ala Ser Trp Met Phe Gly Val 785 790 795
800 Pro Arg Glu Ala Val Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Ile
805 810 815 Asn Phe Gly Val Leu Tyr Gly Met Ser Ala His Arg Leu Ser
Gln Glu 820 825 830 Leu Ala Ile Pro Tyr Glu Glu Ala Gln Ala Phe Ile
Glu Arg Tyr Phe 835 840 845 Gln Ser Phe Pro Lys Val Arg Ala Trp Ile
Glu Lys Thr Leu Glu Glu 850 855 860 Gly Arg Arg Arg Gly Tyr Val Glu
Thr Leu Phe Gly Arg Arg Arg Tyr 865 870 875 880 Val Pro Asp Leu Glu
Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu 885 890 895 Arg Met Ala
Phe Asn Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met 900 905 910 Lys
Leu Ala Met Val Lys Leu Phe Pro Arg Leu Glu Glu Met Gly Ala 915 920
925 Arg Met Leu Leu Gln Val His Asp Glu Leu Val Leu Glu Ala Pro Lys
930 935 940 Glu Arg Ala Glu Ala Val Ala Arg Leu Ala Lys Glu Val Met
Glu Gly 945 950 955 960 Val Tyr Pro Leu Ala Val Pro Leu Glu Val Glu
Val Gly Ile Gly Glu 965 970 975 Asp Trp Leu Ser Ala Lys Glu 980
12958PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Pho/Tth Chimeric polymerase polypeptide 12Met Ile Leu Asp
Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile 1 5 10 15 Arg Ile
Phe Lys Lys Glu Asn Gly Glu Phe Lys Val Glu Tyr Asp Arg 20 25 30
Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Ala Ile 35
40 45 Asp Glu Ile Lys Lys Ile Thr Ala Gln Arg His Gly Lys Val Val
Arg 50 55 60 Ile Val Glu Thr Glu Lys Ile Gln Arg Lys Phe Leu Gly
Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Leu Glu His Pro Gln
Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Glu His Pro Ala
Val Val Asp Ile Phe Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg
Tyr Leu Ile Asp Lys Gly Leu Thr Pro 115 120 125 Met Glu Gly Asn Glu
Lys Leu Thr Phe Leu Ala Val Asp Ile Glu Thr 130 135 140 Leu Tyr His
Glu Gly Glu Glu Phe Gly Lys Gly Pro Val Ile Met Ile 145 150 155 160
Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Ile 165
170 175 Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile
Lys 180 185 190 Arg Leu Ile Arg Val Ile Lys Glu Lys Asp Pro Asp Val
Ile Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu
Leu Lys Arg Ala Glu 210 215 220 Lys Leu Gly Ile Lys Leu Leu Leu Gly
Arg Asp Asn Ser Glu Pro Lys 225 230 235 240 Met Gln Lys Met Gly Asp
Ser Leu Ala Val Glu Ile Lys Gly Arg Ile 245 250 255 His Phe Asp Leu
Phe Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr
Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu 275 280 285
Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu Thr Gly Glu Gly 290
295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr
Tyr 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln
Leu Ala Arg Leu 325 330 335 Val Gly Gln Pro Val Trp Asp Val Ser Arg
Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys
Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu
Lys Glu Tyr Glu Arg Arg Leu Arg Glu Ser 370 375 380 Tyr Glu Gly Gly
Tyr Val Lys Glu Pro Glu Lys Gly Ala Phe Leu Glu 385 390 395 400 Arg
Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu Glu Ala 405 410
415 Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly Ala Phe
420 425 430 Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp Ala Glu
Leu Lys 435 440 445 Ala Leu Ala Ala Cys Arg Asp Gly Arg Val His Arg
Ala Ala Asp Pro 450 455 460 Leu Ala Gly Leu Lys Asp Leu Lys Glu Val
Arg Gly Leu Leu Ala Lys 465 470 475 480 Asp Leu Ala Val Leu Ala Ser
Arg Glu Gly Leu Asp Leu Val Pro Gly 485 490 495 Asp Asp Pro Met Leu
Leu Ala Tyr Leu Leu Asp Pro Ser Asn Thr Thr 500 505 510 Pro Glu Gly
Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu Asp Ala 515 520 525 Ala
His Arg Ala Leu Leu Ser Glu Arg Leu His Arg Asn Leu Leu Lys 530 535
540 Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr His Glu Val Glu
545 550 555 560 Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala Thr
Gly Val Arg 565 570 575 Arg Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu
Glu Leu Ala Glu Glu 580 585 590 Ile Arg Arg Leu Glu Glu Glu Val Phe
Arg Leu Ala Gly His Pro Phe 595 600 605 Asn Leu Asn Ser Arg Asp Gln
Leu Glu Arg Val Leu Phe Asp Glu Leu 610 615 620 Arg Leu Pro Ala Leu
Gly Lys Thr Gln Lys Thr Gly Lys Arg Ser Thr 625 630 635 640 Ser Ala
Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile Val Glu 645 650 655
Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn Thr Tyr Val 660
665 670 Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly Arg Leu His
Thr 675 680 685 Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser
Ser Ser Asp 690 695 700 Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro
Leu Gly Gln Arg Ile 705 710 715 720 Arg Arg Ala Phe Val Ala Glu Ala
Gly Trp Ala Leu Val Ala Leu Asp 725 730 735 Tyr Ser Gln Ile Glu Leu
Arg Val Leu Ala His Leu Ser Gly Asp Glu 740 745 750 Asn Leu Ile Arg
Val Phe Gln Glu Gly Lys Asp Ile His Thr Gln Thr 755 760 765 Ala Ser
Trp Met Phe Gly Val Pro Pro Glu Ala Val Asp Pro Leu Met 770 775 780
Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu Tyr Gly Met Ser 785
790 795 800 Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
Ala Val 805 810 815 Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys
Val Arg Ala Trp 820 825 830 Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys
Arg Gly Tyr Val Glu Thr 835 840 845 Leu Phe Gly Arg Arg Arg Tyr Val
Pro Asp Leu Asn Ala Arg Val Lys 850 855 860 Ser Val Arg Glu Ala Ala
Glu Arg Met Ala Phe Asn Met Pro Val Gln 865 870 875 880 Gly Thr Ala
Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu Phe Pro 885 890 895 Arg
Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gln Val His Asp Glu 900 905
910 Leu Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu Glu Val Ala Ala Leu
915 920 925 Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala Val Pro
Leu Glu 930 935 940 Val Glu Val Gly Met Gly Glu Asp Trp Leu Ser Ala
Lys Gly 945 950 955 1330DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 13tttcccagtc acgacgttgt
aaaacgacgg 301419DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 14gcaccccgct tgggcagag
191520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15tcccgcccct cctggaagac 2016395PRTThermococcus
pacificus 16Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro
Val Ile 1 5 10 15 Arg Ile Phe Arg Lys Glu Lys Gly Glu Phe Lys Ile
Glu Tyr Asp Arg 20 25 30 Asn Phe Glu Pro Tyr Phe Tyr Ala Leu Leu
Lys Asp Asp Ser Ala Ile 35 40 45 Glu Asp Val Lys Lys Ile Thr Ala
Glu Arg His Gly Thr Thr Val Arg 50 55 60 Val Val Arg Ala Glu Lys
Val Lys Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys
Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp
Lys Ile Arg Glu His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115
120 125 Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu
Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile
Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val
Ile Thr Trp Lys Asn Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val Val
Ser Thr Glu Lys Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Val Ile
Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp
Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu 210 215 220 Lys Leu
Gly Val Lys Phe Ile Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235
240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile
245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu
Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly
Gln Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Thr Gln Ala
Trp Glu Thr Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser
Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu
Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Val Gly Gln
Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val
Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360
365 Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Glu Ser Tyr
370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly 385 390 395
17957PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Tpac/Taq Chimeric polymerase polypeptide 17Met Ile Leu
Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile 1 5 10 15 Arg
Ile Phe Arg Lys Glu Lys Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25
30 Asn Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile
35 40 45 Glu Asp Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Thr
Val Arg 50 55 60 Val Val Arg Ala Glu Lys Val Lys Lys Lys Phe Leu
Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro
Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Glu His Pro
Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys
Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asp
Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr
His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155
160 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile
165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met
Ile Lys 180 185 190 Arg Phe Leu Arg Val Ile Lys Glu Lys Asp Pro Asp
Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr
Leu Lys Lys Arg Ser Glu 210 215 220 Lys Leu Gly Val Lys Phe Ile Leu
Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly
Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp
Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr
Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Gln Pro Lys Glu 275 280
285 Lys Val Tyr Ala Glu Glu Ile Thr Gln Ala Trp Glu Thr Gly Glu Gly
290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val
Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Phe Pro Met Glu
Ala Gln Leu Ser Arg Leu 325 330 335 Val Gly Gln Ser Leu Trp Asp Val
Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu
Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro
Asp Glu Lys Glu Leu Ala Arg Arg Arg Glu Ser Tyr 370 375 380 Ala Gly
Gly Tyr Val Lys Glu Pro Glu Lys Gly Ala Phe Leu Glu Arg 385 390 395
400 Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu Glu Ser Pro
405 410 415 Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly Ala
Phe Val 420 425 430 Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala
Asp Leu Leu Ala 435 440 445 Leu Ala Ala Ala Arg Gly Gly Arg Val His
Arg Ala Pro Glu Pro Tyr 450 455 460 Lys Ala Leu Arg Asp Leu Lys Glu
Ala Arg Gly Leu Leu Ala Lys Asp 465 470 475 480 Leu Ser Val Leu Ala
Leu Arg Glu Gly Leu Gly Leu Pro Pro Gly Asp 485 490 495 Asp Pro Met
Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn Thr Thr Pro 500 505 510 Glu
Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu Glu Ala Gly 515 520
525 Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu Trp Gly Arg
530 535 540 Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu Val
Glu Arg 545 550 555 560 Pro Leu Ser Ala Val Leu Ala His Met Glu Ala
Thr Gly Val Arg Leu 565 570 575 Asp Val Ala Tyr Leu Arg Ala Leu Ser
Leu Glu Val Ala Glu Glu Ile 580 585 590 Ala Arg Leu Glu Ala Glu Val
Phe Arg Leu Ala Gly His Pro Phe Asn 595 600 605 Leu Asn Ser Arg Asp
Gln Leu Glu Arg Val Leu Phe Asp Glu Leu Gly 610 615 620 Leu Pro Ala
Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg Ser Thr Ser 625 630 635 640
Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile Val Glu Lys 645
650 655 Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr Tyr Ile
Asp 660 665 670 Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu
His Thr Arg 675 680 685 Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu
Ser Ser Ser Asp Pro 690 695 700 Asn Leu Gln Asn Ile Pro Val Arg Thr
Pro Leu Gly Gln Arg Ile Arg 705 710 715 720 Arg Ala Phe Ile Ala Glu
Glu Gly Trp Leu Leu Val Ala Leu Asp Tyr 725 730 735 Ser Gln Ile Glu
Leu Arg Val Leu Ala His Leu Ser Gly Asp Glu Asn 740 745 750 Leu Ile
Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr Glu Thr Ala 755 760 765
Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met Arg 770
775 780 Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly Met Ser
Ala 785 790 795 800 His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu
Glu Ala Gln Ala 805 810 815 Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro
Lys Val Arg Ala Trp Ile 820 825 830 Glu Lys Thr Leu Glu Glu Gly Arg
Arg Arg Gly Tyr Val Glu Thr Leu 835 840 845 Phe Gly Arg Arg Arg Tyr
Val Pro Asp Leu Glu Ala Arg Val Lys Ser 850 855 860 Val Arg Glu Ala
Ala Glu Arg Met Ala Phe Asn Met Pro Val Gln Gly 865 870 875 880 Thr
Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu Phe Pro Arg 885 890
895 Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His Asp Glu Leu
900 905 910 Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala Arg
Leu Ala 915 920 925 Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val
Pro Leu Glu Val 930 935 940 Glu Val Gly Ile Gly Glu Asp Trp Leu Ser
Ala Lys Glu 945 950 955
* * * * *