Patent application title: THERMOSTABLE CHIMERIC NUCLEIC ACID POLYMERASES AND USES THEREOF
Inventors:
Dirk Löffert (Dusseldorf, DE)
Dirk Löffert (Dusseldorf, DE)
Dirk Löffert (Dusseldorf, DE)
Andreas Missel (Dusseldorf, DE)
Jie Kang (Mettmann, DE)
Assignees:
Qiagen GMBH
IPC8 Class: AC12P1934FI
USPC Class:
435 912
Class name: Nucleotide polynucleotide (e.g., nucleic acid, oligonucleotide, etc.) acellular exponential or geometric amplification (e.g., pcr, etc.)
Publication date: 2014-06-05
Patent application number: 20140154748
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.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--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 α, SONDZEICHEN pol β, 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° 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® and Deep Vent SONDZEICHEN® 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® 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® 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® (exo.sup.-) and Deep Vent SONDZEICHEN® (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° C. or 95° 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° C.) after exposure to 95° C. for three minutes. More preferably, a thermostable chimeric nucleic acid polymerase is able to withstand one minute at 95° 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° C.) after one minute at 95° C. Even more preferably, a thermostable chimeric nucleic acid polymerase is able to withstand three minutes at 95° C. without significant loss in enzymatic activity. A most preferred thermostable chimeric nucleic acid polymerase is able to withstand ten minutes at 90° 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° 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° 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° 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° 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, α-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, α-lactalbumin, β-lactoglobulin, phosphorylase b, myosin, actin, β-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 μM); genomic DNA isolated from the appropriate microorganism containing the source thermostable polymerase; and 200 μM of each dNTP in a 1×PCR buffer (Qiagen, Valencia, Calif.). A 3-step PCR cycling program was run, consisting of an initial denaturation step at 94° 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® 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 μM of each dNTP in 1×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° C. using T4 DNA ligase and an appropriate buffer (Life Technologies, Gaithersburg, Md.) in a final volume of 20 μl.
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 DH5SONDZEICHENα competent cells. Aliquots of the transformation mixture were spread onto agar plates containing ampicillin and kanamycin (for M15[pRep4] cells), or ampicillin only (for DH5α competent cells), and incubated overnight at 37° 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® 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® 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® or MicrosepSONDZEICHEN® 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° 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° 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° 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 μM 30-mer oligonucleotide primer 5'-TTTCCCAGTCACGACGTTGTAAAACGACGG-3' (SEQ ID NO: 13), and 50 μM of each dNTP in 10 μl of 10 mM Tris HCl.
[0109] Polymerization reactions containing Taq DNA polymerase and a thermostable chimeric DNA polymerase were performed in 1×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 μg/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° C.; a 30 sec. annealing step at 55° C.; and a 3 min. polymerization step at 72° C. Heating of the reaction mixture to 94° 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° C.
[0112] Results of primer extension reactions at 72° C. were reproducible. After completing the reaction, reaction products were mixed with 1 μl gel loading solution (50% Glycerol, 1×TAE buffer, 0.02 mg/ml Bromphenol blue) and loaded on a 1% agarose gel containing 0.5 μg/ml ethidium bromide. The gel was run at 80 mA for 15 min in 1×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° C., followed by primer extension at 72° 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 μl gel loading solution (50% Glycerol, 1×TAE buffer, 0.02 mg/ml Bromphenol blue), and loaded on a 1% agarose gel containing 0.5 μg/ml ethidium bromide. The gel was run at 80 mA for 15 min in 1×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° 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° 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° 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° C. for 0, 3, 5, and 10 min. The results, quantified in Table 3 below, are representative of the 95° 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° 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 μM of the appropriate trial primers (wild type vs. mutant primers); 200 μM of each dNTP; 1× Qiagen PCR buffer (Qiagen, Valencia, Calif.) or 1×Pfu reaction buffer (Stratagene, La Jolla, Calif.) and 1.5 mM MgCl2 in a final reaction volume of 50 μl.
[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° C. followed by 40 cycles of a 30 sec. denaturation step 94° C.; a 30 sec. annealing step at 62° C.; and a 1 min. polymerization step at 72° C. for 1 min. The PCR concluded with a final prolonged extension step for 2 min. at 72° C.
[0124] PCR products were analyzed on a 2% agarose gel by gel electrophoresis (approximately 35 min. at 85 volts) in 1×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® Ladder (Life Technologies, Gaithersburg, Md., USA) as standard by gel densitometry. PCR products were purified using QIAquick® 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° C. Digestion products were analyzed on a 4% MetaphorSONDZEICHEN® 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° 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× buffer consisting of 50 mM TrisHCl (pH 8.9 at room temperature), 10 mM (NH4)2SO4, 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 μM of each primer, 200 μM of each dNTP, and a final MgCl2 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° C. for 3 min followed by a denaturation step at 94° C. for 30 sec, an annealing step at 60° C. for 30 sec, and a primer extension step at 72° 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° 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 Aug. 1997).
[0137] Bernard et al., Cell 59:219 (1989).
[0138] Eom et al., Nature 382: 278-281 (1996)
[0139] Flaman et al. N.A.R. 22:3259-3260 (1994).
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[0144] Gelfand et al., EP 0 892 058 A2 (20 Jan. 1999).
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[0146] Ho et al. U.S. Pat. No. 5,023,171 (1991).
[0147] Innes et al. U.S. Pat. No. 5,075,216 (1991).
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[0149] Jacobson et al., Eur. J. Biochem. 45:623 (1974).
[0150] Joyce and Grindley, PNAS USA 80:1830 (1983).
[0151] Joyce and Steitz, Annu. Rev. Biochem. 63:777-822 (1994).
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[0153] Klenow and Henningsen, PNAS USA 65:168 (1970).
[0154] Longley et al. N.A.R. 18:7317-7322 (1990).
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[0163] Silver et al. U.S. Pat. No. 5,104,792 (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
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