Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-28
2003-01-14
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S194000, C435S015000, C435S320100, C435S252300, C435S254110, C435S325000, C435S091100, C435S091200, C435S091500, C435S091510, C536S023100, C536S023200
Reexamination Certificate
active
06506560
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a substantially pure thermostable DNA polymerase. Specifically, the DNA polymerase of the present invention is a Thermotoga DNA polymerase and more specifically a
Thermotoga neapolitana
(Tne) DNA polymerase or
Thermotoga maritima
(Tma) DNA polymerase. Preferably, the polymerase has a molecular weight of about 100 kilodaltons. The present invention also relates to the cloning and expression of the Thermotoga DNA polymerase in
E. coli,
to DNA molecules containing the cloned gene, and to hosts which express said genes. The DNA polymerase of the present invention may be used in DNA sequencing, amplification reactions, and cDNA synthesis.
This invention also relates to mutants of the Thermotoga DNA polymerase, including Tne and Tma DNA polymerase. Specifically, the DNA polymerases of the present invention have mutations which substantially reduce 3′→5′ exonuclease activity; mutations resulting in the ability of the mutant DNA polymerase to incorporate dideoxynucleotides into a DNA molecule about as efficiently as deoxynucleotides; and mutations which substantially reduce 5′→3′ exonuclease activity. The Thermotoga (e.g., Tne and Tma) mutant DNA polymerase of this invention can have one or more of these properties. These DNA polymerase mutants may also be used in DNA sequencing, amplification reactions, and cDNA synthesis.
The present invention is also directed to novel mutants of other DNA polymerases which have substantially reduced 5′→3′ exonuclease activity.
BACKGROUND INFORMATION
DNA polymerases synthesize the formation of DNA molecules which are complementary to a DNA template. Upon hybridization of a primer to the single-stranded DNA template, polymerases synthesize DNA in the 5′ to 3′ direction, successively adding nucleotides to the 3′-hydroxyl group of the growing strand. Thus, in the presence of deoxyribonucleoside triphosphates (dNTPs) and a primer, a new DNA molecule, complementary to the single stranded DNA template, can be synthesized.
A number of DNA polymerases have been isolated from mesophilic microorganisms such as
E. coli.
A number of these mesophilic DNA polymerases have also been cloned. Lin et al. cloned and expressed T4 DNA polymerase in
E. coli
(
Proc. Natl. Acad. Sci. USA
84:7000-7004 (1987)). Tabor et al. (U.S. Pat. No. 4,795,699) describes a cloned T7 DNA polymerase, while Minkley et al. (
J. Biol. Chem.
259:10386-10392 (1984)) and Chatterjee (U.S. Pat. No. 5,047,342) described
E. coli
DNA polymerase I and the cloning of T5 DNA polymerase, respectively.
Although DNA polymerases from thermophiles are known, relatively little investigation has been done to isolate and even clone these enzymes. Chien et al.,
J. Bacteriol.
127:1550-1557 (1976) describe a purification scheme for obtaining a polymerase from
Thermus aquaticus
(Taq). The resulting protein had a molecular weight of about 63,000 daltons by gel filtration analysis and 68,000 daltons by sucrose gradient centrifugation. Kaledin et al.,
Biokhymiya
45:644-51 (1980) disclosed a purification procedure for isolating DNA polymerase from
T. aquaticus
YT1 strain. The purified enzyme was reported to be a 62,000 dalton monomeric protein. Gelfand et al. (U.S. Pat. No. 4,889,818) cloned a gene encoding a thermostable DNA polymerase from
Thermus aquaticus.
The molecular weight of this protein was found to be about 86,000 to 90,000 daltons.
Simpson et al. purified and partially characterized a thermostable DNA polymerase from a Thermotoga species (
Biochem. Cell. Biol.
86:1292-1296 (1990)). The purified DNA polymerase isolated by Simpson et al. exhibited a molecular weight of 85,000 daltons as determined by SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. The enzyme exhibited half-lives of 3 minutes at 95° C. and 60 minutes at 50° C. in the absence of substrate and its pH optimum was in the range of pH 7.5 to 8.0. Triton X-100 appeared to enhance the thermostability of this enzyme. The strain used to obtain the thermostable DNA polymerase described by Simpson et al. was Thermotoga species strain FjSS3-B.1 (Hussar et al.,
FEMS Microbiology Letters
37:121-127 (1986)). Others have cloned and sequenced a thermostable DNA polymerase from
Thermotoga maritima
(U.S. Pat. No. 5,374,553, which is expressly incorporated herein by reference).
Other DNA polymerases have been isolated from thermophilic bacteria including
Bacillus steraothermophilus
(Stenesh et al.,
Biochim. Biophys. Acta
272:156-166 (1972); and Kaboev et al.,
J. Bacteriol.
145:21-26 (1981)) and several archaebacterial species (Rossi et al.,
System. Appl. Microbiol.
7:337-341 (1986); Klimczak et al., Biochemistry 25:4850-4855 (1986); and Elie et al.,
Eur. J. Biochem.
178:619-626 (1989)). The most extensively purified archaebacterial DNA polymerase had a reported half-life of 15 minutes at 87° C. (Elie et al. (1989), supra). Innis et al., In
PCR Protocol: A Guide To Methods and Amplification,
Academic Press, Inc., San Diego (1990) noted that there are several extreme thermophilic eubacteria and archaebacteria that are capable of growth at very high temperatures (Bergquist et al.,
Biotech. Genet. Eng. Rev.
5:199-244 (1987); and Kelly et al.,
Biotechnol. Prog.
4:47-62 (1988)) and suggested that these organisms may contain very thermostable DNA polymerases.
In many of the known polymerases, the 5′→3′ exonuclease activity is present in the N-terminal region of the polymerase. (Ollis, et al.,
Nature
313:762-766 (1985); Freemont et al.,
Proteins
1:66-73 (1986); Joyce,
Cur. Opin. Struct. Biol.
1:123-129 (1991).) There are some ammo acids, the mutation of which are thought to impair the 5′→3′ exonuclease activity of
E. coli
DNA polymerase I. (Gutman & Minton,
Nucl. Acids Res.
21:4406-4407 (1993).) These amino acids include Tyr
77
, Gly
103
, Gly
184
, and Gly
192
in
E. coli
DNA polymerase I. It is known that the 5′-exonuclease domain is dispensable. The best known example is the Klenow fragment of
E. coli
polymerase I. The Klenow fragment is a natural proteolytic fragment devoid of 5′-exonuclease activity (Joyce et. al.,
J. Biol. Chem.
257:1958-64 (1990).) Polymerases lacking this activity are useful for DNA sequencing.
Most DNA polymerases also contain a 3′→5′ exonuclease activity. This exonuclease activity provides a proofreading ability to the DNA polymerase. A T5 DNA polymerase that lacks 3′→5′ exonuclease activity is disclosed in U.S. Pat. No. 5,270,179. Polymerases lacking this activity are particularly useful for DNA sequencing.
The polymerase active site, including the dNTP binding domain is usually present at the carboxyl terminal region of the polymerase (Ollis et al.,
Nature
313:762-766 (1985); Freemont et al.,
Proteins
1:66-73 (1986)). It has been shown that Phe
762
of
E. coli
polymerase I is one of the amino acids that directly interacts with the nucleotides (Joyce & Steitz,
Ann. Rev. Biochem.
63:777-822 (1994); Astatke,
J. Biol. Chem.
270:1945-54 (1995)). Converting this amino acid to a Tyr results in a mutant DNA polymerase that does not discriminate against dideoxynucleotides. See copending U.S. application Ser. No. 08/525,087, of Deb K. Chatterjee, filed Sep. 8, 1995, entitled “Mutant DNA Polymerases and the Use Thereof,” which is expressly incorporated herein by reference.
Thus, there exists a need in the art to develop more thermostable DNA polymerases. There also exists a need in the art to obtain wild type or mutant DNA polymerases that are devoid of exonuclease activities and are non-discriminating against dideoxynucleotides.
SUMMARY OF THE INVENTION
The present invention satisfies these needs in the art by providing additional DNA polymerases useful in molecular biology. Specifically, this invention includes a thermostable DNA polymerase. Preferably, the polymerase has a molecular weight of about 100 kilodaltons. Specifi
Chatterjee Deb K.
Hughes A. John
Hutson Richard G.
Invitrogen Corporation
Prouty Rebecca E.
Sterne Kessler Goldstein & Fox P.L.L.C.
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