Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-15
2001-12-25
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100, C536S023100, C424S094100
Reexamination Certificate
active
06333158
ABSTRACT:
TECHNICAL FIELD
The present invention relates a DNA polymerase-associated factor. More specifically, the present invention relates to a DNA polymerase-associated factor which is useful for a reagent for genetic engineering and a method for producing the same, and further a gene encoding thereof, and the like.
BACKGROUND ART
DNA polymerases are useful enzymes for reagents for genetic engineering, and the DNA polymerases are widely used for nucleotide sequencing of DNA, DNA labeling, site-directed mutagenesis, and the like. Also, thermostable DNA polymerases have recently been remarked with the development of the polymerase chain reaction (PCR) method, and various DNA polymerases suitable for the PCR method have been developed and commercialized.
Presently known DNA polymerases can be roughly classified into four families according to amino acid sequence homologies, among which family A (pol I type enzymes) and family B (&agr; type enzymes) account for the great majority. Although DNA polymerases belonging to each family generally possess mutually similar biochemical properties, detailed comparison reveals that individual enzymes differ from each other in terms of substrate specificity, incorporation efficiency of a substrate analog, primer extensibility and extension rate, mode of DNA synthesis, association of exonuclease activity, optimum reaction conditions of temperature, pH and the like, and sensitivity to inhibitors. Therefore, those possessing most appropriate properties for the applications have been selected among all available DNA polymerases, and the selected DNA polymerase has been used.
A hyperthermophilic archaebacterium
Pyrococcus furiosus
has produced a DNA polymerase belonging to &agr; type, and its gene has already been isolated [
Nucleic Acids Research,
21, 259-265 (1993)].
As DNA polymerases, in addition to ones expressing their functions with only one kind of an enzyme protein, such as the pol I type enzyme or the &agr; type enzyme, there have been known oligomer enzymes constituted by a large number of subunit proteins. In addition to the protein serving as a DNA polymerase, there have also been known some cases where protein molecules for regulating their functions coexist.
DISCLOSURE OF INVENTION
An object of the present invention is to provide a thermostable DNA polymerase-associated factor capable of enhancing DNA synthesizing-activity of a DNA polymerase, and a thermostable DNA polymerase-associated factor possessing an activity of binding to a DNA polymerase.
Another object of the present invention is to provide a gene for the DNA polymerase-associated factor of the present invention.
Still another object of the present invention is to provide a method for producing the DNA polymerase-associated factor of the present invention.
Still another object of the present invention is to provide a method of DNA synthesis by using a DNA polymerase in the presence of the DNA polymerase-associated factor of the present invention.
Still another object of the present invention is to provide a kit comprising the DNA polymerase-associated factor of the present invention.
According to the present invention, there can be provided in vitro DNA synthesis and a DNA amplification system which are more excellent than conventional techniques by utilizing the DNA polymerase-associated factor of the present invention.
Recently, a novel DNA polymerase having completely no structural homology to conventionally known DNA polymerases has been found by the present inventors from hyperthermophilic archaebacterium
Pyrococcus furiosus
(WO 97/24444 Pamphlet). In this DNA polymerase, two kinds of novel proteins form a complex and exhibit a DNA polymerase activity. In addition, the enzyme exhibits a potent 3′→5′ exonuclease activity and excellent primer extension activity. For example, when the enzyme is used for PCR, a DNA fragment of the size of about 20 kb can be amplified. In this novel DNA polymerase derived from
Pyrococcus furiosus,
although at least two kinds of proteins are essential constituents in the enzyme activity, it has not been elucidated whether or not a constituent protein of the enzyme beside the above exists, or whether or not a factor having an influence on the activity of the enzyme exists.
As a result of intensive studies, the present inventors have succeeded in isolating a protein binding to the novel DNA polymerase derived from
Pyrococcus furiosus.
Further, they have found that the production of the protein by genetic engineering is made possible by cloning the gene, and moreover that a DNA synthesizing-activity of a DNA polymerase is enhanced.
In sum, the present invention relates to:
[1] a thermostable DNA polymerase-associated factor capable of enhancing DNA synthesizing-activity of a DNA polymerase;
[2] the DNA polymerase-associated factor according to item [1] above, further possessing an activity of binding to a DNA polymerase;
[3] the DNA polymerase-associated factor according to item [2] above, which possesses an activity of binding to a DNA polymerase comprising a DNA polymerase-constituting protein having the amino acid sequence as shown in SEQ ID NO: 5 or 6 in Sequence Listing;
[4] the DNA polymerase-associated factor according to any one of items [1] to [3] above, comprising at least one of amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 3, 19, 27, 34, 64, 70 and 80 in Sequence Listing, or an amino acid sequence resulting from substitution, deletion, addition or insertion of one or more amino acids in at least one of the amino acid sequences;
[5] a gene encoding a DNA polymerase-associated factor, wherein the factor comprises at least one of amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 3, 19, 27, 34, 64, 70 and 80 in Sequence Listing, or an amino acid sequence resulting from substitution, deletion, addition or insertion of one or more amino acids in at least one of amino acid sequences, and possesses an activity of enhancing DNA synthesizing-activity of a DNA polymerase;
[6] the gene according to item [5] above, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 18, 26, 33, 63, 69 and 79, or a nucleotide sequence resulting from substitution, deletion, addition or insertion of one or more bases in the nucleotide sequence;
[7] a gene capable of hybridizing to the gene of item [5] or [6] above, and encoding a DNA polymerase-associated factor possessing an activity of enhancing DNA synthesizing-activity of a DNA polymerase;
[8] a method for producing a DNA polymerase-associated factor, characterized in that the method comprises culturing a transformant harboring the gene of any one of items [5] to [7] above, and collecting a thermostable DNA polymerase-associated factor capable of enhancing DNA synthesizing-activity of a DNA polymerase from the cultured medium;
[9] a method of DNA synthesis by using a DNA polymerase, characterized in that DNA is synthesized in the presence of the DNA polymerase-associated factor of any one of items [1] to [4] above;
[10] the method of DNA synthesis according to item [9] above, wherein DNA is synthesized in the presence of two or more kinds of DNA polymerase-associated factors;
[11] the method of DNA synthesis according to item [10] above, wherein DNA is synthesized in the presence of F7, PFU-RFC and PFU-RFCLS as a DNA polymerase-associated factor;
[12] the method of DNA synthesis according to any one of items [9] to [11] above, wherein the DNA polymerase is a thermostable DNA polymerase;
[13] the method of DNA synthesis according to item [12] above, wherein the synthesis is carried out by PCR method;
[14] a kit usable for in vitro DNA synthesis, comprising the DNA polymerase-associated factor of any
Asada Kiyozo
Fujita Tomoko
Kato Ikunoshin
Miyake Kazue
Mukai Hiroyuki
Birch & Stewart Kolasch & Birch, LLP
Chakrabarti Arun
Fredman Jeffrey
Takara Shuzo Co. Ltd.
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