Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
1999-07-30
2001-02-13
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C435S070100, C435S252300, C435S325000, C536S022100, C530S350000
Reexamination Certificate
active
06187573
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of amplifying nucleic acid wherein DNA or RNA is amplified within a short reaction time and with a high fidelity, to a method of identifying nucleic acid utilizing said amplifying method and to a DNA polymerase and a reagent kit used for those methods.
PRIOR ART
Many studies have been made already for DNA polymerase of mesophilic microorganism such as
Escherichia coli
and for DNA polymerase derived from phages infectable by the mesophilic microorganisms. In addition, many studies have been also made already for heat stable DNA polymerases which are useful in a recombinant DNA technique by means of nucleic acid amplification such-as a polymerase chain reaction (PCR). Examples of the heat-stable polymerases which are used for the PCR are DNA polymerase (Tth polymerase) mostly derived from
Thermus thermophilus
and DNA polymerase (Taq polymerase) derived from
Thermus aguaticus
. Other known examples are DNA polymerase (Pfu polymerase) derived from
Pyrococcus furiosus
and DNA polymerase (Vent polymerase) derived from
Thermococcus litoralis.
PROBLEMS TO BE SOLVED BY THE INVENTION
However, with the Taq polymerase, fidelity and thermostability upon the synthesis of DNA are not sufficient. Although the Pfu polymerase exhibiting excellent fidelity and thermostability has been developed, said Pfu polymerase has some problems that its DNA extension rate is slow and a processivity is low whereby it has been used only for a specific PCR.
Recently, a PCR whereby 20 kb or more DNA is amplified (hereinafter, referred to as a long-PCR) has been developed. In said long-PCR, both Tag polymerase and Pfu polymerase are mixed whereby properties of both enzymes are utilized. However, when two enzymes having different properties are used in the same reaction system, some discrepancies might occur in their appropriate reaction conditions whereby there is a question whether the high extension rate and fidelity which are the advantages of each of those enzymes can be still maintained. Moreover, because of the difference in the thermostabilities and in the composition of the stock solutions of both enzymes, there is a question as to the stability when they are stored in the same container.
In view of the above, there has been a keen demand for novel thermostable polymerase which exhibits both of those advantages.
SUMMARY OF THE INVENTION
The present inventors have succeeded in preparing a thermostable DNA polymerase from a hyperthermophilic archaeon strain KOD1, and, when its properties are investigated, it has been found that s aid DNA polymerase exhibits the advantages of the above-mentioned two enzymes, i.e. high extension rate and high fidelity, whereby the present invention has been achieved.
Thus, the present invention relates to a method for amplifying a target nucleic acid comprising reacting the target nucleic acid with four kinds of DNTP and primer complementary to said target nucleic acid in a buffer solution which contains a thermostable DNA polymerase having a DNA extension rate of at least 30 bases/second and a 3′-5′ exonuclease activity such that the above mentioned primer is annealed to the target nucleic acid and an extention product is synthesized from the primer.
The present invention further relates to a method for amplifying a target nucleic acid in a sample wherein each target nucleic acid consists of two separate complementary strands which comprises the following steps A to D, characterized in that a thermostable DNA polymerase having a DNA extension rate of at least 30 bases/second and a 3′-5′ exonuclease activity is used as a thermostable DNA polymerase;
A: modifying the target nucleic acid, if necessary, to produce single-stranded nucleic acids;
B: reacting the single-stranded nucleic acids with four kinds of dNTP and primers, wherein said primers are selected so as to be sufficiently complementary to different strands of target nucleic acid to anneal therewith, in a buffer solution which contains a thermostable DNA polymerase such that the above mentioned primers are annealed to the single-stranded nucleic acids and extention products are synthesized from the primers,
C: separating the primer extention products from the templates on which they are synthesized to produce single-stranded nucleic acids; and
D: repeatedly conducting the above mentioned steps B and C.
The present invention further relates to a method for detecting a target nucleic acid in a sample wherein each target nucleic acid consists of two separate complementary strands which comprises the following steps A to E, characterized in that a thermostable DNA polymerase having a DNA extension rate of at least 30 bases/second and a 3′-5′ exonuclease activity is used as a thermostable DNA polymerase;
A: modifying the target nucleic acid, if necessary, to produce single-stranded nucleic acids;
B: reacting the single-stranded nucleic acids with four kinds of dNTP and primers, wherein said primers are selected so as to be sufficiently complementary to different strands of target nucleic acid to anneal therewith, in a buffer solution which contains a thermostable DNA polymerase such that the above mentioned primers are annealed to the single-stranded nucleic acids and extention products are synthesized from the primers,
C: separating the primer extention products from the templates on which they are synthesized to produce single-stranded nucleic acids;
D: repeatedly conducting the above mentioned steps B and C, and
E: detecting an amplified nucleic acid.
The present invention further relates to a reagent kit for amplifying target nucleic acid which comprises primers, wherein said primers are selected so as to be sufficiently complementary to different strands of target nucleic acid to anneal therewith, four kinds of dNTP, divalent cation, thermostable DNA polymerase having a DNA extension rate of at least 30 bases/second and a 3′-5′ exonuclease activity and buffer solution.
The present invention further relates to a reagent kit for detecting target nucleic acid which comprises primers, wherein said primers are selected so as to be sufficiently complementary to different strands of target nucleic acid to anneal therewith, four kinds of dNTP, divalent cation, thermostable DNA polymerase having a DNA extension rate of at least 30 bases/second and a 3′-5′ exonuclease activity, amplifying buffer solution, a probe capable of hybridizing with amplified nucleic acid and a detection buffer solution.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a thermostable DNA polymerase which is obtainable from a strain KOD1 which belongs to a hyperthermophilic archaeon strain.
The present invention relates to an isolated DNA comprising a nucleotide sequence that encodes the thermostable DNA polymerase derived from a KOD1 strain which belongs to hyperthermophilic archaon
The present invention further relates to a recombinant DNA expression vector that comprises the DNA sequence inserted into a vector, wherein the DNA sequence encodes the thermostable DNA polymerase derived from a KOD1 strain which belongs to hyperthermophilic archaeon.
The present invention further relates to a transformed recombinant host cell using a recombinant DNA expression vector that comprises the DNA sequence inserted into a vector, wherein the DNA sequence encodes the thermostable DNA polymerase derived from a KOD1 strain which belongs to hyperthermophilic archaeon.
The present invention relates to a method for producing a DNA polymerase obtainable from a KOD1 strain which belongs to hyperthermophilic archaeon, comprising culturing recombinant host cells which are transformed by a recombinant DNA expression vector that comprises the DNA sequence inserted into a vector, wherein the DNA sequence encodes the thermostable DNA polymerase derived from a KOD1 strain which belongs to hyperthermophilic archaeon, and recovering the produced thermostable DNA polymerase.
The present invention further relates to a method fo
Imanaka Tadayuki
Morikawa Masaaki
Takagi Masahiro
Jones W. Gary
Kenyon & Kenyon
Toyo Boseki Kabushiki Kaisha
Tung Joyce
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