Method for synthesizing DNA

• US 20090098613A1
• Filed: 10/15/2008
• Published: 04/16/2009
• Est. Priority Date: 04/23/1998
• Status: Active Grant

• Current Assignee: Hiroyuki Mukai, Ikunoshin Kato, Kazue Miyake, Kiyozo Asada, Osamu Takeda, Takashi Uemori, Tomoko Fujita, Yoshimi Sato
• Original Assignee: Hiroyuki Mukai, Ikunoshin Kato, Kazue Miyake, Kiyozo Asada, Osamu Takeda, Takashi Uemori, Tomoko Fujita, Yoshimi Sato
• Inventors: Sato, Yoshimi, Mukai, Hiroyuki, Takeda, Osamu, Asada, Kiyozo, Fujita, Tomoko, Miyake, Kazue, Kato, Ikunoshin, Uemori, Takashi
• Granted Patent: US 8,367,328 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: Y10S 435/975 Kit

Abstract

A DNA synthesis reaction-enhancer comprising at least one kind selected from the group consisting of acidic substances and cationic complexes; a DNA synthesis method in which during a DNA synthesis reaction a reaction is carried out in the presence of the above enhancer by using DNA polymerase; a DNA synthesis reaction composition comprising the above enhancer; a DNA synthesis reaction composition comprising two or more kinds of DNA polymerases each having 3′→5′ exonuclease activity; a DNA synthesis method in which during a DNA synthesis reaction two or more kinds of DNA polymerases each having 3′→5′ exonuclease activity are used; a kit for use in in vitro DNA synthesis, comprising two or more kinds of DNA polymerases each having 3′→5′ exonuclease activity; and a kit for use in in vitro DNA synthesis, wherein the kit comprises the DNA synthesis reaction-enhancer and DNA polymerase. According to the present invention, DNA synthesis can be carried out at an efficiency more excellent as compared to conventional DNA synthesis reaction.

Claims

1. A DNA synthesis reaction composition comprising:

1) a DNA polymerase having 3′

→

5′

exonuclease activity;

2) water-soluble acidic macromolecular substances selected from the group consisting of carrageenan, rhamnan sulfate, chondroitin sulfate, dermatan sulfate, hyaluronic acid, pectin, polyvinyl sulfates, polystyrene sulfates, polyglutamic acids, polyacrylic acids, and water-soluble salts thereof; and

3) components necessary for DNA synthesis using DNA polymerase.

2. The DNA synthesis reaction composition according to claim 1, wherein the composition comprises two or more kinds of DNA polymerases.

3. The DNA synthesis reaction composition according to claim 2, wherein the composition comprises one DNA polymerase having 3′

→

5′

exonuclease activity, and one DNA polymerase having no 3′

→

5′

exonuclease activity.

4. A method for synthesizing DNA comprising steps of:

a) preparing a reaction mixture by using the DNA synthesis composition of any one of claims 1 to 3; and

b) reacting a DNA synthesis.

5. A kit for use in in vitro DNA synthesis, comprising:

1) DNA polymerases having 3′

→

5′

exonuclease activity;

2) water-soluble acidic macromolecular substances selected from the group consisting of carrageenan, rhamnan sulfate, chondroitin sulfate, dermatan sulfate, hyaluronic acid, pectin, polyvinyl sulfates, polystyrene sulfates, polyglutamic acids, polyacrylic acids, and water-soluble salts thereof; and

3) components necessary for DNA synthesis using DNA polymerase.

6. The kit according to claim 5, wherein the kit comprises two or more kinds of DNA polymerases.

7. The kit according to claim 6, wherein the kit comprises one DNA polymerase having 3′

→

5′

exonuclease activity, and one DNA polymerase having no 3′

→

5′

exonuclease activity.