Method for synthesizing DNA

• US 7,521,178 B1
• Filed: 04/21/1999
• Issued: 04/21/2009
• Est. Priority Date: 04/23/1998
• Status: Expired due to Fees

• Current Assignee: Takara Bio Incorporated (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Takara Bio Incorporated (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Mukai, Hiroyuki, Takeda, Osamu, Fujita, Tomoko, Miyake, Kazue, Sato, Yoshimi, Kato, Ikunoshin, Uemori, Takashi, Asada, Kiyozo
• Primary Examiner(s): Strzelecka; Teresa E
• Application Number:
  US09/673,884
• Time in Patent Office: 3,653 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536 243- 33, 536/23.2, 536/23.5
• US Class Current: 435/6.12
• 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;

2) water-soluble acidic macromolecular substances or water-soluble salts thereof, wherein said water-soluble acidic macromolecular substances are one or more substances selected from the group consisting of sulfated-fucose-containing polysaccharide F, sulfated-fucose-containing polysaccharide-U, and alginic acid; and

3) components necessary for DNA synthesis using DNA polymerase.

7. The DNA synthesis reaction composition according to claim 1, wherein said water-soluble acid macromolecular substances or water-soluble salts thereof are present in the composition at about 0.1 ng to about 5 μ

g, and wherein the composition is about 50 μ

l in total volume.

8. The DNA synthesis reaction composition according to claim 1, wherein said DNA polymerase is selected from the group consisting of:

pol I-type DNA polymerase, E. coli DNA polymerase I, Klenow fragment, Thermococcus aquaticus-derived DNA polymerase, α

-type DNA polymerase, α

-type Pyrococcus furiosus-derived DNA polymerase, Thermococcus litralis-derived DNA polymerase and Pyrococcus sp.-derived DNA polymerase.

9. The DNA synthesis reaction composition according to claim 1, wherein said DNA polymerase is selected from the group consisting of:

E. coli DNA polymerase I, Klenow fragment, Taq DNA polymerase, VENT DNA polymerase, Pyrobest DNA polymerase, Pfu DNA polymerase I, Pfu DNA polymerase II, Ex-Taq DNA polymerase, KOD dash DNA polymerase, DEEP VENT DNA polymerase, KOD DNA polymerase and LA-Taq DNA polymerase.

11. A method of enhancing DNA synthesis which comprises:

incubating the synthesis reaction composition of claim 1 in the presence of a nucleic acid to be amplified.

2. A DNA synthesis reaction composition comprising:

1) two or more kinds of DNA polymerases;

2) water-soluble acidic macromolecular substances or water-soluble salts thereof, wherein said water-soluble acidic macromolecular substances are one or more substances selected from the group consisting of sulfated-fucose-containing polysaccharide-F, sulfated-fucose-containing polysaccharide-U, and alginic acid; and

3) components necessary for DNA synthesis using DNA polymerase,

wherein the two or more kinds of DNA polymerases comprise a DNA polymerase having 3′

→

5′

exonuclease activity, and a DNA polymerase having no 3′

→

5′

exonuclease activity.

10. The DNA synthesis reaction composition according to claim 2, wherein said two or more kinds of DNA polymerases are selected from the group consisting of:

pol I-type DNA polymerase, E. coli DNA polymerase I, Klenow fragment, Thermococcus aquaticus-derived DNA polymerase, α

-type DNA polymerase, α

-type Pyrococcus furiosus-derived DNA polymerase. Thermococcus litralis-derived DNA polymerase and Pyrococcus sp.-derived DNA polymerase.

12. A method of enhancing DNA synthesis which comprises:

incubating the synthesis reaction composition of claim 2 in the presence of a nucleic acid to be amplified.

3. A kit for use in in vitro DNA synthesis, wherein the kit comprises:

1) a DNA polymerase;

2) a reaction buffer comprising water-soluble acidic macromolecular substances or water-soluble salts thereof, wherein said water-soluble acidic macromolecular substances are one or more substances selected from the group consisting of sulfated-fucose-containing polysaccharide-F, sulfated-fucose-containing polysaccharide-U, and alginic acid; and

3) dNTP, wherein N is a mixture of adenine, thymine, guanine and cytosine.

4. The kit according to claim 3, wherein said DNA polymerase is a thermostable DNA polymerase.

5. A kit for use in in vitro DNA synthesis, wherein the kit comprises:

1) two or more kinds of DNA polymerases, wherein the two or more kinds of DNA polymerases comprise a DNA polymerase having 3′

→

5′

exonuclease activity, and a DNA polymerase having no 3′

→

5′

exonuclease activity

2) a reaction buffer comprising water-soluble acidic macromolecular substances or water-soluble salts thereof, wherein said water-soluble acidic macromolecular substances are one or more substances selected from the group consisting of sulfated-fucose-containing polysaccharide-F, sulfated-fucose-containing polysaccharide-U, and alginic acid; and

3) dNTP, wherein N is a mixture of adenine, thymine guanine and cytosine.

6. The kit according to claim 5, wherein at least one of said two or more kinds of DNA polymerases is thermostable.