Methods for nucleic acid manipulation

• US 7,723,031 B2
• Filed: 08/15/2005
• Issued: 05/25/2010
• Est. Priority Date: 04/20/2001
• Status: Expired due to Fees

• Current Assignee: Penn State Research Foundation
• Original Assignee: The Thepenn State Research Foundation
• Inventors: Salinas, Frank, Benkovic, Stephen J.
• Primary Examiner(s): Strzelecka; Teresa E
• Application Number:
  US11/204,125
• Publication Number:
  US 20060292582A1
• Time in Patent Office: 1,744 Days
• Field of Search: None
• US Class Current: 435/5
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2521/513 Winding/unwinding enzyme, e.g. helicase
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

A method for replicating and amplifying a target nucleic acid sequence is described. A method of the invention involves the formation of a recombination intermediate without the prior denaturing of a nucleic acid duplex through the use of a recombination factor. The recombination intermediate is treated with a high fidelity polymerase to permit the replication and amplification of the target nucleic acid sequence. In preferred embodiments, the polymerase comprises a polymerase holoenzyme. In further preferred embodiments, the recombination factor is bacteriophage T4 UvsX protein or homologs from other species, and the polymerase holoenzyme comprises a polymerase enzyme, a clamp protein and a clamp loader protein, derived from viral, bacteriophage, prokaryotic, archaebacterial, or eukaryotic systems.

Claims

1. A method for amplifying a target nucleic acid sequence comprising reacting said nucleic acid with two primers that are complementary to the flanking ends of said target nucleic acid sequence within a nucleic acid duplex, said reaction being carried out in the presence of a bacteriophage UvsX protein, bacteriophage UvsY protein, and a DNA polymerase having a proofreading/editing function, without previously denaturing said nucleic acid duplex, wherein said nucleic acid, primers and bacteriophase UvsX protein form a recombination intermediate and said polymerase combines with said recombination intermediate to form a polymerase complex.

2. The method according to claim 1 wherein said DNA polymerase is a gene product of a viral, bacteriophage, prokaryotic, or eukaryotic system.

3. The method according to claim 2 wherein said DNA polymerase is a gene product of bacteriophage T4.

4. The method according to claim 1 wherein said DNA polymerase is a holoenzyme complex.

5. The method according to claim 4 wherein said DNA polymerase holoenzyme complex is a gene product of a viral, bacteriophage, prokaryotic, or eukaryotic system.

6. The method according to claim 5 wherein said DNA polymerase holoenzyme complex comprises a polymerase enzyme, a clamp protein, and a clamp loader.

7. The method according to claim 5 wherein said DNA polymerase is a gene product of bacteriophage T4.

8. The method according to claim 1 wherein said method is carried out in the presence of a single stranded nucleic acid binding protein.

9. The method according to claim 1 wherein said method is carried out in the presence of a helicase.

10. The method according to claim 9 wherein said method is carried out in the presence of a helicase accessory factor.

11. The method according to claim 1 wherein said method is carried out in the presence of an ATP regeneration system.