Methods for nucleic acid manipulation

• US 8,759,000 B2
• Filed: 12/20/2012
• Issued: 06/24/2014
• Est. Priority Date: 04/20/2001
• Status: Expired due to Fees

• Current Assignee: Penn State Research Foundation
• Original Assignee: Penn State Research Foundation
• Inventors: Benkovic, Stephen J, Salinas, Frank
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US13/722,765
• Publication Number:
  US 20130143274A1
• Time in Patent Office: 551 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 composition for amplifying a target DNA sequence comprising bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA sequence, and a DNA polymerase.

2. The composition of claim 1, wherein said polymerase is a prokaryotic polymerase.

3. The composition of claim 1, wherein said primers are present in a molar excess relative to said target nucleic acid sequence.

4. The composition of claim 1, further comprising an ATP regeneration system.

5. The composition of claim 1, further comprising a single stranded nucleic acid binding protein.

6. The composition of claim 1, further comprising bacteriophage UvsY protein.

7. The composition of claim 1, further comprising nucleotides in an amount sufficient to support amplification of said target DNA sequence.

8. A composition for amplifying a target DNA sequence comprising bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA sequence, and a DNA polymerase having proofreading activity.

9. The composition of claim 8, wherein said polymerase is a prokaryotic polymerase.

10. The composition of claim 8, wherein said primers are present in a molar excess relative to said target nucleic acid sequence.

11. The composition of claim 8, further comprising an ATP regeneration system.

12. The composition of claim 8, further comprising a single stranded nucleic acid binding protein.

13. The composition of claim 8, further comprising bacteriophage UvsY protein.

14. The composition of claim 8, further comprising nucleotides in an amount sufficient to support amplification of said target DNA sequence.