Methods for nucleic acid manipulation

• US 9,322,058 B2
• Filed: 05/02/2014
• Issued: 04/26/2016
• 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:
  US14/268,444
• Publication Number:
  US 20140234910A1
• Time in Patent Office: 725 Days
• US Class Current: 1/1
• 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 a bacteriophage UvsX protein, a DNA polymerase, two primers that bind to flanking ends of the target DNA sequence, and a buffer system compatible with said bacteriophage UvsX protein and the DNA polymerase.

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

3. The composition of claim 1, wherein said primers are present in a molar excess relative to said target DNA 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 a 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 a bacteriophage UvsX protein, a DNA polymerase having proofreading activity, two primers that bind to flanking ends of the target DNA sequence, and a buffer system compatible with said bacteriophage UvsX protein and the DNA polymerase.

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 DNA 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 a bacteriophage UvsY protein.

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