Methods for nucleic acid manipulation

• US 8,129,116 B2
• Filed: 05/24/2010
• Issued: 03/06/2012
• 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:
  US12/785,974
• Publication Number:
  US 20100279357A1
• Time in Patent Office: 652 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 nucleic acid sequence comprising:

(a) bacteriophage T4 UvsX protein

(b) two primers that are complementary to the flanking ends of said target nucleic acid sequence, and

(c) a polymerase.

2. A composition according to claim 1, further comprising bacteriophage T4 UvsY protein.

3. A composition according to claim 1, wherein said polymerase is a polymerase holoenzyme.

4. A composition according to claim 3, wherein said polymerase holoenzyme comprises a polymerase enzyme, a clamp protein, and a clamp loader protein.

5. A composition according to claim 1, further comprising nucleotide triphosphates in amounts sufficient to permit amplification of said target nucleic acid sequence.

6. A composition according to claim 1, further comprising said target nucleic acid sequence.

7. A composition according to claim 1, further comprising bacteriophage T4 gene product 32 protein.

8. A composition according to claim 1, wherein said primers are present in a molar excess relative to said target nucleic acid sequence.

9. A method for amplifying a target nucleic acid comprising contacting said target nucleic acid sequence with the composition of claim 1.

10. A composition according to claim 1, further comprising a helicase.

11. A composition according to claim 10, wherein said helicase is bacteriophage T4 gene product 41 DNA helicase.

12. A composition according to claim 10, wherein said helicase is bacteriophage T4 replicative helicase complex, comprising bacteriophage T4 gene product 41 and gene product 59.

13. A composition for amplifying a target nucleic acid sequence comprising:

(a) bacteriophage T4 UvsX protein

(b) two primers that are complementary to the flanking ends of said target nucleic acid sequence

(c) a polymerase, and

(d) a helicase.