Methods for nucleic acid manipulation

• US 8,673,561 B2
• Filed: 12/20/2012
• Issued: 03/18/2014
• Est. Priority Date: 04/20/2001
• Status: Expired due to Fees

• Current Assignee: Penn State Research Foundation (Pennsylvania State University)
• Original Assignee: Penn State Research Foundation (Pennsylvania State University)
• Inventors: Benkovic, Stephen J, Salinas, Frank
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US13/722,617
• Publication Number:
  US 20130137146A1
• Time in Patent Office: 453 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 method for replicating a target nucleic acid sequence comprising:

contacting said target nucleic acid sequence with two primers that are complementary to the ends of said target nucleic acid sequence, a polymerase, bacteriophage UvsX protein, and nucleotides in an amount sufficient to support amplification of said target nucleic acid sequence, said replicating being carried out at a temperature below about 45°

C.

2. The method according to claim 1 wherein said polymerase is a gene product of a bacteriophage.

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

4. The method according to claim 3 wherein said polymerase holoenzyme complex is a gene product of a viral, bacteriophage, prokaryotic, or eukaryotic system.

5. The method according to claim 4 wherein said polymerase holoenzyme complex comprises a polymerase enzyme, a clamp protein, and a clamp loader.

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

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

8. The method according to claim 7 wherein said replicating is carried out in the presence of a helicase accessory factor.

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

10. A method according to claim 1 wherein said replicating is carried out at about 37°

C.

11. A method for replicating a target nucleic acid sequence comprising:

contacting said target nucleic acid sequence with two primers that are complementary to the ends of said target nucleic acid sequence, a polymerase, bacteriophage UvsX protein, and nucleotides in an amount sufficient to support amplification of said target nucleic acid sequence, said replicating being carried out independent of using a thermal cycle.

12. A method according to claim 11, wherein said replicating is carried out at room temperature.

13. The method according to claim 11, wherein said replicating is carried out at about 37°

C.

23. The method according to claim 11 wherein said polymerase is a gene product of a bacteriophage.

24. The method according to claim 11 wherein said polymerase is a holoenzyme complex.

25. The method according to claim 11 wherein said replicating is carried out in the presence of a single stranded nucleic acid binding protein.

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

14. A method for replicating a target nucleic acid sequence comprising:

contacting said target nucleic acid sequence with two primers that are complementary to the ends of said target nucleic acid sequence, a polymerase, bacteriophage UvsX protein, and nucleotides in an amount sufficient to support amplification of said target nucleic acid sequence, said primers present in a molar excess relative to said target nucleic acid sequence.

27. The method according to claim 14 wherein said polymerase is a holoenzyme complex.

28. The method according to claim 14 wherein said replicating is carried out in the presence of a single stranded nucleic acid binding protein.

29. The method according to claim 14 wherein said replicating is carried out in the presence of an ATP regeneration system.

30. The method according to claim 14, wherein said replicating is carried out at about 37°

C.

15. A method for replicating a target nucleic acid sequence comprising:

contacting said target nucleic acid sequence with two primers that are complementary to the ends of said target nucleic acid sequence, a prokaryotic polymerase, bacteriophage UvsX protein, and nucleotides in an amount sufficient to support amplification of said target nucleic acid sequence.

16. The method according to claim 15 wherein said polymerase is a holoenzyme complex.

17. The method according to claim 16 wherein said polymerase holoenzyme complex comprises a polymerase enzyme, a clamp protein, and a clamp loader.

18. The method according to claim 15 wherein said replicating is carried out in the presence of a single stranded nucleic acid binding protein.

19. The method according to claim 15 wherein said replicating is carried out in the presence of a helicase.

20. The method according to claim 19 wherein said replicating is carried out in the presence of a helicase accessory factor.

21. The method according to claim 15 wherein said replicating is carried out in the presence of an ATP regeneration system.

22. A method according to claim 15 wherein said replicating is carried out at about 37°

C.