Methods for nucleic acid manipulation

• US 7,906,279 B2
• Filed: 06/15/2005
• Issued: 03/15/2011
• 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:
  US11/153,793
• Publication Number:
  US 20060024709A1
• Time in Patent Office: 2,099 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 reproducing and amplifying a target nucleic acid sequence within a nucleic acid duplex at a temperature below about 45°

C. comprising;

a) reacting said target nucleic acid sequence with two primers that are complementary to said nucleic acid within a nucleic acid duplex in the presence of bacteriophage T4 UvsX protein and bacteriophage UvsY protein without previously denaturing said nucleic acid duplex or said primers in whole or in part to form a recombination intermediate;

b) admixing said recombination intermediate with a polymerase to form a polymerase complex, whereby said polymerase replicates the target nucleic acid sequence.

2. The method of claim 1 wherein said two primers are pretreated with a single stranded nucleic acid binding protein.

3. The method of claim 1 wherein said two primers are pretreated with bacteriophage T4 UvsX protein prior to said reaction with said target sequence.

4. The method of claim 1 wherein said polymerase is bacteriophage T4 gene product 43 DNA polymerase.

5. The method of claim 1 wherein a helicase is used to facilitate replication by said polymerase.

6. The method of claim 5 where said helicase is bacteriophage T4 gene product 41 DNA helicase.

7. The method of claim 5 wherein said helicase is bacteriophage T4 replicative helicase complex, comprising bacteriophage T4 gp 41 and gp 59.

8. The method of claim 1 wherein a combination of a helicase and an accessory factor is used.

9. The method of claim 8 wherein said helicase is bacteriophage T4 gene product 41 and said accessory factor is bacteriophage T4 UvsY.

10. The method of claim 1 wherein said polymerase is a polymerase holoenzyme.

11. The method of claim 10 wherein said polymerase holoenzyme comprises a polymerase enzyme, a clamp protein, and a clamp loader protein.

12. The method of claim 11 wherein said polymerase enzyme, said clamp protein and said clamp loader are obtained from bacteriophage T4.

13. The method of claim 12 wherein said polymerase is bacteriophage T4 gene product 43 polymerase, said clamp protein is bacteriophage T4 gene product 45 clamp protein and said clamp loader is bacteriophage T4 gene product 44/gene product 62 clamp loader complex.

14. The method of claim 10 wherein said polymerase holoenzyme complex comprises a viral, bacteriophage, eukaryote, archeabacteria, or prokaryote polymerase holoenzyme complex.

15. The method of claim 14 wherein said polymerase holoenzyme complex includes E. coli DNA polymerase III holoenzyme.

16. The method of claim 14 wherein said eukaryote is yeast and said polymerase holoenzyme complex includes DNA polymerase delta.

17. The method of claim 14 wherein said eukaryote is yeast and said polymerase holoenzyme complex includes DNA polymerase epsilon.

18. The method of claim 2 wherein said single stranded binding protein is bacteriophage T4 gene product 32.

19. The method of claim 1 wherein a single stranded binding protein is used to destabilize the helix at or near the points of the primer template junctions.

20. A method for reproducing and amplifying a target nucleic acid sequence contained within a nucleic acid duplex at a temperature below about 45°

C. comprising reacting said target nucleic acid with two primers that are complementary to said target nucleic acid sequence in the presence of bacteriophage T4 UvsX protein, bacteriophage T4 UvsY protein, and a polymerase, without previously denaturing said target nucleic acid duplex or said primers whereby said polymerase replicates the target nucleic acid sequence.

21. The method of claim 20 carried out in the presence of bacteriophage T4 gene product 32 protein.

22. The method of claim 20 wherein said polymerase is a polymerase holoenzyme.

23. The method of claim 20 carried out in the presence of nucleotide triphosphates in amounts sufficient to permit amplification of said target nucleic acid sequence.

24. The method of claim 20 carried out in the presence of a helicase.

25. The method of claim 24 wherein said helicase is bacteriophage T4 gene product 41 DNA helicase.

26. The method of claim 24 wherein said helicase is bacteriophage T4 replicative helicase complex comprising bacteriophage T4 gene product 41 and gene product 59.