Methods for nucleic acid manipulation

• US 10,017,803 B2
• Filed: 03/15/2016
• Issued: 07/10/2018
• 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:
  US15/070,529
• Publication Number:
  US 20160237475A1
• Time in Patent Office: 847 Days
• 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 system for nucleic acid replication comprising:

(a) a nucleic acid duplex comprising a target nucleic acid sequence;

(b) two primers, each complementary to a flanking end of the target nucleic acid sequence, which primers have been pretreated with a UvsX recombination factor; and

(c) a DNA polymerase.

2. The system according to claim 1 wherein the primers have been further pretreated with a single stranded nucleic acid binding protein.

3. The system according to claim 2 wherein the single stranded nucleic acid binding protein comprises bacteriophage T4 gene product 32.

4. The system according to claim 1 further comprising a helicase.

5. The system according to claim 1 wherein said DNA polymerase comprises a holoenzyme complex.

6. The system according to claim 5 wherein the holoenzyme complex comprises a holoenzyme complex selected from the group consisting of a bacteriophage T4 holoenzyme complex, a prokaryotic DNA polymerase III complex, a eukaryotic DNA polymerase delta polymerase complex, and eukaryotic DNA polymerase epsilon polymerase complex.

7. The system according to claim 1 wherein the polymerase comprises bacteriophage T4 gene product 43 polymerase, wherein the clamp protein comprises bacteriophage T4 gene product 45 clamp protein, and wherein the clamp loader protein comprises bacteriophage T4 gene product 44/gene product 62 clamp loader protein complex.

8. The system according to claim 1 wherein the primers are present, in molar excess relative to the nucleic acid duplex.

9. The system according to claim 1 wherein the nucleic acid duplex comprises a linear duplex or a circular duplex.

10. The system according to claim 1 further comprising a reaction vessel.

11. The system according to claim 1 further comprising an accessory factor configured to stabilize the UvsX recombination factor.

12. The system according to claim 1 wherein the accessory factor comprises bacteriophage T4 UvsY.

13. The system according to claim 1 further comprising an ATP regeneration system.

14. The system according to claim 1 wherein the primers are pretreated at 37°

C.

15. The system according to claim 1 further comprising nucleoside triphosphates.

16. The system according to claim 1 further comprising a buffer.

17. The system according to claim 1 further comprising a means to visualize a replicated nucleic acid.

18. The system according to claim 17 wherein the means to visualize the replicated nucleic acid comprises a fluorescent molecule configured to interact with a replicated nucleic acid.

19. The system according to claim 1 further comprising a detector configured to detect a replicated nucleic acid.

20. The system according to claim 19 wherein the detector configured to detect the replicated nucleic acid comprises a fluorescent detector.

21. The system according to claim 1 further comprising a means to separate or purify a replicated nucleic acid.

22. The system according to claim 21 wherein the means to separate or purify a replicated nucleic is based on a size of the nucleic acid or a charge of the nucleic acid or a mixture of both size and charge of the nucleic acid.