Methods for nucleic acid manipulation

• US 10,000,781 B2
• Filed: 03/15/2016
• Issued: 06/19/2018
• 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:
  US15/070,401
• Publication Number:
  US 20160265015A1
• Time in Patent Office: 826 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 method for amplifying a target DNA, said method comprising the steps:

i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;

ii) maintaining the reaction mixture at a temperature below about 45°

C., wherein amplified target DNA is produced;

iii) and detecting the presence of amplified target DNA.

2. The method of claim 1, wherein said DNA polymerase is a non-thermostable polymerase.

3. The method of claim 1, wherein said DNA polymerase is a prokaryotic polymerase.

4. The method of claim 1, wherein said DNA polymerase is a polymerase holoenzyme.

5. The method of claim 1, wherein said reaction mixture further comprises bacteriophage UvsY protein, bacteriophage gene product 32 protein, or both.

6. The method of claim 1, wherein said reaction mixture further comprises a helicase.

7. The method of claim 1, wherein said reaction mixture further comprises an ATP regeneration system.

8. A method for amplifying a target DNA, said method comprising the steps:

i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, bacteriophage UvsY protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;

ii) maintaining the reaction mixture at a temperature below about 45°

C., wherein amplified target DNA is produced;

iii) and detecting the presence of amplified target DNA.

9. The method of claim 8, wherein said DNA polymerase is a non-thermostable polymerase.

10. The method of claim 8, wherein said DNA polymerase is a prokaryotic polymerase.

11. The method of claim 8, wherein said DNA polymerase is a polymerase holoenzyme.

12. The method of claim 8, wherein said reaction mixture further comprises bacteriophage gene product 32 protein.

13. The method of claim 8, wherein said reaction mixture further comprises a helicase.

14. The method of claim 8, wherein said reaction mixture further comprises an ATP regeneration system.

15. A method for amplifying a target DNA, said method comprising the steps:

i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;

ii) maintaining the reaction mixture at room temperature, wherein amplified target DNA is produced;

iii) and detecting the presence of amplified target DNA.

16. The method of claim 15, wherein said DNA polymerase is a non-thermostable polymerase.

17. The method of claim 15, wherein said DNA polymerase is a prokaryotic polymerase.

18. The method of claim 15, wherein said DNA polymerase is a polymerase holoenzyme.

19. The method of claim 15, wherein said reaction mixture further comprises bacteriophage UvsY protein, bacteriophage gene product 32 protein, or both.

20. The method of claim 15, wherein said reaction mixture further comprises a helicase, an ATP regeneration system, or both.