Methods for nucleic acid manipulation

• US 8,124,342 B2
• Filed: 05/24/2010
• Issued: 02/28/2012
• Est. Priority Date: 04/20/2001
• Status: Expired due to Fees

• Current Assignee: Penn State Research Foundation (Pennsylvania State University)
• Original Assignee: The PANN State Research Foundation
• Inventors: Benkovic, Stephen J., Salinas, Frank
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US12/786,033
• Publication Number:
  US 20100285481A1
• Time in Patent Office: 645 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 of determining the presence of a target nucleic acid sequence in a sample comprising contacting said sample with a polymerase, bacteriophage T4 UvsX protein, and two primers that are complementary to the flanking ends of said target nucleic acid sequence;

and detecting the presence of amplified target nucleic acid sequence.

2. The method of claim 1, carried out at a constant temperature.

3. The method of claim 1, carried out at a temperature below about 45°

C.

4. The method of claim 1, carried out in the presence of bacteriophage T4 UvsY protein.

5. The method of claim 1, carried out in the presence of bacteriophage T4 gene product 32 protein.

6. The method of claim 1, wherein said primers are present in a molar excess relative to said target nucleic acid sequence.

7. The method of claim 1, carried out in the presence of a helicase.

8. The method of claim 7, wherein said helicase is a bacteriophage T4 gene product 41 DNA helicase.

9. The method of claim 7, wherein said helicase if bacteriophage T4 replicative helicase complex, comprising bacteriophage T4 gene product and gene product 59.

10. The method of claim 1, carried out in the presence of nucleotide triphosphates in amounts sufficient to permit amplification of said target nucleic acid sequence.

11. The method of claim 1, carried out at a temperature of about 37°

C.

12. A method of determining the presence of a target nucleic acid sequence in a sample comprising contacting said sample with a polymerase, bacteriophage T4 UvsX protein, and two primers that are complementary to the flanking ends of said target nucleic acid sequence;

and detecting the presence of amplified target nucleic acid sequence at a temperature of about 37°

C.