Methods for nucleic acid manipulation

• US 8,361,718 B2
• Filed: 05/24/2010
• Issued: 01/29/2013
• 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:
  US12/786,053
• Publication Number:
  US 20100285482A1
• Time in Patent Office: 981 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. In combination:

(1) a composition for use in amplifying a target nucleic acid sequence comprising;

a) bacteriophage T4 UvsX protein

b) two primers that are complementary to the flanking ends of said target nucleic acid sequence, and

c) a polymerase; and

,

(2) a detection system for detecting the presence of said amplified target nucleic acid sequence.

2. The combination of claim 1, said composition further comprising bacteriophage T4 UvsY protein.

3. The combination of claim 1, wherein said polymerase is a polymerase holoenzyme.

4. The combination of claim 1 wherein said polymerase holoenzyme comprises a polymerase enzyme, a clamp protein, and a clamp loader protein.

5. The combination of claim 1, said composition further comprising nucleotide triphosphates in amounts sufficient to permit amplification of said target nucleic acid sequence.

6. The combination of claim 1, said composition further comprising said target nucleic acid sequence.

7. The combination of claim 1, said composition further comprising bacteriophage T4 gene product 32 protein.

8. The combination of claim 7, wherein said primers are present in a molar excess relative to said target nucleic acid sequence.

9. The combination of claim 1, said composition further comprising a helicase.

10. The combination of claim 9, wherein said helicase is bacteriophage T4 gene product 41 DNA helicase.

11. The combination of claim 9 wherein said helicase is bacteriophage T4 replicative helicase complex comprising bacteriophage T4 gene product 41 and gene product 59.