Methods for nucleic acid manipulation

• US 9,562,250 B2
• Filed: 06/24/2014
• Issued: 02/07/2017
• 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:
  US14/313,193
• Publication Number:
  US 20140356936A1
• Time in Patent Office: 959 Days
• US Class Current: 1/1
• 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 system for replicating a target nucleic acid sequence within a nucleic acid molecule, the system comprising:

a reaction vessel containing a reaction mixture, wherein the reaction mixture includes;

(a) a target nucleic acid molecule including a target nucleic acid sequence;

(b) a UvsX recombination factor;

(c) two primers that bind to the flanking ends of the target nucleic acid sequence;

(d) a DNA polymerase; and

(e) nucleotides in an amount sufficient to support replication of the target nucleic acid sequence.

2. The system according to claim 1, wherein the system further includes a detector configured to detect the product produced via replication of the target nucleic acid sequence.

3. The system according to claim 2, wherein the detector is configured to detect fluorescence.

4. The system according to claim 1 wherein said DNA polymerase is a gene product of a viral, bacteriophage, prokaryotic, or eukaryotic system.

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

6. The system according to claim 1 wherein said uvsX recombination factor is a gene product of a bacteriophage.

7. The system according to claim 1, wherein said DNA polymerase is a product of a viral, bacteriophage, prokaryotic, or eukaryotic system.

8. The system according to claim 1 wherein the reaction vessel also includes a single stranded nucleic acid binding protein.

9. The system according to claim 1 wherein the reaction vessel also includes a helicase.

10. The system according to claim 1 wherein the reaction vessel also includes an accessory factor configured to stabilize the UvsX recombination factor.

11. The system according to claim 1 wherein the reaction vessel also includes an ATP regeneration system.

12. The system according to claim 1, wherein the target nucleic acid molecule is double stranded.

13. The system according to claim 1, wherein the target nucleic acid sequence is embedded within the longer target nucleic acid molecule.