Methods for nucleic acid manipulation

Patent metadata

Field	Value
Publication number	US-9562250-B2
Application number	US-201414313193-A
Country	US
Kind code	B2
Filing date	Jun 24, 2014
Priority date	Apr 20, 2001
Publication date	Feb 7, 2017
Grant date	Feb 7, 2017

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.

First claim

What is claimed:

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.

Assignees

• Penn State Res Found

Inventors

• Benkovic Stephen J
• Salinas Frank

Classifications

• C12N15/1027

  by DNA shuffling, e.g. RSR, STEP, RPR · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q2521/513

  Winding/unwinding enzyme, e.g. helicase · CPC title

• C12Q2521/507

  Recombinase · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title