METHODS FOR NUCLEIC ACID MANIPULATION

• US 20140356936A1
• Filed: 06/24/2014
• Published: 12/04/2014
• Est. Priority Date: 04/20/2001
• Status: Active Grant

• Current Assignee: Penn State Research Foundation
• Original Assignee: Frank Salinas, Stephen J. Benkovic
• Inventors: BENKOVIC, Stephen J, Salinas, Frank
• Granted Patent: US 9,562,250 B2
• Time in Patent Office: Days
• US Class Current: 435/287.2
• 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

1-22. -22. (canceled)

23. 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 target nucleic acid sequence;

(d) a DNA polymerase; and

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