Methods for nucleic acid manipulation

• US 9,322,059 B2
• Filed: 06/24/2014
• Issued: 04/26/2016
• Est. Priority Date: 04/20/2001
• Status: Expired due to Fees

• Current Assignee: Penn State Research Foundation (Pennsylvania State University)
• Original Assignee: Penn State Research Foundation (Pennsylvania State University)
• Inventors: Benkovic, Stephen J, Salinas, Frank
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US14/313,295
• Publication Number:
  US 20140308673A1
• Time in Patent Office: 672 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 method for amplifying a target nucleic acid sequence within a nucleic acid molecule, comprising:

providing a target nucleic acid molecule containing a target nucleic acid sequence;

forming, within a reaction vessel, a reaction mixture containing the target nucleic acid molecule, a UvsX recombination factor, two primers that bind to flanking ends of the target nucleic acid sequence, a polymerase, and nucleotides in an amount sufficient to support amplification of the target nucleic acid sequence; and

forming an amplification product by amplifying the target nucleic acid sequence within the reaction vessel.

2. The method according to claim 1, further including contacting the amplification product with a fluorescent molecule.

3. The method according to claim 1, wherein the method further includes detecting the amplification product.

4. The method according to claim 3, further including contacting the amplification product with a fluorescent molecule.

5. The method according to claim 3, wherein the detecting step includes detecting fluorescence.

6. The method according to claim 1 wherein said polymerase is a holoenzyme complex.

7. The method according to claim 6, wherein said holoenzyme complex is a gene product of a viral, bacteriophage, prokaryotic, or eukaryotic system.

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

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

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

11. The method according to claim 1, further including separating the amplification product from the reaction mixture.

12. The method according to claim 1, further including analyzing the amplification product to confirm the identity of the target nucleic acid sequence.

13. The method according to claim 1 wherein the target nucleic acid molecule is double stranded.

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