Methods For Nucleic Acid Manipulation

Patent metadata

Field	Value
Publication number	US-2016265015-A1
Application number	US-201615070401-A
Country	US
Kind code	A1
Filing date	Mar 15, 2016
Priority date	Apr 20, 2001
Publication date	Sep 15, 2016
Grant date	—

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

1 .- 22 . (canceled)

23 . A method for amplifying a target DNA, said method comprising the steps: i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence; ii) maintaining the reaction mixture at a temperature below about 45° C., wherein amplified target DNA is produced; iii) and detecting the presence of amplified target DNA.

24 . The method of claim 23 , wherein said DNA polymerase is a non-thermostable polymerase.

25 . The method of claim 23 , wherein said DNA polymerase is a prokaryotic polymerase.

26 . The method of claim 23 , wherein said DNA polymerase is a polymerase holoenzyme.

27 . The method of claim 23 , wherein said reaction mixture further comprises bacteriophage UvsY protein, bacteriophage gene product 32 protein, or both.

28 . The method of claim 23 , wherein said reaction mixture further comprises a helicase.

29 . The method of claim 23 , wherein said reaction mixture further comprises an ATP regeneration system.

30 . A method for amplifying a target DNA, said method comprising the steps: i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, bacteriophage UvsY protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence; ii) maintaining the reaction mixture at a temperature below about 45° C., wherein amplified target DNA is produced; iii) and detecting the presence of amplified target DNA.

31 . The method of claim 30 , wherein said DNA polymerase is a non-thermostable polymerase.

32 . The method of claim 30 , wherein said DNA polymerase is a prokaryotic polymerase.

33 . The method of claim 30 , wherein said DNA polymerase is a polymerase holoenzyme.

34 . The method of claim 30 , wherein said reaction mixture further comprises bacteriophage gene product 32 protein.

35 . The method of claim 30 , wherein said reaction mixture further comprises a helicase.

36 . The method of claim 30 , wherein said reaction mixture further comprises an ATP regeneration system.

37 . A method for amplifying a target DNA, said method comprising the steps: i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence; ii) maintaining the reaction mixture at room temperature, wherein amplified target DNA is produced; iii) and detecting the presence of amplified target DNA.

38 . The method of claim 37 , wherein said DNA polymerase is a non-thermostable polymerase.

39 . The method of claim 37 , wherein said DNA polymerase is a prokaryotic polymerase.

40 . The method of claim 37 , wherein said DNA polymerase is a polymerase holoenzyme.

41 . The method of claim 37 , wherein said reaction mixture further comprises bacteriophage UvsY protein, bacteriophage gene product 32 protein, or both.

42 . The method of claim 37 , wherein said reaction mixture further comprises a helicase, an ATP regeneration system, or both.

Assignees

• Penn State Res Found

Inventors

• Benkovic Stephen J
• Salinas Frank

Classifications

• C12P19/34 Primary

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

• C12N15/1027

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

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6806

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

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title