Homologous recombination-based DNA cloning compositions

• US 8,501,454 B2
• Filed: 01/31/2012
• Issued: 08/06/2013
• Est. Priority Date: 09/10/2008
• Status: Active Grant

• Current Assignee: Genscript Nanjing, Nanjing Genscript Biotech Company Limited
• Original Assignee: Nanjingjinsirui Science & Technology Biology Corporation
• Inventors: Liu, Weiqiang, Yang, Ping, Wang, Tao, Wang, Zhuying, Chen, Wenzhu, Zhang, Fang Liang
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US13/362,382
• Publication Number:
  US 20120129239A1
• Time in Patent Office: 553 Days
• Field of Search: None
• US Class Current: 435/252.1
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

Methods and compositions for cloning a donor DNA molecule into an acceptor vector at a predetermined location are described. The methods are based on homologous recombination mediated by in vitro treatment of the donor DNA and the acceptor vector with an enzyme cocktail containing an exonuclease and a single-stranded DNA binding protein.

Claims

1. A kit for use in cloning a donor DNA molecule into an acceptor vector at a predetermined location, the kit consisting essentially of:

(1) a composition comprising;

a) an enzyme cocktail consisting essentially of an exonuclease having only 3′

- or 5′

-exonucleolytic single-strand degradation activity, and a single-stranded DNA binding protein; and

b) a reaction buffer;

(2) instructions on using the composition in the cloning, and

(3) a competent cell for use in the cloning.

3. The kit according to claim 1, wherein the exonuclease is selected from the group consisting of Escherichia coli exonuclease I, Escherichia coli exonuclease III, Escherichia coli exonuclease VII, bacteriophage lambda exonuclease, and bacteriophage T7 -exonuclease Gene 6.

4. The kit according to claim 1, wherein the single-stranded DNA binding protein is selected from the group consisting of extreme thermostable single-strand DNA binding protein, RecA, T4 Gene 32 Protein, Thermus thermophilus RecA (Tth RecA), and Escherichia coli single-strand DNA binding protein.

5. The kit according to claim 1, wherein the enzyme cocktail comprises a combination selected from the group consisting of Escherichia coli exonuclease I and RecA;

Escherichia coli exonuclease III and RecA;

Escherichia coli exonuclease VII and RecA;

bacteriophage lambda exonuclease and RecA;

bacteriophage T7 - exonuclease Gene 6 III and RecA;

Escherichia coli exonuclease I and Tth RecA;

Escherichia coli exonuclease III and Tth RecA;

Escherichia coli exonuclease VII and Tth RecA;

bacteriophage lambda exonuclease and Tth RecA;

bacteriophage T7 - exonuclease Gene 6 III and Tth RecA;

Escherichia coli exonuclease I and ET SSB;

Escherichia coli exonuclease I and T4 Gene 32 Protein;

Escherichia coli exonuclease I and Escherichia coli SSB;

Escherichia coli exonuclease III and ET SSB;

Escherichia coli exonuclease III and T4 Gene 32 Protein;

Escherichia coli exonuclease III and Escherichia coli SSB;

Escherichia coli exonuclease VII and ET SSB;

Escherichia coli exonuclease VII and T4 Gene 32 Protein; and

Escherichia coli exonuclease VII and Escherichia coli SSB.

2. A system for use in cloning a donor DNA molecule into an acceptor vector at a predetermined location, the system comprising:

a) the acceptor vector;

b) an extended donor DNA molecule comprising a first sequence and a second sequence at the 5′

-end and the 3′

-end of the donor DNA molecule, respectively, wherein each of the first and second sequences, independently, is at least 12 nucleotides in length and is at least 90% identical to a first region and a second region of the acceptor vector, respectively;

c) a composition comprising a reaction buffer and an enzyme cocktail consisting essentially of an exonuclease having only 3′

- or 5′

- exonucleolytic single-strand degradation activity, and a single-stranded DNA binding protein; and

d) a cell transformable with an intermediate product formed after incubating a reaction mixture comprising (a), (b) and (c), whereby the transformed cell produces a recombinant DNA molecule that comprises the donor DNA located between the first and the second regions.

6. The system according to claim 2, wherein the exonuclease is selected from the group consisting of Escherichia coli exonuclease I, Escherichia coli exonuclease III, Escherichia coli exonuclease VII, bacteriophage lambda exonuclease, and bacteriophage T7 - exonuclease Gene 6.

7. The system according to claim 2, wherein the single-stranded DNA binding protein is selected from the group consisting of extreme thermostable single-strand DNA binding protein, RecA, T4 Gene 32 Protein, Thermus thermophilus RecA (Tth RecA), and Escherichia coli single-strand DNA binding protein.

8. The system according to claim 2, wherein the enzyme cocktail comprises a combination selected from the group consisting of Escherichia coli exonuclease I and RecA;

Escherichia coli exonuclease III and RecA;

Escherichia coli exonuclease VII and RecA;

bacteriophage lambda exonuclease and RecA;

bacteriophage T7 - exonuclease Gene 6 III and RecA;

Escherichia coli exonuclease I and Tth RecA;

Escherichia coli exonuclease III and Tth RecA;

Escherichia coli exonuclease VII and Tth RecA;

bacteriophage lambda exonuclease and Tth RecA;

bacteriophage T7 - exonuclease Gene 6 III and Tth RecA;

Escherichia coli exonuclease I and ET SSB;

Escherichia coli exonuclease I and T4 Gene 32 Protein;

Escherichia coli exonuclease I and Escherichia coli SSB;

Escherichia coli exonuclease III and ET SSB;

Escherichia coli exonuclease III and T4 Gene 32 Protein;

Escherichia coli exonuclease III and Escherichia coli SSB;

Escherichia coli exonuclease VII and ET SSB;

Escherichia coli exonuclease VII and T4 Gene 32 Protein; and

Escherichia coli exonuclease VII and Escherichia coli SSB.