Homologous recombination-based DNA cloning methods and compositions

• US 8,815,600 B2
• Filed: 03/05/2010
• Issued: 08/26/2014
• Est. Priority Date: 09/10/2008
• Status: Active Grant

• Current Assignee: Genscript Nanjing, Nanjing Genscript Biotech Company Limited
• Original Assignee: Genscript Hongkong
• Inventors: Liu, Weiqiang, Yang, Ping, Wang, Tao, Wang, Zhuying, Chen, Wenzhu, Zhang, Fang Liang
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US12/660,885
• Publication Number:
  US 20100167356A1
• Time in Patent Office: 1,635 Days
• Field of Search: None
• US Class Current: 435/471
• 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 method of cloning a donor DNA molecule into an acceptor vector at a predetermined location, the method comprising:

a) preparing an extended donor DNA molecule by adding to the 5′

-end and the 3′

-end of the donor DNA molecule a first sequence and a second sequence, 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;

b) providing a reaction mixture comprising;

i) the acceptor vector;

ii) the extended donor DNA molecule; and

iii) an enzyme cocktail comprising an exonuclease having only 3′

- or 5′

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

c) incubating the reaction mixture to obtain an intermediate product;

d) transforming a cell with the intermediate product to obtain a transformed cell; and

e) culturing the transformed cell under conditions to produce a recombinant DNA molecule comprising the donor DNA located between the first region and the second region.

2. The method according to claim 1, wherein the acceptor vector is a circular vector or a linearized vector.

3. The method according to claim 2, wherein the first region and the second region are present at the 3′

-end and the 5-end of the linearized vector, respectively.

4. The method according to claim 1, wherein the first and the second sequences are 100% identical to the first and the second regions, respectively.

5. The method according to claim 1, wherein the extended donor DNA molecule is prepared by a polymerase chain reaction (PCR), using a first PCR primer comprising the first sequence and a second PCR primer comprising the complement of the second sequence.

6. The method 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, or a combination thereof.

7. The method 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 (ET SSB), RecA, T4 Gene 32 Protein, Thermus thermophilus RecA (Tth RecA) and Escherichia coli single-strand DNA binding protein (SSB), or a combination thereof.

8. The method according to claim 7, 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.

9. The method according to claim 1, wherein the reaction mixture comprises about 1 to about 100 mg/l of the exonuclease and about 1 to about 100 mg/l of the single-stranded DNA binding protein.

10. The method according to claim 1, wherein the reaction mixture further comprises about 1 to about 10 g/l of tris(hydroxymethyl)aminomethane (Tris), about 0.1 to about 10 g/l of NaCl, about 0.1 to about 10 g/l of ethylenediaminetetraacetic acid (EDTA), about 0.1 to about 10 g/l of MgCl₂, about 10 to about 200 g/l of glycerol, about 10 to about 50 g/l of bovine serum albumin (BSA), about 0.1 to about 10 g/l of adenosine-5′

-triphosphate (ATP), about 0.1 to about 10 g/l of dithiothreitol (DTT), and wherein the pH of the reaction mixture is about 5.0 to about 9.0.

11. The method according to claim 1, wherein in step (c), the incubation is conducted at a temperature of about 10°

C. to about 38°

C. for about 15 minutes to about 60 minutes.

12. The method according to claim 1, wherein the cell is an Escherichia coli cell and the acceptor vector is a plasmid comprising an origin of replication that directs replication of the plasmid in the Escherichia coli cell.

13. The method according to claim 1, wherein the transformed cell expresses a marker gene from the recombinant DNA molecule that allows the selection or screening of the transformed cell.