Homologous recombination-based DNA cloning methods and compositions

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.

I/We claim: 
     
         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 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 1 , 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 an enzyme 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 MgCl 2 , 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. 
     
     
         14 . A composition for use in cloning a donor DNA molecule into an acceptor vector at a predetermined location, comprising:
 a) an enzyme cocktail comprising an exonuclease and a single-stranded DNA binding protein; and   b) a reaction buffer.   
     
     
         15 . The composition of  claim 14 , 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. 
     
     
         16 . The composition of  claim 14 , 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. 
     
     
         17 . The composition according to  claim 16 , wherein the enzyme cocktail comprises an enzyme 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. 
     
     
         18 . A kit for use in cloning a donor DNA molecule into an acceptor vector at a predetermined location, the kit comprising:
 a) the composition of  claim 14 ; and   b) instructions on using the composition in the cloning.   
     
     
         19 . The kit according to  claim 18 , further comprising a competent cell for use in the cloning. 
     
     
         20 . 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) an enzyme cocktail comprising an exonuclease 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.