Construction of recombinant DNA by exonuclease recession.
We describe a new exonuclease-based method for joining and/or constructing two or more DNA molecules. DNA fragments containing ends complementary to those of a vector or another independent molecules were generated by the polymerase chain reaction. The 3' ends of these molecules as well as the vector DNA were then recessed by exonuclease activity and annealed in an orientation-determined manner via their complementary single-stranded regions. This recombinant DNA can be transformed directly into bacteria without a further ligase-dependent reaction. Using this approach, we have constructed recombinant DNA molecules rapidly, efficiently and directionally. This method can effectively replace conventional protocols for PCR cloning, PCR SOEing, DNA subcloning and site-directed mutagenesis.
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235-9048.
Y S
YS
Yang
W J
WJ
Watson
P W
PW
Tucker
J D
JD
Capra
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial; D004274:DNA, Recombinant / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D005092:Exonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D005092:Exonucleases