Description of a one step staggered reannealing method for directional cloning of PCR-generated DNA using sticky-end ligation without employing restriction enzymes.
A novel method is described for ligation of PCR products into vectors. It utilizes formation of sticky end sequences compatible with those generated on the plasmid. Four primers are used: two primers are designed to contain the desired sequence of the sticky end plus the sequence of the gene and the other two primers contain the same sequence as the first two primers without the additional bases. The primers are paired to create two PCR products containing the additional bases in a staggered 5' position. Following melting and reannealing, the newly formed products contain the additional sequences as sticky overhangs compatible with the sticky ends of the plasmid.
Ailenberg
M
M
Department of Medicine, University of Toronto, Ontario, Canada.
Silverman
M
M
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D005821:Genetic Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D016914:Ribonuclease H / Q000378:metabolism
D004247:DNA; D016914:Ribonuclease H