TA strategy for rapid and efficient site-directed mutagenesis

摘要

A simple, rapid, and efficient site-directed mutagenesis method using TA strategy with synthetic mutagenic oligonucleotides is described. Briefly, a 3′ A-overhang vector was prepared by polymerase chain reaction (PCR) using a classical Taq polymerase with terminal transferase activity, a reverse vector primer starting the complement nucleotide prior to the 5′ end adenosine of the target, and a forward vector primer starting the nucleotide posterior to the 3′ end thymidine. The 3′ T-overhang mutagenic double-strand oligonucleotide was synthesized and cloned directly into the PCR-amplified 3′ A-overhang vector. Thus, direct ligation of synthetic mutagenic oligonucleotides and PCR-amplified vector via TA sticky ends provides us with simple, rapid, and efficient site-directed mutagenesis.