Yoshifumi Adachi, Chihiro Fukuhara
Department of Molecular Biology and Biochemistry, Shinshu University School of Medicine, Matsumoto, Nagano 390-8621, Japan. adachiy@shinshu-u.ac.jp
Analytical biochemistry 2012 Dec 1A 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. Copyright © 2012 Elsevier Inc. All rights reserved.
Yoshifumi Adachi, Chihiro Fukuhara. TA strategy for rapid and efficient site-directed mutagenesis. Analytical biochemistry. 2012 Dec 1;431(1):66-8
PMID: 22960560
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