title: TA strategy for rapid and efficient site-directed mutagenesis.
abstract: 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.
authors.affiliation: Department of Molecular Biology and Biochemistry, Shinshu University School of Medicine, Matsumoto, Nagano 390-8621, Japan. adachiy@shinshu-u.ac.jp
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Adachi
authors.forename: Chihiro
authors.initials: C
authors.lastname: Fukuhara
mesh_terms: D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers