Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis.

ABSTRACT: To develop a simple, speedy, economical and widely applicable method for multiple-site mutagenesis, we have substantially modified the Quik-Change Site-Directed Mutagenesis Kit protocol (Stratagene, La Jolla, CA). Our new protocol consists of (i) a PCR reaction using an in vitro technique, LDA (ligation-during-amplification), (ii) a DPN:I treatment to digest parental DNA and to make megaprimers and (iii) a synthesis of double-stranded plasmid DNA for bacterial transformation. While the Quik Change Kit protocol introduces mutations at a single site, requiring two complementary mutagenic oligonucleotides, our new protocol requires only one mutagenic oligonucleotide for a mutation site, and can introduce mutations in a plasmid at multiple sites simultaneously. A targeting efficiency >70% was

SUBMITTER: Sawano A 

PROVIDER: S-EPMC108465 | biostudies-literature | 2000 Aug

REPOSITORIES: biostudies-literature