Nucleic Acids Research, 2000, Vol. 28, No. 16 E78-e78
© 2000 Oxford University Press
Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis
1Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan and 2Brain Science Research Division, Brain Science and Life Technology Research Foundation, 1-28-12 Narimasu, Itabashi, Tokyo, 175-0094, Japan
To develop a simple, speedy, economical and widely applicable method for multiple-site mutagenesis, we have substantially modified the Quik-ChangeTM 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 DpnI treatment to digest parental DNA and to make megaprimers and (iii) a synthesis of double-stranded plasmid DNA for bacterial transformation. While the Quik ChangeTM 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 consistently achieved for multiple-site mutagenesis. Furthermore, the new protocol allows random mutagenesis with degenerative primers, because it does not use two complementary primers. Our mutagenesis strategy was successfully used to alter the fluorescence properties of green fluorescent protein (GFP), creating a new-color GFP mutant, cyan-green fluorescent protein (CGFP). An eminent feature of CGFP is its remarkable stability in a wide pH range (pH 4–12). The use of CGFP would allow us to monitor protein localization quantitatively in acidic organelles in secretory pathways.
* To whom correspondence should be addressed. Tel: +81 48 467 5917; Fax: +81 48 467 5924; Email: matsushi@brain.riken.go.jp
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