A simple high-efficiency method for random mutagenesis of cloned genes using forced nucleotide misincorporation.
We describe a simple protocol for introducing random point mutations into cloned DNA fragments via forced misincorporation of deoxynucleoside triphosphates (dNTPs) by either of two polymerases which lack proofreading activity, reverse transcriptase or a mutant T7 DNA polymerase. A high ratio of one nucleotide to the other three is used to enhance the error rate. Mutagenesis is initiated from a specific primer and restricted to a relatively short segment by limiting the amounts of the nonmutagenic dNTPs. Our method is highly efficient, resulting in the isolation of greater than 50% mutant plasmids with most polymerase/forcing dNTP combinations. A wide spectrum of mutations can be obtained, in contrast to commonly used methods for random mutagenesis.
Liao
X B
XB
Department of Biochemistry, University of Illinois, Urbana 61801.
Wise
J A
JA
D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase; D008969:Molecular Sequence Data; D009154:Mutation; D012194:RNA-Directed DNA Polymerase; D014158:Transcription, Genetic
D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase