Mutagenesis of longer inserts by the ligation of two PCR fragments amplified with a mutation primer.
We report a method for efficient mutagenesis of DNA in large vectors without subcloning. Two segments of the target DNA sequence, one having a mutation introduced via a mutant primer, were amplified by PCR and then the purified fragments were ligated to a vector. The mutation efficiency was nearly 100%.
Kato
Yu
Y
Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, Suehiro-cho 1-7-29, Tsurumi-ku, Yokohama 230-0045, Japan.
Arakawa
Noriaki
N
Masuishi
Yusuke
Y
Kawasaki
Hiroshi
H
Hirano
Hisashi
H
D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics*; D017353:Gene Deletion; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D008957:Models, Genetic; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D017931:DNA Primers; D004247:DNA