Random mutagenesis and recombination of sam1 gene by integrating error-prone PCR with staggered extension process.
An efficient method for creating a DNA library is presented in which gene mutagenesis and recombination can be introduced by integrating error-prone PCR with a staggered extension process in one test tube. In this process, less than 15 cycles of error-prone PCR are used to introduce random mutations. After precipitated and washed with ethanol solution, the error-prone PCR product is directly used both as template and primers in the following staggered extension process to introduce DNA recombination. The method was validated by using adenosyl-methionine (AdoMet) synthetase gene, sam1, as a model. The full-length target DNA fragment was available after a single round. After being selected with a competitive inhibitor, ethionine, a mutated gene was obtained that increased AdoMet accumulation in vivo by 56%.
An
Yingfeng
Y
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China.
Ji
Jianfei
J
Wu
Wenfang
W
Huang
Ribo
R
Wei
Yutuo
Y
Xiu
Zhilong
Z
D000963:Antimetabolites / Q000494:pharmacology; D019020:Directed Molecular Evolution / Q000379:methods; D005001:Ethionine / Q000494:pharmacology; D008716:Methionine Adenosyltransferase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic / Q000187:drug effects; D012436:S-Adenosylmethionine / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics
D000963:Antimetabolites; D029701:Saccharomyces cerevisiae Proteins; D012436:S-Adenosylmethionine; D008716:Methionine Adenosyltransferase; D005001:Ethionine