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Indexed by:期刊论文

Date of Publication:2008-07-01

Journal:BIOTECHNOLOGY LETTERS

Included Journals:SCIE、EI

Volume:30

Issue:7

Page Number:1227-1232

ISSN No.:0141-5492

Key Words:AdoMet synthetase; directed evolution; DNA recombination; error-prone PCR; S-adenosylmethionine

Abstract: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%.