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论文类型：期刊论文

发表时间：2008-07-01

发表刊物：BIOTECHNOLOGY LETTERS

收录刊物：SCIE、EI

卷号：30

期号：7

页面范围：1227-1232

ISSN号：0141-5492

关键字：AdoMet synthetase; directed evolution; DNA recombination; error-prone PCR; S-adenosylmethionine

摘要：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%.