An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes |
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Authors: | Zhiqiang Zhang Kun Xu Ying Xin Zhiying Zhang |
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Affiliation: | College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China |
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Abstract: | Site-directed mutagenesis (SDM) methods are very important in modern molecular biology, biochemistry, and protein engineering. Here, we present a novel SDM method that can be used for multiple mutation generation using type IIs restriction enzymes. This approach is faster and more convenient than the overlap polymerase chain reaction (PCR) method due to its having fewer reaction steps and being cheaper than, but as convenient as, enzymatic assembly. We illustrate the usefulness of our method by introducing three mutations into the bacterial Streptococcus thermophilus Cas9 (bStCas9) gene, converting the humanized S. thermophilus Cas9 (hStCas9) gene into nuclease dead or H847A nickase mutants and generating sunnyTALEN mutagenesis from a wild-type TALEN backbone. |
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Keywords: | SDM Golden Gate cloning bStCas9 hStCas9 sunnyTALEN |
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