Site-directed mutagenesis of GC-rich fragment in bovine Dgat1 gene

A 1.8 kb fragment of bovine Dgat1 gene was obtained by PCR and was cloned into pUCm-T vector.Mutation PCR was then performed with the plasmid vector as template.The mutation PCR product was treated with Dpn Ⅰ to digest the parental methylated and hemimethylated DNA template.After purified the digested product was transduced into DH5α competent E.coli.The mutation plasmid was isolated and sequenced.The results of sequence analysis showed that dinucleotide AA had been substituted by GC and the mutation vector had been completed.In this paper,the method of Site-Directed Mutagenesis Kit was improved to complete the site-directed mutagenesis of GC-rich(up to 69.5%) fragment.The modified method was a rapid,simple and economic approach for site-directed mutagenesis which provided a solution for the mutagenesis of GC-rich fragment.