牛Dgat1基因高GC含量片段的定点突变

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

将扩增出的1.8 kb目的片段克隆入pUCm-T载体,以构建好的质粒为模板进行突变反应。DpnⅠ酶切反应产物,去除甲基化及半甲基化DNA模板。酶切产物经纯化后转入DH5α感受态细胞,提取质粒测序。结果表明,双核苷酸碱基AA突变为GC,成功得到突变载体。将定点突变试剂盒方法加以改进,完成GC含量高达69.5%模板定点突变,改进后的方法操作简单、经济实用,有效地解决了高GC含量模板难突变难题。

A 1.8 kb fragment of bovine Dgatl 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. coll. 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.