PCR介导的ret基因体外定点突变

A site-directed mutation at 2753 bp of the ret cDNA induced by PCR

目的:利用PCR技术介导ret基因cDNA上第2753位碱基的体外定点突变。方法:利用PCR技术进行定点突变,采用DpnⅠ进行原始模板消化。经Amp抗性、PCR技术初步筛选,通过序列分析进行确证。结果:Amp抗性及PCR筛选结果均为阳性;序列分析提示,2753处碱基由T→C。结论:成功完成了ret基因的体外定点突变。

Aim ： To fulfil a site-directed mutation at 2 753 bp of the ret cDNA by PCR. Methods ：Using a PCR-based site-directed mutagenesis method, a mutation at 2 753 bp of the ret cDNA was induced. After Dpn I digestion, the products were introduced into E. cell TG1. The positive strain was selected by Amp resistant test, PCR, and DNA sequencing. ReSuits ： The result of Amp resistant test and PCR was positive ; DNA sequence analysis showed that the base has changed at 2 753 bp Of ret cDNA from T to C. Conclusion： Site-directed mutation has been fulfilled successfully.