【摘要】 小规模抽提含有编码小鼠补体Ⅱ型受体(mCR2)cDNA的质粒,体外合成两对寡核苷酸突变引物,利用PCR反应将突变引入mCR2cDNA中,即产生P15S和T68Y两种定点突变。然后用DpnI对消化PCR产物以去除模板DNA,取适量消化产物转化大肠杆菌XL1-Blue,随机挑选克隆进行测序筛选、鉴定所需突变株。结果表明这一新方法不仅操作简单、快速,而且突变率极高,几乎100％。更多还原

【Abstract】 Pure plasmid containing cDNA of murine complement receptor type I gene (mCR2) was mini-prepared. Two pairs of mutagenic primers were synthesized in vitro and the desired two mutations, P15S and T68Y,were introduced into mCR2 cDNA by PCR. After that,DpnI was used to digest the PCR products to remove the methylated,nonmutat-ed parental DNA template. Optimal amount of the digested products were used to transform competent XLl-Blue cells and colonies resistant to ampicillin were picked up randomly followed by being sequenced in order to screen and identify the needed mutant strains. The results show that this novel site -directed mutagenesis method is not only fast and easy to perform,but also very efficient,for the mutagenesis rate is almost更多还原