【摘要】 报道了一种新的PCR突变方法,它不需要纯化大引物或设计特别的旁侧引物.利用一个诱变引物和两个测序引物(Tm≤58℃)作为旁侧引物.第一轮PCR产物12.5μl直接加入到50μl的第二轮PCR反应体系作为模板和大引物,在开始第二轮PCR反应时,增加在68℃退火温度下进行10个循环的不对称PCR,这一步骤大大提高了通过600bp或800bp大引物所导致的突变效率.结果表明,该方法的产物能够达到高保真、97%～98%的突变效率和高产率.更多还原

【Abstract】 A novel PCR-based mutagenesis method was reported,in which there is no need to purify megaprimers or design a special flanking primer.This method used one mutagenic primer and two sequencing primers (T m ≤58℃) as flanking primers.After first round PCR,12.5 μl first PCR production was directly added into 50 μl second PCR system as template and megaprimer,and 10 rounds of asymmetrical PCR at high temperature of annealing (68 ℃) was to add in initiation of second PCR.This additional step greatly has increased the efficiency of mutagenesis via 600 bp or 800 bp long megaprimer.The results demonstrated that this method can achieve high fidelity,97%～98% efficiency,high yield.更多还原