Site-specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site-specific mutagenesis of any genes on plasmids using long distance inverse PCR in the presence of Pfu-DNA polymerase. The efficiency of this method is higher than 90% and the entire procedure can be performed just in one tube. No subcloning is needed. This method is especially useful for obtaining mutant genes on large plasmids such as Ti plasmids used for plant transformation.
一种简易的利用Pfu-DNA聚合酶进行反向长距离PCR获得定点突变的方法
董宇清 于昕 赵进东*
（北京大学生命科学学院，北京100871）
摘要：定点突变是一种常用的分子生物学方法。利用Pfu-DNA聚合酶进行反向长距离PCR可以有效地获得定点突变。方法操作简易 ,突变效率高于 90 %。整个操作过程可在一个试管中完成 ,不需亚克隆。对克隆在大质粒 (如转化植物的Ti-类型质粒 )尤为实用。
关键词： 反向长距离PCR；质粒；定点突变
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