Background and objective In vitro site-directed mutagenesis is a routine technique in molecular biol-ogy labs. However, although there are numbers of related methods available, most of these methods are not suitable for intro-ducing mutations into large vectors. Methods In this report, we describe a method which is highly effective for this purpose. Our method is based on the other site-directed method we recently reported. hTe basic protocol of our method is as follows:(1) Synthesize a pair of vector primers based on the sequences around the region to be mutated, each containing a suitable type IIs endonuclease restriction site;meanwhile, synthesize a pair of short complementary oligonucleotides which forms a mutagenic fragment atfer annealing;(2) Synthesize a pair of bridge primers which can speciifcally bind to a site in the vector sequence, each containing a suitable type IIs endonuclease restriction site;(3) Perform PCR reactions using these Vector primers and Bridge primers;(4) Digest the PCR products with the corresponding type IIs restriction enzyme;(5) Ligate the digested frag-ment with the mutagenic fragment to make the desired mutant. Results Using this protocol, we have introduced mutations into a vector larger than 9 kb. hTe results shows that the mutation rates are more that 90%. Conclusion Our method provides a useful tool for performing site-directed mutagenesis experiment in large vector.%背景与目的体外基因定点突变是分子生物学实验的常用方法。然而，虽然目前已经报道了多种基因突变的方法，但对于在长的载体序列中引入突变，一般的方法并不太容易实现。方法本研究在我们前期报告的基因突变方法的基础上，描述了一种简单易操作的可在长序列中引入定点突变的方法。这个方法的基本实验程序是：①确定待突变区域，合成一对均含有Type IIs类限制性内切酶位点载体引物，并合成一对互补的突变单链；②在突变区域之外的合适位置上，选择一个桥点，并合成一对均含有Type IIs类的限制性内切酶位点的桥点引物；③利用载体引物序列和桥点引物序列做PCR反应，以扩增载体序列中突变区域外的序列；④利用相应的Type IIs类的限制性内切酶，对以上扩增产物进行酶切；⑤将酶切产物和两个突变单链复性成的突变双链连接，形成突变载体，并转化进受体菌作克隆鉴定。结果为证明我们所报告的方法的有效性，我们在长的载体中进行了测试，结果显示，不但实验操作简单易行，而且突变效率可达到90%以上。结论我们提供了一种有效的在长载体中进行定点突变的方法。
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