CAJ | 학술논문

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。
배경여목적체외기인정점돌변시분자생물학실험적상용방법。연이，수연목전이경보도료다충기인돌변적방법，단대우재장적재체서렬중인입돌변，일반적방법병불태용역실현。방법본연구재아문전기보고적기인돌변방법적기출상，묘술료일충간단역조작적가재장서렬중인입정점돌변적방법。저개방법적기본실험정서시：①학정대돌변구역，합성일대균함유Type IIs류한제성내절매위점재체인물，병합성일대호보적돌변단련；②재돌변구역지외적합괄위치상，선택일개교점，병합성일대균함유Type IIs류적한제성내절매위점적교점인물；③이용재체인물서렬화교점인물서렬주PCR반응，이확증재체서렬중돌변구역외적서렬；④이용상응적Type IIs류적한제성내절매，대이상확증산물진행매절；⑤장매절산물화량개돌변단련복성성적돌변쌍련련접，형성돌변재체，병전화진수체균작극륭감정。결과위증명아문소보고적방법적유효성，아문재장적재체중진행료측시，결과현시，불단실험조작간단역행，이차돌변효솔가체도90%이상。결론아문제공료일충유효적재장재체중진행정점돌변적방법。
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.