CAJ | 학술논문

背景与目的在基因序列中引入点突变是研究基因结构和功能及其相关性的重要手段。目前已有多种对基因序列进行突变的方法，然而，这些方法大多对单一位置的基因突变有效，而对在基因序列中引入多位点突变，还有待方法的进一步改进。为适应这一需要，本研究提供了一种高效的可在基因序列的多个位点引入突变的方法。方法该方法依赖于一种Type IIs类的限制性内切酶，例如Esp 3I等。本研究所提供的方法中，针对每一个突变位点，合成一对含有突变点和所选择的Type IIs类的限制性内切酶位点的引物，当需要引入多位点突变时，则利用邻近的两个突变位点的引物做PCR反应，多个位点的突变，就可以得到多个扩增片段，将这些片段用和引物上的限制性内切酶位点对应的酶进行反应，然后用连接反应将片段连接形成突变基因。结果本研究所提供的方法非常简便，主要实验步骤可以在一天之内完成。我们已经利用这种方法，在绿色荧光蛋白（enhanced green lfuorecence protein, EGFP）和nm23基因中，引入3个或4个突变，突变效率几乎为100%。结论本研究所提供的方法可以成为研究基因功能的有用工具。
배경여목적재기인서렬중인입점돌변시연구기인결구화공능급기상관성적중요수단。목전이유다충대기인서렬진행돌변적방법，연이，저사방법대다대단일위치적기인돌변유효，이대재기인서렬중인입다위점돌변，환유대방법적진일보개진。위괄응저일수요，본연구제공료일충고효적가재기인서렬적다개위점인입돌변적방법。방법해방법의뢰우일충Type IIs류적한제성내절매，례여Esp 3I등。본연구소제공적방법중，침대매일개돌변위점，합성일대함유돌변점화소선택적Type IIs류적한제성내절매위점적인물，당수요인입다위점돌변시，칙이용린근적량개돌변위점적인물주PCR반응，다개위점적돌변，취가이득도다개확증편단，장저사편단용화인물상적한제성내절매위점대응적매진행반응，연후용련접반응장편단련접형성돌변기인。결과본연구소제공적방법비상간편，주요실험보취가이재일천지내완성。아문이경이용저충방법，재록색형광단백（enhanced green lfuorecence protein, EGFP）화nm23기인중，인입3개혹4개돌변，돌변효솔궤호위100%。결론본연구소제공적방법가이성위연구기인공능적유용공구。
Background and objective hTe methods for introducing point mutations into target genes are impor-tant for dissecting the relationship of gene structure and function. Up to date, there are numbers of protocols available for the purpose. However, many of them are suited for introducing single site mutation into the target gene. For introducing multiple-site mutations simultaneously into the target genes, it is required to further improve the related methods. Methods In this report, we describe an improvement on the type IIs restriction enzyme-dependent site-directed mutagenesis method and the improved protocol is highly effcient for multiple-site mutagenesis. In our method, a pair of mutagenic primers are synthesized for each desired site and each mutagenic primer contains a selected type IIs restriction site. hTe DNA fragments between two neighboring sites are ampliifed with PCR. All ampliifed fragments are then digested by the selected Type IIs restriction enzyme. hTe expected mutant is eventually generated by ligation of these digested DNA fragments. Results hTe improved protocol is very easy and can be achieved in just one day. As a proof of principle, we have introduced multiple-site, i.e., 3-site or 4-site mu-tations into the fusion gene of nm23 and EGFP (enhanced green lfuorecence protein). hTe mutagenic frequencies are almost reached 100%. Conclusion Our protocol provides a useful tool for gene function research.