Background: The methods for introducing point mutations into target genes are important 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 efficient 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. The DNA fragments between two neighboring sites are amplified with PCR. All amplified fragments are then digested by the selected Type IIs restriction enzyme. The expected mutant is eventually generated by ligation of these digested DNA fragments.
Results: The 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 mutations into the fusion gene of nm23 and EGFP (enhanced green fluorecence protein). The mutagenic frequencies are almost reached 100%.
Conclusions: Our protocol provides a useful tool for gene function research.
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 fluorecence 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。