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Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.重叠延伸 PCR 是基因定点突变的主要方法，但是以该方法制作长基因定点突变时，往往遇到难以获得第二轮PCR 产物或容易引入新的非预期突变等问题。此时，可先以重叠延伸PCR 扩增含突变位点的部分基因片段，再将其连入适当载体获得重组质粒。若该扩增片段两侧的酶切位点在质粒载体上不单一，则可采用双片段连接法构建完整质粒。以制作视网膜母细胞瘤基因S780E 定点突变为例，直接以重叠延伸PCR 扩增全长基因时未能得到理想的目标产物。故先扩增含点突变的F3 片段，再将其与源自原始质粒的F2 片段一起连入含F1 片段的质粒载体而构建完整质粒。两个筛选出的重组质粒经序列检测完全符合目标突变序列特征，验证了该方案的可行性。该方法作为重叠延伸PCR 的补充，可为许多长基因定点突变提供解决方案。. |
