Xiao Juan, Ma Mengqi, Liang Mingxing, He Ruyang, Chen Huabo
Medical College, Hubei University of Arts and Science, Xiangyang 441053, Hubei, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Jun 25;36(6):1232-1240. doi: 10.13345/j.cjb.190476.
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是一种常用的定点诱变方法。随着目的基因序列变长，第二轮PCR中往往难以获得目标产物，并且很有可能通过PCR将意外突变引入长基因片段中。为了规避这些问题，我们可以通过重叠延伸PCR仅扩增包含目标突变的小基因片段，然后将其与载体连接以获得目标质粒。如果扩增片段末端的限制性酶切位点在质粒载体上不是单一的，则可以使用双片段连接法构建目标质粒。部分扩增与双片段连接相结合，可以解决长基因诱变中的许多问题。以视网膜母细胞瘤基因1的S７80E诱变为例，由于片段过长干扰第二轮PCR的重叠延伸，通过重叠延伸PCR扩增整个视网膜母细胞瘤基因1很困难。然而，扩增包含目标突变S７80E的F3（1 968-2 787）片段相对容易。F3片段的5'端有一个可用于连接的Nhe I位点，但质粒上的另一个Nhe I位点阻碍了直接这样做。为了克服这一障碍，我们将F3片段与从野生型质粒消化得到的F2（900-1 968）片段一起与包含该基因F1（1-900）片段的载体连接。两个片段同时与一个载体连接，虽然效率较低，但可以重组为一个完整的质粒。所选的两个重组质粒的序列与目标突变一致，验证了该方案的可行性。作为重叠延伸PCR的一种改进，部分扩增和双片段连接方法可为许多长基因的定点诱变提供解决方案。
