A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning.
A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning.
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DOI：
10.1016/j.ab.2011.09.019
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发表时间：
2012-01
影响因子：
2.9
通讯作者：
Haisu Wan;Yongwen Li;Yu Fan;Fanrong Meng;Cheng Chen;Qinghua Zhou
中科院分区：
文献类型：
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作者：
Haisu Wan;Yongwen Li;Yu Fan;Fanrong Meng;Cheng Chen;Qinghua Zhou
定点突变已成为分子生物学中的常规方法。然而，许多突变体仍然很难创造。复杂的嵌合突变、串联重复序列、反向序列、GC富集区和/或重链二级结构可导致诱变引物与靶序列的无效或不正确结合，并影响后续扩增。理论上，这些问题可以通过使用诱变片段将突变引入靶序列中来避免，从而消除对引物-模板退火的需要。盒式诱变在其方案中使用诱变片段;然而，在大多数情况下，它需要进行两轮基于诱变引物的诱变以将合适的限制性酶切位点引入模板中，并且不适合常规诱变。在这里，我们描述了一种高效的方法，其中除了要突变的区域以外的模板通过聚合酶链式反应（PCR）扩增，并且IIs型限制性酶消化的PCR产物与诱变片段直接连接。我们的方法不需要诱变引物的辅助。我们已经用这种方法创造了各种类型的难以制造的突变体，其诱变频率接近100%。我们的协议有许多优势，流行的QuikChange方法，是一个有价值的工具，研究基因的结构和功能。
Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer–template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function.