Department of Computer Science, University of Bristol, Bristol, UK.
BMC Bioinformatics. 2013 Mar 22;14:105. doi: 10.1186/1471-2105-14-105.
Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing.
We have developed a program - 'SDM-Assist' which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of 'mutated clones' by a simple restriction digest.
The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs.
在过去的几十年中，定点突变（SDM）已成为生物结构功能研究不可或缺的工具。原则上，SDM 在 PCR 反应中使用修饰的引物对在 cDNA 插入物中引入突变。在用大肠杆菌扩增之前，用 DpnI 消化反应混合物以消除模板拷贝; 然而，该过程效率低下，导致未突变的克隆，只能通过测序与突变克隆区分开来。
我们开发了一个程序 - 'SDM-Assist'，它创建 SDM 引物并添加一个特定的标识符：通过额外的沉默突变，引入一个限制位点或去除一个先前的限制位点，从而可以通过简单的限制消化高效地识别“突变克隆”。
直接鉴定 SDM 克隆将为研究人员节省时间和金钱。SDM-Assist 还根据 Tm、GC 含量和二级结构等因素对引物进行评分，从而可以简化最佳引物对的选择。
