Füzik Tibor, Ulbrich Pavel, Ruml Tomáš
Institute of Chemical Technology Prague, Department of Biochemistry and Microbiology, Technicka 5, 166 28 Prague, Czech Republic.
Institute of Chemical Technology Prague, Department of Biochemistry and Microbiology, Technicka 5, 166 28 Prague, Czech Republic.
J Microbiol Methods. 2014 Nov;106:67-71. doi: 10.1016/j.mimet.2014.08.003. Epub 2014 Aug 18.
Site-directed mutagenesis is one of the most widely used techniques in life sciences. Here we describe an improved and simplified method for introducing mutations at desired sites. It consists of an inverse PCR using a plasmid template and two partially complementary primers. The synthesis step is followed by annealing of the PCR product's sticky ends, which are generated by exonuclease digestion. This method is fast, extremely efficient and cost-effective. It can be used to introduce large insertions and deletions, but also for multiple point mutations in a single step. To show the principle and to prove the efficiency of the method, we present a series of basic mutations (insertions, deletions, point mutations) on pUC19 plasmid DNA.
定点诱变是生命科学中使用最广泛的技术之一。在此，我们描述了一种在所需位点引入突变的改进且简化的方法。它包括使用质粒模板和两条部分互补引物进行的反向PCR。合成步骤之后是通过核酸外切酶消化产生的PCR产物粘性末端的退火。该方法快速、极其高效且具有成本效益。它可用于引入大的插入和缺失，也可用于一步引入多个点突变。为了展示该方法的原理并证明其效率，我们在pUC19质粒DNA上展示了一系列基本突变（插入、缺失、点突变）。
