多片段酶切连接反应结合同源重组的DNA组装方法

DNA assembly by multi-fragment digestion/ligation and homologous recombination

摘要:

为了寻找一种准确高效的多片段组装及多点突变方案，将目前常用的多片段组装方法进行了整合与优化，并以果糖-1,6-二磷酸酶1 (fructose-1,6-diphosphatase 1, FBP1)基因4位点突变为例进行验证。通过引入突变位点和Bsa I识别序列连接含有突变位点的片段，经过酶切/连接反应后扩增组装成功的目的片段，并引入与线性化载体两端同源的序列，最后进行片段与线性化载体的重组并转化大肠杆菌。经筛选与测序，得到4位点突变重组质粒。该方案弥补了常规的Gibson组装和Golden Gate组装的部分缺点，是一种高效进行多片段组装和多点突变的实验方案。

Abstract:

To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and Bsa I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into Escherichia coli. After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.