School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Protein Sci. 2010 May;19(5):967-73. doi: 10.1002/pro.374.
Function studies of many proteins are waited to develop after genome sequencing. High-throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation-independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate-modified primers and the digestion of PCR products by lambda exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3' overhangs, which are designed to form complementary double-stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3' overhangs is a better cohesive end for LIC than 5' overhang and the existence of 5'phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.
功能研究的许多蛋白质正在等待发展后的基因组测序。高通量技术的基因克隆将有力地促进蛋白质的功能研究。在这里，我们描述了一种连接独立克隆（LIC）的方法，该方法是基于使用硫代磷酸修饰的引物对靶基因和线性载体进行 PCR 扩增，以及利用 λ核酸外切酶对 PCR 产物进行消化。硫代磷酸酯抑制消化，导致产生 3'突出端，其设计用于靶基因和线性载体之间形成互补的双链 DNA。我们比较了我们的硫代磷酸酯引物克隆方法与几种 LIC 方法，包括 dU 引物克隆、杂交克隆、T4 DNA 聚合酶克隆和体内重组克隆。这些 LIC 方法的克隆效率如下：硫代磷酸酯引物克隆>dU 引物克隆>杂交克隆>T4 DNA 聚合酶克隆>>体内重组克隆。我们的结果表明，3'突出端是 LIC 的一个更好的粘性末端，而 5'磷酸的存在促进了大肠杆菌中的 DNA 修复，从而提高了 LIC 的克隆效率。我们成功地在一周内构建了 156 个 Aeropyrum pernix 基因的表达质粒。
