一种新型的基因定点突变方法及其在DdsA突变研究中的应用

为了发展基因突变技术,介绍一种新型的基因定点突变方法.该方法巧妙利用了基因序列中广泛存在的不完整的平端酶切位点.与传统方法相比，可以迅速地在全基因的任何部位替换核苷酸，并可以在突变实验过程中直接将目的基因克隆到T载体上，便于测序及进一步克隆.利用该方法成功地获得了DdsA（decaprenyl diphosphate synthase，十聚异戊二烯焦磷酸合成酶）在4个氨基酸位点上的19个变体酶.这些位点分布在基因的不同区域内.证明这种新方法的高效性.

Novel Method for Site-directed Mutagenesis and Its Application in DdsA Mutant Research

To develop the gene mutagenesis technique, a novel method of site-directed mutagenesis was introduced. The half-blunt end restriction enzyme sites were used in this new method. Comparing with the traditional overlapping extension mutagenesis, this method could replace any nucleotides covered the gene sequence. Meanwhile, the mutant gene was cloned in the T-vector directly that make sequencing convenient. Nineteen mutants of DdsA (decaprenyl diphosphate synthase), focused on four residues, were generated via this method. The results showed that the new gene mutagenesis method was efficient.