古核嗜热菌组蛋白基因多点突变的新方法

A New Method for Multipoint Mutation of the Histone in Archaic Ther- mophilic Bacterium

嗜热菌组蛋白基因序列中精氨酸密码子在大肠杆菌中的表达率极低,限制了组蛋白的大量获得。运用分子生物学技术,在传统PCR定点突变技术的基础上进行改进,参照古核嗜热菌组蛋白基因序列和大肠杆菌偏爱密码子表,设计了一对含有5个精氨酸突变密码子的单链DNA,全长为115nt,在3’端有20nt互补序列。将单链DNA在94℃变性,45℃复性,使其互补结合,72℃进行延伸反应,得到含有突变位点的组蛋白基因;再以此基因为模板设计相应的一对引物进行PCR扩增反应,得到大量组蛋白突变基因。该研究为一些序列已知,片段较小基因的多点突变提供了一种简单、易行的方法。

The histone gene in Archaic Thermophilic bacterium express poorly, which limits the massive acquisition of the histone. Using the molecular biological technique, modifying the traditional technique of PCR fixed point mutation and according to the sequence for the histone gene of archaic thermophilic bacterium and the table for partial codons of Escherichia coli, this study has desighed a pair of simple chains which contain a mutated point. The entire length is 115nts. At the end of 3', 20nts are complementary. We denaturalize the simple chains of DNA at 94℃ , renaturalize them at 45℃ to unite them and then extend them at 72℃. Thus, we produce histone gene with the mutated point. With this as the template, we design the corresponding primers and end up with the massive histone mutated gene. This study provides a feasible method for multipoint mutation into the small fragment genes whose ger.e sequences are known.