【摘要】 目的:DNA片段中快速构建位点不同的定点双突变体。方法:借鉴DNA shuffling技术中DNA小片段延伸扩增获得全长DNA片段的工作原理,与常规基因定点突变技术相结合,改进重叠延伸PCR技术构建定点双突变。结果:对嗜酸热脂肪杆菌(Alicyclobacillus acidocaldarius)Tc-12-31的甘露聚糖酶基因AamanA中两个可能的活性位点E151和E231进行双点突变,先后经过无引物和有引物两步PCR,扩增获得全长DNA,测序结果表明得到预期的定点双突变体;酶活性检测和薄层层析结果表明双点突变体丧失了酶的活性。结论:改良的重叠PCR技术,能经济、简便、高效地获得双点定点突变体,在酶的催化机理的阐述、蛋白质结构改造等分子生物学领域中具有较高的应用价值。更多还原

【Abstract】 Objective:in order to improve the processing of construction the mutant with double mutations at two different residue sites.Methods:According to the method for construction of the full-length DNA fragment by DNA shuffling,the primer pairs were designed at the mutation site,three small DNA fragments were PCR amplified using wild type gene as template,respectively,then separately mixed three DNA fragments and used as templates to carry out PCR without primer.The PCR product was subjected to the last-ste PPCR with primers to amplify the full-length gene AamanA with double-site mutations.Results:the DNA sequenced results indicates the mutant with double mutations at residues E151 and E231 was succeed to create.The Thin-layer chromatography and enzyme activity assay clearly shown the catalytic activity of the mutant with double mutations was lost.Conclusion:this is a simple,economic,rapid and effective method to construct two mutations in the DNA fragment.It has the potential application value in the field of molecular biology,such as characterization the reaction mechanism of enzyme and modification the structure of the proteins,and so on.更多还原