基孔肯雅病毒非结构蛋白基因合成中多位点缺失突变的校正方法

Correction of Multi-Site Deletions During Synthesis of Chikungunya Virus Non-Structure Protein Gene

目的:建立一种PCR方法,以快速校正基孔肯雅病毒非结构蛋白基因合成过程中发生的多位点缺失突变。方法:用PCR方法合成基孔肯雅病毒非结构蛋白基因;对测序的克隆进行序列比对,分析不同克隆上缺失突变发生的位置,以保守区域互相重叠的寡核苷酸为上下游引物、以该区域测序正确的克隆为模板进行PCR扩增,得到所需片段,再将这些片段用PCR方法进一步组装成完整的基因序列并进行测序。结果:测序结果表明,经过2次PCR扩增,校正了基孔肯雅病毒非结构蛋白基因合成过程中发生的5个位点缺失突变。结论:得到序列正确的基孔肯雅病毒非结构蛋白基因。在进行基因合成过程中如发生多位点缺失突变,可利用该方法同时对以上突变进行校正,无须再合成引物,降低了实验操作难度,并提高了实验效率。

Objective：Considering the multi-site deletions during gene synthesis of Chikungunya virus non-structure protein gene,set up a novel method to correct those mutations simultaneously based on PCR.Methods：The sequence of Chikungunya virus non-structure protein gene was synthesized based on PCR method.The positions of deletion in different clones were identified through alignment.Then the PCR was performed to amplify the correct part of the correct clone using previously synthesized oligo-nucleotides which overlaps and locates in the constant region.The above obtained fragments were further assembled by PCR using the outer oligonucleotides.The full gene was then sequenced.Results：The results of sequencing showed that the five deletion mutations in Chikungunya non-structure protein gene synthesized were corrected after two-round PCR reaction.Conclusion：The correct sequence of Chikungunya virus non-structure protein gene was obtained,and this method could be used to correct the multi-site deletions during gene synthesis effectively and efficiently.