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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: To establish a PCR method to rapidly correct multi-locus deletion mutations during the synthesis of non-structural protein genes of Chikungunya virus. Methods: Chikungunya virus nonstructural protein gene was synthesized by PCR. Sequence alignment was performed on cloned clones. The position of deletion mutation on different clones was analyzed. Oligonucleotides with conserved regions overlapped each other as upstream and downstream primers. The correct clones in the region were used as templates for PCR amplification to obtain the desired fragments, and the fragments were further assembled into the complete gene sequence by PCR and sequenced. Results: Sequencing results showed that five mutations in the non-structural protein gene of chikungunya were deleted after two PCR amplifications. Conclusion: The correct sequence of Chikungunya virus non-structural protein gene was obtained. In the process of gene synthesis, such as the occurrence of multi-locus deletion mutations, this method can be used simultaneously to correct the above mutations without further synthesis of primers, reducing the difficulty of experimental operation and improve the experimental efficiency.