摘要
单核苷酸多态性(SNP)分型可以作为丰富的分子标记用于家蚕群体遗传学、基因组注释以及功能基因研究。利用SNaPshot试剂盒的SNP分型方法,对以单碱基延伸法检测家蚕SNP位点进行了探索。首先对SNP位点进行PCR扩增,然后以PCR产物为模板,以4种荧光标记ddNTPs为底物,采用延伸引物扩增SNP位点处的一个碱基,最后用377测序仪进行基因分型。通过对家蚕2个SNP位点的分析结果表明,利用单碱基延伸法可以准确地检测SNP位点,并且可以知道该位点的碱基。由于单碱基延伸法可以通过多重PCR在1个反应体系检测多至10个位点,因此该方法不仅方便快捷,并且可以实现高通量检测SNP位点。
SNP genotyping plays an important role in population genetics, genome annotation and functional gene study in silkworm, Bombyx mori. The present study employed single base extension （SBE） method to explore SNP （single nucleotide polymorphism） genotyping in the silkworm Bombyx mori by using the SNaPshot kit. SNP locus was amplified at first, and then the PCR product was used as template to amplify the SNP locus by a specific extension primer that locates at the upstream of the SNP site. Only one nucleic acid, which is exactly the SNP locus, could be added to the end of the specific extension primer since the fluorescent labeled ddNTPs were used in the assay. The genotyping was carried out by using an ABI 377 DNA sequencer. An analysis to the results of two silkworm SNP loci turned out that SBE genotyping could detect the SNP locus accurately. Nevertheless, the nucleotide at that locus could be discerned from electrophoretic result. Since SBE genotyping method can interrogate up to 10 SNP loci by multiplex PCR, this makes it convenient and efficient in genotyping, and can also achieve high throughput genotyping of SNP loci.
出处
《蚕业科学》
CAS
CSCD
北大核心
2009年第3期523-527,共5页
ACTA SERICOLOGICA SINICA
基金
国家重点基础研究发展计划"973"项目(编号2005CB12-1001)
关键词
单碱基延伸
单核苷酸多态性
基因分型
家蚕
Single base extension
Single nucleotide polymorphism
Genotyping
Bombyx mori
