一种单核苷酸多态性的单倍型分析技术

运用多步PCR和测序技术，完成基因组中相距较远的单核苷酸多态位点的单倍型构建。通过设计2条等位基因特异性引物，扩增大片段DNA(10kb左右)，以此大片段DNA作为下一轮PCR反应的模板，再在该片段中设计待检测区域的PCR引物，进行第2轮PCR。对PCR产物进行测序分析，确定其多态位点处的等位基因。结合第1轮PCR中的等位基因特异性引物，即可确定该大片段DNA中不同单核苷酸多态性构成的单倍型。以脂蛋白脂酶基因为例，应用其启动子区以及第4外显子区的等位基因特异性引物扩增约16kb的DNA片段，然后检测位于该片段中第2、3外显子的多态性。在￡-尸￡-基因第2内含子中发现了 13557G→A多态性。经分析确定出-421G／ 13557G／ 15222A、-421A／ 13557G／ 15222A、-421G／ 13557G／ 15222G、-421G／ 13557A／ 15222A等4种单倍型。等位基因特异性PCR结合小片段测序是一种快捷高效的对相距较远的多个SNP进行单倍型构建的新策略。

A Method of Haplotype Analysis for Multiple Single-nucleotide Polymorphisms

Haplotypes from multiple single nucleotide polymorphisms(SNPs) spaced in longer DNA were constructed by multi-step PCR and DNA sequencing methods.Two allele-specific primers were synthesized and used for long DNA fragments (～10 kb) amplification from human genome DNA.Fragments within these long DNA fragments were amplified by using these PCR products as templates in the second round PCR.The second round PCR products were subsequently sequenced.Haplotype construction was performed based on the character of nucleotides at the 3′-end of the allele-specific primers and the sequencing results from the second round PCR products.The DNA fragment(～16 kb) from promoter to exon 4 of lipoprotein lipase(LPL) gene was amplified by allele-specific primers,and the DNA fragments including exon 2 or exon 3 of LPL gene were amplified and sequenced.A SNP of +13,557G→A within intron 2 was identified.Four haplotypes including -421G/+13557G/+15222A,-421A/+13557G/+15222A,-421G/+13557G/+15222G,-421G/+13557A/+15222A were detected.The method is effective and relatively simple for construction of haplotypes from multiple single nucleotide polymorphisms.