CAJ | 학술논문

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 (10 kb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16 kb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LPL基因第2内含子中发现了+13557 G→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.
운용다보PCR화측서기술,완성기인조중상거교원적단핵감산다태위점적단배형구건. 통과설계2조등위기인특이성인물,확증대편단DNA (10 kb좌우),이차대편단DNA작위하일륜PCR반응적모판,재재해편단중설계대검측구역적PCR인물,진행제2륜PCR.대PCR산물진행측서분석,학정기다태위점처적등위기인.결합제1륜PCR중적등위기인특이성인물,즉가학정해대편단DNA중불동단핵감산다태성구성적단배형.이지단백지매기인위례,응용기계동자구이급제4외현자구적등위기인특이성인물확증약16 kb적DNA편단,연후검측위우해편단중제2、3외현자적다태성.재LPL기인제2내함자중발현료+13557 G→A다태성.경분석학정출-421G/+13557G/+15222A、-421A/+13557G/+15222A、-421G/+13557G/+15222G、-421G/+13557A/+15222A 등4충단배형.등위기인특이성PCR결합소편단측서시일충쾌첩고효적대상거교원적다개SNP진행단배형구건적신책략.
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.