一种基于磁性纳米粒子PCR的高通量SNP分型方法  

High-throughput SNP Genotyping Method with PCR on Magnetic Nanoparticles

作　　者：刘洪娜[1] 李松[1] 王志飞[2] 何农跃[2] 贺全国[1] 

机构地区：[1]湖南工业大学绿色包装与生物纳米技术应用湖南省重点实验室,株洲412008 [2]东南大学生物电子学国家重点实验室生物科学与医学工程系,南京210096

出　　处：《高等学校化学学报》2007年第6期1035-1038,共4页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金(批准号:60571032;60121101;20505020;90606027);国家'八六三'计划(批准号:2006AA02Z133);高等学校博士学科点专项科研基金(批准号:20050286014);湖南省教育厅科研项目(批准号:05C508)资助

摘　　要：利用磁性纳米粒子PCR扩增(MNPs-PCR)和等位基因特异性双色荧光探针(Cy3,Cy5)杂交,建立了一种单核苷酸多态性(SNP)分型的新方法.应用该方法对9个样本MTHFR基因的C677T多态进行检测,野生和突变型样本正错配信号比大于9·0,杂合型正错配信号比接近1·0,分型结果经测序验证.此方法无须产物纯化、浓缩,扫描分型结果快速、直观,是一种操作简单、快速、高通量、高灵敏度的分型方法.Single nucleotide polymorphisms （SNPs） assay is important for elucidating the genetics of indivi-dual differences in drug response and disease susceptibility. This report described a novel high-throughput SNP genotyping method using magnetic nanoparticles as PCR primers carriers. PCR products were directly am-plified on MNPs and interrogated by hybridization with a pair of dual-color fluorescence （ Cy3, Cy5 ） probes to determine SNP, and then genotype of each sample can be simultaneously identified by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. As the results, the homozygous wild type, homozygous mutant type and heterozygote type yield strongly green, red and yellow fluorescent sig-nals, respectively. The methylenetetrahydrofolate reductase（MTHFR） gene C677T polymorphism of nine di-fferent samples was detected and their fluorescent signals of the nine samples were quantified. The fluorescent ratios （ match probe signal to mismatch probe signal） of homozygous samples were over 9.0, whereas heterozy-gous samples had ratios near 1.0. The genotyping results were additionally validated by sequencing. Without any purification and concentration of PCR products, the approach reported here should be a simple, sensitive, high-throughput and high accurate genotyping method.

关 键 词：磁性纳米粒子 PCR 双色荧光杂交 SNP分型 

分 类 号：O657[理学—分析化学]