基于磁性颗粒微阵列与双色荧光杂交的单核苷酸多态性分型方法研究  

Single Nucleotide Polymorphism Genotyping Based on Magnetic Bead Array and Dual-color Hybridization

作　　者：刘洪娜[1] 李松[1,2] 刘丽赏[2] 田岚[1] 贾英英[1] 李智洋[1] 邓燕[1,2] 何农跃[1,2] 

机构地区：[1]东南大学生物电子学国家重点实验室,南京210096 [2]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

出　　处：《分析化学》2010年第12期1708-1713,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(Nos.60801007;60927001;60971045);863国家高技术研究发展计划(No.2007AA022007);国家重点基础研究发展计划-纳米研究重大科学研究计划(Nos.2007CB936104;2010CB933903);中国博士后科学基金(No.2010471007);国家科技重大专项"十一五"计划科研支撑平台项目(No.2009ZX10004-311);江苏省博士后科研资助计划(No.1001009C);湖南省教育厅科研基金(No.08B018);湖南省自然科学基金(No.09JJ4044;09JJ3017)资助

摘　　要：基于磁性颗粒微阵列与双色荧光杂交,建立了单核苷酸多态性(Single nucleoitide polymorphism,SNP)分型方法。将利用不对称扩增得到的含有待检测位点生物素标记的单链PCR产物固定在链亲和素修饰的金磁纳米颗粒(Gold magnetic nanoparticles,GMNPs)表面;将ssDNA-GMNPs混合物点样在底部固定有磁铁的载玻片上构建磁性颗粒微阵列,然后在基因框中与双色荧光探针杂交;杂交完全后,充分洗涤,通过扫描获得分型结果。通过优化不对称PCR的扩增条件,直接扩增出产量较高的单链DNA作为靶序列用于分型。利用本方法对24个样本MTHFR基因的C677T位点多态性进行了检测。实验证明,本方法步骤简单,易实现自动化操作、非常适用于分子诊断与法医鉴定。An approach to fabricate magnetic bead array using gold coated magnetic nanoparticles was developed.Single strand DNAs(ssDNA) obtained using asymmetry PCR were captured on gold coated magnetic nanoparticles(SA-GMNPs) modified with streptavidin.The GMNPs-ssDNA mixtures were spotted onto a clean glass slide adhered with a magnet to fabricate a bead array,and then interrogated by hybridization with a pair of dual-color probes to determine single nucleotide polymorphism(SNP).The genotype of each sample can be simultaneously identified on the surface of GMNPs by scanning the microarray.The amplification factors were optimized to obtain ssDNA directly by use of polymerase chain reaction(PCR).In this method,the genotyping results were obtained from 24 different samples analyzed for C677T loci.The method described here is simple and easily to be automated,which is suitable for molecular diagnostics and forensic analysis.

关 键 词：磁性颗粒微阵列 不对称扩增 双色荧光杂交 单核苷酸多态性 

分 类 号：Q75[生物学—分子生物学]