基于PLP-LDR-HRCA策略进行微量DNA分析——rs17750303位点分型  

A Pilot Study on PLP-LDR-HRCA Method for Trace DNA Analysis: Genotyping of rs17750303

作　　者：陈宝生[1] 张振[1] 王保捷[2] 

机构地区：[1]河南科技大学法医学院,洛阳471003 [2]中国医科大学法医学院,沈阳110001

出　　处：《中国生物化学与分子生物学报》2010年第8期776-782,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No30772460);高校博士科研启动基金项目(No09001369)资助~~

摘　　要：增强PCR和全基因组扩增是当前微量DNA分析的主要策略,但是,由于DNA模板量过少,受随机效应影响显著,往往不能得到可靠的DNA分型结果.本文提出一种新的检验策略:PLP-LDR-HRCA,尝试微量DNA检材的SNPs分型研究.选择rs17750303位点,并设计等位基因特异性锁式探针,采用连接酶检测反应来识别等位基因,而后采用超分支滚环扩增反应来放大检测信号.结果表明,PLP-LDR-HRCA反应特异性好,灵敏度高,能够直接鉴别微量基因组DNA模板中待测SNP位点,rs17750303纯合型样品(AA型或CC型)和杂合型样品(AC型)准确分型所需最少模板量分别为20pg和30pg.对于增强PCR和全基因组扩增技术不能有效检验的微量检材,PLP-LDR-PCR策略独具优势,可能具有较大的开发价值.Nowadays,enhancing PCR and whole genome amplification（ WGA） represent the two main strategies for trace DNA analysis. However,duo to stochastic variation in amplification of trace DNA template,both methods are liable to preferential amplification of some target fragments,resulting in unexplainable results. Here, for SNP genotyping of trace DNA, a PLP-LDR-HRCA method was developed. Two allele-specific padlock probes were designed for SNP rs17750303 locus. The discrimination of SNP alleles were achieved by padlock probe mediated ligation. Then,by hyper-branched rolling circle amplification,the ligation products were amplified for gel detections. By this method,trace DNA lower to 20 ～ 30 pg could be typed for SNP rs1775030 without pre-amplification. The sensitivity,specificity and reproducibility make it an alternative way for trace DNA analysis. Therefore, SNP genotyping based on the PLP-LDR-HRCA techniques may be a potential candidate for genetic analysis of trace samples.

关 键 词：微量DNA 锁式探针 连接酶检测反应 超分支滚环扩增 单核苷酸多态性 

分 类 号：Q503[生物学—生物化学]