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

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.

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

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.