基于NUPACK预测设计的Toehold诱导链置换反应及其在DNA酶催化微流控化学发光单核苷酸多态性分析中的应用

NUPACK Prediction Assisted of Toehold Induced Strand Displacement Reaction and Its Application in SNPs Genotyping by DNAzyme-catalyzed Microfluidic Chemiluminescence Detection

以阿尔兹海默症相关的基因片段rs242557为研究对象,通过NUPACK软件预测Toehold(黏性末端)长度对链置换反应的影响,设计具有高特异性的双链核苷酸探针;富G的核苷酸序列首先被掩蔽在WatsonCrick双链结构中,当体系加入目标基因后,通过引发链置换反应解开该双链结构,形成的DNA酶可催化氧化鲁米诺化学发光反应,最终采用微流控化学发光法实现单核苷酸多态性(SNP)分析.该方法不仅具有设计简单、免标记及检测通量高等优点,而且在仅消耗2μL试样的条件下实现了单碱基突变的rs242557目标基因的SNP分析(区分因子达56),正常目标基因的检出限为1.7 nmol/L.

Highly sensitive and selective method for single nucleotide polymorphisms（SNPs） genotyping is of great importance for early diagnosis and treatment of various diseases. By taking rs242557, an Alzheimer＇ s disease related gene fragment, as a target model, the impact of toehold length on strand displacement reaction was predicted by NUPACK software simulation in order to rationalize the design of duplex DNA probe for high genotyping efficiency. The G-rich DNA sequence was initially blocked in the duplex strand structure. With the addition of target gene fragment to trigger the blocked-domain dehybridization and the toehold-mediated strand displacement reaction, the DNAzyme was formed. Combined with microfluidic chemiluminescence detection, a novel SNP genotyping method had been developed, which was not only label-free, easy to design and exhibited high throughput ability, but also displayed a remarkable specificity to target rs242557 against single base mutation, achieving a discrimination factor of 56 and a detection limit of 1.7 nmol/L with just 2μL of sample solution consumption.