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

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 2 mu L of sample solution consumption.