Application Of Toehold 's Chain Displacement Reaction In Nucleic Acid Detection

Abstract:Objective We developed a silver enhanced fluorescence method for single nucleotide polymorphisms(SNP) at nanomolar level utilizing toehold-mediated strand-displacement reaction, a highly sensitive and selective method for mutation detection utilizing metal enhanced fluorescence (MEF). Method A hairpin capture probe containing an external toehold region was immobilized on96-well plate coated with silver nanoparticles. Strand-displacement was initiated by introducing the target ssDNA and resulting in the unfolding of the capture probe. A signal probe coupled with FAM was further hybridizing with the unfolding capture probe. The silver coated in the well can significantly enhance the signal of the fluorophore in certain distance. As the distance between silver and fluorophore is a crucial factor in determining the amplification of signal, the capture probe is verified by fluorescence sensing and the final distance was about10nm. Results Comparing to normal method, the toehold design holds noticeable specificity and highly sensitivity with the detection limit of0.1nM in silver-coated96-well plate. Conclusion The MEF assay provided an alternative screening method for mutation detection. Abstract:Object We developed a double-plex hybridizing chain polymerization system through toehold-mediated strand displacement reaction to achieve signal amplification in DNA/RNA detection, and also apply the system to real sample detection. Methods The target hybridized with the capture probes immobilized in96-well plate, and then triggered hybridize chain reaction by opening the hairpin structure probe to form a linear polymer with a hangout single strand DNA in every monomer. The hangout strand coule further trigger the next fold polymerizition, hence, the system could generate double-plex signal amplification. Agarose gel and Biacore were used to demenstrate the formation of hybridization chain reaction products and optimize experimental condition. SYBR Green I can be enbedded in DNAdouble helix and show remarkablely fluorescence signal amplification comparing with free state. Results Comparing with control group, the signal had a130-fold amplification with a target concentration of1nM in experientmental group, and because of the advanture of toehold-mediated strand displacement reaction, the specificity of the system was not decreaed alone with the enhancd sensitivity. We further apply the system in malaria RNA detection, which also showed remarkablely signal amplification. Conclusion The method has great potential in disease diagnosis.