The Detection Method Of Single-nucleotide Mutation Based On Nucleic Acid Transient Hybridization And Magnetic Separation Technology

Single-nucleotide mutation(SNM)has proven to be associated with a variety of human diseases.Development of reliable methods for the detection of SNM is crucial for molecular diagnosis and personalized medicine.The sandwich assays are widely used tools for detecting nucleic acid biomarkers due to their low cost and rapid signaling.However,the poor hybridization specificity of signal probe at room temperature hampers the discrimination of mutant and wild type.Here,we demonstrate a dynamic sandwich assay on magnetic beads for SNM detection based on the transient binding between signal probe and target.By taking the advantage of mismatch sensitive thermodynamics of transient DNA binding,the dynamic sandwich assay exhibits high discrimination factor for mutant with a broad range of salt concentration at room temperature.By investigating the DNA hybridization on magnetic particle surface,we found that the stability of wild type duplex(single-nucleotide mismatched duplex)can be selectively weakened by the high density negative charge on particle surface and have no significant effect on the mutant duplex.Therefore,The beads used in this assay not noly serve as a tool for separation,but also can improve the selectivity of SNM;Flexible design of signal probe and facile magnetic separation allow multiple-mode downstream analysis including colorimetric detection and isothermal amplification.For colorimetric detection,the typical green color and strong absorbance at 410 nm of the oxidation product of ABTS-were found in the presence of mutant,however,not found in presence of wild type.The detection limit of DSA based on G-rich probe was 5 nm.For isothermal amplification,5 amol mutant can be detected within 1 h.As expected,the wild type does not show typical amplification curve.The detection limit of DSA based on isothermal amplification was 0.1 fM;With this method,BRAF D594G(c.1781A>G)and BRAF V600E(c.1799T>A)mutations in the genomic DNA extracted from cancer cell lines were tested,allowing sensitive detection of SNM at very low abundances(0.1-0.5%mutant/wild type).