Ultrasensitive Detection Of Methylated DNA Site Based On Rolling Circle Amplification And Fluorescence Resonance Energy Transfer

Objective:DNA methylation is a common epigenetic modification in eukaryote,which plays an important role in multiple physiology and pathology processes,such as the expression and regulation of genes,cell proliferation,et al.On the other hand,the abnormal methylation in certain regions of genome may lead to the silence of tumor suppressor genes and thus was regarded as a biomarker of malignant tumor.The sensitive and accurate detection of DNA methylation is required in clinical practice.However,existing method of DNA methylation detection was restrained by sorts of obstacles.In this study,an ultrasensitive and specific method for DNA methylation site detection was established.Methods:1.Bisulfite treatment of DNA,rolling circle amplification and digestion.The target methylated DNA was treated with bisulfite to generate single base variation.By introducing padlock probe,DNA ligase and DNA polymerase,the target was amplified.The G-rich fragment was separated by enzyme digestion.The RCA reaction and digestion were confirmed by agarose gel electrophoresis.2.Preparation of oligonucleotide-modified quantum dots.EDC was used to activate the carboxylic acid group on the surface of QDs,which allows the surface modification of oligonucleotide.The non-conjugated oligonucleotide was removed by ultra-filtration.3.Self-assembly of G-quaruplex/hemin and fluorescence detection.QD-probe and digestion product were hybridized and forms G-quadruplex/hemin in the presence of hemin.The fluorescence signal was detected after introducing H₂O₂ to enlarge the signal.Results:1.The RCA reaction and enzyme digestion were characterized by agarose gel electrophoresis.The successful amplification indicates the correct identification of padlock probe and the correct enzyme digestion2.By a series of experiments,we optimized the experimental conditions.2 hours of incubation,20nM of primer concentration and 600?M of dNTP concentration were chosen as the optimal condition for RCA.100nM of hemin,0.5?M of H₂O₂ and 1 hour of incubation for FRET were chosen as the optimal condition for G-quaruplex/hemin self-assembly and signal detection.3.The biosensing system exhibits good analytical performance:under the optimal conditions,the biosensing system beholds ultrahigh sensitivity and specificity,with a low limit of the detection of142fM,and also showed good linearity from 1pM to 100nM.Conclusion:In summary,an ultrasensitive fluorescence sensing system was established based on RCA and FRET.Two signal enlargement mechanism was employed in this sensing system,RCA replicates the target and H₂O₂ assisted FRET enhanced the detected signal.By which the designed method demonstrated ultrahigh sensitivity,acceptable reproducibility and low matrix effect.In particular,the designed method presented an ultrahigh selectivity comparing to previous methods.On the other hand,this proposed method is completely independent on expensive equipment and agents,thus it's accessible to basic medical institution and laboratory.The easy-to-achieve platform of DNA methylation site detection demonstrated great potential for clinic application.