Detection Of DNA Methylation Based On The Kinetics Of DNA Strand Displacement Reaction And GFET

DNA methylation,as a new type biomarker,shows a great potential for application in human disease detection and prognosis.Traditional testing and biosensors have been developed to detect DNA methylation levels,but these detection techniques limit their application in clinical diagnosis due to its drawbacks such as expensive equipment or complicated operation processes.Graphene,due to its excellent electrical conductivity,it has been widely used in the field of sensing,specially for Graphene Field Effect Transistor(GFET)which made of single-layer graphene.As its high detection sensitivity,it has been used in detection of a variety of biological molecules.To date,it is still lack of research on the use of GFET to detect DNA methylation levels.Therefore,in this study,we mainly developed a method based on label-free GFET to detect DNA methylation.In this study,we selected a sequence in the promoter region of Glutathione S-transferase pi 1(GSTP1),which was used as the target sequence of this experiment by methylation modification of cytosine in the sequence with different numbers and different sites.Firstly,in order to understand the kinetics reaction between the target sequence modified at different methylation levels and DNA probe,we used real-time fluorescence quantitative PCR to analyze the hybridization process between the target sequence modified at different methylation modification levels and the DNA probe,and we observed the effect of the sequences modified at different methylation levels on the rate of DNA probe chain replacement by using the principle of fluorescence quenching.The results showed that,whether hybridization with DNA probe or chain replacement reaction,the numbers and sites methylated in target sequence all have an effect on the above reactions.Secondly,through using the strand displacement reaction between DNA probe modified on the GFET surface and the difference methylation levels,and due to the offset of GFET current curve and the resistance change,we achieved the detection of the target sequence of different methylation levels,and the detection sensibility is 10pM.The results of this experiment provide a new,label-free and simple detection method for DNA methylation detection.Meanwhile,we also achieved detection of different methylation modification levels by GFET for the first time.