Detection Of Methyltransferase Activity By Using Isothermally Amplification Reaction

DNA methylation is one of the most important epigenetic approaches.The expression of genes,interaction of proteins and development of tumors are closely related to DNA methylation level and DNA methyltransferase(MTase)activity.It has a great significance to develop a simple and sensitive new method for the detection of MTase activity in the field of biochemical analysis and clinical disease diagnosis,which relate to the activity of MTase.Here,we develop two new simple,sensitive methods for detection of the MTase activity by using isothermally amplification reaction.The main works are summarized as follows:1.Detection of DNA MTase activity based on isothermally exponential amplification reaction(EXPAR).First,we design a label-free hairpin probe.The stem of the hairpin probe was designed to incorporate the specific recognition sequence of DNA adenine methylation Dam MTase and the loop was designed with a primer sequence,which can trigger the EXPAR.In the presence of Dam MTase,the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease of Dpn I.The cleaved hairpin probe then acts as a primer to initiate EXPAR.In the absence of Dam MTase,the hairpin probe cannot be methylated and cleaved,leading to a no effective EXPAR.Combined with the specific DNA fluorescence dye,we can achieve the detection of MTase by signal amplification.The method is simple,fast,and can be used for MTase activity detection,molecular diagnosis and disease treatments.2.Integration of rolling circle amplification and cationic conjugated polymer for the homogeneous detection of MTase activity.In the presence of Dam MTase and endonuclease,the hairpin probe that incorporates the specific recognition sequence of Dam MTase in the stem was methylated and cleaved.The cleaved sequence in the loop can act as the primer to initiate the RCA reaction.In the process of RCA,we introduce the FITC-modified dUTP(dUTP-FITC),which can be combined to DNA along with reaction.By adding PFP,high efficient fluorescence resonance energy transfer(FRET)from PFP to FITC binding to the DNA strand can occur.By contrast,when there is just Dam TMase in solution,the hairpin probe will be remained intact,so it cannot trigger RCA.Then,when dUTP-FITC free in solution,they will be degradated by alkaline phosphatase,the efficient FRET from PFP to FITC will not take place owing to the weak electrostatic interaction between PFP and FITC.We demonstrated that this method could be utilized to detect MTase activity based on different FRET efficiency.It has been proved that this method possesses the advantage of simplicity of instrument and high sensitivity with a detection limit of 0.36 U/mL.The method may open a new way for the detection of the MTase activity related to the clinical diagnosis and drug screening.