A Method Of Assessment Of Bisulfite Conversion Rate For DNA Methylation Analysis

Background:DNA methylation, an important molecular modification associated with epigenetic regulation of the genome, is involved in many normal biological processes and human diseases. Bisulfite conversion of genomic DNA has been widely applied in the examination of DNA methylation. However, there is no approach to assess the efficiency of bisulfite conversion of genomic DNA, and the converted rate can affect the accuracy of DNA methylation analysis. In this study, we report a novel but simple method to detect the conversion rate of bisulfite treatment of genomic DNA based on quantitative Real-time PCR technology.Aims:A novel and simple method detect the conversion rate of bisulfite treatment of different species DNA based on quantitative Real-time PCR (Real time PCR) technology.Methods:Taqman Real time PCR assay was performed using probes and primers that are specific for bisulfite-converted or-unconverted DNA templates. Two linear standard curves were generated by plotting Ct values against logarithm of absolute DNA amount with serial dilutions of the bisulfite-converted or unconverted DNA templates. The bisulfite-treated DNA samples were detected by TagMan Real time PCR as described above. Then the absolute copy numbers of converted DNA and unconverted DNA in unknown samples were calculated using the standard curves. The rate of bisulfite conversion can be calculated by using the formula [The rate of bisulfite conversion=Conv/(unconv+Conv) X 100%]. DNA fragment that is not homology with any known species was constructed as a reference sequence. The DNA of samples and the reference sequence can be converted by bisulfite in the same tube, and mixed DNA was detected by SYBR Green Real time PCR to assess the rate of bisulfite conversion.Results:After generating two standard curves, the unknown bisulfite-treated genomic DNA samples were analyzed by specific Taqman probes and primers for the conversion rate of genomic DNA. This method could be verified by Real time PCR using known DNA templates with serial dilutions of the bisulfite-converted and-unconverted DNA.Conclusion:This approach can effective detect the conversion rate of bisulfite treatment of genomic DNA from different species, and improve the accuracy of DNA methylation analysis.