Abstract: Objective To develop the single-tube one-step methylation variable position （MVP） analysis technology——single-tube post-digestion PCR-melting curve analysis （PDP-MCA）. Methods Based on differentially methylated region （DMR） reported previously as the model, a set of primers with different melting temperatures of products in the two sides of MVP were designed. By using the FastDigest methylation-sensitive restriction enzyme （MSRE）, DNA digestion, multiplex amplification, MCA detection and MCA profiles were performed in a single reaction tube. Same samples （peripheral venous blood, semen, and vaginal fluid, 5 samples each type） were tested by single-tube one step MVP and traditional MSRE- PCR MCA technology. To verify the feasibility of this method, the results were compared with that of the traditional technology. The MCA/HRM profiles of different samples were analyzed and compared. Results When the melting temperature of the fragments had a differential of 2 ℃, the MCA melting peaks separated well, and MCA detection after multiplex amplification was successful. The single-tube PDP-MCA assay was developed, which integrated multiple reactions （digestion, amplification and detection） into one tube. By this method, the sample-specific profiles and data were analyzed in 2 h, which is similar to that of the traditional method. The rapid classifications of the samples were also realized. Conclusion Multiplex MVPs can be analyzed in a single closed-tube. The single-tube PDP-MCA technology is a simple, fast, and automatable method. It can be used for detection of DNA methylation variations.

Key words: forensic genetics, DNA methylation, melting curve, methylation-sensitive restriction enzyme, methylation variable position