title: [One-step methylation variable position analysis technology in single-tube].
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 degrees C, 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.
  
  
  CONCLUSIONS
  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.
authors.forename: Yang-Yang
authors.initials: YY
authors.lastname: Yue
authors.forename: Gui-Sen
authors.initials: GS
authors.lastname: Zhao
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhang
authors.forename: Di
authors.initials: D
authors.lastname: Lu
authors.forename: Xian-Dun
authors.initials: XD
authors.lastname: Zhai
authors.forename: Yao-Nan
authors.initials: YN
authors.lastname: Mo
mesh_terms: D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019175:DNA Methylation / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D009691:Nucleic Acid Denaturation
chemical_list: D017931:DNA Primers; D004247:DNA