One-step methylation variable position analysis technology in single-tube.
- Author:
Yang-Yang YUE
;
Gui-Sen ZHAO
;
Qian ZHANG
;
Di LU
;
Xian-Dun ZHAI
;
Yao-Nan MO
- Publication Type:Journal Article
- MeSH:
DNA/isolation & purification*;
DNA Methylation/genetics*;
DNA Primers/genetics*;
Humans;
Multiplex Polymerase Chain Reaction/standards*;
Nucleic Acid Denaturation
- From:
Journal of Forensic Medicine
2013;29(6):419-424
- CountryChina
- Language:Chinese
-
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.
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.
