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Fa Yi Xue Za Zhi. 2013 Dec;29(6):419-24.

[One-step methylation variable position analysis technology in single-tube].

[Article in 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.

PMID:
24665611
[Indexed for MEDLINE]

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