A simple modification to improve the accuracy of methylation-sensitive restriction enzyme quantitative polymerase chain reaction

Magdalena Krygier; Justyna Podolak-Popinigis; Janusz Limon; Paweł Sachadyn; Anna Stanisławska-Sachadyn

doi:10.1016/j.ab.2016.01.020

A simple modification to improve the accuracy of methylation-sensitive restriction enzyme quantitative polymerase chain reaction

Anal Biochem. 2016 May 1:500:88-90. doi: 10.1016/j.ab.2016.01.020. Epub 2016 Feb 4.

Authors

Magdalena Krygier¹, Justyna Podolak-Popinigis², Janusz Limon¹, Paweł Sachadyn², Anna Stanisławska-Sachadyn³

Affiliations

¹ Department of Biology and Genetics, Medical University of Gdańsk, 80-210 Gdańsk, Poland.
² Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, 80-233 Gdańsk, Poland.
³ Department of Biology and Genetics, Medical University of Gdańsk, 80-210 Gdańsk, Poland. Electronic address: astanisl@gumed.edu.pl.

PMID: 26853744
DOI: 10.1016/j.ab.2016.01.020

Abstract

DNA digestion with endonucleases sensitive to CpG methylation such as HpaII followed by polymerase chain reaction (PCR) quantitation is commonly used in molecular studies as a simple and inexpensive solution for assessment of region-specific DNA methylation. We observed that the results of such analyses were highly overestimated if mock-digested samples were applied as the reference. We determined DNA methylation levels in several promoter regions in two setups implementing different references: mock-digested and treated with a restriction enzyme that has no recognition sites within examined amplicons. Fragmentation of reference templates allowed removing the overestimation effect, thereby improving measurement accuracy.

Keywords: Assay; MSRE–qPCR; Methylation; Methylation-sensitive restriction enzyme; Validation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Methylation*
DNA Restriction Enzymes / metabolism*
Polymerase Chain Reaction / methods*

Substances

DNA Restriction Enzymes