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

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.

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Authors (5)

Magdalena Krygier
- Gdański Uniwersytet Medyczny Katedra i Zakład Biologii i Genetyki
Justyna Podolak-Popinigis dr inż.
Janusz Limon Prof. dr hab.
- Gdański Uniwersytet Medyczny Katedra i Zakład Biologii i Genetyki
Paweł Sachadyn prof. dr hab. inż.
Anna Stanisławska-Sachadyn
- Gdański Uniwersytet Medyczny Katedra i Zakład Biologii i Genetyki

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Category:: Articles
Type:: artykuł w czasopiśmie wyróżnionym w JCR
Published in:: ANALYTICAL BIOCHEMISTRY no. 500, pages 88 - 90,
ISSN: 0003-2697
Language:: English
Publication year:: 2016
Bibliographic description:: Krygier M., Podolak-Popinigis J., Limon J., Sachadyn P., Stanisławska-Sachadyn A.: A simple modification to improve the accuracy of methylation-sensitive restriction enzyme quantitative polymerase chain reaction// ANALYTICAL BIOCHEMISTRY. -Vol. 500, (2016), s.88-90
DOI:: Digital Object Identifier (open in new tab) 10.1016/j.ab.2016.01.020
Verified by:: Gdańsk University of Technology