Premium
Determination of DNA methylation by COBRA: A comparative study of CGE with LIF detection and conventional gel electrophoresis
Author(s) -
Goedecke Simon,
Schlosser Sabrina,
Mühlisch Jörg,
Hempel Georg,
Frühwald Michael C.,
Wünsch Bernhard
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200900204
Subject(s) - dna methylation , gel electrophoresis , microbiology and biotechnology , dna , sybr green i , methylation , ethidium bromide , biology , epigenetics , chemistry , real time polymerase chain reaction , gene , biochemistry , gene expression
DNA methylation as an epigenetic modification of the human genome is under emphatic investigation. Several studies have demonstrated a role of DNA methylation in oncogenesis. In conjunction with histone modifications, DNA methylation may cause the formation of heterochromatin and thus mediate the inactivation of gene transcription. It is important to develop methods that allow for an accurate quantification of the amount of DNA methylation in particular DNA regions, to gain information concerning the threshold of methylation levels necessary for gene inactivation. In this article, a CGE method with on‐column LIF detection using SYBR Green is compared with a conventional slab‐gel electrophoresis. We thus investigate the validity to analyze DNA methylation in the samples of a combined bisulfite restriction analysis. It is demonstrated that CGE is superior to gel electrophoresis in means of linearity, precision, accuracy, automatization (high throughput), and sample consumption. However, gel electrophoresis is easier to perform (simple devices, no PC usage), and the running costs are comparatively low. A further advantage of CGE is the sparse use of toxic compounds (MeOH and SYBR Green), whereas gel electrophoresis is performed in polyacrylamide gels with ethidium bromide staining.