Detection of genomic DNA methylation with denaturing high performance liquid chromatography.
DNA methylation contributes to the epigenetic control of gene expression. Variations in the methylation status can result in the silencing of genes. DNA methyltransferase converts cytosine to 5-methyl cytosine in CpG islands located in the promoter regions of genes. When CpG islands are hypermethylated, the gene is repressed/silenced, and similarly when it is hypomethylated, transcription can take place and the gene is expressed. The classical methods to detect DNA methylation require labor-intensive and time-consuming steps. As a result of large-scale expression profiling studies, high-throughput techniques are needed to screen for alterations in the methylation patterns. Denaturing high performance liquid chromatography (DHPLC) is a reliable, highly sensitive technique for mutation discovery. In the present study we examined the suitability of DHPLC technology to detect alterations in methylation pattern of the promoter regions of several genes. We report reliable and reproducible results in distinguishing methylated and unmethylated promoter regions of human PCDHGB6, c-MYC, MGMT1, CDKN2A/p16, and ATM genes. These DHPLC profiles were independently confirmed with bisulfite genomic sequencing. In conclusion, DHPLC technology serves as a rapid screening tool to monitor the genomic DNA methylation and could be used to increase the throughput efficiency of methylation analysis.
Omaruddin
Romaica A
RA
Department of Medical Laboratory and Radiation Science, University of Vermont, Burlington, Vermont 05405, USA.
Chaudhry
M Ahmad
MA
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019175:DNA Methylation / Q000502:physiology*; D015894:Genome, Human; D006801:Humans; D011401:Promoter Regions, Genetic