Discovery of DNA hypermethylation using a DHPLC screening strategy.
Promoter hypermethylation is recognized as a hallmark of human cancer, in addition to conventional mechanisms of gene inactivation. As such, many new technologies have been developed over the past two decades to uncover novel targets of methylation and decipher complex epigenetic patterns. However, many of these are either labor intensive or provide limited data, confined to oligonucleotide hybridization sequences or enzyme cleavage sites and cannot be easily applied to screening large sets of sequences or samples. We present an application of denaturing high performance liquid chromatography (DHPLC), which relies on bisulfite modification of genomic DNA, for methylation screening. We validated DHPLC as a methylation screening tool using GSTP1, a well known target of methylation in prostate cancer. We developed an in silico approach to identify potential targets of promoter hypermethylation in prostate cancer. Using DHPLC, we screened two of these targets LGALS3 and SMAD4 for methylation. We show that DHPLC has an application as a fast, sensitive, quantitative and cost effective method for screening novel targets or DNA samples for DNA methylation.
Perry
Antoinette S
AS
Department of Haematology and Academic Unit of Clinical and Molecular Oncology, Institute of Molecular Medicine, St. James's Hospital and Trinity College, Dublin, Ireland.
Liyanage
Hema
H
Lawler
Mark
M
Woodson
Karen
K
D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics* / Q000302:isolation & purification; D051549:Glutathione S-Transferase pi / Q000235:genetics*; D006801:Humans; D008297:Male; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000235:genetics
D004273:DNA, Neoplasm; D004247:DNA; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi