A microarray to analyze methylation patterns of p16 Ink4a gene 5′-CpG islands

Abstract

Objectives Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation on the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput approach for analyzing methylation patterns. The aim of this study was to develop a new method to analyze methylation patterns of p16 Ink4a gene. Design and methods We selected a 336 bp segment of the 5′ untranslated region and the first exon of the p16 Ink4a gene, as the target sequence, which include the most densely packed CpG fragment of the islands containing 32 CpG sites. A set of oligonucleotide probes was designed to assemble a DNA microarray to discriminate the methylation patterns of several adjacent CpG sites. Results Methylation patterns of human p16 Ink4a gene were mapped and the results were validated by bisulphite DNA sequencing. A good reproducibility was observed in several parallel experiments. Conclusions The methylation oligonucleotide microarray can be applied as a useful and powerful tool to map methylation patterns in multiple CpG island sites.