Screening Hypermethylated Regions by Methylation-Sensitive Single-Strand Conformational Polymorphism

Abstract

DNA methylation is an important epigenetic modification that alters transcription in those genes containing CpG islands. In this report we demonstrate that the familiar technique single-strand conformation polymorphism (SSCP) can be easily applied to bisulfite-treated DNA to detect methylation differences over large (∼250 bp) CG-enriched regions. In the methylation-sensitive SSCP (Ms-SSCP) technique, sodium bisulfite modification of DNA converts unmethylated cytosine to uracil under conditions in which 5-methylcytosine remains unaltered. Amplification of these regions to be tested is then performed using primers specific for bisulfite-treated DNA. The resulting products are then subjected to nondenaturing gel analysis. Based on differential band mobility, Ms-SSCP is able to sensitively detect alterations in methylation density and determine if unmethylated alleles are present in a sample. Ms-SSCP is a rapid and simple technique for screening multiple samples for methylation changes. It has several advantages over existing techniques, including the utilization of nanogram amounts of DNA, the avoidance of difficulties in sequencing CG-enriched regions, and the screening of multiple sites for methylation across large regions of DNA.