NotI-MseI methylation-sensitive amplified fragment length polymorphism forDNA methylation analysis of human cancers

We have applied a methylation-sensitive restriction endonuclease, Notl, to the existing amplified fragment length polymorphism (AFLP) method and devel oped Notl-Msel methylation-sensitive-AFLP (MS-AFLP). Notl-Msel MS-AFLP allo ws the analysis of DNA methylation alterations at the Notl sites scattered over the genome. Hypermethylation and hypomethylation are visualized by the decrease and increase in the band intensity of DNA fingerprints. Identific ation of consistent changes can be facilitated through parallel electrophor esis of multiple samples. DNA fragments exhibiting alterations can be clone d from fingerprint bands by amplification of gel-eluted DNA with the same p air of primers used for radioactive fingerprint presentation. Fluorescent N otl-Msel MS-AFLP offers a safer method of studying the alterations in DNA m ethylation, and may be applied to the hybridization of DNA microarrays in t he future. Using Notl-Msel MS-AFLP, we observed frequent hypomethylation of a satellite DNA repeat sequence in a majority of breast tumors.