Promoter methylation analysis on micro-dissected paraffin-embedded tissuesusing bisulfite treatment and PCR-SSCP

Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a ne w method of screening for DNA methylation changes. The combination of bisul fite modification and PCR results in the conversion of unmethylated cytosin es to thymines, whereas methylated cytosines remain unchanged This sequence conversion can lend to methylation-dependent alterations of single-strand conformation, which can be detected by SSCA. An analysis of mixtures of met hylated and unmethylated DNA nr known ratios revealed that the relative int ensities of the corresponding banns following MS-SSCA were maintained. MS-S SCA was applied for methylation analysis of human p16 promoter region using genomic DNA obtained from either frozen, fixed, or microdissected fixed ti ssue sections. MS-SSCA is a rapid specific, and semiquantitative approach t hat allows the detection of methylation of the p16 gene promoter: In recons truction experiments, the method permits the detection of 10% or less of ce lls harboring a methylated p16 promoter. We have been successful in analyzi ng by MS-SSCA almost all (96%) tumor samples microdissected from archival p araffin-embedded fixed tissue sections and obtaining reproducible results. in addition, when microdissection was performed, the clonality of this gene tic alteration could be identified.