DNA Methylation Analysis Using Bisulfite Treatment and PCR–Single-Strand Conformation Polymorphism in Colorectal Cancer Showing Microsatellite Instability

• Published in: Biochemical and biophysical research communications 1999-09, Vol.262 (3), p.671-676
• Main Authors: Maekawa, Masato, Sugano, Kokichi, Kashiwabara, Hidefumi, Ushiama, Mineko, Fujita, Shin, Yoshimori, Masayoshi, Kakizoe, Tadao
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing; Base Sequence; Calibration; Carrier Proteins; Colorectal Neoplasms - genetics; Colorectal Neoplasms - surgery; DNA Methylation; DNA Repair - genetics; DNA, Neoplasm - chemistry; DNA, Neoplasm - genetics; Humans; Indicators and Reagents; Microsatellite Repeats; Molecular Sequence Data; MutL Protein Homolog 1; Neoplasm Proteins - genetics; Nuclear Proteins; Polymerase Chain Reaction - methods; Polymorphism, Single-Stranded Conformational; Promoter Regions, Genetic; Sulfites
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1999.1230

The combination of bisulfite treatment and PCR–single-strand DNA conformation polymorphism (SSCP) analysis is proposed for quantitative methylation assay. We applied this procedure to the methylation analysis of the hMLH1 promoter region in colorectal cancer. An analysis of mixtures of known amounts of methylated and unmethylated DNA revealed a linear relation. Using a calibration curve, proportions of methylated DNA were calculated. The hMLH1 promoter region was highly methylated in about 80% of microsatellite instability (MSI) (+) colorectal cancers, but in none of the MSI(−) colorectal cancers. A significant correlation existed between hypermethylation of the hMLH1 promoter and MSI, as in previous reports. In conclusion, bisulfite-PCR-SSCP (BiPS) analysis could be applied to the rapid identification of methylation status in multiple samples, quantification of methylation differences, and detection of methylation heterogeneity in amplified DNA fragments.