Journal: British Journal of Cancer
Article Title: CpG island methylation is a common finding in colorectal cancer cell lines
Figure Lengend Snippet: Analysis of methylation of p16 and MINT loci in colorectal cell lines. ( A ) For p16 promoter analysis, bisulphite-modified cell line DNA was amplified in separate reactions using primers specific for unmethylated (U) or methylated (M) template (methylation-specific PCR - MSP). Examples of methylated cell lines are T84 and RKO; these show amplification in the methylated reaction only. Presence of PCR product in both unmethylated and methylated reactions of HCT116 is indicative of partial methylation at p16 . LIM1215 is shown as an example of a cell line unmethylated at p16 . ( B ) For analysis of methylation at MINT loci, bisulphite-modified DNA was first PCR amplified using appropriate loci-specific primers, and then the PCR products were digested with restriction enzymes that only cut amplicons generated from the methylated template. MINT2 is shown as a representative MINT locus. Resistance to digestion indicates an unmethylated template. Shown are cell lines with partial methylation at MINT 2, CACO2 and RKO, a fully methylated cell line, COLO205, and an unmethylated cell line LIM1215. Undigested amplicons are shown in the last lane. Molecular weight (MW) marker in A and B is pUC19/ Msp I.
Article Snippet: Genomic DNA (500 ng) was used in PCR with 0.5 μ M of each of the primers 5′-ATATAAACTTGTGGTAGTTCCAGCTGGT-3′ and 5′-ATCAAAGAATGGTCCTGCACC-3′, 2.5 mM MgCl2 , 100 μ M dNTPs, 1.25 U FastStart TaqDNA polymerase (Roche Diagnostics GmBH, Mannheim, Germany) in the reaction buffer provided by the manufacturer.
Techniques: Methylation, Modification, Amplification, Polymerase Chain Reaction, Generated, Molecular Weight, Marker