The Retinoblastoma Protein

Javed Hussain Choudhury, Raima Das, Shaheen Laskar, Sharbadeb Kundu, Manish Kumar, Partha Pratim Das, Yashmin Choudhury, Rosy Mondal, Sankar Kumar Ghosh

DNA methylation plays a decisive role in the regulation and control of gene expression. DNA methylation is a covalent modification, in which a methyl group is attached to the 5th carbon of the cytosine ring of a CpG dinucleotide that is located upstream from the promoter region of a gene. Promoter hypermethylation (gain of DNA methylation) of the p16 gene may cause silencing of gene expression and plays an important role in cancer. Therefore, detection of the methylation status of p16 gene is an important tool in epigenetic studies of various human cancers. The methylation-specific PCR (MSP) is the most commonly used technique for studying DNA methylation. This technique is based on bisulfite modification of DNA, which converts unmethylated cytosine (C) into uracil (U) and leaving methylated cytosine (Cm) unchanged. Here we describe the bisulfite modification of DNA samples and detection of promoter methylation of p16 gene from bisulfite-treated DNA using MSP. In MSP, modified DNA samples are subjected to PCR amplification using methylated and unmethylated specific primers for the p16 gene separately. The PCR amplified products are then analyzed in a 2.5-3% agarose gel containing ethidium bromide. The PCR amplified band generated by specific sets of primers is used to determine the methylation status of the p16 gene.