Detection of hypermethylation of p16ink4a by rolling circle amplification

Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancerrelated genes may be useful for cancer diagnosis or the detection of recurrence. p16, an inhibitor of the cyclin D-dependent protein kinases, is a classical tumor suppressor gene, and its inactivation is closely associated with carcinogenesis. p16 hypermethylation could be detected in each stage, which is consistent with the finding that aberrant methylation of p16 is a very early event in carcinogenesis. We have developed an new procedure for detecting DNA methylation of the human p16(Ink4a) gene. The procedure is based on the chemiluminescent detection with rolling cycle amplifyication DNA from human hepatocellular tumor cell lines and whole blood cells of healthy human. Methylation status of human p16(Ink4a) gene was detected and a good reproducibility was observed in several parallel experiments. Our experiments successfully demonstrated that the rolling circle amplification based chemiluminescent methodology could be applied as a highthroughput tool to determine hypermethylation status of the investigated genes.