A Simplified Method to Determine Methylated Cytosines in a Target Gene

Abstract-In this paper we discuss methylation sequencing of DNA target in E-Cadherin gene in head and neck cancer and colorectal cancer by bisulfite-PCR. Bisulfite converted genomic DNA were purified prior to amplification by PCR. Primers are designed to amplify both methylated and unmethylated strands of target DNA sequence. Status of methylation was ascertained by native PAGE. Intriguingly, we have observed methylated, unmethylated and partially methylated amplicons in the tissue samples. Occurrence of positive results in unmethylated primers of cancer tissues as well as positive results for both methylated and unmethylated primers in some tissue samples reveal the limitation of existing methodology in capturing real methylation status. In partially overcoming this limitation, we have designed and implemented specific fluorescence detection based sequencing protocol namely, methylation-sequencing which capture complete methylation status of target gene. In that we have combined dideoxy termination at specific methylation site with fluorescently tagged primer in the PCR reaction which allows the fluorescence detection of specifically terminated oligos. Using a target template DNA and custom-built fluorescence detection apparatus, we demonstrate utility of this approach in obtaining accurate methylation status of given gene target. By design, the methodology eliminates need to sequence entire target DNA.