Abstract 152: Methylated-DNA enrichment and PCR array technology: A qPCR-based method to rapidly screen for changes in promoter DNA methylation patterns

Abstract

Abstract As the only known enzymatic modification of DNA in mammals, methylation of cytosines at CpG dinucleotides is recognized as a “third pathway” in Knudson's two-hit hypothesis for cancer which suggests that at least two DNA insults (hits) are required for cancer progression. This epigenetic event alters gene expression patterns associated with normal development and disease progression without changing the actual DNA sequence. In cancer, hypermethylation of tumor suppressor gene promoters abolishes their transcription, while hypomethylation of proto-oncogene promoters increases their transcription, effectively driving tumorigenesis in either case. Given the importance of this event for tumorgenesis, various techniques have been developed to identify DNA methylation based diagnostic biomarkers. Unfortunately, these methods often fall short in terms of throughput capacity, cost, or equipment requirements. This work presents a novel combination of technologies to efficiently screen for changes in DNA methylation patterns within a panel of genes simultaneously. Using lung cancer as an experimental model, this study establishes that methylated DNA enrichment combined with real-time PCR (qPCR) technology offers great potential to identify putative biomarkers. This one-day assay is quite simple, and with very little hands-on time it can be easily adapted for high-throughput screening. A genomic DNA sample of interest is sheared, denatured, and incubated with a highly specific anti-5-methylcytosine antibody immobilized to a resin. The methylated DNA is then released, purified over a column, and applied to a pathway-focused qPCR array. The sensitivity of the method is very high; even one methylated CpG dinucleotide is enough to selectively enrich for a methylated target of interest, and the assay detection limit is well below 2.5% of a target in a mixed genomic DNA background. Importantly, data derived from this method has been validated with data derived from the Illumina® Infinium® Methylation Assay platform, further attesting to the potential for discovering methylation biomarkers with this system. In conclusion, the combination of methylated-DNA enrichment and qPCR array technology has been extensively optimized to discriminate methylated-CpG dinucleotides with low background. With this method, researchers will be able to quickly determine whether changes in gene expression (upregulation or downregulation) are associated with promoter methylation changes. This combination of methylated-DNA enrichment with qPCR array technology offers great potential as a methylated CpG dinucleotide screening tool, without the high cost. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 152.