Abstract 163: DTMETH: A novel method for increased length quantitative CpG methylation analysis

Abstract

Abstract DNA methylation analysis has been blooming in the last decade as there is increasing evidence for epigenetic control of new pathways involved in normal cell function as well as the pathogenesis of human disease. A large number of methods have been devised to address the needs of DNA methylation detection in clinical samples. A DNA methylation detection method of preference should be able to interrogate multiple CpGs over long stretches of DNA, quantify DNA methylation, include multiple bisulphite conversion controls, provide increased sensitivity and low background. Here we present DTMETH, a novel method for DNA methylation analysis that bears the above characteristics. In principle, the desired locus is amplified from bisulphite treated-DNA using primers at CpG-free areas. Primers bear short 5’ tails which contain two cytosines (forward) or guanines (reverse) that will serve as normalisation tools. After a spin-column cleanup process, the PCR product is subjected to DTMETH. This is a thermostable reaction utilising FS Taq and labelled ddC (Applied Biosystems). The latter will be only incorporated at methylated cytosines, as post-bisulphite unmethylated cytosines are converted to uracils. The DTMETH products are cleaned by ethanol precipitation and subjected to analysis on a capillary sequencer. ddC peak areas corresponding to methylated Cs are normalised by the peak areas of the control Cs on the primer tail. As proof of principle we present DNA methylation analysis for the p16 gene. We amplified a 269 bp region of the gene's 5’UTR containing 28 CpGs. The assays provide excellent linearity and reproducibility. We used it to screen 90 non-small cell lung carcinomas for p16 methylation in comparison to an established pyrosequencing assay. p16 hypermethylation was detected in 23 samples with both methods, 3 samples with DTMETH only, while 64 samples were negative. DTMETH combines the read lengths of dideoxy terminator sequencing, and enables quantitation without the need of cloning. It is highly versatile, offering easy assay design and due to its thermostable nature can easily overcome secondary structure issues arising from the low complexity bisulphite DNA sequence composition. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 163.