DMMC Course: EPIGENETICS: FROM MECHANISMS TO MEDICINES

1650-1730 Monday 25 June 2007. O’Reilly Hall, University College Dublin.

A Novel Method for Analysis of 5-Methylcytosine in DNA by Capillary Electrophoresis Mobility Shift
Dr Mark Lynch (Application Specialist, Applied Biosystems)

Among many methods available for studying methylation in genomic DNA ‘bisulfite / PCR / sequencing’ offers the highest degree of resolution of the methylation status of a given sample, allowing us to determine the positional CpG genotype for individual samples. While automatable, the success of bisulfite/sequencing is in large part dependent on successful execution of critical steps: bisulfite modification, DNA recovery, desulfonation, bisulfite-specific PCR (relies heavily on the in silico design of adequate primers) and finally the DNA sequencing of the PCR product. PCR- and cycle sequencing reactions are both challenging because of the unusual composition of the amplicon, the sparsity of C- and abundance of T-residues in the forward strand of the PCR amplicon. PCR bias - favoring the amplification of non-methylated DNA over methylated DNA - and enzyme slippage on stretches of poly-T during the cycle sequencing reaction are just two of many effects to be taken into account when practicing bisulfite/sequencing. Here we discuss a number of protocols and standardized tools for the generation of sequencing data under these challenging conditions.