Affinity-Based Enrichment Techniques for the Genome-Wide Analysis of 5-Hydroxymethylcytosine

Abstract

Since its initial characterization in 2009 there has been a great degree of interest in comparative profiling of 5-hydroxymethylcytosine (5hmC) nucleotides in vertebrate DNA. Through a host of genome-wide studies the distribution of 5hmC has been mapped in a range of cell lines, tissue types and organisms; the majority of which have been generated through affinity-based methods for 5hmC enrichment. Although recent advances in the field have resulted in the ability to investigate the levels of both methylated and hydroxymethylated cytosines at single base resolution, such studies are still relatively cost-prohibitive as well as technically challenging. As such affinity-based methods for the enrichment of 5hmC-modified DNA fragments represent a cost-effective and highly informative method for profiling 5hmC residency in genomic DNA. Here we will outline protocols for two independent affinity based methods to generate 5hmC enriched fractions for subsequent locus specific and genome-wide analysis; immunoprecipitation using highly specific 5hmC antibodies as well as a chemical capture based method.