The Hydroxymethyl Collector™ Kit is designed to detect and capture DNA fragments containing 5-hydroxymethylcytosine (5-hmC) DNA methylation. The Hydroxymethyl Collector kit utilizes a β-glucosyltransferase enzyme to transfer a modified glucose moiety to 5-hydroxymethylcytosine residues in double-stranded DNA. This modified glucose is then used to chemically attach a biotin conjugate for capture and enrichment with streptavidin magnetic beads. Enriched DNA can be used in the analysis of individual genes by PCR, or in combination with microarrays and sequencing for geome-wide analysis of 5-hydroxymethylcytosine.
By utilizing chemical labeling of 5-hmC residues, the Hydroxymethyl Collector Kit is extremely specific in its capture of hydroxymethylated DNA fragments. The biotin-streptavidin binding reaction also allows for stringent binding and wash conditions to reduce any non-specific background. The technique is sensitive enough to enrich DNA fragments containing two or more 5-hmC residues.
Advantages of the Hydroxymethyl Collector Method for 5-hmC Enrichment:
- Chemical labeling ensures specific modification of 5-hmC DNA without cross-reactivity of 5-mC sites
- β-Glucosyltransferase modifies 5-hmC regardless of sequence context, enabling detection of non-CpG methylation (CpA or CpT)
- Uses dsDNA throughout the process which makes it easier to prepare libraries for downstream analysis by Next-Gen sequencing techniques
- Biotin/streptavidin binding enables more stringent binding and wash conditions to reduce non-specific background without losing the sensitivity of the assay
- Analysis is not constrained by the properties or consensus sequence of glucosyl-sensitive restriction enzymes (GSREs)
- Simple, fast procedure can be completed in less than 4 hours
- Downstream applications include individual gene analysis by PCR/qPCR or genome-wide analysis by sequencing or microarray
Hydroxymethyl Collector™ contains all the reagents needed to perform the glucosylation reaction, biotin conjugation, streptavidin capture, elution and purification of DNA containing 5-hydroxymethylcytosine. Protocols are provided for the fragmentation of sample DNA all the way through to real time PCR analysis of a locus of interest. For added convenience, the kit also includes positive control 5-hmC DNA, which is a 338 bp sequence of the APC locus in which 25% of the cytosine residues have been hydroxymethylated. PCR primers for APC are provided to confirm the efficiency of the enrichment reactions.View Hydroxymethyl Collector on Active Motif’s website