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Method

Hydroxymethyl Collector™ Method and Advantages

 

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 5 hours
  • Downstream applications include individual gene analysis by PCR/qPCR or genome-wide analysis by sequencing or microarray

Flow Chart of Hydroxymethyl Collector™ Method

Below is the process of how Hydroxymethyl Collector is able to specifically modify, capture and enrich for double-stranded DNA fragments containing 5-hydroxymethylcytosine (5-hmC) residues.

Flow Chart of the Hydroxymethyl Collector method to enrich for 5-hydroxymethylcytosine DNA using a biotin capture method
Figure 1: Hydroxymethyl Collector Method.

Hydroxymethyl Collector Kit works with fragmented (100-500 bp) double-stranded DNA. The DNA is combined with our β-Glucosyltransferase enzyme in the presence of a UDP-Azide-Glucose donor. The β-Glucosyltransferase enzyme will add the modified glucose moiety onto 5-hydroxymethylcytosine residues leaving unmethylated and 5-methylcytosine residues untouched. A chemical labeling reaction then attaches a biotin conjugate to the modified glucosyl-5-hmC DNA. Streptavidin magnetic beads are used for capture. Elution buffer is added to release the enriched DNA from the biotin linker, resulting in DNA fragments that are enriched for 5-hydroxymethylcytosine. Using the included purification reagents, the DNA is cleaned up prior to use in downstream applications.