The Hydroxymethyl Collector™ Kits are designed to detect and capture DNA fragments containing 5-hydroxymethylcytosine (5-hmC) DNA methylation* for analysis. They utilize 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.
* Song, C.X., et al., Nature Biotechnology. 29, 68-72 (2011).
Patent US 8,741,567 B2.
Active Motif's Hydroxymethyl Collector™ and Hydroxymethyl Collector™-Seq Kits are currently unavailable.
Please check out our hMeDIP Kit (Catalog No. 55010) as an alternative for the enrichment of DNA fragments containing 5-hydroxymethylcytosine using antibody-based enrichment.
Hydroxymethyl Collector™ Data on 5-hmC Enrichment
Hydroxymethyl Collector Range of Detection
Hydroxymethyl Collector is designed for locus-specific anlaysis of 5-hmC by real time PCR. We recommend using 5 ng - 2.5 µg fragmented genomic DNA per enrichment reaction.
Hydroxymethyl Collector–Seq is deisgned for genome-wide analysis of 5-hmC with Next-Generation sequencing. We recommend using 1 µg - 10 µg fragmented genomic DNA per enrichment reaction, with 5 µg as the suggested starting quantity. Samples containing very low levels of 5-hmC may require larger quantities of DNA. Suggested input amounts include 8-10 µg from stem cells, 20 µg from cancer tissues, 1-3 µg from brain and 5-10 µg from normal tissue.
To determine the range of detection for Hydroxymethyl Collector–Seq, various quantities of mouse and human tissues were assayed in the Hydroxymethyl Collector–Seq Kit. The enriched 5-hydroxymethylcytosine DNA fragments were analyzed by real time PCR using species-specific and tissue-specific PCR primer sets. The amount of enriched 5-hmC DNA was plotted as fold enrichment. From this analysis it was determined that Hydroxymethyl Collector–Seq is sensitive over the range of 1 - 10 µg fragmented genomic DNA.
To determine the range of detection for Hydroxymethyl Collector, various quantities of mouse brain DNA were assayed in the Hydroxymethyl Collector Kit. The enriched 5-hydroxymethylcytosine DNA fragments were analyzed by real time PCR across multiple loci. The amount of enriched 5-hmC DNA was plotted as a percentage of the starting material. From this analysis it was determined that Hydroxymethyl Collector is sensitive over the range of 5 ng - 2.5 µg fragmented genomic DNA. For downstream analysis requiring larger inputs, e.g. microarray, it may be necessary to pool multiple enrichment reactions or perform whole-genome amplification following 5-hmC DNA enrichment.
Enrichment Efficiency of Hydroxymethyl Collector using the 5-hmC Control DNA
The Hydroxymethyl Collector Kit includes a 5-hmC positive control DNA, MseI digested human genomic DNA and PCR primers that can be used to determine the efficiency of the enrichment reactions. The 5-hmC Control DNA consists of a 338 base pair DNA fragment derived from the APC gene. The 5-hmC control DNA has been modified so that 25% of the cytosine residues contain 5-hydroxymethylcytosine. When the positive control fragment is spiked into human genomic DNA and enriched, the included APC PCR primers can be used to confirm the enrichment efficiency.
Applications of Hydroxymethyl Collector Enrichment
Hydroxymethyl Collector–Seq was performed on 5 µg each of mouse brain, mouse kidney and mouse liver genomic DNA and submitted for Nex-Gen sequencing to identify the localization of 5-hmC within each sample.
Human brain DNA was used in the Hydroxymethyl Collector Kit to enrich for 5-hydroxymethylcytosine DNA fragments. Enriched DNA was amplified by whole-genome amplification and hybridized to a Human tiling array in an effort to perform genome-wide 5-hydroxymethylcytosine DNA methylation analysis.
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.
Contents & Storage
Hydroxymethyl Collector™ contains all thre 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. The enriched dsDNA is compatible with Next-Generation sequencing library preparation. 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.
Please note that the Hydroxymethyl Collector™ Kits are shipped on dry ice and contain reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Do not refreeze the streptavidin beads. Once thawed, store at their proper temperature. This kit includes the following components:
- β-glucosyltransferase enzyme (200 U/µl); Store at -20°C
- UDP-Azide-Glucose (3 mM); Store at -20°C
- 10X Reaction Buffer AM1; Store at -20°C
- 1 M DTT; Store at -20°C
- Biotin Conjugate Solution; Store at -20°C
- 5-hmC Control DNA (50 ng/µl); Store at -20°C
- APC PCR Primer Mix (2.5 µM); Store at -20°C
- Human genomic DNA, MseI digested (20 ng/µl); Store at -20°C (only in Hydroxymethyl Collector Catalog No. 55013)
- Streptavidin Beads; Store at 4°C
- Binding Buffer AM13; Store at 4°C
- 10X Elution Buffer AM2; Store at 4°C
- DNA Purification Binding Buffer; Store at RT
- DNA Purification Wash Buffer; Store at RT
- DNA Purification Elution Buffer; Store at RT
- ChIP Filtration Columns; Store at RT
- 3M Sodium Acetate; Store at RT
- DNA purification columns & collection tubes; Store at RT
- Bar magnet & glue dots; Store at RT