biotin-based enrichment of 5-hydroxymethylcytosine
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.
Figure 1: Sensitivity of the Hydroxymethyl Collector–Seq Kit for enrichment of 5-hmC 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.
Figure 2: Sensitivity of the Hydroxymethyl Collector Kit for enrichment of 5-hmC DNA.
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.
Figure 3: Enrichment of 5-hmC Control DNA included in the Hydroxymethyl Collector Kit.
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.
Figure 4: Genome-wide localization of 5-hmC in mouse tissue using Hydroxymethyl Collector–Seq.
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.
Figure 5: Human tiling array using DNA enriched with Hydroxymethyl Collector.
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.
Figure 1: Hydroxymethyl Collector Method.
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