biotin-based enrichment of 5-hydroxymethylcytosine
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 genome-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. To learn more about the Hydroxymethyl Collector method, click on the Method tab below. To download the manual, please click on the Documents tab below.
* Song, C.X., et al., Nature Biotechnology. 29, 68-72 (2011).
Hydroxymethyl Collector™ Data on 5-hmC Enrichment
Hydroxymethyl Collector Range of Detection
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 1: 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 2: Enrichment of 5-hmC Control DNA included in the Hydroxymethyl Collector Kit.
Applications of Hydroxymethyl Collector Enrichment
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 3: Human tiling array using DNA enriched with Hydroxymethyl Collector.
Hydroxymethyl Collector™ Method and Advantages
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.
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
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. 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.
Each kit contains β-glucosyltransferase enzyme, UDP-Azide-Glucose, 10X Reaction Buffer AM1, 1 M DTT, Biotin Conjugate Solution, Streptavidin Beads, Binding Buffer AM13, 10X Ellution Buffer AM2, DNA Purification Binding Buffer, DNA Purification Wash Buffer, DNA Purification Elution Buffer, 3 M Sodium Acetate, DNA purification columns, 5-hmC Control DNA, APC PCR primer mix, 0.2 ml PCR tubes, a bar magnet and glue dots.
Storage conditions range from room temperature to -20°C; please refer to the product manual for proper storage temperatures of each component.