The EpiQuik™ Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit is a complete set of optimized buffers and reagents to carry out a successful hMeDIP procedure for the selective enrichment of DNA fragments containing 5-hydroxymethylcytosine (5-hmC) in a high throughput format using DNA isolated from any species. The hydroxymethylated DNA that is enriched with this kit can be used in various downstream applications including PCR (hMeDIP-PCR) and microarrays (hMeDIP-chip) for investigating gene-specific hydroxymethylation in cells or tissues.

About 5-Hydroxymethylcytosine
5-hmC is a modified form of cytosine, recently discovered in animal tissues, and plays an important and different role than 5-mC in regulation of DNA methylation, chromatin remodeling, and gene expression, particularly in brain-specific gene regulation. Quite recently, 5-hmC was observed to be linked to epigenetic reprogramming in mammalian zygotes. However, the exact functions of 5-hmC have not yet been fully identified since gene-specific distribution of 5-hmC is unknown due to the inability of currently used DNA methylation analysis methods to distinguish between 5-hmC and 5-mC.

Product Features
Because of the presence of 5-hmC in DNA with unclear functions in gene regulation and because of the discovery of enzymes that produce 5-hmC, it is crucial to identify hydroxymethylation status in specific gene loci, which would help to better understand methylation-based epigenetic regulation of gene functions. To achieve this, Epigentek has developed an innovative method to capture DNA fragments containing 5-hmC, and incorporated this method into its EpiQuik™ Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit. This kit uses a high affinity 5-hmC antibody to selectively capture double-stranded or single stranded DNA fragments containing 5-hmC. The kit has the following features:

• Extremely fast and convenient protocol with a total procedure time (from input sample to ready-to-use hydroxymethylated DNA) of less than 3 hours, which includes a minimal handling time of less than 20 minutes.
• Flexible 96 stripwell microplate format makes the assay very easy to handle: manual method with one reaction at a time or high throughput method with 96 reactions at a time.
• Highly efficient enrichment ratio of positive/negative control > 1000.
• Low DNA input requirement of as low as 0.1 µg per reaction.
• High reproducibility through pre-optimized hMeDIP conditions.
• Compatible with various downstream analysis workflows including hMeDIP-PCR and hMeDIP-chip.

Principle and Procedure
The EpiQuik™ Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit contains all reagents required for carrying out a successful hMeDIP procedure using DNA isolated from mammalian cells or tissues. This kit includes a positive control DNA fragment, a negative control non-immune IgG, and control primers that can be used with the positive control to demonstrate the enrichment efficacy for hydroxymethylated DNA with the kit reagents and protocol. The positive control DNA containing 5-hmC can be immunoprecipitated by a 5-hmC antibody but not by a non-immune IgG. In this hMeDIP, immunoprecipitation of 5-hmC-enriched DNA fragments is processed in a microplate under optimized reaction conditions, which enables hMeDIP to be completed within 3 hours with high efficiency. Immunoprecipitated hydroxymethylated DNA is then cleaned, released, and eluted. Eluted DNA can be used for various downstream applications including PCR (hMeDIP-PCR) and microarray (hMeDIP-chip).

Easy, Fast, and Flexible
The entire hMeDIP procedure has easy-to-follow steps for convenience and speed, which can be completed in 3 hours from DNA shearing to hMeDNA elution. The 96 stripwell microplate format permits manual handling by doing 1 reaction at a time or high throughput handling by doing 96 reactions at a time. Hydroxymethylated DNA fragments and non-immune IgG are included with the kit as the positive control and negative control, respectively, for hydroxymethylated DNA enrichment from any species including mammalian, plant, fungal, bacterial, and viral types.

Selective and High Enrichment
The unique procedure and proprietary kit compositions allow for a selective and high enrichment of hydroxymethylated DNA from genomic DNA samples for gene-specific hydroxymethylation analysis. Combining real time PCR with this kit, 5-hmC enrichment in the OCT4 promoter region was detected to be 110 times higher than that in the GAPDH promoter region (see Fig. 2), indicating that the OCT4 promoter is hydroxymethylated. There is no cross-reactivity to methylcytosine and unmethylated cytosine so that only hydroxymethylated DNA (5-hmC) is enriched (see Fig. 3).

Compatible With MeDIP
The EpiQuik™ hMeDIP kit can be used with MeDIP procedures for a simultaneous analysis of hydroxymethylated and methylated statuses of specific gene loci.

Responsive, Reliable, and Practical
Based on its working principle and the microplate format, the kit can be used in a high throughput manner with DNA from any species in a variety of forms including cultured cells, fresh and frozen tissues, paraffin-embedded tissues, blood samples, and body fluid samples. To demonstrate the capabilities of the kit, it has been successfully used for capturing hydroxymethylated DNA fraction from genomic DNA isolated from human brain tissues. The percent content of hydroxymethylated DNA in human brains enriched with the kit (0.80%) is comparable to that measured with the EpiQuik™ Hydroxymethylated DNA Quantification Kit (0.67%), suggesting that the hydroxymethylated DNA fraction is fully captured with the hMeDIP kit.