The EpiQuik™ MeDIP Ultra Kit is a complete set of optimized reagents to enrich and capture methylated DNA fragments in a convenient microplate-based format, and is a further refinement of the predecessor Methylamp™ MeDIP Kitby significantly improving sensitivity and specificity while reducing background signals. The method, methylated DNA immunoprecipitation (MeDIP), uses a monoclonal antibody specific to 5-methylcytosine to immunoprecipitate methylated genomic DNA. The enriched methylated fractions can then be used for gene-specific DNA methylation analysis on a genome wide scale. The highly sensitive and specific format of the kit can use DNA isolated from various species. The methylated DNA that is enriched with this kit can be used for various downstream applications including qualitative and quantitative PCR (MeDIP-PCR), microarray (MeDIP-chip) and especially sequencing (MeDIP-seq).The kit has the following advantages and features:

• Extremely fast and convenient protocol with a total procedure time (from input sample to ready-to-use methylated DNA) of less than 3 hours, which includes a minimal handling time of less than 20 minutes.
• Optimized buffers and protocol allow minimal background by overcoming the weaknesses that cause non-specific enrichment.
• A highly specific 5-mC monoclonal antibody included in the kit can strongly bind both single and double stranded DNA fragments containing 2 or more 5-mCs which enables highly sensitive enrichment of methylated DNA with >99% specificity.
• Flexible 96 strip-well microplate format makes the assay very easy to handle: manual method with one reaction at a time or high throughput method with 24-48 reactions at a time.
• Spin columns and collection tubes conveniently included for a DNA purification step.
• Low DNA input requirement as low as 50 ng (10,000 cells) per reaction.
• High reproducibility using pre-optimized MeDIP conditions.
• Compatible with various downstream analysis workflows including MeDIP-PCR and MeDIP-chip, and specifically for MeDIP-seq.

Background InformationCore mechanisms for epigenetic alteration of genomic DNA are hypermethylation of CpG islands in specific genes and global DNA hypomethylation. Region-specific DNA methylation plays an important role in the repression of gene transcription and is mainly found in 5’-CpG-3’dinucleotides within promoters or in the first exon of genes. Global DNA hypomethylation is likely caused by methyl-deficiency due to a variety of environmental influences. It has been demonstrated that alterations in DNA methylation are associated with many diseases, especially cancer.Highly specific isolation of methylated DNA combined with next generation sequencing for genome-wide methylation analysis should provide an advantage for convenient and comprehensive identification of methylation status of normal and diseased cells, such as cancer cells [1]. Such analysis requires the isolated methylated DNA to contain minimal background in order to achieve high specificity (>98%) for reliably identifying true methylated regions. The major method for enriching methylated DNA used for genome-wide methylation profiling is methylated DNA immunoprecipitation (MeDIP) [2]. However, currently used MeDIP methods, represented by most commercially available kits, have significant weaknesses including highly non-specific enrichment (amount of enriched DNA is >75% of the amount of input DNA) [3], time consuming, labor intensive, and has low throughput. Thus, for effectively and specifically capturing methylated DNA used for next generation sequencing analysis, an ideal MeDIP method requires maximum sensitivity with minimal background levels. Epigentek’s EpiQuik™ MeDIP Ultra Kit is designed to achieve these goals by maximizing sensitivity and minimizing non-specific background signals, and is a significant improvement over previous MeDIP kits.

1. Ruike Y et al: BMC Genomics, 11: 137, 2010.2. Weber M et al: Nature Genetics, 37: 853-862, 2005.3. Brebi-Mieville P et al: Epigenetics, 7: 106-112, 2012.

Principle & ProcedureThis kit includes a methylated DNA control and an unmethylated DNA control, a negative control non-immune IgG, and control primers that can be used with the control DNA to demonstrate the enrichment efficacy and specificity for methylated DNA.The 5-methylcytosine monoclonal antibody provided in this kit is highly specific against methylated DNA fragments, both single and double stranded, and is not cross-reactive to hydroxymethylated and unmethylated DNA fragments. This antibody can capture >50% of DNA fragments containing as few as two 5-mCs and enriches all DNA fragments containing four or more 5-mCs.The positive control DNA containing 5-mC can be immunoprecipitated by the 5-mC antibody but not by the non-immune IgG. In this MeDIP, immunoprecipitation of 5-mC-enriched DNA fragments is processed in a microplate under optimized reaction conditions, which enables MeDIP to be completed within 3 hours with high efficiency. Immunoprecipitated methylated DNA is then cleaned, released, and eluted. Eluted DNA can be used for various downstream applications including PCR (MeDIP-PCR) and microarray (MeDIP-chip), and is especially suitable for MeDIP-seq.

Starting Materials, Input Amount, & Expected YieldThe starting material should be good quality purified DNA. The amount of DNA for each reaction can be 50 ng (approximately 10,000 cells) to 500 ng. For an optimal reaction, the input DNA amount should be 100-200 ng per well. The yielded methylated DNA is about 4 ng for 100 ng input DNA (4%), which is consistent with the expected percentage (4-5%) at which the highest sensitivity and specificity for enriched methylated DNA has been demonstrated by bisulfite sequencing.

Fig. 1.Schematic procedure of the EpiQuik™ MeDIP Ultra Kit.

Fig. 2.Selective enrichment of methylated DNA with the EpiQuik™ MeDIP Ultra Kit | 50 pg of unmethylated or methylated DNA control were each spiked into fragmented human genomic DNA (100 ng). MeDIP was processed with the included 5-mC monoclonal antibody and non-immune IgG included in the kit. Eluted DNA was analyzed by real time PCR with the control primers included in the kit to detect the presence of spiked control DNA. Fold-enrichment represents the amount of recovered control DNA and was calculated based on the real time PCR Ct value.

Fig. 3.Sensitive detection of gene-specific methylation by MeDIP-qPCR | Fully methylated HeLa DNA (500 ng) was fragmented to 100-500 bps using an EpiSonic™ 1100. The fragmented DNA was used for methylated DNA enrichment with the EpiQuik™ MeDIP Kit. Eluted DNA was analyzed by real time PCR with primers specific for MLH1 sequences in the promoter regions. Results show the specificity of the 5-mC antibody and low background for non-immune IgG. Fold-enrichment represents the amount of recovered DNA and was calculated based on the real time PCR Ct value.