MeDIP Ultra Kit

Product details

The MeDIP Ultra Kit is a complete set of optimized reagents to enrich and capture methylated DNA fragments in a convenient microplate-based format. 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).

Starting Materials, Input Amount, & Expected Yield
The 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.

Core 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. 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). 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), 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. TheMeDIP 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.