BisulPlus Loci 5mC & 5hmC Detection PCR Kit

The BisulPlus™ Loci 5mc & 5hmC Detection PCR Kit is a complete set of optimized buffers and reagents to identify both 5mC and 5hmC on a loci- or gene-specific level by qPCR using bisulfite conversion and cytosine deaminase treatment.

This kit has the following advantages and features:

• Simultaneous Reading: DNA treatment is prepared simultaneously for both 5hmC and 5mC, which allows for parallel identification in a loci- or gene-specific manner simultaneously by qPCR.
• Innovative Method: A combination of bisulfite and subsequent cytosine deaminase (APOBEC) treatment allows for tactical conversion of 5mC in order to identify 5hmC.
• High Specificity: 5hmC is discriminated from C and 5mC as well as from other modified cytosines 5fC and 5caC.
• Flexibility: Perform 24 reactions for 5hmC and 24 reactions for 5mC analysis simultaneously, or perform 24 reactions for just 5hmC or 48 reactions for 5mC
• Fast and Streamlined Procedure: The procedure from DNA bisulfite treatment to PCR products can be completed within 5 hours.
• Complete Conversion: Completely converts C into uracil (>99%) and 5mC to thymine (>95%)
• Broad sample suitability: Starting materials can be genomic DNA isolated from various tissue/cell samples such as fresh and frozen tissue, cultured cells from a flask or microplate, microdissection samples, paraffin-embedded tissue, biopsy, embryonic cells, plasma/serum samples, and body fluid samples, etc. DNA enriched from various enrichment reactions such as ChIP, MeDIP/hMeDIP, or exon capture may also be used as starting material.

Background
DNA methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring, resulting in 5-methylcytosine (5mC). In somatic cells, 5mC is found almost exclusively in the context of paired symmetrical methylation of the dinucleotide CpG, whereas in embryonic stem (ES) cells, a substantial amount of 5mC is also observed in non-CpG contexts. The biological importance of 5mC as a major epigenetic modification in phenotype and gene expression has been widely recognized. 5-hydroxymethylcytosine (5hmC), as a sixth DNA base with functions in transcription regulation, has been detected to be abundant in human and mouse brains and embryonic stem (ES) cells. In mammals, it can be generated by the oxidation of 5mC, a reaction mediated by the ten-eleven translocation (TET) family of 5mC-hydroxylases. 5hmC has been demonstrated to be tissue specific, ranging from undetectable levels in cultured cell lines to 0.6% in human brain tissues, and can be as high as 8% of total DNA in some other species. The biological significance of 5hmC as an important epigenetic modification in phenotype and gene expression has been recently recognized. For example, global decrease in 5hmC content (DNA hypohydroxymethylation) has been exhibited in nearly all cancers and has been proposed as a molecular marker and therapeutic target in cancer as well. More importantly, in contrast to 5mC that is generally considered as a gene repression marker, 5hmC is regarded as an intermediate of DNA demethylation and associated with expressed genes with a potential role in activating genes. Like 5mC, 5hmC remains the same after bisulfite-conversion of cytosine to uracil (C to U), which makes 5hmC indistinguishable from 5mC. As consequence, all 5hmC will be misread as 5mC in the assay with using MS-PCR or bisulfite-seq, which results in that the identified DNA methylation status is incorrect as it in fact includes both 5mC and 5hmC. 

Principle & Procedure
This kit includes all reagents required for successful qPCR using converted DNA generated from a tiny amount of input DNA. In this preparation, DNA is simultaneously bisulfite modified and fragmented to the appropriate length during the bisulfite process. During the bisulfite treatment, unmodified cytosine (C) is converted to uracil and will be read as T in the sequencing. 5-methylcytosine (5mC) remains the same and 5-hydroxymethylcytosine (5hmC) forms cytosine 5-methylenesulfonate (CMS). The bisulfite modified DNA is further treated with specific APOBEC deaminase, which converts 5mC to thymine but not affect CMS. During the PCR and sequencing CMS will still be read as C so that the 5hmC can be discriminated not only from C, 5mC but also other modified cytosines such as 5fC and 5caC (see the table 1). The bisulfite-enzyme converted DNA can then be used for qPCR for loci specific detection of 5mC and 5hmC. 

Principle of BisulPlus™ Loci 5mC & 5hmC Detection PCR Kit.

Starting Materials
Starting materials can be genomic DNA isolated from various tissue/cell samples such as fresh and frozen tissue, cultured cells from a flask or microplate, microdissection samples, paraffin-embedded tissue, biopsy, embryonic cells, plasma/serum samples, and body fluid samples, etc. DNA enriched from various enrichment reactions such as ChIP, MeDIP/hMeDIP, or exon capture may also be used as starting material. The input amount of DNA can be from 10 ng to 200 ng. For optimal preparation, the input amount should be 50-100 ng.