BisulPlus Loci 5mC and 5hmC Detection PCR Kit

Product Description

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.

• Simultaneous prepared DNA treatment for both 5hmC and 5mC allows for parallel identification in a loci- or gene-specific manner simultaneously by qPCR.
• Innovative 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 48 reactions for just 5mC.
• Fast and Streamlined Procedure - From DNA bisulfite treatment to PCR products within 5 hours.
• Completely converts C into uracil (>99%) and 5mC to thymine (>95%)
• Broad sample suitability

Workflow

Principle and 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.

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. 

Fig.1: Principle of BisulPlus™ Loci 5mC & 5hmC Detection PCR Kit

Performance Data

Fig.2: Discrimination of 5hmC from cytosine and 5mC by qPCR Unmodified, methylated and hydroxymethylated DNA standards are used for bisulfite-enzyme treatment using the he BisulPlus™ Loci 5mc & 5hmC Detection PCR Kit followed by real-time amplification with use of primers targeting to CpG converted or unconverted sequences: A: BisulPlus; B: Bisulfite standalone. Amplification lines are highlighted for visibility.