EpiMark® 5-hmC and 5-mC Analysis Kit
The EpiMark® 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a specific locus. Complete conversion of 5-hmC to glucosylated 5-hmC in DNA by T4 β-glucosyltransferase (T4-BGT).
- Discrimination between 5-mC and 5-hmC in CCGG sequences using enzymatic digestion and PCR amplification
- Relative quantitation of 5-mC and 5-hmC
- Easy-to-use protocol
- This kit contains enough material for 20 reactions
5-methylcytosine (5-mC) is the predominant epigenetic mark in mammalian genomic DNA. 5-hydroxymethylcytosine (5-hmC) is a newly discovered epigenetic modification that is presumably generated by oxidation of 5-mC by the TET family of cytosine oxygenasesrefref.
Techniques exist that can identify 5-mC in genomic DNA, but the most commonly used method, bisulfite sequencing, is laborious and cannot distinguish between 5-mC from 5-hmCref.
The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing T4 β-glucosyltransferase (T4-BGT). When 5-hmC occurs in the context of CCGG, this modification converts a cleavable MspI site to a noncleavable one.
An overview of the detection procedure is summarized in Figure 1.
Control DNA Sequence
Control DNA substrates are synthetic 100 bp double stranded fragments containing a single MspI/HpaII site (CCGG). The three fragments are identical except for modification of the internal C in this site.
FW Primer Sequence
5´- CA GTG AAG TTG GCA GAC TGA GC -3´
REV Primer Sequence
5´- CTG ACT TGC CAC CTA TAG ACA GC -3´
The following reagents are supplied with this product:
Store at (°C) Concentration NEBuffer™ 4 -20 10 X T4 Phage β-glucosyltransferase (T4-BGT) -20 10000 units/ml Unmodified Control DNA -20 0.1 ng/µl 5-mC Control DNA -20 0.1 ng/µl 5-hmC Control DNA -20 0.1 ng/µl Forward and Reverse Control Primer Mix -20 10 µM each Proteinase K, Molecular Biology Grade -20 800 mg/ml MspI -20 100000 units/ml HpaII -20 50000 units/ml Uridine Diphosphate Glucose -20 25 X
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