About Quest 5-hmC Detection Kit™
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Quest 5-hmC Detection Kit™ and Quest 5-hmC Detection Kit™- Lite Workflows
Treatment of DNA containing 5hmC with 5-hmC Glucosyltransferase specifically adds a glucose moiety yielding glucosyl-5-hydroxymethylcytosine. Subsequent digestion with GSREs (see Table 1 below) will specifically cleave DNA having cytosine, 5-methylcytosine, or 5-hydroxymethylcytosine in the recognition sequence. Glucosyl-5-hydroxymethylcytosine in the DNA will remain uncleaved. |
The Quest 5-hmC Detection Kit™ from Zymo Research allows for sequence specific detection of 5-hydroxymethylcytosine within DNA using a simple and efficient reaction setup. Utilizing a robust and highly specific 5-hmC Glucosyltransferase enzyme, 5-hydroxymethylcytosine in DNA is specifically tagged with a glucose moiety yielding a modified base, glucosyl-5-hydroxymethylcytosine (Figure 1).
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Figure 1: 5-hmC Glucosyltransferase transfers a glucose moiety from uridine diphosphoglucose (UDPG) onto preexisting 5-hydroxymethylcytosines within DNA.
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After glucosylation of 5-hydroxymethylcytosine, digestion of DNA with glucosyl-5-hydroxymethylcytosine sensitive restriction endonucleases (GSREs) allow differentiation of 5-methylcytosine from 5-hydroxymethylcytosine according to the context of a GSRE's recognition sequence (see Table 1). GSRE's can efficiently digest DNA when cytosine, 5-methylcytosine, or 5-hydroxymethylcytosine is within their recognition sequence. However, if 5-hydroxymethylcytosine is glucosylated (i.e., glucosyl-5-hydroxymethylcytosine), GSREs can no longer digest the DNA. Therefore, by exploiting this sensitivity to glucosyl-5-hydroxymethylcytosine, effective detection of 5-hydroxymethylcytosine can be achieved by a number of downstream applications (e.g.- qPCR, NextGen Sequencing, Southern blotting, microarray, etc.). QPCR is recommended to assess locus-specific context of 5-hydroxymethylcytosine within the DNA (see Figure 3). Controls and protocols for qPCR 5-hydroxymethylcytosine detection are included with the kit.
MspI is included in the Quest 5-hmC Detection Kit™. MspI is a GSRE that cleaves DNA when cytosine, 5-methylcytosine, or 5-hydroxymethylcytosine is located at the inner C position (underlined) within its recognition sequence (i.e., CCGG). Glucosylation at this position (i.e., glucosyl-5-hydroxymethylcytosine) inhibits cleavage by the enzyme.
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Table 1: Glucosyl-5hmC Sensitive Restriction Endonucleases - GSREs
* Quest 5-hmC Detection Kit™- Lite can be used with any GSRE of your choice (includes all reagents except for GSRE). * Detection of 5-hmC with MspI and HaeIII can only be used for the C indicated (by an underline). * TaqaI and MboI display incomplete sensitivity to Glucosyl-5hmC. Titration of the enzyme amount and incubation time will be required to yield optimal results. |
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Figure 3: Loci Specific 5-hydroxymethylcytosine Detection
Genomic DNA from Mouse 5-hmC & 5-mC DNA Set was processed according to Quest 5-hmC Detection Kit™ protocol. qPCR results were quantified as percentage of 5-hydroxymethylcytosine at each loci. 5-hydroxymethylcytosine was detected at Locus 1 and Locus 2 with the use of MspI (CCGG) and HaeIII (GGCC), respectively. Use of HaeIII allows for greater coverage as well as detection of 5-hmC in CpG and non-CpG contexts. |
Specifications
| 5-hmC Glucosyltransferase | 5-hmC Glucosyltransferase is guaranteed for 1 year at -20°C. Long term storage at -80°C is recommended. Avoid multiple freeze thawing. |
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| MspI Restriction Enzyme | MspI is guaranteed for 1 year at -20°C. Long term storage at -80°C is recommended. Avoid multiple freeze thawing. |
| Quest 5-hmC Control DNA | A 90 bp DNA standard used for qPCR. [10 ng/μl] in storage buffer (10mM Tris-HCl; 0.1mM EDTA; pH 8.0) supplied in 30 μl volume. See Appendix I (pg. 6) for more details. |
| Quest qPCR Primers | Control primers specific to Quest 5-hmC Control DNA validated for qPCR. See Appendix II (pgs. 7, 8) in Protocol for detailed information. |
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