About Quest 5-hmC Detection Kit™
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).
Figure 1: 5-hmC Glucosyltransferase transfers a glucose moiety from uridine diphosphoglucose (UDPG) onto preexisting 5-hydroxymethylcytosines within DNA.
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., C
* 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.
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.
* 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.
|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.|
|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.|
The Quest 5-hmC Detection Kit from Zymo Research was used by researchers to investigate DNA hydroxymethylation in the development of osteoclast cells. Osteoclasts are derived from monocytes in the hematopoietic system, and these cells can degrade bones, which is important in the healing process of broken bones. The researchers observed that certain genes exhibited DNA hypermethylation, and other genes showed a decrease in DNA methylation during the cellular differentiation process from monocytes to osteoclasts. A corresponding increase in the levels of DNA hydroxymethylation was also observed at the genes where active demethylation was seen. The decreases in DNA methylation appeared to be mediated by the enzyme TET2 and were directed by the transcription factor PU.1, resulting in increased levels of DNA hydroxymethylation at these loci.
The Quest 5-hmC Detection Kit was used to detect the presence of 5-hmC in mammalian mitochondrial DNA (mtDNA). Both 5-mC and 5-hmC were found in mtDNA, and these modifications are thought to be regulated by mitochondrial DNA methyltransferase 1 to help maintain healthy mitochondrial function.