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PRODUCT OVERVIEWQUEST 5-hmC DETECTION KIT™ | |
| 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 an “ultra” 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.
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| After glucosylation of 5-hydroxymethylcytosine, digestion of DNA with GSRE (glucosyl-5-hydroxymethylcytosine sensitive restriction endonucleases, such as GlaI, MspI and TaqI), allows for effective differentiation of 5-methylcytosine from 5-hydroxymethylcytosine. Identification of 5-hydroxymethylcytosine in a sequence specific context can then be deduced from the restriction endonuclease recognition sequence. | |
 Click image for larger view Figure 2: Quest 5-hmC Detection Kit™ Workflow Treatment of DNA containing 5-hmC with 5-hmC Glucosyltransferase specifically adds a glucose moiety, yielding glucosyl-5-hydroxymethylcytosine. Subsequent GSRE (glucosyl-5-hydroxymethylcytosine sensitive restriction endonucleases, such as MspI and GlaI) digestion, will cut DNA with 5-methylcytosine or 5-hydroxymethylcytosine in their recognition sequence, but will be blocked by the glucosyl-5-hydroxymethylcytosine DNA. | |
SCIENTIFIC FOCUS5-HYDROXYMETHYLCYTOSINE | |
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What is 5-hydroxymethylcytosine? The 5-hmC is also referred to as the 6th base of DNA in reference to the 5-methylcytosine as the 5th base by some authors. The 5-hmC can be further modified by certain glucose transferase at the hydroxyl position to form glucosyl-5-hydroxymethylcytosine. See the structure of cytosine and it's naturally occuring derivatives below.  The exact function of this modified base is still under investigation, but it is thought to be involved in DNA demethylation or gene expression regulation. However, the lack of a valid technique which can accurately detect 5-hmC sensitively has hindered researchers to further explore its potential biological function. This results from the fact that 5-hmC behaves very similarly to its precursor 5-mC in bisulfite conversion and restriction enzyme digestion[4, 5]. Zymo Research overcame this obstacle and is introducing the first kit available for sequence specific determination of 5-hmC, Quest 5-hmC Detection Kit™.
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Quest 5-hmC Detection Kit™ and Quest 5-hmC Detection Kit™ - Lite contain patent pending technologies.