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Epigenetics |
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| Epigenetics>【GeneMarkBio】 |
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Quest 5-hmC Detection Kit™ (A-D5410 A-D5411) |
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Quest 5-hmC Detection Kit™ 货号：A-D5410
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Method to distinguish 5-hydroxymethylcytosine in sequence- and locus -specific context within DNA. |
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Convenient and reliable single tube reaction format. |
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DNA is eluted in water or low salt buffer and is suitable for analysis by a variety of downstream applications. |
| Product |
Catalog No. |
Size |
Price |
Protocol |
Quest 5-hmC Detection Kit
Include MspI GSRE |
A-D5410 |
25 preps. |
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| A-D5411 |
50 preps. |
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| For basic module version of Quest 5-hmC Detection Kit™ please see Quest 5-hmC Detection Kit™ - Lite. |
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| Quest 5-hmC Detection Kit™ and Quest 5-hmC Detection Kit™ - Lite contain patent pending technologies. |
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Components Available
 ：艾德科技生命科学旗舰店,是世界各大知名生物公司在中国区域的网络销售平台,代理和销售世界各大知名生物公司的各种生化试剂,实验仪器与实验耗材,依托于艾德科技（北京）有限公司的强大实力，引入国外先进技术与实验解决方案,产品与服务经客户使用后，引起中国科学院，中国医学科学院，北京大学，复旦大学生，协和医院，肿瘤医院等科研单位的高度关注,并给予充分的肯定与好评，此店已经成为世界各大知名生物公司的首选平台，艾德科技生命科学旗舰店,始终把客户的需求当作实验的需求，我们坚信：客户的需要就是我们不断前进的动力，客户的需求就是我们的不断前进的方向。
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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. |
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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. |
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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. |
Descriptions as follows:
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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.
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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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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).
* TaqαI 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.
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