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【产品名称】
 MethylFlash羟甲基化DNA定量试剂盒(Colorimetric)
【订购货号】
 E14267
【产品CAS号码】
 无
【规格/价格】
 
【预计到货时间】
 30 天
【产品详细说明】
【本产品仅供科学研究使用】
【更新日期】 2015-4-8  
MethylFlash Hydroxymethylated DNA 5-hmC Quantification Kit (Colorimetric)
 
货号:P-1036
 
产品背景
 MethylFlash™羟甲基化DNA定量试剂盒(比色法)提供了一整套优化的缓冲液和试剂,运用比色检测法,以微孔板形式通过特异地检测板子上的5-hmC的水平定量检测总的DNA羟甲基化。而与甲基胞嘧啶和未甲基化的胞嘧啶无交叉反应。
 
此款试剂盒尤其适合与MethylFlash甲基化DNA定量试剂盒(比色法)配合使用,可同时定量甲基化DNA和羟甲基化DNA
 
关于5-羟甲基化胞嘧啶
最近在动物组织中发现的5-hmC(5-羟甲基化胞嘧啶)是胞嘧啶的一种修饰形式。在表观遗传中5-hmC的功能与其前体5-甲基胞嘧啶是不同的,到目前为止仍是一个谜。但人们相信,5-hmC在转换基因的激活或关闭状态方面起着重要作用。5-hmC的存在不仅对于现有的DNA甲基化数据进行再评估是必要的,而且在决定健康或生病的人组织中的5-hmC的相对分布和变化也是必要的。之前的Epigentek的MethylFlashTM技术,没有方法可以鉴定5-hmC和区别5-hmC和5-mC。
 
区分5-hmC5-mC
目前包括限制性酶消化,亚硫酸盐或MeDIP介导的MS-PCR和测序等在内的所用的甲基化DNA分析方法,都不能真正地区分5-hmC和5-mC。为了解决这个问题,Epigentek公司提供了MethylFlash™ Hydroxymethylated DNA Quantification Kit (MethylFlash™羟甲基化DNA定量试剂盒)运用独特的方法对总的DNA羟甲基化进行定量。此试剂盒提供了一种经济有效的方法检测5-羟甲基化的水平并能区分5-hmC,5-mC和C。这使研究者就DNA羟甲基化而言对DNA甲基化进行重新评估,并且在一个新的DNA样品中有效地找到DNA羟甲基化。
 
此试剂盒有以下优势和特色:
1.       简单易操作的步骤荧光法分析便捷快速,整个流程在3小时45分钟内完成。
2.       高灵敏性,其检测极限可低至40pg的羟甲基化DNA,接受低至50ng的输入基因组DNA。
3.       高特异性,与甲基胞嘧啶和未甲基化的胞嘧啶无交叉反应。单独检测5-羟甲基化胞嘧啶。
4.       试剂盒里包括通用的阳性和阴性对照,适用于定量任何物种里的羟甲基化DNA。
5.       微孔板形式使分析更具灵活性:手动或高通量分析。
6.       简单,可靠且分析条件一致。
 
原理与程序
MethylFlashTM羟甲基化DNA定量试剂盒(比色法)包含了总DNA羟甲基化定量用的所有必需的试剂。在此试剂盒的分析中,DNA被结合到经特殊处理对DNA具有高亲和度的微孔板上。用捕获和检测抗体对羟甲基化的DNA片段进行检测并通过ELISA-like反应和荧光微孔板分光光度计在450nm处的吸光度值对羟甲基化DNA进行定量。羟甲基化DNA的量与测量的OD值强度是成正比的,试剂盒里提供的公式可计算出两种不同的DNA样品的相对羟甲基化状态或用一个标准曲线对5-hmC胞嘧啶进行绝对定量。
 
简单,快速,灵活
整个比色分析方法的简单易操作的步骤,便捷快速,能在3小时45分钟内完成。微孔板形式使得分析更灵活,适用于手动或高通量分析。试剂盒里提供的通用阳性和阴性对照用于定量任何物种如哺乳动物,植物,真菌,细菌和病毒中的羟甲基化DNA。
 
高灵敏性和高特异性
新颖的程序和专有的试剂盒组分可精确地高特异性和高灵敏性地定量甲基化DNA。输入DNA检测极限可低至50pg的甲基化DNA。
 
 

 

The MethylFlash™ Hydroxymethylated DNA Quantification Kit (Colorimetric) is a complete set of optimized buffers and reagents to colorimetrically quantify global DNA hydroxymethylation by specifically measuring levels of 5-hydroxymethylcytosine (5-hmC) without cross-reactivity to methylcytosine and unmethylated cytosine.

This kit is also specifically optimized for paired use with the MethylFlash Methylated DNA Quantification Kit (Colorimetric) for simultaneously quantifying both methylated DNA and hydroxymethylated DNA.

About 5-Hydroxymethylcytosine
5-hmC is a modified form of cytosine, recently discovered in animal tissues. The function of 5-hmC in epigenetics may be different from its forerunner 5-methylcytosine (5-mC) and currently remains a mystery. It is believed, though, that 5-hmC plays an important role in switching genes on and off. The presence of 5-hmC makes it necessary to not only re-evaluate existing DNA methylation data, but also necessary to determine the relative distribution and changes of 5-hmC in human tissues of healthy and diseased statuses. Prior to Epigentek’s MethylFlash™ technology, there were no methods that could be used for practically and routinely identifying 5-hmC and discriminating this base from 5-mC.

Distinguishing Between 5-hmC and 5-mC
Currently used methylated DNA analysis methods including restriction enzyme digestion and bisulfite or MeDIP-mediated MS-PCR and sequencing are not suitable for 5-hmC detection as 5-hmC and 5-mC are virtually indistinguishable between each other with these methods. To address this problem, Epigentek offers the MethylFlash™ Hydroxymethylated DNA Quantification Kit which uses a unique procedure to quantify global DNA hydroxymethylation. This product provides a cost-effective way to measure levels of 5-hydroxymethylcytosine and to distinguish between 5-hmC, 5-mC, and C. This allows for researchers to re-evaluate their DNA methylation data for DNA hydroxymethylation and to efficiently look for DNA hydroxymethylation in new DNA samples. The kit has the following advantages and features:

  • Colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be completed within 3 hours and 45 minutes.
  • High sensitivity, of which the detection limit can be as low as 40 pg of hydroxymethylated DNA from just 50 ng of input genomic DNA.
  • High specificity with no cross-reactivity to methylcytosine and unmethylated cytosine. 5-hydroxymethylcytosine is separately detected.
  • Universal positive and negative controls are included, which are suitable for quantifying hydroxymethylated DNA from any species.
  • Strip-well microplate format makes the assay flexible for either manual or high throughput analysis.
  • Simple, reliable, and consistent assay conditions.

Fig. 1. Schematic procedure of the MethylFlash™ Hydroxymethylated DNA Quantification Kit.

 

Product Details


Principle & Procedure
The MethylFlashHydroxymethylated DNA Quantification Kit (Colorimetric) contains all reagents necessary for the colorimetric quantification of global DNA hydroxymethylation. In this assay, DNA is bound to strip wells that are specifically treated to have a high DNA affinity. The hydroxymethylated fraction of DNA is detected using capture and detection antibodies and then quantified through an ELISA-like reaction by reading the absorbance in a microplate spectrophotometer at 450 nm. The amount of hydroxymethylated DNA is proportional to the OD intensity measured, which can be calculated with the kit's included formulas for relative hydroxymethylation status of two different DNA samples or absolute quantification of 5-hydroxymethylcytosine (5-hmC) using a standard curve.

Easy, Fast, and Flexible
The entire colorimetric assay has easy-to-follow steps for convenience and speed, which can be completed in 3 hours and 45 minutes. The strip-well microplate format allows for a flexible assay in manual or high throughput analysis. Universal positive and negative controls are included with the kit for quantifying hydroxymethylated DNA from any species such as mammals, plants, fungi, bacteria, and viruses.

Safe and Convenient
All the needed reagents, including negative controls and positive controls, for quantification of global DNA hydroxymethylation are conveniently packaged in the kit. The direct colorimetric quantification of DNA samples replaces obsolete or inferior methods and eliminates the need for DNA digestion/denaturation, radioactivity, extraction, or chromatography.

Highly Sensitive and Specific
The novel procedure and proprietary kit compositions allow for accurate quantification of hydroxymethylated DNA to be achieved with high sensitivity and specificity. The detection limit of the input DNA can be as low as 40 pg of hydroxymethylated DNA with a dynamic range from 40 pg to 20 ng (see Fig. 2). Analytic sensitivity is about ten-fold higher than that obtained by HPLC (0.008% vs 0.08%). There is no cross-reactivity to methylcytosine and unmethylated cytosine so that only hydroxymethylated DNA (5-hmC) is detected (see Fig. 3).

Responsive, Reliable, and Practical
Based on its working principle and the microplate format, the kit can be practically and routinely used for any species from a variety of forms including from cultured cells, fresh and frozen tissues, paraffin-embedded tissues, plasma/serum samples, and body fluid samples. To demonstrate the capabilities of the kit, it has been successfully used for quantifying the content of 5-hmC in DNA from mouse brains and HeLa cells. The percentage of 5-hmC measured by the MethylFlash kit is parallel and comparable to that detected by HPLC and LC-MS methods (see Table 1). Through the use of the kit, a high abundance of 5-hmC in normal human brains and colon tissues is discovered for the first time. Furthermore, it shows that 5-hmC accounts for 32% and 16% of total methylated cytosine (5-mC plus 5-hmC) in normal human brains and colon tissues, respectively (see Table 2). In contrast, 5-hmC is low or absent in colon cancer cell lines (HCT116) and cervical cancer cell lines (HeLa) (see Fig. 4A and 4B).

 

  MethylFlash HPLC1 LC-MS2
Mouse Brain 0.16% 0.55% 0.13%
HeLa Cells < 0.02% undetectable not determined

Table 1. Comparison of the MethylFlash™ Hydroxymethylated DNA Quantification Kit and chromatography-based methods for the detection of 5-hydroxymethylcytosine, shown in percentage of 5-hmC present in two DNA sample types.

1 Kriaucionus S et al: Science, 324: 929-930, 2009
2 Munzel M et al: Angewandte Chemie, 122: ePub, 2010

  % 5-hmC % 5-mC 5-hmC/(5-mC + 5-hmC)
Human Brain 0.67% 1.00% 40.00%
Mouse Brain 0.16% 1.53% 10.00%
Human Placenta 0.06% 0.67% 8.00%
Human Colon 0.47% 1.46% 24.00%
HCT116 < 0.01% 1.30% < 1.00%
HeLa < 0.02% 1.62% < 1.00%

Table 2. Percentage of 5-hmC and 5-mC in different tissues/cells, measured with the MethylFlash™ Hydroxymethylated DNA Quantification Kit and the MethylFlash™ Methylated DNA Quantification Kit, respectively.


Fig. 2. Demonstration of high sensitivity of 5-hydroxymethylcytosine detection achieved by the MethylFlash™ kit. Synthetic hydroxymethylated DNA was added into the assay wells at different concentrations and then measured with the MethylFlash™ Hydroxymethylated DNA Quantification Kit.

Fig. 3. Demonstration of high specificity of 5-hydroxymethylcytosine detection achieved by the MethylFlash™ kit. Synthetic unmethylated DNA (contains only cytosine), methylated DNA (contains only 5-methylcytosine), and hydroxymethylated DNA standard (contains only 5-hydroxymethylcytosine) were added into the assay wells at different concentrations and then measured with the MethylFlash™ Hydroxymethylated DNA Quantification Kit.

 


Fig. 4. Demonstration of a high abundance of 5-hydroxymethylcytosine in human brain and colon tissues. 200 ng of DNA isolated from different tissues or cells were added into the assay wells and the hydroxymethylated fraction of DNA was measured using the MethylFlash™ Hydroxymethylated DNA Quantification Kit. A: OD intensity measured in variety of DNA samples. B: Percentage of hydroxymethylated DNA in various tissues and cells.

Product Components


HC1 (10X Wash Buffer)
HC2 (Binding Solution)
HC3 (Negative Control I, 20 µg/ml)*
HC4 (Negative Control II, 20 µg/ml)*
HC5 (Positive Control, 20 µg/ml)*
HC6 (Capture Antibody, 1000 µg/ml*
HC7 (Detection Antibody, 400 µg/ml)*
HC8 (Enhancer Solution)*
HC9 (Developer Solution)
HC10 (Stop Solution)
8-Well Assay Strips (With Frame)
User Guide

* Spin the solution down to the bottom prior to use.

Note: The HC3 Negative Control I is an unmethylated polynucleotide containing 20% of cytosine. The HC4 Negative Control II is a methylated polynucleotide containing 20% of 5-methylcytosine. The HC5 Positive Control is a hydroxymethylated polynucleotide containing 20% of hydroxymethylcytosine.

User Guide & MSDS


[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

Product Citations


Li, W. et. al. (2011). Distribution of 5-hydroxymethylcytosine in different human tissues. J Nucleic Acids. 2011:870726. PubMed Abstract

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