总体DNA甲基化极易定量检测试剂盒（比色法）                                                      货号：A-P-1030

MethylFlash  Global DNA Methylation (5-mC) ELISA Easy Kit (Colorimetric)

背景资料：DNA methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring by DNA methyltransferases, resulting in 5-methylcytosine (5-mC). In somatic cells, 5-mC is found almost exclusively in the context of paired symmetrical methylation of the dinucleotide CpG, whereas in embryonic stem (ES) cells, a substantial amount of 5-mC is also observed in non-CpG contexts. Levels of 5-mC are variable in animal genomes, ranging from undetectable amounts in some insects to about 2% of total DNA in vertebrates. The level of 5-mC in plants generally accounts for 0.5-2% and can be as high as 8% of total DNA in some other species.  The biological importance of 5-mC as a major epigenetic modification in phenotype and gene expression has been recognized widely. For example, global decrease in 5-mC content (DNA hypomethylation) is likely caused by methyl-deficiency due to a variety of environmental influences, and has been proposed as a molecular marker in multiple biological processes such as cancer. It has been well demonstrated that the decrease in global DNA methylation is one of the most important characteristics of cancer. A few novel modified nucleotides, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) have been detected in human and mouse tissues as well as embryonic stem cells. In mammals, these modified nucleotides can be generated by iterative oxidation of 5-methylcytosine, a reaction mediated by the TET family of enzymes。

产品描述：This kit contains all reagents necessary for the quantification of global DNA methylation. In this assay, DNA is bound to strip-wells that are specifically treated to have a high DNA affinity. The methylated fraction of DNA is detected using capture and detection antibodies and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer. The percentage of methylated DNA is proportional to the OD intensity measured。

产品特点：

1·Fast - Reduced steps so that the entire procedure only needs 2 hours；

2·Robust - Improved kit composition allows the assay to have a greater "signal window" with reduced variation between replicates ；

3.Convenient - Inherently low background noise, thereby eliminating the need for DNA denaturation and plate blocking steps；

4.Sensitive - Detection limit can be as low as 0.05% methylated DNA from 100 ng of input DNA ；

5.Specific - High specificity to 5-mC, with no cross-reactivity to unmethylated cytosine and no cross-reactivity to hydroxymethylcytosine within the indicated concentration range of the sample DNA；

6.Universal - Positive and negative controls are included and allow for detection of DNA methylation in any species from either single-stranded or double-stranded input DNA；

7.Accurate - Optimized positive controls that can be fractionalized in percentage scale, allowing the assay to be more accurate and highly comparable with HPLC-MS analysis；

8.Flexible - Strip-well microplate format makes the assay available for manual or high throughput analysis。

参考文献：

1.Koestel ZL et. al. (December 2016). Bisphenol A (BPA) in the serum of pet dogs following short-term consumption of canned dog food and potential health consequences of exposure to BPA. Sci Total Environ.

2.Khot VV et. al. (September 2016). Variable Methylation Potential in Preterm Placenta: Implication for Epigenetic Programming of the Offspring. Reprod Sci.

3.AM Manzardo and MG Butler et. al. (September 2016). Examination of Global Methylation and Targeted Imprinted Genes in Prader- Willi Syndrome Imedpub. 2(3)

保存建议：在接收到艾德寄出的试剂盒后请按照说明书建议，使用不同的保存条件或温度来保存试剂盒内组分。

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关于5-hmC
5-hmC是近年来在动物组织中发现的，由胞嘧啶修饰而来。5-hmC在表观遗传学上的功能可能与5-甲基化胞嘧啶(5-mC)不同。尽管到现在为止还不确知其功能，有研究者猜测它在调控基因的表达与关闭过程中起着重要的作用。5-mC的发现让我们不得不重新评估DNA甲基信息，也不得不监测人类的健康组织和病理组织之间5-mC相对分布的差异。在EPI公司的MethylFlash技术之前，我们还没有发现任何直接的常规方法来检测5- hmC,以及区分5-hmC和5-mC

5-hmC 和 5-mC的区别

时下常用的DNA甲基化分析方法包括限制内切酶酶切和亚硫酸氢盐或MeDIP介导的MS-PCR和测序，这些技术都不适合用来检测5-hmC，因为它与5-mC事实上很难用这类方法区分开来。为了解决这个问题，EPIk研制了MethylFlash羟甲基化DNA定量试剂盒(荧光法)。本试剂盒提供了一种很经济的方法来检测5-羟甲基化胞嘧啶，并且区分5-hmC, 5-mC, 和 C，使得研究者能够重新评估他们的DNA甲基化信息，也能够在新样品中寻找DNA羟甲基化。