·产品描述

   DNA甲基化是DNA甲基转移酶把一个甲基共价连接到胞嘧啶的5号碳原子上，形成5-甲基胞嘧啶（5-mC）。在体细胞中，5-mC 只会对称地出现在CpG双核苷酸上。而在胚胎肝细胞里，大量的5-mC 则出现在非CpG核苷酸上。现在已经广泛认识到5-mC作为一个表型和基因表达的表观遗传修饰。例如，全局性的5-mC减少（DNA去甲基化）可能是由于环境因素导致的甲基缺乏，并被认为是很多过程的生物标志，如癌症。

   MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit（P-1030）是原有试剂盒P-1034的升级款，经过全新优化的、完整的的试剂组合，可以利用抗体结合和ELISA原理方式，定量分析DNA中5-mc。它可以直接检测哺乳动物，植物，真菌细菌，病毒，细胞，组织，血清/血浆以及其它体液中的总DNA样本的5-mc的甲基化程度。

·产品特点

1.快速实验：优化实验操作，完成实验仅需2h 

2. 灵敏度：最低可检测50 pg的甲基化DNA 

3.特异性：不与未修饰的胞嘧啶或者其它修饰的胞嘧啶有任何交叉反应 

4.精确性：最优化的阳性对照标准品，提供堪比HPLC-MS的可靠结果 5.一致性：简化的操作流程，有效降低复孔间的变异系数（CV%）

·产品说明书

The MethylFlash™ Global DNA Methylation (5-mC) ELISA Easy Kit  is a complete set of optimized buffers and reagents to colorimetrically quantify global DNA methylation status by specifically measuring levels of 5-methylcytosine (5-mC) in a simplified, "one-step" ELISA-like reaction. This allows global DNA methylation to be directly measured more quickly than other techniques such as LUMA, LINE-1, Alu or LTR-based assays. As a fourth generation technology of EpiGentek's patented global DNA methylation technique, it is a further refinement of the popular, predecessor MethylFlash kit by improving upon speed, simplicity, sensitivity, and reproducibility.

This kit is also specifically optimized for paired use with the MethylFlash Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (Colorimetric) for simultaneously quantifying both methylated DNA and hydroxymethylated DNA.

This kit has the following advantages and features:

• Fast - Reduced steps so that the entire procedure only needs 2 hours
• Robust - Improved kit composition allows the assay to have a greater "signal window" with reduced variation between replicates
• Convenient - Inherently low background noise, thereby eliminating the need for DNA denaturation and plate blocking steps
• Sensitive - Detection limit can be as low as 0.05% methylated DNA from 100 ng of input DNA
• 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
• 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
• 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
• Flexible - Strip-well microplate format makes the assay available for manual or high throughput analysis

What Your Peers Are Saying Online:

• "For low 5mc amounts (<0.5%) my bet is on Epigentek."
• "I recommend you to use Epigentek's new kit, MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit instead that we optimized in our lab."
• "The MethylFlash kit was indeed the best one I tried."

Background
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.

Principle & Procedure
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.

Starting Materials
Input DNA should be relatively pure with 260/280 ratio >1.6 and can be diluted with water or TE buffer. The DNA amount can range from 20 ng to 200 ng per reaction. However, we recommend using 100 ng of DNA, which is the optimized input amount for the best results. DNA can be isolated from any species such as mammals, plants, fungi, bacteria, and viruses in a variety of forms including, but not limited to, cultured cells, fresh and frozen tissues, paraffin-embedded tissues, plasma/serum samples, and body fluid samples. Both single stranded DNA and double stranded DNA with a size of 200 bps to full length is suitable for use.

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