双位点5mC&5hmC定量检测试剂盒 又名:特定位点甲基化和羟甲基化qPCR定量检测试剂盒 货号：A-P-1067
BisulPlus Loci 5mC & 5hmC Detection PCR Kit
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背景资料：DNA methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring, resulting in 5-methylcytosine (5mC). In somatic cells, 5mC 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 5mC is also observed in non-CpG contexts. The biological importance of 5mC as a major epigenetic modification in phenotype and gene expression has been widely recognized. 5-hydroxymethylcytosine (5hmC), as a sixth DNA base with functions in transcription regulation, has been detected to be abundant in human and mouse brains and embryonic stem (ES) cells. In mammals, it can be generated by the oxidation of 5mC, a reaction mediated by the ten-eleven translocation (TET) family of 5mC-hydroxylases. 5hmC has been demonstrated to be tissue specific, ranging from undetectable levels in cultured cell lines to 0.6% in human brain tissues, and can be as high as 8% of total DNA in some other species. The biological significance of 5hmC as an important epigenetic modification in phenotype and gene expression has been recently recognized. For example, global decrease in 5hmC content (DNA hypohydroxymethylation) has been exhibited in nearly all cancers and has been proposed as a molecular marker and therapeutic target in cancer as well. More importantly, in contrast to 5mC that is generally considered as a gene repression marker, 5hmC is regarded as an intermediate of DNA demethylation and associated with expressed genes with a potential role in activating genes. Like 5mC, 5hmC remains the same after bisulfite-conversion of cytosine to uracil (C to U), which makes 5hmC indistinguishable from 5mC. As consequence, all 5hmC will be misread as 5mC in the assay with using MS-PCR or bisulfite-seq, which results in that the identified DNA methylation status is incorrect as it in fact includes both 5mC and 5hmC。
产品描述：This kit includes all reagents required for successful qPCR using converted DNA generated from a tiny amount of input DNA. In this preparation, DNA is simultaneously bisulfite modified and fragmented to the appropriate length during the bisulfite process. During the bisulfite treatment, unmodified cytosine (C) is converted to uracil and will be read as T in the sequencing. 5-methylcytosine (5mC) remains the same and 5-hydroxymethylcytosine (5hmC) forms cytosine 5-methylenesulfonate (CMS). The bisulfite modified DNA is further treated with specific APOBEC deaminase, which converts 5mC to thymine but not affect CMS. During the PCR and sequencing CMS will still be read as C so that the 5hmC can be discriminated not only from C, 5mC but also other modified cytosines such as 5fC and 5caC (see the table 1). The bisulfite-enzyme converted DNA can then be used for qPCR for loci specific detection of 5mC and 5hmC。
产品特点：
1·Simultaneous Reading:DNA treatment is prepared simultaneously for both 5hmC and 5mC, which allows for parallel identification in a loci- or gene-specific manner simultaneously by qPCR；
2·Innovative Method:A combination of bisulfite and subsequent cytosine deaminase (APOBEC) treatment allows for tactical conversion of 5mC in order to identify 5hmC；
3.High Specificity:5hmC is discriminated from C and 5mC as well as from other modified cytosines 5fC and 5caC.
4.Flexibility: Perform 24 reactions for 5hmC and 24 reactions for 5mC analysis simultaneously, or perform 48 reactions for just 5mC；
5.Fast and Streamlined Procedure:The procedure from DNA bisulfite treatment to PCR products can be completed within 5 hours；
6.Complete Conversion: Completely converts C into uracil (>99%) and 5mC to thymine (>95%)；
7.Broad sample suitability:Starting materials can be genomic DNA isolated from various tissue/cell samples such as fresh and frozen tissue, cultured cells from a flask or microplate, microdissection samples, paraffin-embedded tissue, biopsy, embryonic cells, plasma/serum samples, and body fluid samples, etc. DNA enriched from various enrichment reactions such as ChIP, MeDIP/hMeDIP, or exon capture may also be used as starting material.
8.Simple, reliable, and consistent assay conditions。
数据分析：
| 操作流程 |
 |
| Discrimination of 5hmC from cytosine and 5mC by qPCR: Unmodified, methylated and hydroxymethylated DNA standards are used for bisulfite-enzyme treatment using the he BisulPlus™ Loci 5mc & 5hmC Detection PCR Kit followed by real-time amplification with use of primers targeting to CpG converted or unconverted sequences: A: BisulPlus; B: Bisulfite standalone. Amplification lines are highlighted for visibility. |
参考文献：
1.Lewinska A et. al. (February 2017). Downregulation of Methyltransferase Dnmt2 Results in Condition-dependent Telomere Shortening and Senescence or Apoptosis in Mouse Fibroblasts. J Cell Physiol.
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。
定购信息：
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操作手册 |
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100ug |
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C-HM2078 |
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100ug |
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C-HM2022F |
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96 次 |
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50 次 |
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A-P-9003-050 |
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50 次 |
英文PDF--中文PDF |
A-R5001 |
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表观遗传学产品全面解决方案列下：
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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。 |
