羟甲基化收集试剂盒 货号：E-55013
Hydroxymethyl Collector™
背景资料：The Hydroxymethyl Collector™ Kit is designed to detect and capture DNA fragments containing 5-hydroxymethylcytosine (5-hmC) DNA methylation*. The Hydroxymethyl Collector Kit utilizes a β-glucosyltransferase enzyme to transfer a modified glucose moiety to 5-hydroxymethylcytosine residues in double-stranded DNA. This modified glucose is then used to chemically attach a biotin conjugate for capture and enrichment with streptavidin magnetic beads. Enriched DNA can be used in the analysis of individual genes by PCR, or in combination with microarrays and sequencing for genome-wide analysis of 5-hydroxymethylcytosine..
By utilizing chemical labeling of 5-hmC residues, the Hydroxymethyl Collector Kit is extremely specific in its capture of hydroxymethylated DNA fragments. The biotin-streptavidin binding reaction also allows for stringent binding and wash conditions to reduce any non-specific background. The technique is sensitive enough to enrich DNA fragments containing two or more 5-hmC residues. To learn more about the Hydroxymethyl Collector method, click on the Method tab below. To download the manual, please click on the Documents tab below
产品描述：To determine the range of detection for Hydroxymethyl Collector, various quantities of mouse brain DNA were assayed in the Hydroxymethyl Collector Kit. The enriched 5-hydroxymethylcytosine DNA fragments were analyzed by real time PCR across multiple loci. The amount of enriched 5-hmC DNA was plotted as a percentage of the starting material. From this analysis it was determined that Hydroxymethyl Collector is sensitive over the range of 5 ng - 2.5 µg fragmented genomic DNA. For downstream analysis requiring larger inputs, e.g. microarray, it may be necessary to pool multiple enrichment reactions or perform whole-genome amplification following 5-hmC DNA enrichment.
产品特点：
1.Chemical labeling ensures specific modification of 5-hmC DNA without cross-reactivity of 5-mC sites；
产品应用：Hydroxymethyl Collector Kit works with fragmented (100-500 bp) double-stranded DNA. The DNA is combined with our β-Glucosyltransferase enzyme in the presence of a UDP-Azide-Glucose donor. The β-Glucosyltransferase enzyme will add the modified glucose moiety onto 5-hydroxymethylcytosine residues leaving unmethylated and 5-methylcytosine residues untouched. A chemical labeling reaction then attaches a biotin conjugate to the modified glucosyl-5-hmC DNA. Streptavidin magnetic beads are used for capture. Elution buffer is added to release the enriched DNA from the biotin linker, resulting in DNA fragments that are enriched for 5-hydroxymethylcytosine. Using the included purification reagetns, the DNA is cleaned up prior to use in downstream applications.The Hydroxymethyl Collector Kit includes a 5-hmC positive control DNA, MseI digested human genomic DNA and PCR primers that can be used to determine the efficiency of the enrichment reactions. The 5-hmC Control DNA consists of a 338 base pair DNA fragment derived from the APC gene. The 5-hmC control DNA has been modified so that 25% of the cytosine residues contain 5-hydroxymethylcytosine. When the positive control fragment is spiked into human genomic DNA and enriched, the included APC PCR primers can be used to confirm the enrichment efficiency
保存建议：Hydroxymethyl Collector™ contains all thre reagents needed to perform the glucosylation reaction, biotin conjugation, streptavidin capture, elution and purification of DNA containing 5-hydroxymethylcytosine. Protocols are provided for the fragmentation of sample DNA all the way through to real time PCR analysis of a locus of interest. For added convenience, the kit also includes positive control 5-hmC DNA, which is a 338 bp sequence of the APC locus in which 25% of the cytosine residues have been hydroxymethylated. PCR primers for APC are provided to confirm the efficiency of the enrichment reactions..
Each kit contains β-glucosyltransferase enzyme, UDP-Azide-Glucose, 10X Reaction Buffer AM1, 1 M DTT, Biotin Conjugate Solution, Streptavidin Beads, Binding Buffer AM13, 10X Ellution Buffer AM2, DNA Purification Binding Buffer, DNA Purification Wash Buffer, DNA Purification Elution Buffer, 3 M Sodium Acetate, DNA purification columns, 5-hmC Control DNA, APC PCR primer mix, 0.2 ml PCR tubes, a bar magnet and glue dots.
Storage conditions range from room temperature to -20°C; please refer to the product manual for proper storage temperatures of each component
定购信息：
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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。
2.β-Glucosyltransferase modifies 5-hmC regardless of sequence context, enabling detection of non-CpG methylation (CpA or CpT)；
3.Uses dsDNA throughout the process which makes it easier to prepare libraries for downstream analysis by Next-Gen sequencing techniques;
4.Biotin/streptavidin binding enables more stringent binding and wash conditions to reduce non-specific background without losing the sensitivity of the assay;
5.Analysis is not constrained by the properties or consensus sequence of glucosyl-sensitive restriction enzymes (GSREs);
6.Simple, fast procedure can be completed in less than 4 hours;
7.Downstream applications include individual gene analysis by PCR/qPCR or genome-wide analysis by sequencing or microarray. |
