亚硫酸氢盐的完全转化和DNA回收,用于甲基化分析

  • 简化的流程,获得快速、可靠的结果
  • 胞嘧啶完全转化
  • 独特的DNA保护体系,用于高度敏感的分析
  • 优化的试验方案可处理FFPE组织样本DNA
  • 离心柱和高通量96孔板方式
EpiTect Bisulfite Kit可在6小时内将DNA 序列中非甲基化的胞嘧啶完全转化为尿嘧啶。EpiTect 96 Bisulfite操作时间少于7小时。高灵敏度的方法采用全新的保护机制防止DNA降解,确保即使是1 ng的DNA也可获得灵敏的结果,转化率可高达99%。得益于独特的DNA保护技术,EpiTect Bisulfite转化后DNA高度适用于EpiTect Whole Bisulfitome Kits转化后全基因组扩增。因此可在转化后DNA量较少的情况下确保可靠的扩增。EpiTect Bisulfite Kits包括离心柱或96孔板形式的扩增和脱磺酸基过程,确保快速可靠的实验结果用于下游应用。两个试剂盒都可用于离心机或真空装置,另外,EpiTect Bisulfite Kit可在QIAcube全自动核酸纯化仪上全自动运行。
Product Product no. Cat. no. List price:
 
 
Show details
    varies
Can't order online?
To place an order via phone, email or for requesting a quote, please provide the product’s name, number and catalog number.
Product Cat. no. List price:
EpiTect Bisulfite Kit (48)
48 EpiTect Bisulfite Spin Columns, Reaction Mix, DNA Protect Buffer, Carrier RNA, Buffers
59104
Inquire
EpiTect 96 Bisulfite Kit (2)
2x EpiTect Bisulfite 96-well Plates, Reaction Mix, DNA Protect Buffer, Carrier RNA, Buffers
59110
Inquire

EpiTect Bisulfite Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.



  • Main Image Navi
Long-term DNA storage.|Highly efficient cytosine conversion.|Complete cytosine conversion.|Unique DNA Protect Buffer and pH indicator system.|Methylation detection from FFPE tissue samples.|EpiTect Bisulfite Conversion procedure.|Fast and easy bisulfite conversion.|Time-saving procedure.|Standardized workflows in epigenetics.|
Bisulfite converted DNA generated with EpiTect Kits was stored at –20°C for up to 36 months, and amplified at several time intervals. The resulting threshold cycle values at all time points for various DNA amounts were comparable to those obtained with freshly converted DNA (time point 0).|Over 900 clones generated by PCR from 6 independent EpiTect Bisulfite reactions (1 μg DNA each) and cloned using the QIAGEN PCR Cloning Kit were sequenced. Over 150,000 of the 450,000 sequenced nucleotides were unmethylated cytosine residues (DNA 1) that were converted into uracil residues (DNA 2). Cytosine to uracil conversion rates of 99.4–99.8% were obtained. The sequence shown is a small portion of the total sequence analyzed. DNA 1: untreated DNA; DNA 2: converted DNA.|A 1 μg aliquot of genomic DNA was converted using the EpiTect Bisulfite Kit or bisulfite kits from alternative suppliers (Supplier E and Supplier Z) according to the manufacturer's instructions. Next, the presence of unconverted DNA in comparable amounts of each sample was determined by [A] end-point PCR using the HotStarTaq Plus Master Mix Kit and [B] SYBR® Green based real-time PCR using the QuantiTect SYBR® Green PCR Kit. The EpiTect Bisulfite Kit treated DNA was completely converted, while DNA treated using kits from Suppliers E and Z contained a significant proportion of unconverted DNA.|[A] DNA Protect Buffer prevents DNA fragmentation during bisulfite treatment of DNA and facilitates the formation of single-stranded DNA, enabling complete bisulfite conversion. [B] The pH indicator dye in the DNA Protect Buffer enables visualization of complete reaction mixing, thereby ensuring the correct pH is achieved for complete cytosine conversion.|DNA (1 µg) isolated from an FFPE sample using the QIAamp DNA Mini Kit was converted with the EpiTect Bisulfite Kit using the FFPE protocol. 1 μl of the 40 μl eluate was analyzed by [A] end-point PCR and [B] TaqMan probe-based real-time PCR. Both assays for the Glutathione S-Transferase gene, were specific for converted DNA. In each application the unconverted genomic DNA (control) was not amplified.||Human genomic DNA was purified from blood using the QIAamp DNA Blood Mini Kit, and various amounts (1 ng – 1 µg) were converted using the EpiTect Bisulfite Kit. PCR was performed using the HotStarTaq Master Mix Kit and 2 sets of primers designed to amplify converted DNA. A 5 µl aliquot of each PCR was loaded onto a 1.3% agarose gel. As little as 1 ng DNA is sufficient for conversion using the EpiTect Bisulfite Kit. C: untreated genomic DNA (negative control). M: marker.|The complete EpiTect Bisulfite Kit centrifugation or vacuum procedure — from DNA to pure bisulfite converted DNA ready for analysis — takes less than 6 hours. Traditional bisulfite conversion procedures can take over 18 hours and require extensive user interaction.|  |
Performance
快速得到实验结果

完整的EpiTect离心和真空过程只需6小时,包括从DNA到待分析的亚硫酸氢盐转化后的高纯DNA。EpiTect 96 Bisulfite操作时间少于7小时。传统的亚硫酸氢盐转化过程需要18个小时,且需要大量的操作(参见"快速简单的亚硫酸氢盐转化"和"Time saving procedure")。EpiTect Bisulfite Kit为成功进行亚硫酸氢盐转化和DNA回收提供所需的全部试剂。

快速简单的亚硫酸氢盐转化
EpiTect流程 传统流程
反应构建 10分钟;预制溶液 40分钟;需反应构建
亚硫酸氢盐转化 5小时 16小时
纯化 30–45分钟; EpiTect离心柱或孔板 120分钟;纯化、脱璜、沉淀和洗涤
完全的DNA转化

每个EpiTect Bisulfite Kit可以相同的效率转化低至1 ng到2 µg DNA。全新的实验步骤和优化的缓冲液可回收得到高产量的转化DNA。当通过real-time PCR扩增经转化的DNA时,低CT值的实验结果显示了高效的胞嘧啶转化(参见"Complete cytosine conversion")。对转化的DNA的分析显示,EpiTect Bisulfite Kit可转化超过99%的非甲基化的胞嘧啶(参见"Highly efficient cytosine conversion")。高转化率确保了高重复性和可靠的下游分析结果。

Principle
特殊样本的亚硫酸氢盐转化

从珍稀样本和少量样本(如:福尔马林固定、FFPE石蜡包埋或显微切割组织)中确定甲基化模式,由于DNA的量很少,实验尤其困难。但是这些样本类型是成功鉴定有价值的疾病,预测生物标记物的专门研究对象。 EpiTect Bisulfite Kit 为这些类型的样本提供优化的操作流程,从而解决了难题(参见"Methylation detection from FFPE tissue samples")。该试剂盒含有实验所需的全部缓冲液和试剂,只需少量的手动操作。

对于从FFPE样本、细胞、血液及组织中直接进行亚硫酸氢盐转化而无需预先分离DNA,我们推荐使用EpiTect Plus FFPE Bisulfite Kit和EpiTect Plus LyseAll Bisulfite Kit。

防止DNA降解

DNA保护缓冲液采用独特的配方,防止DNA过度降解(在高温、低pH值条件下使用亚硫酸氢盐处理DNA时经常出现严重的DNA降解),并实现有效的DNA变性,从而产生完全转化为胞嘧啶时所需的单链DNA(参见"Unique DNA Protect Buffer and pH indicator system")。防止DNA降解的发生可以保证后续大PCR片段的扩增和分析(参见"Fast and easy bisulfite conversion")。DNA保护缓冲液含有PH指示剂染料,从而可保证用于胞嘧啶转化的正确的pH条件。有效的完整DNA回收可保证转化后的DNA至少保存12个月而不影响DNA的质量(参见"Long-term DNA storage")。

Procedure

EpiTect Bisulfite操作流程基于QIAGEN简单直接的结合、洗涤和洗脱体系,仅需几个简单的步骤(参见"Conversion procedure")。DNA经过热变性和亚硫酸氢盐转化后,被上样到EpiTect离心柱或96孔板上,使用优化的缓冲液和标准化的离心步骤或真空装置,洗去所有的亚硫酸氢钠并洗脱DNA。洗脱的DNA可用于各种下游应用,如:甲基特异性PCR、real-time PCR分析、亚硫酸氢盐转化后测序(bisulfite sequencing)、结合亚硫酸氢盐的限制性内切酶法分析(COBRA)和焦磷酸测序(Pyrosequencing)。

表观遗传学信息对于生物医学研究(尤其是肿瘤学)、干细胞研究以及发育生物学等领域都非常重要。但是分析DNA甲基化的变化目前仍具有挑战性,因为缺乏标准化的方法可从有限的样本中得到可重现性的数据。QIAGEN推出新的表观遗传学解决方案,使样本分析前处理到分析整个过程实现标准化,即从DNA样本收集、稳定和纯化,到亚硫酸氢盐转化和real-time或终点式PCR甲基化分析或测序(参见"Standardized workflows in epigenetics")。

Applications

经EpiTect Bisulfite Kit转化和纯化的DNA适用于多种下游应用,如:

甲基化特异性PCR 、real-time PCR
结合亚硫酸氢盐的限制性内切酶法分析(COBRA) 
测序/焦磷酸测序(Pyrosequencing)/NGS
高分辨率熔解
甲基化芯片
Feature
Specifications
Applications Multiplex PCR, real-time PCR, sequencing
Conversion efficiency 99.4–99.8%
Elution volume Varies
Format Spin column and 96-well plate
Processing Manual (spin and vacuum)
Sample amount 1–2 µg
Sample types Blood, (FFPE) tissue, DNA samples
Time per run or per prep <6 hours (1-48 samples), ~ 7 hours (48-96 samples)
Yield Depends on input amount of purified genomic DNA used for conversion

You are not authorized to download the resource

Kit Handbooks
2
For complete bisulfite conversion and cleanup of DNA for methylation analysis
Show details
For complete bisulfite conversion and cleanup of DNA for methylation analysis in 96-well format
Show details