EpiTect Whole Bisulfitome Kit

For whole genome amplification of bisulfite converted DNA for PCR analyses
  • High yield of bisulfite converted DNA for multiple PCRs
  • Representative amplification from bisulfite converted DNA
  • Convenient one-step reaction setup

The EpiTect Whole Bisulfitome Kit provides DNA polymerase, buffers, and reagents for amplification of the entire bisulfite converted genome — the bisulfitome — using multiple displacement amplification technology (MDA). The EpiTect Whole Bisulfitome Kit has been specially developed for the amplification of DNA after conversion with EpiTect Bisulfite Kits. It uses proven REPLI-g technology and has been adapted to the specific requirements of bisulfite converted DNA (smaller DNA fragment size and altered nucleotide composition due to bisulfite conversion), while maintaining the converted sequence representation. Typical DNA yields from the EpiTect Whole Bisulfitome Kit are 1–3 μg per reaction, sufficient for up to 300 PCR analyses from just a single EpiTect bisulfite reaction.

产品 货号 目录价:
EpiTect Whole Bisulfitome Kit (25)
REPLI-g Midi DNA Polymerase, EpiTect WBA Reaction Buffer, nuclease-free water for 25 whole bisulfitome amplification reactions
59203
询价

EpiTect Whole Bisulfitome Kit适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


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Reliable whole bisulfitome amplification.|High amplification efficiency.|WBA procedure.|EpiTYPER MALDI-TOF analysis of WBA DNA.|Standardized workflows in epigenetics.|

Real-time PCR was used to measure the representation of four different loci in 10 ng bisulfite converted DNA, before (–WBA) and after (+WBA) amplification with the EpiTect Whole Bisulfitome Kit. DNA was first bisulfite converted using either the EpiTect Bisulfite Kit or a kit from supplier Z, and was then amplified; qPCR was performed using the QuantiTect SYBR® Green PCR Kit. In the EpiTect converted DNA, the four loci were represented similarly before and after amplification. Higher CT values for the DNA converted with the competitor’s bisulfite kit indicate higher DNA fragmentation prior to WBA. Following amplification, the relative representation of the four loci was increased and different, indicating lower whole bisulfitome amplification and unequal amplification.|A starting amount of 50 ng bisulfite converted DNA was used for the whole bisulfitome amplification reaction. The amplification was performed using the EpiTect Whole Bisulfitome Kit, as well as kits from suppliers A and S. DNA yields following the amplification reactions were measured with PicoGreen. The EpiTect Whole Bisulfitome Kit resulted in a 50-fold increase in DNA yield, which was much higher than the yields from the other suppliers' kits.||Methylation of the MP6 locus was measured in unmethylated DNA (UMDNA 1), methylated DNA (VIAL A 1), and a mixture of both (HTXA 1), before and after amplification (WBAUMDNA1, WBAHTXA 1, WBAVIAL A 1) using the EpiTect Whole Bisulfitome Kit. The methylation pattern prior to amplification is very similar to that of the amplified DNA, demonstrating representative amplification. (Data kindly provided by Hany Ezzeldin, Mayo Clinic, Rochester, USA).|  |

Performance
The EpiTect Whole Bisulfitome Kit is the ideal tool to provide sufficient DNA for all downstream methylation analyses, whenever bisulfite converted DNA is limited. This whole bisulfitome amplification (WBA) procedure provides high yields of bisulfite converted DNA with accurate locus representation, when used in conjunction with EpiTect Bisulfite Kits. The EpiTect Whole Bisulfitome Kit has outperformed amplification kits tested from other suppliers (see figures "High amplification efficiency" and "Reliable whole bisulfitome amplification").
Principle

Methylation analysis of genomic DNA sequences can be limited by the small amount of sample available. The EpiTect Whole Bisulfitome Kit provides highly uniform amplification across the entire bisulfite converted genome, with negligible sequence bias (see figure "EpiTYPER MALDI-TOF analysis of WBA DNA"). The method is based on isothermal multiple displacement amplification (MDA) technology. A uniquely processive DNA polymerase with a 3'→5' exonuclease proofreading activity maintains high fidelity during the replication process. 

The EpiTect Whole Bisulfitome Kit has been specially developed using proven REPLI-g technology and has been adapted to the special requirements of bisulfite converted DNA (smaller DNA fragment size and changed nucleotide composition due to bisulfite conversion), while maintaining the converted sequence representation. Best results are obtained when used in combination with EpiTect Plus Bisulfite Kits, which contain a uniquely formulated DNA Protect system. This enables effective DNA denaturation, which is necessary for complete cytosine conversion, and prevents the excessive DNA fragmentation usually associated with high temperature and low pH conditions of the bisulfite conversion reaction. Preventing DNA fragmentation enables subsequent amplification of large DNA fragments. Typically, DNA yields of 1–3 μg are generated per reaction using the EpiTect Whole Bisulfitome Kit.

Procedure

The convenient one-step reaction setup involves incubating bisulfite converted template DNA with a mixture of reaction buffer and REPLI-g DNA polymerase. After heat inactivation of the polymerase, the amplified DNA is ready for analysis or storage (see figure "WBA procedure").

Accessing epigenetic information is of prime importance for many areas of biological and medical research — particularly oncology, but also stem cell research and developmental biology. However, the analysis of changes in DNA methylation is challenging due to the lack of standardized methods for providing reproducible data particularly from limited sample material. With its newly introduced EpiTect products, QIAGEN makes available standardized, pre-analytical and analytical solutions, from DNA sample collection, stabilization and purification, to bisulfite conversion and real-time or end-point PCR methylation analysis or sequencing (see figure "Standardized workflows in epigenetics").

Applications

The EpiTect Whole Bisulfitome Kit is the ideal tool to provide sufficient DNA for all downstream methylation analyses, whenever bisulfite converted DNA is limited. The amplification reaction produces enough DNA for 100–300 real-time PCR assays (e.g., using EpiTect MethyLight PCR Kits) or end-point PCRs (e.g., using EpiTect MSP Kits), estimating 10 ng of amplified DNA per reaction.

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For whole genome amplification of bisulfite
converted DNA for PCR analysis
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Reliable whole bisulfitome amplification
可靠的全转化组扩增。
在使用EpiTect Whole Bisulfitome Kit进行扩增前(–WBA)和扩增后(+WBA),使用Real-time PCR检测10 ng亚硫酸氢盐转化DNA的四个不同位点。先使用EpiTect Bisulfite Kit或来自供应商Z的试剂盒对DNA进行亚硫酸氢盐转化,再扩增;使用QuantiTect SYBR® Green PCR Kit进行qPCR。在EpiTect转化DNA中,四个位点在扩增 前后的水平类似。使用竞争者的亚硫酸氢盐试剂盒转化的DNA CT值更高,说明WBA前DNA片段化更高。扩增后,四个位点的水平升高且不同,表明全亚硫酸氢盐转化物扩增能力更低,且扩增不平等。
High Amplification Efficiency in Comparison to Other Suppliers
扩增效率高。
使用起始量为50 ng的亚硫酸氢盐转化DNA进行全亚硫酸氢盐转化物扩增反应。使用EpiTect Whole Bisulfitome Kit和来自供应商A和S的试剂盒进行扩增。扩增后使用PicoGreen测定DNA产量。EpiTect Whole Bisulfitome Kit得到的DNA产量升高50倍,比使用其他供应商的试剂盒得到的产量高很多。
Amplification procedure
WBA实验流程。
EpiTYPER® MALDI-TOF Analysis of Bisulfite Converted and Whole Bisulfitome Amplified (WBA)
对WBA DNA进行EpiTYPER MALDI-TOF分析。
对未甲基化DNA(UMDNA 1)、甲基化DNA(VIAL A 1)和二者混合物(HTXA 1),在使用EpiTect Whole Bisulfitome Kit扩增前后(WBAUMDNA1, WBAHTXA 1, WBAVIAL A 1)进行MP6位点的甲基化检测。扩增前的甲基化模式与扩增后的DNA非常类似,说明是有扩增的位点全面。(数据由美国Hany Ezzeldin, Mayo Clinic, Rochester提供)。
Standardized Workflows in Epigenetics
表观遗传学研究的标准工作流程。