对亚硫酸氢盐转化的DNA进行全基因组扩增,用于PCR分析
  • 获得高产量的亚硫酸氢盐转化DNA,可用于多个PCR
  • 专用于扩增亚硫酸氢盐转化后DNA
  • 方便的一步建立反应体系

EpiTect Whole Bisulfitome Kit包含DNA聚合酶、缓冲液和试剂,采用多重置换扩增(MDA)技术,用于经亚硫酸氢盐转化后DNA的全基因组扩增。EpiTect Whole Bisulfitome Kit使用经验证的REPLI-g技术,专为扩增经亚硫酸氢盐转化DNA研发(亚硫酸氢盐转化后其核苷酸组成已发生改变,且DNA片段更小),同时维持转化后序列。EpiTect Whole Bisulfitome Kit的常规DNA产量是每次反应1–3 μg,因此,单次亚硫酸氢盐反应获得的模板DNA可进行多达300次PCR分析。

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EpiTect Whole Bisulfitome Kit (25)
REPLI-g Midi DNA Polymerase, EpiTect WBA Reaction Buffer, nuclease-free water for 25 whole bisulfitome amplification reactions
59203
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The EpiTect Whole Bisulfitome Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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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
EpiTect Whole Bisulfitome Kit是为所有甲基化分析提供足够DNA的理想工具,而不受亚硫酸氢盐转化后DNA量较少的限制。亚硫酸氢盐转化后的全基因组扩增(WBA)流程可获得高产的亚硫酸氢盐转化后DNA,同时配合含有独特DNA保护体系的EpiTect Bisulfite Kits维持转化后序列的表现。EpiTect Whole Bisulfitome Kit经检测优于其他供应商提供的扩增试剂盒(参见"High amplification efficiency"和"Reliable whole bisulfitome amplification")。
Principle

基因组DNA序列的甲基化分析正面临着挑战,尤其在样本量较少的情况下。使用EpiTect Whole Bisulfitome Kit可高度一致的扩增亚硫酸氢盐转化后DNA的全基因组,无序列偏向性(参见"EpiTYPER MALDI-TOF analysis of WBA DNA")。该方法基于等温多重置换扩增(MDA)技术。复制过程中通过带有3'→5'外切酶校正活性的行进性DNA聚合酶确保其高保真扩增。

EpiTect Whole Bisulfitome Kit应用经验证的REPLI-g技术,专为扩增经亚硫酸氢盐转化后DNA研发(亚硫酸氢盐转化后其核苷酸组成已发生改变,且DNA片段更小),同时维持转化后序列的表现。配合含有独特DNA保护体系的EpiTect Bisulfite Kits,可获得最佳结果。这样可确保DNA有效变性,生成胞嘧啶完全转化所必需的单链DNA,同时避免由于高温、低pH值的亚硫酸氢盐处理引起的DNA过度断裂。通过预防DNA断裂可确保后续较长DNA片段的扩增。使用EpiTect Whole Bisulfitome Kit,通常每个反应获得的DNA的量为1–3 μg。

Procedure

方便的一步反应体系,即使用反应缓冲液和REPLI-g DNA聚合酶的混合液孵育亚硫酸氢盐转化后的模板DNA。加热失活聚合酶后,扩增的DNA可直接进行分析或贮存(参见"WBA procedure")。

生物学和医药研究的很多领域中,尤其是肿瘤学、干细胞研究和发育生物学研究中,获得表观遗传学信息是首要任务。但是,由于缺乏提供可重复性数据的标准化方法,尤其是样本材料的限制,分析DNA的甲基化变化非常困难。QIAGEN最新推出的EpiTect方案,建立了从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甲基化分析或测序的标准化预分析和分析方案(参见"Standardized workflows in epigenetics")。

Applications

EpiTect Whole Bisulfitome Kit是为所有甲基化分析提供足够DNA的理想工具,而不受亚硫酸氢盐转化后DNA量较少的限制。每次使用10 ng DNA模板扩增获得的DNA,可进行100–300次real-time PCR分析(如使用EpiTect MethyLight PCR Kits)或终点法PCR(如使用EpiTect MSP Kits)。

Feature
Specifications
Amplification Whole bisulfite converted DNA
Applications Methylation specific PCR
Maximum input volume 10 µl bisulfite converted DNA
Reaction time 8h at 28°C
Reaction volume 40 µl
Starting amount of DNA > 50 ng bisulfite converted DNA
Starting material Epitect bisulfite converted DNA
Technology Multiple Displacement Amplification (MDA)
Yield 1-3 µg per reaction

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Kit Handbooks
1
For whole genome amplification of bisulfite
converted DNA for PCR analysis
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