EpiTect HRM PCR Kit

For reliable screening of changes in methylation status by HRM analysis
  • Accurate detection of changes in CpG methylation status
  • Highly specific PCR amplification without the need for optimization
  • Distinct melting curves due to saturating EvaGreen fluorescent dye
  • Convenient master mix format and optimized protocol
  • Fast and easy development of new HRM methylation assays
The EpiTect HRM PCR Kit uses high-resolution melting (HRM) technology for fast screening and accurate detection of changes in the CpG methylation status of bisulfite converted DNA. The optimized kit is available in a convenient master mix format and contains the novel double-stranded DNA-binding fluorescent dye EvaGreen, as well as an optimized HRM buffer, HotStarTaq Plus DNA Polymerase, and dNTPs. The kit is compatible with real-time instruments suitable for HRM analysis, particularly the Rotor-Gene Q, and also the Rotor-Gene 6000 and the LightCycler 480.
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产品 货号 目录价:
EpiTect HRM PCR Kit (100)
For 100 reactions: 2x EpiTect HRM Master Mix containing HotStarTaq Plus DNA Polymerase, EpiTect HRM PCR Buffer (with EvaGreen dye), dNTP Mix, and RNase-Free Water
59445
询价

EpiTect HRM PCR 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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高度灵敏的结果。|在多种real-time PCR仪上进行甲基化分析。|利用HRM技术对APC启动子进行甲基化分析的原理。|HRM技术的原理。|
用不同比例甲基化和非甲基化DNA(EpiTect Control DNA)的混合物作为模板。扩增APC基因(结肠腺瘤样息肉蛋白)启动子区域的CpG岛,在Rotor-Gene Q实时荧光定量PCR分析仪上使用EpiTect HRM PCR Kit通过HRM甲基化分析确定甲基化水平。[A]标准化的熔解曲线和[B]以50%甲基化样本标准化的区别图。
|不同比例的甲基化(M)和未甲基化(U)DNA混合物作为模板。扩增PTPN6基因(酪氨酸磷酸酶,非受体类型6)启动子区域的CpG岛,通过HRM甲基化分析确定甲基化程度。[A]使用EpiTect HRM PCR Kit和Rotor-Gene Q实时荧光定量PCR分析仪的差异图。[B]使用EpiTect HRM PCR Kit和LightCycler 480时,100%未甲基化和甲基化DNA的差异图和熔解峰。[C]在LightCycler 480上使用Supplier R的HRM试剂盒(配合3.5 mM MgCl2)时,100%未甲基化和甲基化DNA的差异图和熔解峰。使用1.5 mM和3.5 mM MgCl2(推荐的Mg2+最低和最高浓度)配合试剂盒检测。使用1.5 mM MgCl2,扩增失败(数据未展示);使用3.5 mM MgCl2,甲基化DNA作模板时产生非特异性产物(红色的两个峰值),限制HRM分析。|[A]基因组DNA——所有不是CpG岛上的胞嘧啶被标记为X。可能含有甲基化胞嘧啶的CpG岛指示为CG。[B]亚硫酸氢盐转化的基因组DNA——所有未甲基化胞嘧啶残基转化为尿嘧啶。CpG岛内的胞嘧啶标记为Y,被转化为U(若未甲基化)或保持未修饰(若甲基化)。|HRM分析是基于温度升高造成的dsDNA解离行为。dsDNA的熔解取决于GC含量。AT丰富的区域熔解更快。|
性能
Novel fluorescent dye EvaGreen

EvaGreen is a dsDNA-binding dye. The spectral properties of EvaGreen are similar to those of SYBR® Green I. The absorbance maximum is 500 nm, and the emission maximum is 530 nm. In contrast to SYBR® Green I, EvaGreen can be used at higher, saturating concentrations without PCR inhibition and shows equal binding affinity for GC-rich and AT-rich regions with no apparent sequence preference. This makes EvaGreen an ideal dye for HRM analyses of all types of PCR product.

Highly sensitive detection of changes in CpG methylation status

For CpG methylation analysis by HRM, the DNA has to undergo complete bisulfite conversion before the melting behavior of DNA can be measured by HRM analysis. Unknown samples can be compared with a standard control, based on their melting characteristics. The sensitivity ensured by the EpiTect HRM PCR Kit means that even low amounts of methylated DNA can be detected (see figure Highly sensitive results).

Consistent performance on various real-time instruments

The EpiTect HRM PCR Kit delivers highly accurate methylation results with real-time instruments suitable for HRM (see figure "Methylation analysis on various real-time PCR instruments"). The kit includes an optimized protocol that has been tested on the Rotor-Gene Q, Rotor-Gene 6000, and LightCycler 480. The combination of advanced HRM technology on the Rotor-Gene Q with the benefits of the EpiTect HRM PCR Kit delivers exceptional HRM analysis.

原理

HRM is an innovative technique that characterizes double-stranded PCR products based on their dissociation (melting) behavior as they transition from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) with increasing temperature. Amplification of bisulfite-converted DNA is carried out using primers that are conversion-specific but not methylation-specific. The primers should therefore comprise several converted cytosines and should flank CpG sites (see figure "Principle of methylation analysis of the APC promoter by HRM"). This is to ensure amplification of bisulfite-converted DNA only and enable distinction between methylated and unmethylated CpG sites during HRM analysis.

The fluorescence of EvaGreen is measured continuously and is plotted against increasing temperature. EvaGreen is only measured as long as it is bound to dsDNA. This results in high fluorescence at the start of HRM analysis. As the increasing temperature gives rise to DNA melting, EvaGreen is released and the fluorescence reduces to a background level. GC-rich stretches of DNA are more stable and therefore melt more slowly compared with AT-rich regions and remain double stranded at higher temperatures for longer (see figure "Principle of HRM technology").

HRM analysis is an easy and cost-effective alternative to probe-based methylation assays. PCR products can be discriminated according to sequence, length, GC content, or strand complementarity — down to single base pair changes.

Convenient master mix format

The EpiTect HRM PCR Kit is provided in a convenient master mix format for increased ease-of-use. The optimized 2x EpiTect HRM PCR Master Mix ensures highly specific amplification. In addition, it enables flexible, rapid, and sensitive analysis of methylation status of CpG dinucleotides in bisulfite converted DNA via HRM. The master mix consists of optimized concentrations of HotStarTaq Plus DNA Polymerase, EpiTect HRM PCR Buffer, dNTPs, and EvaGreen dye.

HotStarTaq Plus DNA Polymerase

HotStarTaq Plus DNA Polymerase, a modified form of QIAGEN Taq DNA Polymerase, is supplied in an inactive state that has no polymerase activity at ambient temperature. This prevents extension of nonspecifically annealed primers and primer dimers formed at low temperatures during PCR setup and the initial PCR cycle.

Optimized EpiTect HRM PCR Buffer

EpiTect HRM PCR Buffer maintains specific amplification in every cycle of PCR. The balanced combination of KCl and (NH4)2SO4 in the buffer promotes specific primer–template annealing. Simultaneously, nonspecific annealing is reduced, maximizing yields of specific PCR product, which allows optimal HRM analysis. Specific amplification with high product yield is maintained without the need for time-consuming optimization of the Mg2+ concentration.

操作流程

The target sequence is PCR amplified to a high copy number in the presence of the dsDNA binding fluorescent dye, EvaGreen. The dye does not interact with single-stranded DNA (ssDNA), but actively binds to dsDNA and fluoresces brightly when bound. Following the amplification, the temperature is increased, and the fluorescence of EvaGreen is measured continuously and plotted against the temperature.

应用

The EpiTect HRM PCR Kit provides a highly reliable and cost-effective method for methylation analysis using HRM technology and can be successfully used for methylation studies in epigenetic research. The optimized protocol means that information on the methylation status of CpG sites can be derived quickly and efficiently. This simplifies methylation assay development.

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图片
Highly sensitive results with detection of even low percentages of methylated DNA
高度灵敏的结果。
用不同比例甲基化和非甲基化DNA(EpiTect Control DNA)的混合物作为模板。扩增APC基因(结肠腺瘤样息肉蛋白)启动子区域的CpG岛,在Rotor-Gene Q实时荧光定量PCR分析仪上使用EpiTect HRM PCR Kit通过HRM甲基化分析确定甲基化水平。[A]标准化的熔解曲线和[B]以50%甲基化样本标准化的区别图。
Methylation analysis on various real-time PCR instruments
在多种real-time PCR仪上进行甲基化分析。
不同比例的甲基化(M)和未甲基化(U)DNA混合物作为模板。扩增PTPN6基因(酪氨酸磷酸酶,非受体类型6)启动子区域的CpG岛,通过HRM甲基化分析确定甲基化程度。[A]使用EpiTect HRM PCR Kit和Rotor-Gene Q实时荧光定量PCR分析仪的差异图。[B]使用EpiTect HRM PCR Kit和LightCycler 480时,100%未甲基化和甲基化DNA的差异图和熔解峰。[C]在LightCycler 480上使用Supplier R的HRM试剂盒(配合3.5 mM MgCl2)时,100%未甲基化和甲基化DNA的差异图和熔解峰。使用1.5 mM和3.5 mM MgCl2(推荐的Mg2+最低和最高浓度)配合试剂盒检测。使用1.5 mM MgCl2,扩增失败(数据未展示);使用3.5 mM MgCl2,甲基化DNA作模板时产生非特异性产物(红色的两个峰值),限制HRM分析。
Principle of methylation analysis of the APC promoter by HRM
利用HRM技术对APC启动子进行甲基化分析的原理。
[A]基因组DNA——所有不是CpG岛上的胞嘧啶被标记为X。可能含有甲基化胞嘧啶的CpG岛指示为CG。[B]亚硫酸氢盐转化的基因组DNA——所有未甲基化胞嘧啶残基转化为尿嘧啶。CpG岛内的胞嘧啶标记为Y,被转化为U(若未甲基化)或保持未修饰(若甲基化)。
Principle of HRM technology
HRM技术的原理。
HRM分析是基于温度升高造成的dsDNA解离行为。dsDNA的熔解取决于GC含量。AT丰富的区域熔解更快。