应用HRM对甲基化状态的变化进行可靠的筛查
  • 准确检测CpG位点甲基化状态的变化
  • 高度特异性的PCR扩增,无需优化
  • EvaGreen荧光染料可明显区分熔解曲线
  • 方便的预混液形式,经优化的操作步骤
  • 快速方便地开发新的HRM甲基化分析方法
EpiTect HRM PCR Kit应用HRM技术,快速筛查和准确检测亚硫酸氢盐转化后的DNA CpG甲基化位点的状态变化。经优化的试剂盒以方便的预混液形式提供,包含新型的结合双链DNA的荧光染料EvaGreen,以及经优化的HRM缓冲液、HotStarTaq Plus DNA Polymerase和dNTPs。该试剂盒与适合进行HRM分析的real-time仪器兼容,尤其是Rotor-Gene Q实时荧光定量PCR分析仪,以及Rotor-Gene 6000和LightCycler 480。
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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
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The EpiTect HRM PCR 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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Highly sensitive results.|Methylation analysis on various real-time PCR instruments.|Principle of methylation analysis of the APC promoter by HRM.|Principle of HRM technology.|
Mixtures of methylated and unmethylated DNA of varying ratios were used as template. A CpG island from the promoter region of the APC gene (adenomatosis polyposis coli) was amplified, and the degree of methylation was determined by HRM methylation analysis on the Rotor-Gene Q using the EpiTect HRM PCR Kit. [A] A standard normalized melt curve and [B] a difference plot normalized to the 50% methylated sample are shown. Even low percentages of methylated DNA are detected.|Mixtures of methylated (M) and unmethylated (U) DNA in various ratios were used as template. A CpG island from the promoter region of the PTPN6 gene (protein tyrosine phosphatase, nonreceptor type 6) was amplified, and the degree of methylation was determined by HRM methylation analysis. [A] Difference plot when using the EpiTect HRM PCR Kit and the Rotor-Gene Q. [B] Difference plot and melting peaks of 100% unmethylated and methylated DNA, respectively (inset) when using the EpiTect HRM PCR Kit and the LightCycler 480. [C] Difference plot and melting peaks of 100% unmethylated and methylated DNA, respectively (inset) when using Supplier R's HRM kit (with 3.5 mM MgCl2) on the LightCycler 480. The kit was tested with 1.5 mM and 3.5 mM MgCl2 (within the lower and upper limits of the recommended Mg2+ concentration). Amplification with 1.5 mM MgCl2 failed (data not shown); amplification with 3.5 mM MgCl2 resulted in the generation of nonspecific products when using methylated DNA as template (red double peaks), limiting the HRM analysis.|[A] Genomic DNA — all cytosines that are not part of CpG sites are symbolized by X. CpG sites containing potentially methylated cytosines are indicated as CG. [B] Bisulfite conversion of genomic DNA — all unmethylated cytosine residues are converted to uracil. Cytosines within CpG sites are indicated by Y and are either converted to U (if unmethylated) or remain unmodified (if methylated).|HRM analysis is based on the dissociation behavior of dsDNA due to increasing temperature. Melting of dsDNA depends on GC-content. AT-rich regions melt faster.|
Performance
新型荧光染料EvaGreen

EvaGreen是一种dsDNA结合染料。EvaGreen的光谱特性与SYBR® Green I相似。最大吸收波长500 nm,发射波长530 nm。与SYBR® Green I不同的是EvaGreen可使用更高饱和浓度而不会抑制PCR反应,且对富含GC和富含AT的区域有同等的结合能力,无明显偏向性。这使EvaGreen非常适合用于PCR产物的HRM分析。

高度灵敏的检测CpG甲基化状态的变化

使用HRM进行CpG甲基化分析,DNA必须在其熔解行为被HRM检测到之前进行完全的亚硫酸氢盐转化。基于他们的熔解参数,未知样本可与标准对照样本进行比较。EpiTect HRM PCR Kit的高灵敏度,确保即使少量的甲基化DNA也可被检测到(参见"Highly sensitive results")。

在各种real-time仪器上始终如一的性能

EpiTect HRM PCR Kit配合适合进行HRM分析的real-time仪器,获得高度精确的甲基化结果(参见"Methylation analysis on various real-time PCR instruments")。该试剂盒提供经优化的实验方案,已在Rotor-Gene Q实时荧光定量PCR分析仪、Rotor-Gene 6000和LightCycler 480上进行过试验。在Rotor-Gene Q实时荧光定量PCR分析仪上将先进的HRM技术与EpiTect HRM PCR Kit结合,提供优越的HRM分析。

Principle

HRM是一种新型的技术,基于升温过程中双链DNA(dsDNA)转变为单链DNA(ssDNA)时的熔解行为,用于双链PCR产物。使用转化特异性而非甲基化特异性的引物扩增亚硫酸氢盐转化的DNA。因此,该引物需含有几个转化的胞嘧啶,并且在CpG位点的旁边(参见"Principle of methylation analysis of the APC promoter by HRM")。这是为了确保只扩增亚硫酸氢盐转化的DNA和在HRM分析时区分甲基化和非甲基化CpG位点。

EvaGreen荧光可连续检测,随着温度的升高信号减弱。只有结合dsDNA后,EvaGreen才能被检测到。这样在HRM分析刚开始时有很强的荧光。随着温度升高DNA熔解增加,EvaGreen被释放,荧光信号减弱到背景水平。富含GC的DNA更稳定,因此与富含AT的区域相比熔解更加缓慢,在温度时维持双链的时间更长(参见"Principle of HRM technology")。

HRM是进行基于探针的甲基化分析的方便、经济选择。PCR产物可通过序列、长度、GC含量或链补充,甚至单个碱基对的改变进行区分。

方便的预混液形式

EpiTect HRM PCR Kit提供方便的预混液形式,提高其易用性。经优化的2x EpiTect HRM PCR Master Mix确保高度特异性扩增。此外,该试剂盒能够通过HRM灵活、快速和灵敏的分析亚硫酸氢盐转化DNA中CpG二核苷酸的甲基化状态。该预混液包括浓度处于最佳状态的HotStarTaq Plus DNA Polymerase、EpiTect HRM PCR Buffer、dNTPs和EvaGreen荧光染料。

HotStarTaq Plus DNA Polymerase

HotStarTaq Plus DNA Polymerase,一种经修饰的QIAGEN Taq DNA Polymerase,以非活性形式状态提供,在室温下无聚合酶活性。这就避免了在PCR设置和PCR循环起始时低温状态下非特异性退火引物的延长和引物二聚体的形成。

优化的EpiTect HRM PCR Buffer

EpiTect HRM PCR Buffer可在任何PCR循环中进行特异性扩增。缓冲液中平衡的KCl和(NH4)2SO4组合促进特异性引物模板退火。同时,非特异性退火减少,获得最大产量的可进行HRM分析的特异性PCR产物。可持续特异性扩增获得高产量DNA,无需费时优化Mg2+的浓度。

Procedure

dsDNA与荧光染料EvaGreen结合,PCR扩增目标序列获得高拷贝片段。该染料不与ssDNA结合,只与dsDNA结合,结合后显荧光。然后进行扩增,EvaGreen荧光可连续检测,随着温度的升高信号减弱。

Applications

EpiTect HRM PCR Kit使用HRM技术,提供高度可靠、经济的方法进行甲基化分析,可成功用于表观遗传学研究的甲基化分析。经优化的实验方案能够快速、高效的检测CpG位点的甲基化状态。该试剂盒简化了甲基化分析的研发。

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