用于PCR扩增模板DNA,专为焦磷酸测序优化

  • 高特异性扩增亚硫酸氢盐转化的DNA和基因组DNA
  • 专为焦磷酸测序分析优化的试剂盒
  • 高产量的PCR产物,无需优化
  • 方便的预混液形式,室温条件下构建反应体系
  • 经优化的实验方案

PyroMark PCR Kit专为焦磷酸测序优化,确保高特异性和无偏差的扩增DNA模板,适合多种应用,包括:突变检测、SNP分析、甲基化分析和测序。该试剂盒提供使用方便的预混液,其中含有HotStarTaq DNA Polymerase和经优化的PyroMark Reaction Buffer,可高特异性扩增。此外,一种新型的添加剂Q-Solution确保难扩增模板(富含GC的模板)的高效扩增。该试剂盒也提供CoralLoad Concentrate,推荐与PyroMark PCR Master Mix配合使用,用于高特异性PCR和获得高产量的扩增DNA。CoralLoad Concentrate含有两种胶示踪染料,所得的PCR产物可直接上样至凝胶。

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PyroMark PCR Kit (200)
For 200 reactions: 2x PyroMark PCR Master Mix (includes HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer containing 3 mM MgCl2 and dNTPs), 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-Free Water
978703
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PyroMark PCR Kit (800)
For 800 reactions: 2x PyroMark PCR Master Mix (includes HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer containing 3 mM MgCl2 and dNTPs), 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-Free Water
978705
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The PyroMark 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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CoralLoad Concentrate.|PCR optimized for Pyrosequencing.|Superior results with PyroMark PCR Kit.|Increased specificity of primer annealing.|
[A] CoralLoad Concentrate contains [B] 2 gel-tracking dyes, enabling immediate gel loading of PCR products for easy visualization of DNA migration.|The PyroMark PCR Kit ensures greater amplification efficiency with high specificity. This results in [A] higher Pyrogram peaks and more reliable Pyrosequencing results, compared with [B] standard PCR.|Template DNA (10 ng) was amplified with the PyroMark PCR Kit in parallel with PCR master mixes from Suppliers R, I, and AII. Primers specific for Cyclin A1, BARHL1, MINT1, or DNMT3b were designed with the PyroMark Assay Design Software 2.0, and an annealing temperature gradient ranging from 48.4°C to 60.1°C was used. The PyroMark PCR Kit consistently produced higher amplicon yields and fewer nonspecific PCR products than the other master mixes, ensuring reliable results from downstream Pyrosequencing analyses. M: 50 bp ladder|K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing principally the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains the high ratio of specific to nonspecific primer-template binding over a wide temperature range.|
Performance
PyroMark PCR Master Mix,独特的钾盐和钠盐平衡体系,促进特异性引物和模板退火,同时减少非特异性退火。即使使用少量的模板,也可获得最大产量的特异性产物(参见"Superior results with PyroMark PCR Kit")。优化的模板扩增具有更高的Pyrogram峰值,能获得更可靠的焦磷酸测序结果(参见"PCR optimized for Pyrosequencing")。

Principle
PyroMark PCR Master Mix

PyroMark PCR Kit针对焦磷酸测序分析进行了优化,用于模板DNA的PCR扩增。使用方便的预混液可特异性扩增来源于各种起始样本的亚硫酸氢盐转化后DNA或基因组DNA,可用同一个流程处理。亚硫酸氢盐处理后,基因组DNA将未甲基化胞嘧啶转化为尿嘧啶,形成主要由三种碱基组成的DNA。这种更简单的DNA是相对困难的PCR模板,且PCR产量可能较低。DNA复杂性的降低引起错配率的提高,从而产生非特异性产物。独特的预混液中PCR缓冲液平衡的盐离子组成确保特异性引物结合,可避免错误的引物结合。特制的预混液可避免形成生物素酰化引物,并将人造产物的生成降至最低,防止其对焦磷酸测序反应的干扰。高产量的特异性PCR产物确保可靠的焦磷酸测序结果。 

在每个PCR循环的退火步骤,独特的缓冲液成分确保高比例的特异性产物(参见"Increased specificity of primer annealing")。由于KCl和(NH4)2SO4独特的离子平衡组成,该PCR缓冲液确保引物特异性退火的温度范围和Mg2+浓度范围均比传统PCR缓冲液的范围大。因此,通常无需通过调节退火温度或Mg2+浓度来优化PCR。

HotStarTaq DNA Polymerase

预混液中包含的HotStarTaq DNA Polymerase是对QIAGEN 94 kDa Taq DNA Polymerase修饰后的聚合酶。在室温条件下该聚合酶没有活性,因此可避免低温下非特异性引物的延伸和引物二聚体的形成。95°C条件下只需15分钟即可激活HotStarTaq DNA Polymerase,这一步骤可整合入已现有的热循环操作中。HotStarTaq DNA Polymerase确保PCR高度特异性,可提高特异性PCR产物的产量。可方便、快速的构建PCR体系,在室温下可混合各反应组分。

Q-Solution

PyroMark PCR Master Mix同时提供Q-Solution,一种新型的PCR添加剂,通过改变DNA熔解行为促进难扩增模板的扩增。这种独特的试剂可进一步优化由模板引起的表现不理想的PCR,如过多的二级结构和富含GC的模板。不同于DMSO和其他PCR添加剂,Q-Solution的浓度是确定的,没有毒性,并且保证PCR的纯度。存在Q-Solution的条件下扩增的PCR产物可成功用于焦磷酸测序分析。

CoralLoad Concentrate

该试剂盒还含有CoralLoad Concentrate。CoralLoad Concentrate包含胶上样试剂和两个凝胶示踪染料,一个橘色,一个红色(参见"CoralLoad Concentrate")。在CoralLoad Concentrate存在的条件下扩增出的RT-PCR产物可直接上样至琼脂糖凝胶,无需事先添加上样缓冲液。CoralLoad Concentrate可加入PCR体系中,不会影响扩增的灵敏度和特异性。存在CoralLoad Concentrate的条件下扩增的PCR产物可成功用于焦磷酸测序分析。

Procedure

反应组成和循环条件都经过优化,用于扩增焦磷酸测序分析的模板DNA。模板DNA与方便的PyroMark PCR Master混合液一起,配合Q-Solution、CoralLoad concentrate和扩增引物(其中一个是荧光素标记的)使用。HotStarTaq DNA Polymerase在室温下无活性,所以反应体系建立时无需在冰上进行反应。

Applications
焦磷酸测序技术对各个领域的研究应用至关重要,焦磷酸测序技术能够灵敏的分析遗传学和表观遗传学的变化,如检测病原体中药物抗性的发展,基因表达分析中表观遗传学DNA甲基化的作用、牲畜特异性表现型的遗传标记,以及法医检材样本中线粒体DNA的多态性。PyroMark PCR Kit提供可靠的高特异性模板扩增所需的各种试剂,用于焦磷酸测序分析。

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Application Notes
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A Pyrosequencing method for the forensic identification of tissue source using QIAGEN’s PyroMark Q24 Advanced system.
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