Type-it Mutation Detect PCR Kit

用于对突变进行准确、可靠的多重PCR分析

S_1084_5_GEN_V2

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Type-it Mutation Detect PCR Kit (200)

Cat. No. / ID:  206343

For 200 x 25 μl reactions: Type-it Multiplex PCR Master Mix, 5x Q-Solution, RNAse-Free Water, and 10x CoralLoad Dye
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Type-it Mutation Detect PCR Kit 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • 有效、可重复的多重突变分析
  • 无需优化即可建立多重PCR反应
  • 高特异性、灵敏的同时扩增所有片段
  • 优化的操作流程,得到快速、可靠的结果
  • 简单的操作指南,用于多种下游分析平台

Product Details

Type-it Mutation Detect PCR Kit适用于快速、可靠的检测缺失、插入和异位等突变。该试剂盒基于高度特异的HotStarTaq Plus DNA Polymerase和获得专利的缓冲液系统,从而确保对突变位点进行可靠多重PCR扩增,无需优化。后续分析简单方便,可在琼脂糖凝胶、自动化电泳仪以及高分辨率毛细管测序仪上进行。

Performance

Type-it Mutation Detect PCR Kit优于其他供应商的试剂盒,确保可靠的突变分析。该试剂盒专门研发并经功能验证,可用于对缺失或插入等突变进行多重PCR分析,也可检测遗传修饰的有机体或微生物(参见" Sensitive detection of a mutated cancer-related gene)。该试剂盒也适合用作商业化SNP检测PCR体系的预扩增方法,如Applied Biosystems提供的SNaPshot体系(参见" Superior preamplification of SNPs")。
See figures

Principle

Type-it Mutation Detect PCR Kit基于专有的QIAGEN Multiplex Technology,以即用型预混液形式提供。预混液含有浓度经优化的HotStarTaq Plus DNA Polymerase、MgCl2、dNTPs和一种创新的PCR缓冲液,专用于对突变进行多重PCR检测,或是对基因组SNP位点进行预扩增。该试剂盒还包含创新的添加剂Factor MP,以及平衡比例的盐和添加剂。这些组分使反应中的所有引物都具有相当的退火和延伸效率(参见" Stable and efficient annealing"),可平行地对所有片段进行可靠的高产量多重扩增。该试剂盒包括专门用于检测突变的操作流程,以及对模板量、循环数、PCR条件和不同下游分析平台仪器细节的建议,分析平台包括琼脂糖凝胶、毛细管测序仪、Agilent Bioanalyze和QIAxcel Advanced System。

预混液中所有组分配合专有的操作流程,可平行地对所有片段进行高特异性扩增。后续分析简单、直接,可在琼脂糖凝胶、自动化电泳仪上进行,也可进行高分辨率毛细管测序。

Type-it Mutation Detect PCR Buffer

Type-it Mutation Detect PCR Buffer确保高级别多重实验中的所有扩增子具有相当的扩增效率,可整合多个突变靶点,不会丢失任何突变靶点的扩增效率。与常规PCR试剂不同,Type-it Mutation Detect PCR Buffer含有特别研发的、比例平衡的盐和添加剂,确保反应中所有引物的退火和延伸具有同等的效率。KCl和(NH4)2SO4独特的结合比例,使PCR缓冲液与常规PCR缓冲液相比,可在更宽范围的退火温度和Mg2+浓度下提供严格的引物退火条件。极大的减少通过改变退火温度或Mg2+浓度的PCR优化过程,有时甚至不需要。通常使用的多重PCR优化步骤几乎不需要。缓冲液中还含有合成的Factor MP(参见" Stable and efficient annealing"),不论引物序列如何都可进行有效的引物退火和延伸。Factor MP可提高DNA模板处的引物浓度,稳定特定结合的引物。

Q-Solution

Type-it Mutation Detect PCR Kit提供Q-Solution。这种创新的PCR添加剂通过修饰DNA的熔解行为,便于扩增难扩增的模板。使用这种独特的试剂常常能完成或改进不理想的PCR。与DMSO和其他PCR添加剂不同,Q-Solution可在多种引物-模板体系中使用特定工作浓度,而不会产生毒性作用。

CoralLoad Dye

Type-it Mutation Detect PCR Kit提供CoralLoad Dye(参见" CoralLoad Dye"),这种染料含有一种胶上样试剂和两种胶示踪染料,提高移液的可见性,便于估计DNA迁移距离及优化琼脂糖凝胶电泳时间。在多重PCR反应中使用CoralLoad Dye,扩增产物可直接上样到琼脂糖凝胶或QIAxcel Advanced System中,无需提前加入上样缓冲液。CoralLoad染料不会干扰大部分下游酶应用。但是,为获得可重复性的结果,建议在进行酶处理前纯化PCR产物。                                                    如果使用毛细管测序仪进行检测,不能使用CoralLoad Dyes。

See figures

Procedure

Type-it Mutation Detect PCR Kit包括专有的、特异性的操作流程,可同时扩增和后续检测含突变的位点或SNPs,保证在常规分析中获得可靠结果,也可用于新检测的建立。可在室温下建立反应,确保更大的便利性和易用性。

快速、简单的方法,获得可靠的基因分型结果

Type-it Mutation Detect PCR Kit确保快速、简单的多重分析构建,以获得准确、可重复的基因分型结果。无论检测易位、缺失、插入或预扩增基因组位点用于SNP分析,如SNaPshot(Applied Biosystems),只需加入模板和引物后,根据优化方案开始热循环程序即可。反应混合物含有突变特异性多重PCR所需的所有试剂,与目前的其他方法相比,无需冗长的优化步骤即可获得准确的结果(参见" Successful multiplex PCR-based genotypic analysis")。

简单的操作流程,节约时间和成本

有些研究需要分析疾病相关的某个基因的很多不同突变(如缺失、易位或插入),如果进行单重或低级别PCR分析,需要众多的PCR反应,增加时间和成本。Type-it Mutation Detect PCR Buffer确保高级别多重实验中的所有扩增子具有相当的扩增效率,可整合多个突变靶点,大大节约时间和成本(参见" Sensitive detection of a mutated cancer-related gene")。此试剂盒含有专门的、应用特异性的操作流程,可同时扩增和后续检测含突变的位点或SNPs,保证在常规分析中获得可靠结果,也可用于新检测的建立。

可靠的SNP预扩增 

Type-it Mutation Detect PCR Kit非常适合对SNPs进行多重PCR预扩增(如来自Applied Biosystems的SNaPshot系统)。此试剂盒优于来自其他供应商的试剂盒,可持续获得可靠的结果,无需繁琐的优化步骤(参见" Superior preamplification of SNPs")。

See figures

Applications

Type-it Mutation Detect PCR Kit可用于分析缺失、插入、重复和易位的突变,以及进行SNP预扩增(如SNaPshot Multiplex Kit)。该试剂盒可用于多种研究领域:

  • 疾病基因位点的分型
  • 转基因生物分析
  • 转基因植物或动物的分型

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsMultiplex PCR, detection of mutations, preamplification of SNPs
MastermixYes
Reaction typePCR amplification
Product useFunctionally validated and developed for reliable mutation analyis
Real-time or endpointEndpoint
Sample/target typeGenomic DNA
Single or multiplexMultiplex
With/without hotstartWith

Resources

产品介绍与指南 (2)
Second edition — innovative tools
Addressing critical factors and new solutions
试剂盒操作手册 (1)
For optimization-free multiplex PCR-based mutation detection or preamplification of SNPs
安全数据表 (1)
Download Safety Data Sheets for QIAGEN product components.
Certificates of Analysis (1)
Brochures & Guides (2)
Second edition — innovative tools
Addressing critical factors and new solutions
Kit Handbooks (1)
For optimization-free multiplex PCR-based mutation detection or preamplification of SNPs

FAQ

What is the advantage of using the Type-it Mutation Detect PCR Kit?

The Type-it Mutation Detect PCR Kit was specifically developed and validated for the detection of mutations such as deletions, insertions, and translocations. It is also highly suited for multiplex PCR-based preamplification of SNPs as preparation for genotyping systems like ABI's SNaPshot. Dedicated protocols for various detection platforms (Agarose gels, QIAxcel System, Agilent BioAnalyzer, and Capillary Sequencers) are available in the Type-it Mutation Detect PCR Handbook.

 

 

FAQ ID -2064
How do I setup and validate a multiplex PCR assay with QIAGEN PCR kits?

Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.

 

Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.

 

FAQ ID -9093
Why is CoralLoad included in the Type-it Mutation Detect, but not in the Type-it Microsatellite PCR Kit?

CoralLoad Dye unfortunately interferes with Capillary Sequencers and increases the risk of damaging the capillaries of these detection platforms. It is not included in the Type-it Microsatellite PCR Kit, since microsatellite loci are analyzed predominantly on Capillary Sequencers due to their high-resolution capability.

By comparison, mutations are very often analyzed on agarose gels, or the QIAxcel System, compatible with CoralLoad dye included in the Type-it Mutation Detect PCR Kit.

 

FAQ ID -2068
Are there recommendations for sensitive detection of underrepresented fragments using the Type-it Mutation Detect PCR Kit?

Yes, special recommendations for detecting underrepresented fragments with the Type-it Mutation Detect PCR Kit can be found in Appendix E of the Type-it Mutation Detect PCR Handbook.

 

 

FAQ ID -2066
Can I use uracil-N-glycosylase (UNG) with the QuantiFast and Rotor-Gene PCR kits?

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

FAQ ID -9092
Why do I see multiple high-intensity peaks in my qPCR dissociation curve at temperatures less than 70ºC?

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

 

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

 

FAQ ID -90990
How do I quantify gene expression levels if the amplification efficiencies are different between the genes of interest and endogenous reference gene?

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

FAQ ID -9095
What do I do if no fluorescent signal is detected in a real-time PCR assay?

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

 

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line.  Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

 

If the issue persists, please send the original run file to QIAGEN Technical Services.

FAQ ID -9091
How do I select appropriate reporter and quencher combinations for multiplex PCR?

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

 

FAQ ID -9094
Why is the Type-it Mutation Detect PCR Kit recommended for preamplification of SNPs?

Preamplification of SNPs in preparation for genotyping systems like Applied Biosytem's SNaPshot requires single PCRs to be amplified and pooled, before input into this system. Using the Type-it Mutation Detect PCR Kit, all fragments can be obtained at once saving time and costs. Please follow the protocol 'Amplification of Mutations (Detection on Agarose Gels or the QIAxcel System or Agilent 2100 Bioanalyzer)' in the Type-it Mutation Detect Handbook.

 

 

 

 

FAQ ID -2067
Can the Type-it Mutation Detect PCR kit be used for multiplex PCR of fragments longer than 500 bp?

Yes, the Type-it Mutation Detect PCR kit can be used for fragments >500 bp by following the recommendations in the Type-it Mutation Detect PCR Handbook (increase extension time by 30 seconds for each additional 0.5 kb).

 

 

 

FAQ ID -2065
Can I skip the gDNA wipeout buffer treatment step for the QuantiTect Reverse Transcription Kit?

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

FAQ ID -9098
How should I handle and store absolute quantitation standards for real-time experiments?
Store the standards at a high concentration in aliquots at -20oC to -70oC. If using low concentrations, stabilize standards with carrier nucleic acid. It is always best to use freshly diluted standards for each experiment. If possible, use siliconized tubes for standard (and target) dilutions. This will prevent any unspecific binding of nucleic acids to the plastic.
FAQ ID -9099
How do I ensure reliable results for High Resolution Melting (HRM) assays?

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

 

  • Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
  • DNA template concentrations should be normalized using the same dilution buffer. Ensure the CT values are below 30 and differ no more than 3 CT values across individual samples.
  • Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
  • Always start with 0.7 µM primer concentration

 

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

FAQ ID -9097
How do I avoid collecting a fluorescence reading from primer-dimer with the QuantiTect SYBR Green PCR Kit?

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

 

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (Tm) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer Tm, but approximately 3ºC lower than the specific amplicon Tm.

FAQ ID -9096