Type-it Fast SNP Probe PCR Kit

应用TaqMan或TaqMan MGB探针进行准确可靠的SNP基因分型

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Type-it Fast SNP Probe PCR Kit (800)

Cat. No. / ID: 206045

For 800 x 25 μl reactions: 6 x 1.7 ml of 2x Type-it Fast SNP Probe PCR Master Mix, 5x Q-Solution, RNase-Free Water

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Reactions

800

4000

See trademarks.

Type-it Fast SNP Probe 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

经TaqMan SNP Genotyping Assays验证
自动等位基因分型，致密的荧光光束
适用于难以分型的SNP位点和少量模板
快速循环可节约多达40%的时间

Product Details

Type-it Fast SNP Probe PCR Kit基于高度特异性的HotStarTaq Plus DNA Polymerase和新研发的缓冲液体系，可获得可靠、清晰的等位基因分型。预混液中所有组分的配合可提高探针结合的准确性和特异性。Type-it Fast SNP Probe PCR Kit经TaqMan SNP Genotyping Assays验证，能获得可重复的SNP基因分型结果，也可用于难处理的SNP位点（如富含GC）或起始量低的模板。

Performance

Type-it Fast SNP Probe PCR Kit确保持续获得高度准确的SNP基因分型。

等位基因特异性的探针可进行高度严谨和特异的结合

Type-it Fast SNP Genotyping PCR Master Mix基于高度特异性的HotStarTaq Plus DNA Polymerase和新研发的SNP基因分型PCR缓冲液体系，可进行高度特异性的探针结合和持续强的荧光信号。与其他商业化SNP基因分型预混液相比，可获得更广泛、更清晰的等位基因群分离（参见" Highly specific probe binding"）。

低模板量也能获得高信号率

对于有挑战性的靶点或难处理的SNP位点，Type-it Fast SNP Probe PCR Kit提供可靠的SNP基因分型，可完全分开等位基因并使相同的等位基因紧密成群，产生较高的信号率（参见" Successful automated allele calling"）。Type-it Fast SNP Probe PCR Kit优于其他供应商的试剂盒，保证等位基因紧密成群，即使1 ng的DNA模板（参见 " Reliable SNP genotyping with small amounts of template"）。

See figures

Highly specific probe binding.

Mechanism of fast cycling during annealing.

Reliable SNP genotyping with small amounts of template.

Principle

Type-it Fast SNP Probe PCR Kit采用方便的预混液规格，含有高度特异性的HotStarTaq Plus Polymerase和独特的SNP基因分型缓冲液体系，可快速、高效的扩增。独特的试剂盒组分可使等位基因紧密成群，完全分开确保高信号率，获得可重复的、准确的结果（见表）。

高特异性和灵敏度

预混液中提供的HotStarTaq Plus DNA Polymerase确保高特异性扩增，即使处理低模板量。HotStarTaq Plus DNA Polymerase处于非活化状态，在环境温度下没有聚合酶活性。这避免了在低温下引物错搭产物和引物二聚体的形成。

独特的缓冲液体系

预混液中含有的Type-it Fast SNP PCR Buffer专用于通过序列特异性5'核酸酶探针进行快速循环的SNP基因分型。Type-it Fast SNP PCR Buffer的独特组分确保非常严格和特异性的等位基因特异性探针的结合。改变探针的熔解行为可形成更窄的探针熔解温度窗口。基于原始的QIAGEN PCR Buffer，这种创新的缓冲液使每个PCR循环的退火步骤都有较高比率的特异性引物结合。KCl和(NH₄)₂SO₄独特比例结合，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。

Type-it Fast SNP PCR缓冲液中的添加剂，如Q-Solution，可提供反应条件，用于难处理基因组区域和SNP位点的扩增。创新的Q-Bond技术（用于快速循环），可更快获得SNP基因分型结果（参见" Mechanism of fast cycling during annealing"）。预混液中含有的ROX染料，其浓度使其能在Applied Biosystems的多种仪器上进行SNP基因分型。而且，ROX染料也能在不需要ROX作为阴性参照染料的仪器上使用（见表）。

试剂盒的特征
试剂盒组分	特征
2x Master Mix format*	用于配合TaqMan MGB探针进行SNP基因分型。能与多种循环仪配合使用，用于SNP基因分型
HotStarTaq Plus DNA Polymerase	室温下进行快速、简单的反应体系构建。即使使用少量模板，也能高度特异性扩增
Type-it Fast SNP Probe PCR Buffer	广泛分离基因群。紧密的等位基因群，高等位基因信号率。探针结合具有更高特异性。使用Q-Bond Molecule，快速循环
Q-Solution	对于高GC含量的SNP位点，在散点图分析中具有更紧密的集群和更强信号。可进一步提高不理想的等位基因信号

See figures

Successful automated allele calling.

Procedure

Type-it Fast SNP Probe PCR Kit使用商业化的SNP基因分型检测进行了功能验证，能与TaqMan MGB Probes配合使用，也可配合用户自行设计的探针法检测，可使用TaqMan MGB、TaqMan或其他双标记探针。此试剂盒包括简化的、预优化的操作流程，用于快速、可靠的分析。

快速循环步骤，可直接获得结果

Type-it Fast SNP Probe PCR Master Mix提供的反应条件，配合序列特异性探针进行快速PCR循环。缓冲液包含专有化的Q-Bond Molecule，可在标准循环仪或具有快速升温速率的快速循环仪上缩短循环时间。快速循环步骤可减少至多40%的PCR运行时间，提高通量（参见" Genotyping workflow"）。

方便的试剂盒规格

该试剂盒采用即用型、预优化预混液形式，具有更大便利性。使用预混液可节约时间，简化反应体系构建的手工操作，通过消除移液误差和污染的可能来源，提高可重复性，尽量减少移液步骤，无需繁琐的计算。预混液中含有的HotStarTaq Plus DNA Polymerase 95°C，5分钟被激活，可在热循环程序中简单设置。使用预混液进行室温下的反应体系构建快速、简单。

适合的仪器

Type-it Fast SNP Probe PCR Kit经优化，可在多种合适的标准和快速升温的循环仪上进行SNP PCR分析，光学PCR孔板配合real-time PCR仪可用于荧光板阅读分析（见表）。

Suitable instruments
循环仪	模式
Real-time PCR cyclers	QIAGEN: Rotor-Gene Q ABI PRISM 7900 (all series) Applied Biosystems 7500 (all series) Applied Biosystems 7300 ABI PRISM 7700 ABI PRISM 7000 ABI StepOne and StepOnePlus Bio-Rad: iCycler iQ Roche: LightCycler 480 Stratagene: Mx3000P and Mx3005P
Standard cyclers	采用标准升温模式的所有循环仪（如GeneAmp 9700）采用快速升温模式的所有循环仪（如GeneAmp 9800）

See figures

Genotyping workflow.

Applications

Type-it Fast SNP Probe PCR Kit应用TaqMan MGB探针检测SNPs，可用于多个研究领域，包括：

疾病基因位点的分型
生物标记物的发现

Supporting data and figures

Successful automated allele calling.

Average automated allele call rates for a panel of different DNAs were analyzed using 6 different TaqMan® MGB-based SNP genotyping assays and 1 ng of genomic DNA from each of the 60 samples. PCR was performed with the indicated products in 384-well plates in a 5 μl reaction volume. Allelic discrimination plate read was performed on an Applied Biosystems 7900HT instrument. The Type-it Fast SNP Probe PCR Kit consistently resulted in the highest call rates and the lowest error rates.

Specifications

Features	Specifications
Applications	Probe-based SNP Genotyping
Product use	Functionally validated and developed for reliable SNP Genotyping
Mastermix	Yes
Real-time or endpoint	Both
With or without ROX	ROXincludedinMasterMix
With/without hotstart	With
Sample/target type	GenomicDNA
Enzyme activity	5'->3'Exonucleaseactivity
Sequence specific Probe	TaqMan®GenotypingAssays,TaqMan®orTaqManMGB®probes
Reaction type	PCRamplification

Resources

Second edition — innovative tools

Now with even more applications!

Addressing critical factors and new solutions

Download Safety Data Sheets for QIAGEN product components.

Second edition — innovative tools

Now with even more applications!

Addressing critical factors and new solutions

Download Safety Data Sheets for QIAGEN product components.

FAQ

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

The Type-it Fast SNP Probe PCR Kit was developed and validated for use with 5'-3' nuclease (hydrolysis) probes like TaqMan® MGB or standard TaqMan® probes. We have not yet tested other probe technologies with this kit.

See trademarks.

FAQ ID -2058

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

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

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

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

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

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

Store the standards at a high concentration in aliquots at -20^oC to -70^oC. 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

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 C_T values are below 30 and differ no more than 3 C_T 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

SNP Genotyping using TaqMan® probes is a unique application requiring the development of a unique reaction chemistry. The Type-it Fast SNP Probe PCR Kit allows wide separation of genotype clusters and reliable amplification of any difficult genomic region for high call rates.

See trademarks.

FAQ ID -2057

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 (T_m) 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 T_m, but approximately 3ºC lower than the specific amplicon T_m.

FAQ ID -9096