Type-it Microsatellite PCR Kit

快速可靠的微卫星位点多重PCR分析

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Type-it Microsatellite PCR Kit (200)

Cat. No. / ID: 206243

For 200 x 25 μl reactions: Type-it Multiplex PCR Master Mix (3 x 0.85 ml), 5x Q-Solution (1 x 2 ml), and RNAse-Free Water (2 x 1.9 ml)

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Reactions

200

2000

Type-it Microsatellite 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

通过多重PCR进行可靠的微卫星分析
无需优化即可建立微卫星实验
可成功地高特异性共同扩增所有片段
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 Microsatellite PCR Kit基于具有高特异性的HotStarTaq Plus DNA Polymerase和获得专利的缓冲液系统，从而确保多重PCR的微卫星分析结果可靠且无需优化。预混液中所有成分和独特的配方确保所有位点同步的高特异性扩增。优化的操作流程使后续分析可应用高分辨率毛细管测序仪或其他电泳仪器。

Performance

Type-it Mutation Detect PCR Kit优于其他供应商的试剂盒，保证可靠的微卫星分析。该试剂盒专门研发并经功能验证，可用于STRs或VNTRs等微卫星或小卫星的多重PCR分析，如关系分析或群体遗传学（参见""）。

See figures

Reliable 13-plex STR analysis without the need for optimization.

Principle

Type-it Microsatellite PCR Kit使用微卫星、STR或VNTR标记对人类、动物和细菌进行快速、可靠的基因分型分析，无需冗长的优化步骤。Type-it Microsatellite PCR Kit基于专有化的QIAGEN多重技术，包括高度特异性的HotStarTaq Plus DNA Polymerase（一种化学修饰的热启动酶）和特别研发的包括Factor MP的多重缓冲液体系。这些组分确保高产量，并对所有目的微卫星位点进行可靠的多重扩增。该试剂盒包括专门用于微卫星或STRs扩增的操作流程，也包括对模板量、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。

Type-it Microsatellite PCR Buffer

独特的Type-it Microsatellite PCR Buffer便于多重PCR产物的扩增。与常规PCR试剂不同，Type-it Microsatellite 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。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 Microsatellite 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。

See figures

Stable and efficient annealing.

Procedure

Type-it Microsatellite PCR Kit中专有的、特异性的操作流程，经优化后可在高分辨毛细管测序仪上进行微卫星分析，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 Microsatellite 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。无论哪种应用，包括微卫星、STR、SSR或VNTR分析，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。反应混合物含有微卫星特异性的多重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 genotyping analysis"）。

优化后用于毛细管测序仪

在毛细管测序仪等高分辨率检测体系上进行的微卫星分析，常常会面临产物产量不平等及荧光信号强度巨大差异等问题。Type-it Microsatellite PCR Kit突破了这些限制，保证在多重实验中对所有扩增子都有高产量。该试剂盒包括优化的操作流程，可与其他供应商的荧光引物和更好的酶溶液配合使用（参见" Reliable 13-plex STR analysis without the need for optimization"）。

See figures

Successful multiplex PCR-based genotyping analysis.

Reliable 13-plex STR analysis without the need for optimization.

Applications

Type-it Microsatellite PCR Kit用于分析微卫星、STRs（短串联重复片段）、SSRs（简单序列重复）和VNTRs（数目可变串联重复序列），可用于多种研究领域，如：

物种和个体鉴定
关系分析
微卫星不稳定性分析
群体遗传学

Supporting data and figures

Reliable 13-plex STR analysis without the need for optimization.

Only 2 out of 4 channels (FAM and PET) of a 3730 xl Capillary Sequencer (ABI), representing 6 of 13 analyzed STR loci are shown. [A] Using the Type-it Microsatellite PCR Kit under standard conditions, all 6 STR loci are reliably amplified. [B] Despite the increased number of cycles and preoptimized PCR conditions for the hot-start method of Supplier A — compared with 25 cycles and standard conditions for the Type-it Microsatellite Kit — several specific peaks are missing and nonspecific signals are observed (arrows).

Specifications

Features	Specifications
Applications	Multiplex PCR, Microsatellites, STRs, VNTRs, SSRs
Reaction type	PCR amplification
Product use	Functionally validated and developed for reliable Microsatellite Analysis
Mastermix	Yes
Real-time or endpoint	Endpoint
Sample/target type	Genomic DNA
Single or multiplex	Multiplex
With/without hotstart	With

Resources

Second edition — innovative tools

Addressing critical factors and new solutions

For optimization-free and reliable multiplex PCR based analysis of microsatellites

Download Safety Data Sheets for QIAGEN product components.

Second edition — innovative tools

Addressing critical factors and new solutions

For optimization-free and reliable multiplex PCR based analysis of microsatellites

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

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

The Type-it Microsatellite PCR Kit was optimzed and validated for this size range since microsatellite analysis typically only uses fragments of up to 500 bp in length.

FAQ ID -2060

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

All capillary sequencers can be used for detection of PCR product amplified with the Type-it Microsatellite PCR Kit.

FAQ ID -2063

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

Yes. We tested the Type-it Microsaltellite PCR Kit on different commonly used fast and normal PCR cyclers. No significant differences in performance were observed with the systems tested.

FAQ ID -2062

Microsatellite analysis on high resolution detection systems such as capillary sequencers is often associated with the problem of uneven product yield and huge intensity differences of fluorescent signals. The Type-it Microsatellite PCR technology was specifically developed and validated to overcome this limitation and ensure high product yields for all amplicons up to 500 bp long in a multiplex experiment. The kit includes optimized protocols for use with fluorescent primers and outperforms enzyme solutions from other suppliers.

FAQ ID -2059

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

For established systems, previously optimized annealing temperatures can be used with the Type-it Microsatellite PCR Kit. For new PCR systems, follow the guidelines in Appendix B, 'Design of Multiplex Primers', in the Type-it Microsatellite PCR Handbook.

FAQ ID -2061

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