Type-it Microsatellite PCR Kit

マイクロサテライト遺伝子座の迅速で確実なマルチプレックスPCR解析

S_1084_5_GEN_V2

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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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$338.00
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Type-it Microsatellite PCR Kitは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • マルチプレックスPCRによる確実なマイクロサテライト解析
  • 至適化なしにマイクロサテライトアッセイを確立
  • 全フラグメントを良好かつ特異的に同時増幅
  • 至適化済みプロトコールで正確な結果を迅速に実現
  • 各種解析用装置を使用する場合の簡単なインストラクション

Product Details

特異性の高いHotStarTaq Plus DNA Polymerase と特許を持つバッファーシステムをベースにしたType-it Microsatellite PCR Kitは、マルチプレックスPCRによるマイクロサテライト解析を確実に行ない、至適化は不要です。マスターミックスの全成分とその特殊な組み合わせにより、すべての遺伝子座を高い特異性で同時に増幅できます。本キットはマルチプレックスPCR を用いた迅速で信頼できるマイクロサテライト解析専用キットです。至適化済みのプロトコールには、高解像度キャピラリーシークエンシングあるいは電気泳動装置による解析についても記載されています。

Performance

Type-it Mutation Detect PCR Kitは他社に比べて高性能で、信頼性の高いマイクロサテライト解析を実現します。キットは、血縁解析や集団遺伝子学などで使用されるSTRやVNTRのようなマイクロサテライトまたはミニサテライトをマルチプレックスPCRベースで分析するために特別に開発され、検証されています(図“ 至適化不要の正確な 13-plex STR 解析”)。
See figures

Principle

Type-it Microsatellite PCR Kitは、マイクロサテライト、STRあるいはVNTRマーカーを用いてヒト、動物、植物、バクテリアの迅速かつ正確なジェノタイピング解析を実現し、時間のかかる至適化実験は不要です。Type-it Microsatellite PCR Kitは、独自のQIAGENマルチプレックステクノロジーをベースにし、化学的修飾した特異性の高いホットスタート酵素、HotStarTaq Plus DNA Polymeraseおよびマルチプレックス用に特別に開発したFactor MPを含むバッファーシステムを含んでいます。これらの成分により高い収量が得られ、目的のマイクロサテライト遺伝子座がすべて確実に同時増幅できます。本キットは、マイクロサテライトまたはSTRの増幅のための専用プロトコルを含み、テンプレート量、サイクル数、およびPCR条件に関するステップごとのアドバイスと同様に、アガロースゲル、キャピラリーシークエンサー、Agilent Bioanalyzer、QIAxcel Advanced Systemなどのダウンストリーム解析のプラットフォーム用ごとに機器の詳細が含まれています。 

Type-it Microsatellite PCR Buffer

画期的なType-it Microsatellite PCR Bufferにより複数のPCR増幅産物の増幅が容易になります。Type-it Microsatellite PCR Buffer は、特別に開発された塩と添加物のバランスの取れた組み合わせにより、従来のPCR試薬に比べ、反応中の全プライマーのアニーリングとエクステンションを同等の効率で増幅できます。このバッファーはユニークな配合比のKClと (NH4)2SO4を含み、従来のPCRバッファーに比べ、幅広い温度やMg2+濃度の範囲で厳密で特異的なプライマー・アニーリングを実現します。異なるアニーリング温度あるいはMg2+ 濃度を用いて行なうPCRの至適化は最小限ですみ、または不要なこともあります。従来のマルチプレックスPCR操作で行なう至適化操作は不要です。バッファーに入っている合成 Factor MP (図“ 安定で効果的なアニーリングの促進”)は、プライマー配列に関係なくプライマーのアニーリングとエクステンションを効率的に行ないます。Factor MPは、DNAテンプレート付近のプライマーの濃度を上げ、特異的に結合したプライマーを安定化します。

Q-Solution

Type-it Microsatellite PCR KitにはQ-Solutionが含まれています。この革新的なPCR 添加剤はDNAの変性環境を改善することにより、増幅困難なテンプレートの増幅を可能にします。このユニークな試薬を使用すると、PCR条件が最適でない場合でもPCR が可能になり、改良されることがあります。DMSOや他の他のPCR 添加物とは異なり、Q-Solution はどのプライマー・テンプレートシステムでも一定のワーキング濃度で使用し、毒性はありません。

See figures

Procedure

ルーチン解析のための信頼性の高い結果を保証するだけでなく、新しいアッセイの確立のために、高分解能キャピラリーシークエンサーでのマイクロサテライト解析用に最適化されたアプリケーション特有のプロトコールがType-it Microsatellite PCR Kitに含まれています。反応は室温で設定することができるので、使いやすく便利です。

迅速かつ簡単な方法で信頼できるジェノタイピング結果を実現

Type-it Microsatellite PCR Kit は、マルチプレックス・アッセイを迅速かつ簡単に確立でき、正確で再現性のあるジェノタイピング結果が得られます。マイクロサテライト、STR、SSRやVNTR解析など、どのようなアプリケーションでも、テンプレートとプライマーを添加し、至適化済みプロトコールに従ってサーマルサイクラープログラムをスタートするだけで実験ができます。反応ミックスには、マイクロサテライトに特異的なマルチプレックスPCRに必要な試薬が全て入っており、本キットを用いると、通常の方法のように時間がかかる至適化操作なしに正確な結果が得られます(図“ マルチプレックスPCRによる良好なジェノタイピング解析”)。

キャピラリーシークエンサーを用いた解析に至適化済み

キャピラリーシークエンサーのような高分離能検出システムを用いたマイクロサテライト解析では、不均一な増幅産物量や蛍光シグナル強度が非常に異なるという問題が頻繁におこります。Type-it Microsatellite PCR Kit はこのような限界を克服し、マルチプレックス実験で全ての産物を高い収量で増幅できます。本キットは蛍光標識プライマーを使用する場合の至適化済みプロトコールが入っており、他社の酵素製品より性能が優れています(図“ 至適化不要の正確な 13-plex STR 解析”)。

See figures

Applications

Type-it Microsatellite PCR Kitはマイクロサテライト、STRs(short tandem repeats)、SSRs(simple sequence repeats)、VNTRs(variable number of tandem repeats)を解析するために使用し、以下のような様々な研究分野で最適です:

  • 生物種や個人の同定    
  • 血縁関係の解析
  • マイクロサテライトの不安定性
  • 集団遺伝学

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsMultiplex PCR, Microsatellites, STRs, VNTRs, SSRs
Reaction typePCR amplification
Product useFunctionally validated and developed for reliable Microsatellite Analysis
MastermixYes
Real-time or endpointEndpoint
Sample/target typeGenomic DNA
Single or multiplexMultiplex
With/without hotstartWith

Resources

パンフレット (2)
Second edition — innovative tools
Addressing critical factors and new solutions
キットハンドブック (1)
For optimization-free and reliable multiplex PCR based analysis of microsatellites
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
クイックスタートプロトコール (1)
Certificates of Analysis (1)
Brochures & Guides (2)
Second edition — innovative tools
Addressing critical factors and new solutions
Kit Handbooks (1)
For optimization-free and reliable multiplex PCR based analysis of microsatellites
Quick-Start Protocols (1)

FAQ

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
Why is maximum amplicon size for the Type-it Microsatellite PCR Kit limited to 500 bp?

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
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
Which capillary sequencers can be used for detection of amplicons from the Type-it Microsatellite PCR Kits?

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

 

 

FAQ ID -2063
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
Can the Type-it Microsaltellite PCR Kit be used on cyclers with fast ramping rates?

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

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
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
Does the Type-it Microsatellite PCR Kit always require an annealing temperature of 60°C, or can pre-optimized annealing temperatures for target-primer systems be used?

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
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