QuantiTect Probe PCR Kits
For real-time PCR and two-step RT-PCR using sequence-specific probes
- Highly sensitive detection of low-copy targets
- Accurate quantification over several logs of template
- Use of any sequence-specific probe on any real-time cycler
- Available with or without uracil-N-glycosylase (UNG)
- No need to optimize reaction and cycling conditions
QuantiTect Probe PCR Kits enable sensitive quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using sequence-specific probes. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mix can be stored at 2–8°C.
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QuantiTect Probe PCR Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Probe PCR Master Mix, 2 x 2 ml RNase-Free Water
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204343
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QuantiTect Probe PCR Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Probe PCR Master Mix, 20 ml RNase-Free Water
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204345
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QuantiTect Probe PCR +UNG Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Probe PCR Master Mix, 100 ul UNG Solution, 2 x 2 ml RNase-Free Water
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204363
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QuantiTect Probe PCR Kits は分子生物学実験用です。疾病の診断、治療または予防の目的には使用することはできません。
See trademarks.
Effective UNG digestion.|Two-step RT-PCR.|Highly specific amplification.|Wide dynamic range in two-step PCR.|Wide dynamic range in real-time PCR.|High sensitivity and efficiency, and wide dynamic range.|Specific primer annealing.|
106 copies of two dUMP-containing PCR amplicons were treated with or without UNG and then amplified by real-time PCR. UNG was from various suppliers, and real-time PCR was performed using the master mix from the QuantiTect Probe PCR +UNG Kit. ΔCT on the Y-axis indicates CT values for non-UNG-treated samples subtracted from CT values for UNG-treated samples. The UNG supplied with the QuantiTect Kit provided the greatest ΔCT (9-12 cycles). This indicates that QIAGEN UNG digests carryover PCR products more effectively than UNG from other suppliers.|The QuantiTect Probe PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and cDNA template to the ready-to-use PCR master mix, and start the reaction on any real-time cycler (see also the table "Components of 2x QuantiTect Probe PCR Master Mix").|In comparison with other DNA polymerases, only HotStarTaq DNA Polymerase in combination with a unique buffer specifically amplified a 497 bp fragment (from 50 copies of an HIV-pol-gene construct in a background of 1 µg human genomic DNA). M: Markers.|Duplicate reactions were performed on the Mx3005P using tenfold dilutions of human keukocyte cDNA (10 ng to 0.01 ng) and a TaqMan assay for IL1R2 (a cytokine). The QuantiTect Probe PCR Kit provided accurate gene expression analysis from low to high template amounts with a PCR efficiency of 101%.|Probe-based real-time PCR with UNG pretreatment was carried out using the QuantiTect Probe PCR +UNG Kit. Reactions were run in duplicate on the ABI PRISM 7900 using 10-fold dilutions of human leukocyte cDNA (100 ng to 10 pg) and a TaqMan assay for IL8 (a cytokine). The amplification plots were evenly spaced, leading to a high PCR efficiency of 94%.|Tenfold serial dilutions of leukocyte cDNA (100 ng to 1 pg) were analyzed in duplicate on the Mx3005 using the QuantiTect Probe PCR Kit with primers and a FAM-labeled probe specific for IL8 (interleukin 8). A PCR efficiency of 97% over 6 logs of template dilution was achieved, and as little as 1 pg of IL8 transcript was sensitively detected.|
A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers and probes to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing annealing of primers and probes. NH4+ destabilizes weak hydrogen bonds between mismatched bases.
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Performance
The QuantiTect Probe PCR Kit delivers sensitive and reliable results in combination with the QuantiTect Reverse Transcription Kit (see figure "High sensitivity and efficiency, and wide dynamic range"). The unique PCR buffer composition enables QuantiTect Probe PCR Kits to provide sensitive quantification of low-copy DNA targets, as well as accurate quantification over a wide linear range (see figures "High sensitivity and efficiency, and wide dynamic range" and "Wide dynamic range in two-step RT-PCR"). HotStarTaq DNA Polymerase further increases the specificity of the PCR reaction by providing the most stringent hot start compared with other polymerases (see figure "Highly specific amplification").
A major source of PCR contamination is the carryover of PCR products from previous reactions. Pretreatment with UNG prior to starting PCR ensures that any contaminating PCR products do not affect subsequent PCR. The combination of the specially optimized UNG solution and the proven PCR master mix in the QuantiTect Probe PCR +UNG Kit ensures effective elimination of carried-over PCR products together with reliable quantification of target sequences (see figures "Effective UNG digestion" and "Wide dynamic range in real-time PCR").
Principle
QuantiTect Probe PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of gDNA and cDNA targets using sequence-specific probes. The kits are designed for use with all types of sequence-specific probes, including hydrolysis probe detection (e.g., TaqMan® and other dual-labeled probes), FRET probes, and Molecular Beacons. QuantiTect Probe PCR Kits contain a unique PCR buffer that contains a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure "Specific primer annealing"). In addition, HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.
QuantiTect Probe PCR Master Mix also contains dUTP, enabling pretreatment with uracil-N-glycosylase (UNG) prior to starting PCR, which ensures that any contaminating PCR products do not affect subsequent PCR reactions.
| HotStarTaq DNA Polymerase |
15 min activation at 95ºC |
Set-up of qPCR reactions at room temperature |
| QuantiTect Probe PCR Buffer |
Balanced combination of NH4+ and K+ ions |
Specific primer annealing ensures reliable PCR results |
| dNTP mix |
Includes dUTP, which partially replaces dTTP and enables optional UNG treatment of reactions |
Eliminates contamination from carryover of PCR products by optional UNG treatment |
| ROX dye |
For normalization of fluorescent signals on Applied Biosystems and, optionally, Agilent instruments |
Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers |
Procedure
QuantiTect Probe PCR Kits overcome the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and DNA template to the ready-to-use PCR master mix, and start the reaction (see flowchart "Two-step RT-PCR"). Follow the protocol in the handbook to get fast and reliable results on any real-time cycler. If required, reactions can be pretreated with uracil-N-glycosylase (UNG) to eliminate carryover of PCR products from previous reactions.
For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiTect Reverse Transcription Kit. The kit provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination.
Applications
QuantiTect Probe PCR Kits can be used for probe-based gene expression analysis of cDNA targets or quantitative gDNA analysis on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Probe PCR Kit, which has been specially developed for fast cycling on these instruments.
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Feature
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Specifications
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Applications
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Real-time quantification of DNA, cDNA, or RNA targets
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Reaction type
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PCR and two-step RT-PCR
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Real-time or endpoint
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Real-time
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Sample/target type
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DNA, cDNA, RNA
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Single or multiplex
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Single
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SYBR Green I or sequence-specific probes
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Sequence-specific probes
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Thermal cycler
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Most real-time cyclers (e.g. LC, RG, ABI)
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With or without ROX
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With ROX
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FAQ ID -1056
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For use with QuantiTect PCR Kits to eliminate carryover of PCR products
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詳細を表示
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For quantitative, real-time PCR and two-step RT-PCR using sequence-specific probes
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詳細を表示
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配列特異的プローブを用いたリアルタイム定量PCRおよび2ステップRT-PCR用
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詳細を表示
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Download Safety Data Sheets for QIAGEN product components.
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画像
効果的なUNG 前処理
dUMP を含むPCRアンプリコン(106 コピー)2 種類をUNG の存在下/非存在下で処理した後、リアルタイムPCRにより増幅した。様々なメーカーのUNGを使用し、QuantiTect Probe PCR +UNG Kit に添付のマスターミックスでリアルタイムPCR を行なった。Y 軸のΔCTは、UNG 処理を行なったサンプルの CT値からUNG 処理を行なっていないサンプルのCT値を差し引いた値である。QuantiTect Kit(QIAGEN)に添付のUNG では最大の ΔCT(9~12 サイクル)が得られた。このことは、他のメーカーのUNGよりもQIAGEN のUNGが最も効率的にキャリーオーバーしたPCR 産物を分解したことを示す。
2ステップRT-PCR
QuantiTect Probe PCR Kit は面倒で時間のかかる反応条件の至適化が不要。プライマー、プローブ、DNAテンプレートを即使用可能なPCRマスターミックスに添加するだけで、ほとんどのリアルタイムサイクラーで反応を開始することができる(表 "2x QuantiTect Probe PCR Master Mixの成分")。
特異性の高い増幅
他社のDNAポリメラーゼと比較した結果、ユニークなバッファーと組み合わせたHotStarTaq DNA Polymeraseのみが497 bp フラグメントを特異的に増幅した(1 µgのヒトゲノムDNAに添加した50 コピーのHIV-pol-遺伝子コンストラクトからのフラグメント)。M:マーカー。
幅広いダイナミックレンジをもつ2ステップPCR
ヒト白血球cDNA の10 倍段階希釈液(0.01ng ~ 10 ng)とIL1R2(サイトカイン)用TaqMan アッセイを用いてMx3005P 上でduplicate で反応を行なった。QuantiTect Probe PCR Kit により、101%のPCR 効率で幅広いテンプレート量からの正確な遺伝子発現解析が行なえた。
幅広いダイナミックレンジをもつリアルタイムPCR
QuantiTect Probe PCR +UNG Kitを用いて、UNG 前処理およびプローブによるリアルタイムPCRを行なった。ヒト白血球cDNAの10倍段階希釈液(10 pg ~ 10 ng)とIL8(サイトカイン)用TaqMan アッセイを用いてABI PRISM 7900 上で反応をduplicateで行なった。増幅プロットは各濃度の間隔が均等であり、PCR 効率は94 %であった。
幅広いダイナミックレンジにわたり効率的で感度の高い定量を実現
IL8 (interleukin 8)に特異的なプライマーおよびFAM標識プローブおよびQuantiTect Probe PCR Kitを用いて、白血球cDNA(100 ng~1 pg)の10倍連続希釈液をMx3005でduplicateで解析した。テンプレートを106倍に希釈した溶液で97%のPCR効率が得られ、わずか1 pgのIL8転写物を高感度に検出した。
特異的なプライマーアニーリング
バランスの取れたKCl および (NH4)2SO4 の組み合わせは、プライマーおよびプローブのPCRテンプレートへの特異的なアニーリングを促進する。K+は二本鎖DNAのリン酸基に結合し、プライマーとプローブのアニーリングを安定化する。NH4+は、ミスマッチな塩基対間の水素結合を不安定化する。
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