HotStarTaq DNA Polymerase

最小限の至適化で特異性の高い増幅反応
  • 最小限の至適化
  • 高いPCR特異性
  • 取り扱いが簡単、室温でのセットアップが可能

HotStarTaq DNA Polymerase は抗体によるホットスタートシステムではなく、化学修飾によるホットスタートを利用しているため、最初の熱活性化ステップまではポリメラーゼ活性は全くありません。HotStarTaq DNA Polymerase には、非特異的な増幅産物、プライマーダイマー、バックグラウンドを最小限に抑える画期的なQIAGEN PCR Buffer が入っています。また増幅困難なテンプレート(例;GC リッチ)の効率的な増幅を実現するQ-Solutionも含まれています。

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HotStarTaq DNA Polymerase (250 U)
250 units HotStarTaq DNA Polymerase, 10x PCR Buffer, 5x Q-Solution, 25 mM MgCl2
203203
$236.00
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HotStarTaq DNA Polymerase (1000 U)
4 x 250 units HotStarTaq DNA Polymerase, 10x PCR Buffer, 5x Q-Solution, 25 mM MgCl2
203205
$805.00
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HotStarTaq DNA Polymerase (5000 U)
1 x 5000 units HotStarTaq DNA Polymerase, 1 x HotStarTaq Buffer Set (1 x 22 ml PCR Buffer, 1 x 40 ml Q-Solution, 1 x 22 ml MgCl2)
203207
$2,932.00
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HotStarTaq DNA Polymerase (25000)
100 x 250 units HotStarTaq DNA Polymerase, 100 x 1.2 ml HotStarTaq Buffer Set, 100 x 2.0 ml Q-Solution, 100 x 1.2 ml MgCl2
203209
$12,289.00
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The HotStarTaq DNA Polymerase is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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HotStarTaq procedure.|Superior performance.|Amplification of difficult templates.|Higher specificity with different primer–template systems.|Effect of hot start on RT-PCR performance.|Highly sensitive single-cell PCR.|Tolerance to variable temperature and magnesium concentrations.|Increased specificity of primer annealing.|
The HotStarTaq procedure is fast and easy for maximum convenience.|A 497 bp fragment was amplified from 50 copies of an HIV-pol-gene construct which had been added to 1 µg human genomic DNA. Different hot-start enzymes were employed: HotStarTaq DNA Polymerase from QIAGEN (HotStarTaq); hot-start enzyme from Supplier AII (Hot-start enzyme); Taq-antibody mixture from Supplier L (Antibody-mediated); enzyme without hot start from Supplier R (No hot start). Specific PCR product is indicated by the arrow. Equal volumes of the reaction were analyzed on a 2% agarose gel. M: markers.|Two different primer–template systems were amplified in duplicate using QIAGEN PCR Buffer and Taq DNA Polymerase in the absence () or presence (+) of 1x Q-Solution. Q-Solution enables specific amplification of difficult templates. [A] human angiotensin receptor II gene; [B] mouse protein kinase C gene; M: markers.|Three different primer–template systems were amplified under the same conditions with either Taq DNA polymerase from Supplier R (R) or with HotStarTaq DNA Polymerase (H). System 1: A 1.1 kb fragment of a D-IgI homolog was amplified from human genomic DNA. System 2: A 296 bp fragment from the chromosomal region correlated with X-linked juvenile retinoschisis was amplified from human genomic DNA. System 3: A 214 bp fragment of the β-actin gene was amplified from cDNA synthesized from total RNA. M: markers.|A 1.1 kb fragment of the human interleukin 1 receptor (type II) gene was amplified from cDNA. Amplification reactions were prepared in triplicate using Taq DNA polymerase and buffer from Supplier L(No hot start); antibody-mediated hot start using enzyme and buffer from Supplier L (Antibody-mediated); and HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq). M: markers.|A 500 bp fragment of the murine p53 gene was amplified from single cells isolated by flow cytometry, and directly sorted into individual PCR tubes. Reactions were prepared in parallel using HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq), a hot-start enzyme and buffer from Supplier AII (Hot-start enzyme), or antibody-mediated hot start and buffer from Supplier L (Antibody-mediated). M: markers.|[A] PCR amplification at the indicated annealing temperatures using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human cystic fibrosis gene was amplified. M: markers. [B] Tolerance to Variable Magnesium Concentration. PCR amplification at the indicated Mg2+ concentrations using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human prion protein gene was amplified. M: markers.|Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.|
Performance

HotStarTaq DNA Polymeraseは全てのロットで、低コピー数のターゲットを増幅する実験によりPCR特異性の厳密さや再現性をチェックするなど、広範囲の品質管理テストを受けています。HotStarTaq DNA Polymeraseは他社のキットに比べて高性能で、特異性と感度の高いホットスタートPCRを実現します (図“異なるプライマー/テンプレートシステムにおける高特異性”、“卓越した性能”および表)。キットに付属の画期的なPCRバッファーは、様々なPCR条件において特異性を実現し、至適化は最小限に抑えられます(図 “幅広い至適アニーリング温度” および“異なったマグネシウム濃度への適応”)。また、キットに付属のQ - Solutionにより、不適切なPCR条件を改善することができます(図 “増幅困難なテンプレートの増幅”)。これらの成分により様々なアプリケーションで特異的な増幅を実現します(図 “RT-PCRにおけるホットスタートの効果”および “高感度のシングルセルPCR”)。

ホットスタート法の比較 
HotStarTaq DNA Polymerase ホットスタート酵素AII 抗体利用 マニュアル ワックスバリア
特異的な増幅 ++ + + +/– +/–
PCR至適化の不要性 ++ +/– +/–
取り扱いの簡便さ ++ ++ +
HotStarTaq DNA Polymerase 詳細データ

濃度:5 units/µl
組み換え酵素:Yes
基質アナログ:dNTP、ddNTP、dUTP、biotin-11-dUTP、DIG-11-dUTP、 fluorescent-dNTP/ddNTP
エクステンション速度:72℃ で2~4 kb/min 
半減期:97℃で10分、94℃で60分 
増幅効率:≥105
5'–>3' エキソヌクレアーゼ活性:Yes
余分なA付加:Yes
3'–>5' エキソヌクレアーゼ活性:No
ヌクレアーゼの混入:No
RNasesの混入:No
プロテアーゼの混入:No
自己プライマー活性:No 

  

Principle

Taq DNA Polymeraseを修飾したHotStarTaq DNA Polymeraseは、ホットスタートPCR において高い特異性を実現します。本キットには、2種類の陽イオンを含む画期的なPCRバッファー、Q-Solution、MgCl2が入っています。

HotStarTaq DNA Polymerase

HotStarTaq DNA Polymeraseは、常温では不活性状態でポリメラーゼ活性はありません。この特性により、PCR セットアップや最初のPCR サイクル中の低温での非特異的なプライマーのアニーリングやプライマーダイマーの形成を回避できます(図“ホットスタートPCRで最高のパフォーマンスを実現”および“異なるプライマー/テンプレートシステムにおける高特異性”)。HotStarTaq DNA Polymeraseは、95℃、5 分間のインキュベーションステップで活性化され、このステップは既存のサーマルサイクリングのプログラムに容易に導入できます。

QIAGEN PCR Buffer

QIAGEN PCR Buffer は、各PCRサイクルでアニーリングステップ中にプライマー特異的な結合の割合を非特異的な結合に対して高めることにより、各PCRサイクルの特異的な増幅を維持します(図“プライマーアニーリングの特異性増大”)。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、幅広いアニーリング温度や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。従って、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(図“幅広い至適アニーリング温度”および“異なったマグネシウム濃度への適応”)。 

Q-Solution

HotStarTaq DNA Polymeraseに入っているQ-Solutionは、DNAの融解を変更することにより増幅困難なテンプレートの増幅を促進する革新的なPCR添加物です。このユニークな試薬により、高度な二次構造をもつテンプレートやGC リッチなテンプレートなどにより生じる不適切なPCR 条件が改善されることがあります(図“増幅困難なテンプレートの増幅”)。DMSOのような汎用されているPCR添加物と異なり、Q-Solutionは一定の濃度で作用し、毒性もなく、PCR純度は保証されています。Q-Solution の添加により、PCR のフィデリティは損なわれません。

Procedure
HotStarTaq DNA Polymeraseに付属の至適化済みプロトコールを用いてPCRセットアップを迅速かつ容易に行なえます。HotStarTaq DNA Polymeraseは、95℃、15 分間のインキュベーションステップで活性化され、このステップは既存のサーマルサイクリングのプログラムに容易に導入できます。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(図"HotStarTaq procedure")。
Applications

HotStarTaq DNA Polymeraseは、増幅などの高度なアプリケーションを含む様々なアプリケーションに最適です:

  • 複雑なゲノムテンプレート
  • 複雑なcDNAテンプレート(例;RT-PCR)
  • 低コピーのターゲット(例;シングルセルPCR)
  • マルチプレックスPCR
Feature
Specifications
Applications PCR, RT-PCR, Complex genomic templates, very low-copy targets
Enzyme activity 5' -> 3' exonuclease activity
Mastermix No
Reaction type PCR amplification
Real-time or endpoint Endpoint
Sample/target type Genomic DNA and cDNA
Single or multiplex Single
With/without hotstart With hotstart

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Kit Handbooks
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HotStarTaq DNA Polymerase; HotStarTaq Master Mix Kit - For highly specific hot-start PCR without optimization  
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Highly specific PCR results with both manual and automated PCR setup
HotStarTaq procedure.

The HotStarTaq procedure is fast and easy for maximum convenience.

Superior Performance in Hot-Start PCR
Superior performance.
A 497 bp fragment was amplified from 50 copies of an HIV-pol-gene construct which had been added to 1 µg human genomic DNA. Different hot-start enzymes were employed: HotStarTaq DNA Polymerase from QIAGEN (HotStarTaq); hot-start enzyme from Supplier AII (Hot-start enzyme); Taq-antibody mixture from Supplier L (Antibody-mediated); enzyme without hot start from Supplier R (No hot start). Specific PCR product is indicated by the arrow. Equal volumes of the reaction were analyzed on a 2% agarose gel. M: markers.
Amplification of Difficult Templates with Q-Solution
Amplification of difficult templates.
Two different primer–template systems were amplified in duplicate using QIAGEN PCR Buffer and Taq DNA Polymerase in the absence () or presence (+) of 1x Q-Solution. Q-Solution enables specific amplification of difficult templates. [A] human angiotensin receptor II gene; [B] mouse protein kinase C gene; M: markers.
Higher Specificity with Different Primer–Template Systems
Higher specificity with different primer–template systems.
Three different primer–template systems were amplified under the same conditions with either Taq DNA polymerase from Supplier R (R) or with HotStarTaq DNA Polymerase (H). System 1: A 1.1 kb fragment of a D-IgI homolog was amplified from human genomic DNA. System 2: A 296 bp fragment from the chromosomal region correlated with X-linked juvenile retinoschisis was amplified from human genomic DNA. System 3: A 214 bp fragment of the β-actin gene was amplified from cDNA synthesized from total RNA. M: markers.
Effect of Hot Start on RT-PCR Performance
Effect of hot start on RT-PCR performance.
A 1.1 kb fragment of the human interleukin 1 receptor (type II) gene was amplified from cDNA. Amplification reactions were prepared in triplicate using Taq DNA polymerase and buffer from Supplier L(No hot start); antibody-mediated hot start using enzyme and buffer from Supplier L (Antibody-mediated); and HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq). M: markers.
Single-Cell PCR
Highly sensitive single-cell PCR.
A 500 bp fragment of the murine p53 gene was amplified from single cells isolated by flow cytometry, and directly sorted into individual PCR tubes. Reactions were prepared in parallel using HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq), a hot-start enzyme and buffer from Supplier AII (Hot-start enzyme), or antibody-mediated hot start and buffer from Supplier L (Antibody-mediated). M: markers.
Wide Annealing-Temperature Window
Tolerance to variable temperature and magnesium concentrations.
[A] PCR amplification at the indicated annealing temperatures using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human cystic fibrosis gene was amplified. M: markers. [B] Tolerance to Variable Magnesium Concentration. PCR amplification at the indicated Mg2+ concentrations using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human prion protein gene was amplified. M: markers.
NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing
Increased specificity of primer annealing.
Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.