HotStarTaq DNA Polymerase

最小限の至適化で特異性の高い増幅反応

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HotStarTaq DNA Polymerase (250 U)

Cat. No. / ID: 203203

250 units HotStarTaq DNA Polymerase, 10x PCR Buffer, 5x Q-Solution, 25 mM MgCl₂

Quantity

250 U

1000 U

5000 U

25,000 U

Inquire

HotStarTaq DNA Polymerase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。

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特徴

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

製品詳細

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。

パフォーマンス

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A_II社	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 倍
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

図参照

Higher specificity with different primer–template systems.

Superior performance.

Tolerance to variable temperature and magnesium concentrations.

Amplification of difficult templates.

Effect of hot start on RT-PCR performance.

Highly sensitive single-cell PCR.

原理

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。

図参照

Superior performance.

Higher specificity with different primer–template systems.

Increased specificity of primer annealing.

Tolerance to variable temperature and magnesium concentrations.

Amplification of difficult templates.

操作手順

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""）。

図参照

HotStarTaq procedure.

アプリケーション

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

裏付けデータと数値

HotStarTaq procedure.

Specifications

Features	Specifications
Applications	PCR, RT-PCR, Complex genomic templates, very low-copy targets
With/without hotstart	With hotstart
Reaction type	PCR amplification
Sample/target type	Genomic DNA and cDNA
Real-time or endpoint	Endpoint
Enzyme activity	5' -> 3' exonuclease activity
Mastermix	No
Single or multiplex	Single

リソース

HotStarTaq DNA Polymerase; HotStarTaq Master Mix Kit - For highly specific hot-start PCR without optimization

マイクロダイセクションにより採取した組織サンプルからのDNA分離およびHotStarTaq DNA Polymeraseを用いた増幅

PCR 実験における重要項目と新技術

Addressing critical factors and new solutions

Download Safety Data Sheets for QIAGEN product components.

Publications

MALDI-TOF mass spectrometry for multiplex genotyping of CYP2B6 single-nucleotide polymorphisms.

Blievernicht JK; Schaeffeler E; Klein K; Eichelbaum M; Schwab M; Zanger UM;

Clin Chem; 2006; 53 (1):24-33 2006 Nov 2 PMID:17082249

Age-related urinary excretion of BK polyomavirus by nonimmunocompromised individuals.

Zhong S; Zheng HY; Suzuki M; Chen Q; Ikegaya H; Aoki N; Usuku S; Kobayashi N; Nukuzuma S; Yasuda Y; Kuniyoshi N; Yogo Y; Kitamura T;

J Clin Microbiol; 2006; 45 (1):193-8 2006 Nov 8 PMID:17093017

Transcriptional regulation of human CYP2A13 expression in the respiratory tract by CCAAT/enhancer binding protein and epigenetic modulation.

Ling G; Wei Y; Ding X;

Mol Pharmacol; 2006; 71 (3):807-16 2006 Dec 5 PMID:17148654

Seizures and enhanced cortical GABAergic inhibition in two mouse models of human autosomal dominant nocturnal frontal lobe epilepsy.

Klaassen A; Glykys J; Maguire J; Labarca C; Mody I; Boulter J;

Proc Natl Acad Sci U S A; 2006; 103 (50):19152-7 2006 Dec 4 PMID:17146052

Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation.

Candiloro IL; Mikeska T; Dobrovic A;

Epigenetics; 2011; 6 (4):500-7 2011 Apr 1 PMID:21364322

FAQ

Touchdown PCR uses a cycling program with varying annealing temperatures. It is a useful method to increase the specificity of PCR. The annealing temperature in the initial cycle should be 5–10°C above the Tm of the primers. In subsequent cycles, the annealing temperature is decreased in steps of 1–2°C/cycle until a temperature is reached that is equal to, or 2–5°C below, the Tm of the primers. Touchdown PCR enhances the specificity of the initial primer–template duplex formation and hence the specificity of the final PCR product.

To program your thermal cycler for touchdown PCR, you should refer to the manufacturer’s instructions. For additional hints and tips for successful PCR, review the Appendix Sections in our PCR Kit handbooks, and our Brochures and Application Guides for PCR and RT-PCR.

FAQ ID -75

Both the quality and quantity of nucleic acid starting template affect PCR, in particular the sensitivity and efficiency of amplification. PCR sensitivity and efficiency can be reduced by the presence of impurities in nucleic acid preparations or in biological samples. These PCR inhibitors are completely removed when template is prepared using QIAGEN Kits for nucleic acid purification. Please refer to the Brochure "Maximizing PCR and RT-PCR success" for additional information.

The optimal primer–template ratio has to be determined empirically. If too little template is used, primers may not be able to find their complementary sequences. Too much template may lead to an increase in mispriming events. Generally, no more than 1 ug of template DNA should be used per PCR reaction. As an initial guide, spectrophotometric and molar conversion values for different nucleic acid templates are listed below.

Spectrophotometric conversions for nucleic acid templates

1 A₂₆₀ unit*	Concentration (ug/ml)
Double-stranded DNA	50
Single-stranded DNA	33
Single-stranded RNA	40

*Absorbance at 260 nm = 1

Molar conversions for nucleic acid templates

Nucleic Acid	Size	pmol/ug	Molecules/ug
1 kb DNA	1000 bp	1.52	9.1 x 10¹¹
pUC 19 DNA	2686 bp	0.57	3.4 x 10¹¹
pTZ18R DNA	2870 bp	0.54	3.2 x 10¹¹
pBluescript II DNA	2961 bp	0.52	3.1 x 10¹¹
Lambda DNA	48,502 bp	0.03	1.8 x 10¹⁰
Average mRNA	1930 nt	1.67	1.0 x 10¹²
Genomic DNA
Escherichia coli	4.7 x 10⁶*	3.0 x 10^-4	1.8 x 10⁸**
Drosophila melanogaster	1.4 x 10⁸*	1.1 x 10^-5	6.6 x 10⁵**
Mus musculus (mouse)	2.7 x 10⁹*	5.7 x 10^-7	3.4 x 10⁵**
Homo sapiens (human)	3.3 x 10⁹*	4.7 x 10^-7	2.8 x 10⁵**

* Base pairs per haploid genome

** For single-copy genes

FAQ ID -74

PCR products that will be cloned using the QIAGEN PCR Cloning Kit should be generated using a thermostable DNA Polymerase without proofreading activity, such as Taq DNA Polymerase. Such polymerases attach a single A overhang to their reaction products, which can hybridize to the U overhang of the pDrive Cloning Vector. For efficient addition of an A overhang during the PCR procedure, we recommend a final extension step for 10 min at 72°C as described in the standard protocols of the Taq PCR- and HotStarTaq PCR handbook.

FAQ ID -165

Please see the following factors that can contribute to unspecific, smeared PCR products, and suggestions how to avoid it:

too much starting template
Check the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions.
carry-over contamination
If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable pipet tips containing hydrophobic filters to minimize cross-contamination. Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.
enzyme concentration too high
When using HotStarTaq or Taq DNA Polymerase, use 2.5 units per 100 µl reaction.
too many PCR cycles
Reduce the number of cycles in steps of 3 cycles.
Mg2+ concentration not optimal
Perform PCR with different final concentrations of Mg2+ from 1.5–5.0 mM (in 0.5 mM steps) using the 25 mM MgCl2 solution provided (see table below):

Final Mg2+ concentration in reaction (mM)	1.5	2.0	2.5	3.0	3.5	4.0	4.5	5.0
Required volume of 25 mM MgCl2 per reaction (ul)	0	2	4	6	8	10	12	14

Primer concentration not optimal or primers degraded
Repeat the PCR with different primer concentrations from 0.1–0.5 µM of each primer (in 0.1 µM steps). In particular, when performing highly sensitive PCR, check for possible degradation of the primers on a denaturing polyacrylamide gel.
Primer design not optimal

Review your primer design, and design new primers

For additional information on optimization of PCR results, please refer to the Appendix sections of the Taq PCR and HotStarTaq DNA Polymerase Handbook, and our comprehensive Brochure Critical Factors for Successful PCR.

FAQ ID -87

No, we do not sell Q-Solution separately. It is available only as a component of the Taq DNA Polymerase, Taq PCR Core, HotStarTaq DNA Polymerase, QIAGEN Multiplex PCR-, and the QIAGEN OneStep RT-PCR Kits.

FAQ ID -204

CoralLoad dyes supplied in PCR Kits such as, e.g., Taq, HotStarTaq, and TopTaq DNA Polymerase and TopTaq Master Mix do not interfere with most downstream enzymatic applications.

However, for reproducible results, purification of PCR products using the QIAquick or MinElute PCR Purification Kits prior to enzymatic manipulation is recommended.

FAQ ID -1745

Yes, please follow the Supplementary Protocol 'Polyacrylamide_gel_analysis_of_oligonucleotides' (PCR03).

FAQ ID -961

Taq DNA Polymerase has an intrinsic RNA-dependent DNA polymerase activity (reverse transcriptase activity). However, this activity is very low and is only present under buffer conditions that are completely different from those present during PCR. Therefore, "no-RT" controls would not give false positive results due to reverse transcription activity of the Taq polymerase.

FAQ ID -523

Not necessarily. In a lot of cases, the uniquely formulated PCR Buffer provided in the HotStarTag Plus DNA Polymerase, HotStar HiFidelity Polymerase, Taq DNA Polymerase, HotStarTaq DNA Polymerase, and QIAGEN Multiplex PCR Kits provides optimal amplification of specific PCR products. The usefulness of Q-Solution needs to be determined empirically for each primer/template setup, by running parallel PCR reactions with and without Q-Solution under the same cycling conditions.

Q-Solution changes the melting behavior of DNA and will often improve a suboptimal PCR caused by templates that have a high degree of secondary structure or high GC-contents. For more details on the effects of Q-Solution on PCR amplification, please see the Q-Solution sections of the HotStarTaq Plus DNA Polymerase, HotStar HiFidelity Polymerase, Taq DNA Polymerase, HotStarTaq DNA Polymerase, and the QIAGEN Multiplex PCR Handbooks.

FAQ ID -380

Prerequisites for avoiding primer-dimer formation during PCR include the design of optimal primer pairs, and the use of appropriate primer concentrations. Complementarity of two or three bases at the 3' ends of primer pairs and complementary sequences within a primer sequence and between the primer pair should be avoided. Reduce the primer concentration to the lowest amount at which product amplification can be achieved by conducting test runs with primer concentration gradients.

FAQ ID -544

In quantitative (real-time) PCR, primer-dimers will appear as a peak with a Tm lower than the Tm of the specific product. This peak will also appear in the no-template control (NTC). In non-quantitative endpoint PCR, primer dimer will appear as a more or less faint smear on an agarose gel, below the product band of interest.

FAQ ID -552

No. Neither the PCR Buffer nor the Enzyme Storage Buffer contain Triton.

FAQ ID -327

The QIAGEN 10x Taq and HotStarTaq DNA Polymerase PCR buffer contains a uniquely balanced combination of KCl and (NH4)2SO4. It provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers.

FAQ ID -566

Yes. Please see Table 3 in our brochure Maximizing PCR and RT-PCR success. We tested the effects of different inhibitory substances in a number of PCR systems. We also analyzed the effect of including different volumes of reverse transcription (RT) reaction mixtures in PCR. Please see the table below for a list of commonly encountered template impurities and their inhibitory effects on PCR.

Impurities showing inhibitory effects on PCR

Substance	Inhibitory concentration
SDS	>0.005% (w/v)
Phenol	>0.2% (v/v)
Ethanol	>1% (v/v)
Isopropanol	>1% (v/v)
Sodium Acetate	≥5 mM
Sodium Chloride	≥25 nM
EDTA	≥0.5 mM
Hemoglobin	≥1 mg/ml
Heparin	≥0.15 i.U./ml
Urea	>20 mM
RT reaction mixture	≥15%

FAQ ID -818

No, the initial activation time of 15 minutes at 95°C is crucial. Enzyme activation will be incomplete when using shorter activation times, resulting in inefficient PCR product amplification.

FAQ ID -565

QIAGEN's 10x PCR Buffer provided in the Taq DNA Polymerase, Taq PCR Core, and HotStarTaq DNA Polymerase Kits contains:

Tris-Cl
KCl
(NH₄)₂SO₄
15 mM MgCl₂ ; at pH 8.7 (20°C).

Note that further details on the composition of the 10x PCR Buffer are proprietary.

FAQ ID -606

The DNA yield obtained in a PCR reaction depends on the size of the amplicon, design of the primers, starting amount of template and primers, amplification efficiency, reaction volume, numbers of PCR cycles etc. Therefore it is really difficult to predict what yield to expect. Nevertheless, in our experience, approximately 1 µg is a good guess for most cases.

FAQ ID -750

Taq DNA Polymerase and HotStarTaq DNA Polymerase are compatible with cycle sequencing. However, our buffer system is not optimized for this purpose. Optimization of reaction conditions is therefore required when using these Polymerases for cycle sequencing. Unfortunately, we do not have any protocols for this application. An initial activation of the enzyme is necessary if HotStarTaq DNA Polymerase is used.

FAQ ID -741

To determine the optimal annealing temperature for a PCR assay, a Temperature Gradient experiment should be performed. To do this, you will set up several PCR reactions in duplicate for the same primer/template combination, using the same PCR chemistry, and subject each of the reactions to a slightly different annealing temperature within a specified range. If a thermal cycler with a temperature gradient function can be used, you can simply program a temperature range for adjacent wells in the cycling block. If no cycler with a gradient function exists in your lab, you will either have to perform duplicate reactions at different temperatures in different machines (if available), or back to back in the same machine.

FAQ ID -288