HotStarTaq DNA Polymerase

HotStarTaq DNA Polymerase, a modified form of QIAGEN Taq DNA Polymerase, provides high specificity in hot-start PCR.

Features and benefits

• Higher PCR specificity
• Reduced nonspecific amplification
• Easy handling

Product Format Options
Components	HotStarTaq DNA Polymerase	HotStarTaq Master Mix Kit (premixed solution)*
HotStarTaq DNA Polymerase
PCR Buffer†
Q-Solution		—
dNTP Mix‡	—
MgCl2 solution
Distilled water	—

Principle

HotStarTaq DNA Polymerase, a modified form of QIAGEN Taq DNA Polymerase, is supplied in an inactive state that has no polymerase activity at ambient temperatures. This prevents extension of nonspecifically annealed primers and primer–dimers formed at low temperatures during PCR setup and the initial PCR cycle (see figures "Superior Performance in Hot-Start PCR" and "Higher Specificity with Different Primer–Template Systems", and see table "Comparison of hot-start methods"). HotStarTaq DNA Polymerase is activated by a 15-minute incubation at 95°C which can be incorporated into any existing thermal-cycler program. Every lot of HotStarTaq DNA Polymerase is subjected to a comprehensive range of quality control tests, including a stringent PCR specificity and reproducibility assay in which low-copy targets are amplified.

Comparison of hot-start methods

	HotStarTaq DNA Polymerase	Hot-start enzyme from Supplier AII	Antibody- mediated	Manual	Wax barrier
Specific amplification
Minimal PCR optimization
Easy to use

Superior Performance in Hot-Start PCR

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); no hot start with enzyme from Supplier R (No hot start). Arrow indicates the specific PCR product. Equal volumes of the reaction were analyzed on a 2% agarose gel. M: markers.

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 homologue 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.

QIAGEN PCR Buffer maintains specific amplification in every cycle of PCR. The balanced combination of KCl and (NH4)2SO4 in the buffer promotes specific primer–template annealing. Simultaneously, nonspecific annealing is reduced, maximizing yields of specific PCR product. Specificity is maintained over a wide range of temperatures and Mg2+ concentrations, without the need for time-consuming optimization (see figures "Wide Annealing-Temperature Window and Tolerance to Variable Mg2+ Concentration").

Q-Solution facilitates amplification of difficult templates by modifying the melting behavior of DNA. Use of this unique reagent will often enable or improve suboptimal PCR (see Figure: "Amplification of Difficult Templates with Q-Solution"). Unlike DMSO and other PCR additives, Q-Solution is used at a defined working concentration with any primer–template system and is not toxic.

HotStarTaq Master Mix is a ready-to-use mixture of HotStarTaq DNA Polymerase, QIAGEN PCR Buffer, and dNTPs. Room-temperature reaction setup using this Master Mix is fast and easy — simply pipet 25 µl HotStarTaq Master Mix into each PCR tube and add 25 µl of primers and template DNA diluted in the distilled water provided with the kit. Pipetting steps are minimized, reducing the possibility of errors and contamination. The combination of high specificity and easy handling makes the HotStarTaq Master Mix Kit ideal for use with complex genomic or cDNA templates, multiple primer pairs (see figure "Specific Amplification in Multiplex PCR"), templates isolated from difficult sources, and projects such as genetic screening, in which large numbers of samples are amplified.

Specific Amplification in Multiplex PCR

Fragments from the murine p53 gene were amplified from genomic DNA in multiplex PCR. Parallel reactions were prepared using standard reaction conditions and an enzyme from Supplier R (No hot start) or using HotStarTaq Master Mix Kit from QIAGEN (HotStarTaq Master Mix). M: markers.

HotStarTaq DNA Polymerase specifications

Concentration: 5 units/µl Recombinant enzyme: Yes Substrate analogs: dNTP, ddNTP, dUTP,   biotin-11-dUTP,   DIG-11-dUTP,   fluorescent-dNTP/ddNTP Extension rate: 2–4 kb/min at 72°C Half-life: 10 min at 97°C   60 min at 94°C

Amplification efficiency: ≥105 fold 5'3' exonuclease activity: Yes Extra A addition: Yes 3'5' exonuclease activity: No Contaminating nucleases: No Contaminating RNases: No Contaminating proteases: No Self-priming activity: No

Applications

HotStarTaq DNA Polymerase is suitable for:

• Complex genomic templates
• Complex cDNA templates (e.g., RT-PCR, see figure "Effect of Hot Start on RT-PCR Performance")
• Very low-copy targets (e.g., single-cell PCR, see figure "Single-Cell PCR")
• Reactions with multiple primer pairs

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

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.

操作手册:HotStarTaq PCR Handbook 03/02 (PDF version, 179 KB)

HotStarTaq DNA Polymerase

For robust amplification in all applications

Component Included Concentration HotStarTaq DNA Polymerase 5 units/µl   PCR Buffer 10x solution* Q-Solution 5x solution   dNTP Mix — — MgCl2 25 mM solution