FastGene® HiFi HS (Hot Start) Polymerase 2x Master Mix: High-Fidelity PCR for Demanding Applications
The FastGene® HiFi HS (Hot Start) Polymerase 2x Master Mix is a ready-to-use, 2x concentrated PCR reagent formulated for researchers who require both high accuracy and robust performance from a single, convenient format. It addresses the core challenge of high-fidelity PCR: the need to minimise amplification errors whilst maintaining sensitivity, yield, and specificity across a wide range of template types and fragment sizes, including templates that are notoriously difficult, such as those with high GC or AT content or those requiring long-range amplification up to 17.5 kb.
At the heart of the master mix is the FastGene® HiFi polymerase, a high-fidelity enzyme engineered with an enhanced 3’‑5′ exonuclease (proofreading) activity that delivers an error rate approximately 100 times lower than standard Taq DNA polymerase. The hot start function is conferred by an aptamer-like molecule, a technology distinct from the antibody-mediated hot start approach commonly used, which reversibly binds and inhibits both the 3’‑5′ exonuclease and 5’‑3′ polymerase activities of the enzyme at temperatures below 40 °C. This inhibition is fully released upon the initial denaturation step, permitting room-temperature reaction setup without risk of primer dimer formation or non-specific amplification. The 2x formulation contains the polymerase together with an advanced buffering system, dNTPs, Mg²⁺, and reaction enhancers; users need only add template and primers.
Key Benefits:
- Exceptional Fidelity: ~100x more accurate than Taq. The improved 3’‑5′ exonuclease proofreading activity dramatically reduces the error rate, making this master mix the appropriate choice wherever sequence accuracy is critical, such as cloning, sequencing, and site-directed mutagenesis.
- Aptamer-Based Hot Start: Maximum sensitivity and specificity. A proprietary aptamer-like molecule reversibly inhibits polymerase activity below 40 °C, preventing primer dimer formation and non-specific amplification. This enables straightforward room-temperature reaction setup and delivers cleaner results compared to standard cold-setup PCR.
- Long-Range Amplification: Reliably amplifies templates up to 17.5 kb. The enzyme has been engineered to handle particularly long templates with high sequence accuracy, whilst offering improved extension times of 10–30 seconds per kilobase and high yields even from difficult templates.
- Broad Template Compatibility: Handles AT- and GC-rich sequences. Templates with GC content ranging from approximately 30 % to 80 % can be successfully amplified, making the master mix suitable for a wide range of genomic regions that would challenge standard polymerases.
- Multiplex PCR Ready: Proven performance in multiplex formats. The optimised buffer system and hot start technology combine to support clean, specific amplification of multiple targets simultaneously, ideal for genotyping and panel-based workflows.
- Streamlined Workflow: Ready-to-use 2x format. All components necessary for PCR, polymerase, dNTPs, Mg²⁺, and reaction enhancers, are pre-combined at 2x concentration, significantly reducing pipetting steps, minimising variability, and accelerating reaction setup.
Technical Insights
Figure 1: Long-Range Amplification Capability
The FastGene® HiFi polymerase is capable of amplifying a broad range of fragment sizes, with confirmed performance up to 17,500 bp. This demonstrates the enzyme’s suitability for long-range PCR applications such as the amplification of large genomic regions, BAC/plasmid verification, or the generation of long inserts for cloning. Gel electrophoresis data generated using MIDORIGreen Xtra safe DNA dye and the FastGene® FAS-DIGI PRO gel documentation system confirms discrete, clean bands across the full fragment-size range tested.
Figure 2: GC-Content Tolerance
Templates with varying GC content, ranging from approximately 30 % to 80 %, have been successfully amplified using the FastGene® HiFi polymerase. This breadth of GC-content tolerance is particularly valuable for amplifying promoter regions, CpG islands, and other genomic sequences that are refractory to standard Taq-based systems. Results were confirmed by gel electrophoresis with MIDORIGreen Xtra and the FastGene® FAS-DIGI PRO system.
Figure 3: Multiplex PCR Performance
Six phage DNA fragments, four mouse genomic DNA fragments, and a combination of both template types were successfully co-amplified in a single multiplex PCR reaction using the FastGene® HiFi polymerase. The clean banding pattern obtained confirms that the hot start technology and optimised buffer effectively suppress non-specific amplification even when multiple primer pairs are present simultaneously — a critical requirement for reliable multiplex genotyping and diagnostic-style workflows.
Specifications
- Format: 2x Master Mix (ready-to-use; add template and primers only)
- Fidelity: Approximately 100x lower error rate than Taq DNA polymerase
- Maximum Amplicon Size: Up to 17,500 bp (17.5 kb)
- GC Content Range: Approximately 30 %–80 % GC successfully amplified
- Extension Rate: 10–30 seconds per kb
- PCR Product Ends: Blunt-ended (proofreading activity)
- Components Included: FastGene® HiFi HS polymerase, dNTPs, Mg²⁺, reaction enhancers, optimised buffer (2x)
- Pack Size: 100 reactions (2 × 1.25 ml, for 50 µl reactions)
- Applications: Standard PCR, long-range PCR, multiplex PCR, cloning, site-directed mutagenesis, sequencing prep
- Storage: ‑20 °C