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KAPA2G Fast DNA Polymerase is a second-generation enzyme derived through a process of molecular evolution. The KAPA2G Fast DNA Polymerase was engineered for higher processivity and speed, offering significantly faster extension rates and improved PCR success rates than wild-type Taq polymerase. KAPA2G Fast is well suited for both routine and high-throughput PCR, achieving higher yields and improved sensitivity than competitor enzymes across a wide range of amplicon types.
KAPA2G Fast DNA Polymerase and optimized buffer system offers:
- Extension times as low as 1 sec per kilobase.
- Outstanding performance when compared to standard and hot start Taq polymerase.
- 20 - 70% reductions in total PCR time.
- No requirement for specialist PCR consumables or instrumentation.
Product PerformanceThe ultimate in speed and performanceKAPA2G Fast HotStart and optimized buffer system is capable of dramatically reduced extension times and exceptional performance. Hot start formulations of wild-type Taq do not perform satisfactorily when cycling times are reduced. In contrast, the unique properties of KAPA2G Fast HotStart allows for efficient amplification in total reaction times that are typically reduced by 40 - 70%. + Fast amplification of 7 human gene fragments using KAPA2G Fast HotStart or competitor hot start Taq formulations. Reactions (25 µl) contained 5 ng human genomic DNA and 0.5 units (KAPA2G Fast HotStart or Competitor I) or 0.625 units (Competitor A) enzyme. Cycling was performed on an EppendorfMastercyclerepgradient S, using a 3-step (35 cycle) profile with 15 sec denaturation (95ºC), 15 sec annealing (60ºC) and 1 sec extension (72ºC) per cycle. Initial denaturation/ enzyme re-activation time (95ºC) was 2 min for KAPA2G Fast HotStart and Competitor I and 7.5 min for Competitor A. The total reaction time was 29.5 min for KAPA2G Fast HotStart and Competitor I and 37 min for Competitor A. The gene target, length and GC content of each amplicon is as follows: 1 = 5,10-methylene tetrahydrofolatereductase, 150 bp, 54.0% GC; 2 = zinc finger protein 638, 318 bp, 30.9% GC; 3 = attractin, 373 bp, 37.1% GC; 4 = zinc finger protein 41, 502 bp, 42.5% GC; 5 = PCI domain, 526 bp, 39.3% GC; 6 = chromosome 6 ORF167, 599 bp, 35.6% GC and 7 = epidermal growth factor receptor, 626 bp, 42.0% GC.
KAPA2G Fast HotStart outperforms hot start formulations of wild-type Taq, even when the latter are used at their optimal, slow extension rates (1 min/kb per cycle). The unique properties of this second-generation enzyme, combined with a proprietary HotStart antibody and optimized buffer system allows for efficient fast amplification of a larger variety of amplicon types than competitor enzymes. In the example below, competitor enzymes failed or performed poorly with 40 - 60% of a set of single copy human gene fragments, particularly in cases were amplicons are short or their GC content is high (>65%).
+ Amplification of 5 human gene fragments using KAPA2G Fast HotStart or competitor hot start Taq formulations. Reactions (25 µl) contained 5 ng human genomic DNA and 0.5 units (KAPA2G Fast HotStart or Competitor I) or 0.625 units (Competitor A or Competitor Q) enzyme. For amplicons with a GC content >65% (#2 and 3), 7.5% DMSO was included in reaction mixes. Cycling was performed on an EppendorfMastercyclerepgradient S, using 3-step cycling profiles (35 cycles) with 15 sec denaturation (95ºC) and 15 sec annealing (60ºC) per cycle for all enzymes. Extension (72ºC) was 1 sec per cycle for KAPA2G Fast HotStart and 60 sec per cycle for competitor enzymes. Initial denaturation/enzyme re-activation and final extension times used for each enzyme were as per the manufacturers recommendations. The total reaction time for each enzyme is as indicated. The gene target, length and GC content of each amplicon is as follows: 1 = 5,10-methylene tetrahydrofolatereductase, 150 bp, 54.0% GC; 2 = guanine nucleotide binding protein, 241 bp, 83.8% GC; 3 = RET proto-oncogene, 392 bp, 79.1% GC; 4 = zinc finger protein 41, 502 bp, 42.5% GC and 5 = epidermal growth factor receptor, 626 bp, 42.0% GC.
High speed and sensitivity
KAPA2G Fast HotStart is based on a second-generation polymerase with the intrinsic ability to synthesize DNA faster than wild-type Taq and other DNA polymerases. Fast PCR protocols using KAPA2G Fast HotStart are primarily based on reduced extension times that allow for a significant reduction in PCR cycling time without the risk of compromising reaction performance. Using KAPA2G Fast HotStart, a total extension time of 1 second per cycle is sufficient for the reproducible amplification of fragments <1 kb from as little as a single target copy of human genomic DNA. For amplicons >1 kb, 15 sec/kb extension time per cycle is used. + Amplification of a 626 bp fragment of the epidermal growth factor receptor (left) and a 1.3 kb fragment of the macrophage stimulating 1 receptor (right) from a 10-fold dilution series of human genomic DNA using KAPA2G Fast HotStart. 0.5 units of enzyme were used per 25 µl reaction. Cycling was performed on the G-Storm GS1 thermocycler with a fast block, using a standard 3-step cycling profile (35 cycles). For the 626 bp fragment, a total extension time of 1 sec per cycle was used and for the 1.3 kb fragment an extension time of 15 sec/kb (20 sec per cycle). |
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