PCR & qPCR Applications
High-Fidelity Pfu
A high-fidelity antibody-mediated hot-start DNA polymerase from the bacteria Pyrococcus furiosus (Pfu). The Pfu polymerase enzyme replicates DNA in the 5´ → 3´ direction and also possesses 3´ → 5´ exonuclease (proofreading) activity. Base misincorporations that may occur during polymerization are rapidly excised by this proofreading activity. Meridian’s High-Fidelity has an error rate of 3.0 x 10 -6 generating blunt-ended amplicons up to 5 kb in length. It is supplied in a novel buffer system containing dNTPs and enhancers with MgCl 2 provided separately to enable flexibility for applications such as target enrichment, NGS library amplification, or cloning.
Figure 8. High-Fidelity Pfu: NGS library amplification using E.coli DNA
> Antibody hot-start Pfu polymerase for increased specificity > Error rate of 3.0 x 10 -6 > Ideal for NGS library amplification
PRODUCT
CAT NO.
VOLUME REACTIONS
Four NGS libraries were created using E. coli DNA. The libraries were amplified using High-Fidelity Pfu (MDX003) prior to being placed on flow cells and sequenced. For each sample, the fraction of uniquely mapped (mapped to only one place on a reference sequence), ambiguously mapped and unmapped reads relative to the total number of reads per sample is shown. The results illustrate that with E. coli DNA, High-Fidelity Pfu allowed for over 90% unique reads that could be aligned to the reference sequence.
0.2 mL 500 Rxns
MDX003
High-Fidelity Pfu
10 mL 25,000 Rxns
Glycerol-Free High-Fidelity Pfu
A high‑concentration (20 U/ µ L), glycerol‑free, high-fidelity thermostable Pfu enzyme supplied in a novel buffer system containing magnesium, dNTPs and PCR excipients. With an error rate of 3.0 x 10 -6 , this enzyme is ideal for enhancing DNA target enrichment in NGS library fragments and minimizing the incorporation of mutations into the NGS library. Combined, these characteristics facilitate the need for less extensive sequence coverage, leading to notable savings in both time and costs.
Figure 9. Glycerol-Free High-Fidelity Pfu: Efficient multiplex amplification used as wet mix or after lyophilization
> High concentration (20 U/ µ L) Pfu polymerase allowing for bulk production of PCR master mixes > Ideal for NGS library amplification > Compatible with lyophilization for developing ambient-temperature stable assays
PRODUCT
CAT NO.
VOLUME REACTIONS
0.1 mL 500 Rxns
Glycerol-Free High- Fidelity Pfu (HC)
MDX203
Glycerol-Free High-Fidelity Pfu (HC, MDX203), was lyophilized with Lyo-Ready ™ Pfu Reaction Buffer, 5x and stored at 37°C for 7 and 28 days. Lyophilized samples were tested in a multiplex PCR reaction amplifying 7 targets (793 bp, 649 bp, 548 bp, 418 bp, 332 bp, 196 bp, 135 bp) against the same enzyme and reaction buffer without lyophilization (Wet Reference) to demonstrate the enzyme’s stability after lyophilization.
10 mL 50,000 Rxns
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