High-Specificity Pfu HS Mix

In nature, the ability of a DNA polymerase to correct misincorporation of nucleotides in the DNA strand being elongated is often crucial to the survival of the host organism. This ability is termed “proofreading activity” and occurs in the 3′ to 5′ direction. This activity also leads to the polymerase removing unpaired nucleotides overhanging at the 3’end (A-overhangs), creating blunt ends.

High-Specificity Pfu HS Mix fidelity was determine using the method developed by Lee et al. ( Mapping DNA polymerase errors by single-molecule sequencing. Lee F. D., et al. Nucleic Acids Research, 44 (13) e118 2016 (doi: 10.1093/nar/gkw436)) using a barcode strategy and NGS to characterize the average error rate, error hotspots and lesion bypass fidelity of the DNA polymerase.

High-Specificity Pfu HS Mix is designed to work in the presence of inhibitors. If there is too much interference the concentration of magnesium can be increased, for example, up to 4 mM (in the final reaction) should be added in presence of more than 10% of whole blood.

High-Specificity Pfu HS Mix has been validated for templates up to 5 kb.

DNA target enrichment is a pre-sequencing DNA preparation step where DNA sequences are either directly amplified (multiplex PCR-based) or captured (hybrid capture-based). These enriched DNA fragments can then be sequenced using DNA sequencers.

Aptamers are engineered oligonucleotides that bind to the active site of the high-fidelity DNA polymerase but are denatured and released as the temperature is increased during the PCR amplification cycle. This aptamer-mediated inhibition/activation process is however fully reversible, and so at the end of thermal cycling, when the temperature of the reaction is decreased, the aptamer refolds and rebinds to high-fidelity DNA polymerase, inhibiting any further activity in the sample. This has proven to be important in workflows where undesired polymerase activity after reaction completion can disturb baseline readings, such as NGS following amplification of a library.

No, the ends of the aptamer are blocked to prevent them being sequences.

High-Specificity Pfu HS Mix should be stored at -20°C for optimal retention of activity. We do not recommend the storage of High-Specificity Pfu HS Mix at -80 °C as ice crystals could form on the active site, which may affect or destroy the activity of the enzyme.

Specifications:

Description: High-fidelity PCR master mix containing a reversible, temperature-dependent hot start Taq DNA polymerase and buffer
Concentration: 2 x
Appearance: Clear, colorless solution
Hot Start. Aptamer mediated
Application: PCR, NGS target enrichment, NGS library preparation
Sample type: cDNA, DNA
Presentation: 1 vial
Storage: -20 °C
Mix stability: See outer label
Consistency: ± 0.5 Ct variance between test and reference sample
DNA Contamination: None detected in PCR amplification with traces overlay with the negative control on E. coli and mouse genomic DNA specific targets.
DNase Contamination: No detectable degradation