Highly accurate and easy to use
- 40 times more accurate than Taq DNA polymerase
- Easy to use
- No need to protect primers 3'end
- Highly thermostable
Isis is a new proofreading DNA polymerase with high-performance features. Isolated from Pyrococcus abyssi, a hyperthermophilic archaea bacteria living in hydrothermal vents at a depth of 2000 m in the Fiji Basin. It is an excellent choice for PCR applications that require very low probability of base mis-incorporation. Fidelity studies using the highly reliable Flaman method showed that Isis DNA Polymerase is able to amplify DNA with 40 times more accuracy than Taq DNA polymerase with a error rate of 0.66 x 10-6 mutation frequency/bp/duplication.
Easy to use
Isis DNA Polymerase shows very robust activity over a range of different amplification conditions (see Fig. 1) and does not require primer 3'-end protection, unlike many other proofreading enzymes. This means much less time is needed for reaction set-up and optimization.
Isis DNA Polymerase is the most thermostable proofreading polymerase available (80% of the activity remains after 5 hours incubation at 95°C and 50% after 5 hours at 100°C) and therefore will retain more activity during PCR, giving more product. This also means denaturation time and temperature can both be increased if necessary (see Fig. 2), e.g., for reading through difficult secondary structure.
|Storage buffer:||TrisHCl 20 mM, pH 8.0, KCl 100 mM, EDTA 0.1 mM, Dithiothreitol 1mM, Tween 20 0.5%, Nonidet P40 0.5%, Glycerol 50%|
|Incubation conditions:||TrisHCl 20 mM, pH(25°C) 9.0, KCl 25 mM, (NH4)2SO4 10 mM, MgSO4 1.5 mM, Tween 20 0.1%, BSA 0.1 mg/ml|
|Quality control:||Activity, absence of nickases and endonucleases, absence of ribonucleases, specific PCR on genomic and phage templates.|
|Unit definition:||One unit is the amount of enzyme required to catalyze the incorporation of 10 nmol of nucleosides into acid insoluble material in 30 min. at 74 °C under assay conditions.|
Amplification of human β-globin DNA 400 bp (lane 2), 900 bp (lane 3) and mitochrondrial human DNA (4.0 kb lane 4) with Isis DNA polymerase. 10 ng of each DNA template, with 50 pmol of each primer, 100 µM of each dNTP and 1.5 mM MgSO4. 0.5 U of Isis DNA polymerase was used foramplifying 400 bp and 1 U for 900 bp and 4.0 kb. Lane 1: Leon™, Molecular weight marker.
Performance comparison of Pfu, Taq and Isis DNA polymerases. Amplification of human β-globin DNA (400bp) with 1 U of each DNA Polymerase, 50 pmol of primers, 100 µM of dNTPs and 1.5 mM MgSO4. Molecular Weight Marker: pBR HaeIII/Taq I Amplification program: 5 min at 95°C-[1 min at 91 or 97°C, 1 min at 62°C, 1 min 15 sec at 72°C) x 30]
400 bp : 5 min at 93°C-(1 min at 91°C, 1 min at 62°C, 1min 15 sec at 72°C) x 30
900 bp : 5 min at 93°C-(1 min at 91°C, 1 min at 62°C, 1 min 30 sec at 72°C) x 30
4.0 kb : 5 min at 93°C-(30 sec at 94°C, 2 min at 62°C, 5' at 72°C) x 20.
Patent pending on the enzyme Isis DNA polymerase. Isis DNA Polymerase has been developed in collaboration with IFREMER (Institut Francais de Recherche pour l'Exploitation de la Mer).