Substitution of Asn for the conserved Ser543 in the thumb subdomain of the Taq DNA polymerase large fragment (KlenTherm™ DNA polymerase) prevents pausing during DNA synthesis and allows the enzyme to overcome template regions with a complex secondary structure. The mutant enzyme, KlenThermN™ DNA poly-merase (patent pending), provides specific PCR amplification and sequencing of difficult templates, e.g. those with a high GC content or complex secondary structure. Furthermore this substitution increases several times the efficiency of synthesis of long (over 2 kb) DNA molecules. The difference in the DNA synthesis efficiencies by the mutant and native enzymes increases with the increase in the DNA fragment length.
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTPs into acid-insoluble form in 30 min at 73°C under the assay conditions 25 mM TAPS (tris-(hydroxy-methyl)-methyl-amino-propanesul-fonic acid, sodium salt) pH 9.3 (at 25°C), 50 mM KCl, 3.5 mM MgCl2 , 1 mM .-mercaptoethanol) and activa-ted salmon sperm DNA as substrate.
10 mM K-phosphate buffer pH 7.0, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 0.01% Tween 20; 50% glycerol (v/v)
Store KlenThermN™ DNA polymerase, preferably at -20°C, in a constant temperature freezer.
10X REACTION BUFER
500 mM KCl, 100 mM Tris-HCl (pH 9 at 25°C), 1% Triton X100
Extra solution: 50 mM MgCl 2 , add MgCl 2 to a final concentration of 3.5 mM.
Please note the difference between KlenThermTM and BioThermTM reaction buffers!
1.5 ml 10x reaction buffer Cat. No GC-001-006
KlenTherm™ DNA polymerase, Synergy™ DNA polymerase, SynergyN™ DNA polymerase, dNTPs
- Ignatov, K.B., Miroshnikov, A.I., Kramarov, V.M. (1998) FEBS Lett. 425, 249-250. Substitution of Asn for Ser543 in the large fragment of Taq DNA polymerase increases the efficiency of synthesis of long DNA molecules.