Stoffel DNA Polymerase

• Exhibits maximum performance with improved reaction buffer formulation
• No exonuclease activity
• High thermostability; the half-life of the enzyme is 20 minutes at 97.5°C
• Amplifies fragments of up to 5 kb and leaves ‘A’ overhangs

Stoffel DNA Polymerase is a 62.7 kDa recombinant fragment of thermostable Taq DNA polymerase isolated from Thermus aquaticus. It is recommended for a wide range of applications that require DNA synthesis in extremely high temperatures.

The thermostability of Stoffel DNA Polymerase is about twice as high as the TaqNova DNA Polymerase. It requires a higher MgCl₂ concentration level and lower ionic strength for optimum enzymatic activity. The high magnesium optimum for Nova Stoffel DNA Polymerase reduces the need for magnesium optimization experiments. It increases the ease of multiplex PCR optimization which is the simultaneous amplification of multiple targets in the same reaction.

The Stoffel deletion (5’→3’ exo-) makes the enzyme slightly more thermostable with slightly greater fidelity than full length Taq and is strongly suggested for GC rich and secondary structure templates. The hybridization probes in Light-Cycler assays must not be hydrolyzed, therefore, accuracy of quantitative measurements is improved using the Stoffel Fragment that lacks endonucleolytic activity.

The increased thermal stability of the TaqNova Stoffel may lead to superior amplification of excessively GC-rich templates and templates with secondary structure by allowing denaturation temperatures as high as 98°C.

It is supplied with 20 mM Tris-HCl (pH 8.0 at 25°C), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% (v/v) Nonidet P40, 0.5% (v/v) Tween 20, 50% (v/v) glycerol.

One unit is defined as the amount of enzyme required to incorporate 10 nmol of dNTPs to an insoluble DNA fraction in 30 minutes at 72°C in a 50 μL reaction.

Stoffel fragment is encoded by a modified form of the Thermus aquaticus DNA polymerase gene, which has been inserted into an Escherichia coli host. The modified gene encodes a 540 amino acid fragment lacking the N-terminal 292 amino acid portion of the full-length TaqNova DNA Polymerase.

The activity of Stoffel DNA Polymerase is optimal at low ionic strength, thus 10x Stoffel Buffer is optimized and recommended reaction buffer for this enzyme. The use of a different reaction buffer may significantly reduces the enzyme activity. Stoffel DNA Polymerase has a broad MgCl₂ optimum range (2.5–5mM) and generally requires higher concentrations of magnesium ions than TaqNova DNA Polymerase. A 3 mM MgCl₂ concentration is a suggested starting point for PCR protocol optimization.

Quality Control

DNase contamination is evaluated through extensive PCR reactions.

This is used for applications such as:

• Amplification of short and medium-size sequences
• Diagnostic and multiplex PCR
• Genotyping – shows great performance in genetic mapping using primers with arbitrary sequences (RAPD)
• Allele-Specific Amplification PCR (ASA PCR) – amplification depends on 3’ terminal bases complementing the primer
• Primer extension
• Light-cycler assays due to the lack of 5’-3’ exonuclease in the enzyme