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Archaea DNA polymerases as molecular biology tools

- Improvement of Pab Pol B (already marketed by MPBiomedicals under the Isis brand name.

Experiments to optimize PabPol B incubation buffer, indicated that PCR performances and fidelity parameters were the highest in presence of 20mM Tris-HCl, pH = 9,0, 1.5mM MgSO4, 25mM KCl, 10mM (NH4)2 SO4 and 40µM of each dNTP. Under these conditions, the error rate was 0.66x10-6 mutation/nucleotide/duplication (m/n/d), similar to Pfu Pol B, issued from P. furiosus. Fidelity is 40 x higher than classical Taq DNA Pol whose error rate is 24x10-6 m/n/d. Fidelity of PabPol B depends on concentrations of MgSO4, as error rate increases to 1.39x10-6 m/n/d at 3mM MgSO4. Fidelity of PabPol B also depends on dNTPs concentration, as error rate increases from 0.66x10-6 m/n/d to 1.41x10-6m/n/d and 3.05x10-6m/n/d, when 100µM and 200µM of each dNTP are used respectively.

Variation of ionic strength has an obvious effect on fidelity of PabPol B. When KCl concentration decreases from 25mM to 10mM, the error rate increases from 0.66x10-6m/n/d to 1.25x10-6m/n/d. Moreover, if (NH4)2 SO4 concentration is brought to 0 mM, the error rate is 2.0 x 10-6 m/n/d. This value drastically increases to 18.7x10-6 when dNTPs are brought from 40 µM to 200 µM each. The effect of ionic strength was shown to behave similarly on PCR performances of PabPol B. The optimal composition of the PCR incubation buffer has been modified. The decrease of ionic strength, from 25mM to 10mM KCl or from 10mM to 0mM (NH4)2 SO4 , simply leads to inefficient amplification, in parallel with lower fidelity. That dependence of the accuracy and efficiency of elongation and amplification, on ionic strength, have conducted our subsequent studies on PabPol D.

The RF-C/PCNA complex enhanced the speed of PabPol B in vitro. That represents a big step for the development of a Proofreading Ultra Fast PCR complex. Shorter times of elongation still have to be explored.

We have shown that a blend of PabPol B 3exo(+)/ 3exo (-) at a ratio 50/1, amplifies up to 28. kb on lambda DNA, but with peculiar requirements of salt conditions (20 mM Tris-HCl pH = 9.0; 60 mM KCl). No evidence was detected on PCR assays made on a 21.7kb that the presence of either PCNA or/and RF-C, can improve long range PCR, except that the non-specific bands migrating around 3-4kb get weaker in presence of RF-C. In all cases, presence of PCNA generates inhibition even when associated with clamp loader RF-C. Further experiments still have to be made to show whether replication factors can stabilise PabPol B on the DNA strand for full length amplification up to 30-40kb.

- Pab Pol D as molecular biology tool.
PabPol D was shown to prefer primed DNA when performing single run on either circular or linear M13 DNA, but not exclusively, unlike PabPol B. Single run assays with PabPol D were successfully performed in conditions (20mM Bis Tris pH 6.8; 10mM MgCl2; 1mM DTT; 0.4 mg/ml BSA) which cannot be adapted to PCR assays, knowing that a minimal pH = 8.3 is required to avoid depurination at denaturation steps and that 1.5mM MgCl2 is required to reduce misincorporations. First PCR assays using PabPol D were performed on lambda DNA, using the optimised incubation buffer developed for PabPol B. PabPol D showed a bigger flexibility to variation of ionic strength than PabPol B, which was inefficient at 10mM KCl. On the contrary, at 10mM KCl and 10mM Tris HCl. PabPol D remained as efficient as in higher ionic strength. Nevertheless, absence of either KCl or (NH4)2 SO4, drastically reduced the efficiency of PabPol D, but this absence of KCl can be balanced by higher concentration of Tris HCl up to 25mM and (NH4)2 SO4. PCR assays in similar conditions were successfully performed on a human actin gene. In parallel, PabPol D did not stand higher temperatures than 95°C. It presented a much lower sensitivity than PabPol B and failed in Ultra Fast PCR assays, being unable to amplify a 400 bp under the required 45 seconds of elongation per cycle. At this state, no inhibition or activation has been observed when Pab PCNA was added to the PCR using PabPol D, while addition of RF-C still decreased the sensitivity of PabPol D. PabPol D displayed high efficiency in PCR on all types of template, showing less dependance to ionic strength than PabPol B which disliked low ionic strength.

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