Editing Mutagenic PCR

exo− Pfu DNA polymerase substantially lacks the 3′ to 5′ exonuclease activity, and therefore is error prone. exo−Pfu DNA polymerase exhibits an error prone rate of 4.7×10&lt;/span&gt;−5 which is about 6 fold higher than the error rate of Taq DNA polymerase (Cline, J., Braman, J. C. and Hogrefe, H. H. (1996) PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24(18):3546-3551).

[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC545472/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC545472/]

exo- Pfu D473G

, Pfu-Pol B exo– (D141A/E143A) and Pfu-Pol B exo– error prone (D141A/E143A/D473G): error rate is 1.8 10-5


Loop mutations of Pfu-Pol were prepared using ‘overlap extension PCR’ (21) on the wild-type exo− (D215A) gene, excised from the plasmid pET-17b[Pfu-Pol]. Altered genes were inserted into pET-17b and the protein overexpressed in E.coli BL21 (DE3) pLysS. Purification involved a heat step and chromatography on DEAE–Sephacel and Heparin–Sepharose (20). All protein samples were >95% pure by Coomassie blue stained SDS–PAGE. Concentrations were determined by absorbance at 280 nm with an extinction coefficient of 1.2 × 105 M−1 cm−1 ([http://ca.expasy.org/cgi-bin/protparam http://ca.expasy.org/cgi-bin/protparam]). Determination of the specific activity of Pfu-Pol used an assay based on the incorporation of dNTPs into activated calf thymus DNA (22). Approximately 0.5–1.5 μg Pfu-Pol, in a volume of 15 μl, was added to 135 μl of pre-warmed reaction buffer [20 mM Tris–HCl (pH 8.0), 10 mM KCl, 2 mM MgSO4, 10 mM (NH4)2SO4, 0.1% Triton X-100, 100 μg/ml BSA, 66.67 μM each dNTP, 200 μg/ml activated calf thymus DNA and 1 μCi [α-32P] dATP (3000 Ci/mmol) and incubated at 72°C.&nbsp;
[[Category:QPCR RT-PCR]]