Читать книгу PCR – diagnostics - Aizhan Zhussupova - Страница 5

There are 2 common polymerases used for PCR, Taq and Pfu. The typical concentration is 2.5-5.0 units of enzyme per 100 pL reaction for targets below 10 kb. Larger targets may require up to 10 units of enzyme per 100 pL of reaction volume. Note: 1 mL 1000 jL, 1 jg – 1000 ng. The most critical parameter affecting yield of PCR product is the extension time. Taq polymerase can amplify DNA faster than Pfu polymerase but it is not nearly as efficient. Normal extension time for Pfu polymerase is 2 min/kb of template whereas Taq polymerase can be as low as 0.5-1 min/kb.

However, the mutated PCR product percentage per 1 kb fragment is 2.6 % for Pfu polymerase and 16 % for Taq polymerase. 72 °C is the optimal temperature for extension, as Pfu is most active and efficient under this condition. On December 22, 1989 the journal Science awarded Taq polymerase (and PCR) its first «Molecule of the Year». The Saiki R.K., Gelfand D.H., Stoffel S. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase // Science 1988; 239 (4839): 487-491. Paper became the most cited publication in biology for several years.