Pwo DNA Polymerase

for routine high fidelity amplification of up to 3.5 kb DNA targets Cat. No. 1 664 947 100 units
Cat. No. 1 664 955 2 x 250 units

Description

Pwo DNA polymerase was originally isolated from hyperthermophilic archaebacterium Pyrococcus woesei. The enzyme has a molecular weight of about 90 kD. It is a highly processive 5'–3' DNA polymerase and possesses an 3'–5' exonuclease activity also known as proofreading activity. The enzyme has no detectable 5'–3' exonuclease activity. Pwo DNA polymerase exhibits increased thermal stability with a half life of greater than 2 h at 100°C compared to Taq DNA polymerase with a half life of less than 5 min at this temperature. The inherent 3'–5' exonuclease proofreading activity Pwo results in an over 18-fold increased fidelity of DNA synthesis compared to Taq DNA polymerase. Pwo DNA polymerase generated PCR products are blunt-ended and can therefore be used directly for blunt-end ligation without any pretreatment of the ends. For further information please download the Pwo DNA Polymerase
pack insert.

Application

Fidelity of in vitro DNA polymerization is one of the most important subjects in PCR. For many applications of PCR, where a homogenous DNA population is analyzed (i.e. direct sequencing or restriction endonu-clease digestion), the mutations that are induced by the polymerase during PCR are of little concern. However if only a small amount of template DNA or RNA is used as starting material and if after PCR single DNA molecules are analyzed, PCR artifacts can be a significant problem. Fidelity of DNA polymerization is for instance important for:

cloning of PCR products
study of allelic polymorphism in individual RNA transcripts (1, 2)
characterisation of the allelic stage of single cells (3) or single DNA molecules (4, 5)
characterisation of rare mutations in tissue (6)
characterisation of a population of cells in culture

Operating parameters

Thermal stability: Pwo DNA Polymerase is stable for >2 h at 95°C
Divalent ion requirement: Mg²⁺ (standard concentration, 2.00 mM)
dNTP concentration: at least 200 µM for each dNTP
Caution: Lower deoxynucleotide concentrations can increase fidelity but can also activate the 3'–5' exonuclease proofreading activity, which might degrade prime

Experimental results

Key advantages

Maximum PCR fidelity, because the proofreading activity of Pwo DNA Polymerase excises misincorporated nucleotides
High PCR yield, because the polymerase is very resistant to the high denaturing temperatures used in PCR
Simplifies cloning of PCR products because it generates blunt-ended products which can be directly used for blunt-end ligation

Note: Pwo DNA Polymerase cannot amplify targets longer than approx. 3.5 kb. For high fidelity amplification of longer templates use one of the Expand PCR Systems.

Roche Contact Information

Roche

Roche Diagnostics Corporation
P.O. Box 50414
9115 Hague Road
Indianapolis, IN 46250-0414 USA

Customer Service: 800-262-1640

Tech Support: 800-262-4911

Fax Number: 800-428-2883

Web Site: http://www.roche-applied-science.com

Pwo DNA Polymerase