ProofStart DNA Polymerase

ProofStart DNA Polymerase is a hot-start proofreading polymerase uniquely modified to prevent primer degradation during PCR setup, providing robust, high-fidelity PCR.

Features and benefits

• Hot-start enzyme prevents primer degradation, ensuring robust PCR
• Efficient exonuclease activity for high fidelity
• Specially developed buffer for minimal PCR optimization
• Easy handling

Principle

Proofreading enzymes utilize a 3'5' exonuclease activity to remove incorrectly incorporated bases. However, this activity can also degrade primers during setup and the start of PCR, which can cause nonspecific priming, smearing, or even failure to amplify any product at all (see figure "Comparison of Different Proofreading Polymerases").

ProofStart DNA Polymerase, isolated from a new Pyrococcus spp. source, is chemically modified to inactivate both the polymerase and the exonuclease activity. A simple hot start activates both enzymatic components, allowing PCR to start with high proofreading ability. This unique modification ensures that primers remain intact during setup, preventing mispriming (see figure "Primer Degradation Eliminated with ProofStart DNA Polymerase"). The robust performance of ProofStart DNA Polymerase gives trouble-free and highly specific amplification. Enzyme fidelity varies from 2.3 x 10–6 to 3.6 x 10–6 misincorporated bases per PCR product doubling. These values are more than 10 times better than Taq DNA polymerase, as determined by several measurements and are comparable to or better than those of other proofreading enzymes (see figure "Very Low Error Rate of ProofStart DNA Polymerase").

Comparison of Different Proofreading Polymerases

PCR was carried out using ProofStart DNA Polymerase (QIAGEN), proofreading polymerases from the indicated suppliers, or a commercial Taq/proofreading polymerase mixture from Supplier R (R’). Parallel reactions were performed following suppliers’ recommendations, using 200 ng HeLa genomic DNA. A 1.5 kb fragment of the human hypoxanthine ribosyl transferase gene was amplified in 35 PCR cycles. M: markers.

Primer Degradation Eliminated with ProofStart DNA Polymerase

Schematic of primer degradation by proofreading polymerases. Proofreading enzymes contain an exonuclease site (E) and a polymerase site (P). M: markers.

Very Low Error Rate of ProofStart DNA Polymerase

Error rates were determined for ProofStart DNA Polymerase, proofreading enzymes from Suppliers SIII and N, a commercial Taq/proofreading polymerase mixture from Supplier R (R’), and Taq DNA polymerase from Supplier AII. Rates were determined using a beta-galactosidase PCR mutation assay and are reported as the fraction of misincorporated bases per PCR product doubling.

As with other buffer formulations from QIAGEN, ProofStart PCR Buffer contains a uniquely balanced combination of KCl and (NH4)2SO4, providing stringent primer-annealing conditions over a wide range of annealing temperatures and Mg2+ concentrations (see figures "Wide Annealing-Temperature Window and Tolerance to Variable Mg2+ Concentration"). Q-Solution provided with ProofStart DNA Polymerase, improves the amplification of difficult templates, such as those containing a high degree of secondary structure or a high GC content (see figure "Amplification of Difficult Templates with Q-Solution").

ProofStart DNA Polymerase specifications

Concentration 2.5 units/µl 5'3' exonuclease activity No 3'5' exonuclease activity Yes Extra A addition (terminal transferase activity) Minimal

Half-life >4 hours at 95°C Substrate analogs Use of dUTP is not recommended Nuclease contamination No RNase contamination No

Applications

ProofStart DNA Polymerase provides the lowest error rates as well as optimal PCR product yield for applications such as:

• Cloning
• Site-directed mutagenesis
• Mutation analysis

操作手册: ProofStart PCR Handbook 02/02 (PDF version, 145 KB)

For high-fidelity PCR