Maximizing PCR and RT-PCR Success

4 PCR Brochure 10/2016 High-fidelity DNA polymerase Unlike standard DNA polymerases (such as Taq DNA polymerase), high-fidelity PCR enzymes generally provide a 3 ’–5 ’exonuclease activity for removing incorrectly incorporated bases. High-fidelity PCR enzymes are ideally suited to applications requiring a low error rate, such as cloning,sequencing,andsite-directedmutagenesis.However, the 3 ’–5 ’exonuclease activity can degrade primers during PCR setup and the early stages of PCR. Nonspecific priming caused by shortened primers can result in smearing or amplification failure — especially when using low amounts of template (Figures 3 and 4). It should be noted that the proofreading function often causes high-fidelity enzymes to work more slowly than other DNA polymerases. In addition, the A-addition function required for direct UA- or TA-cloning is strongly reduced, resulting in the need for blunt-end cloning with lower ligation and transformation efficiency. These limitations have been overcome with HotStar HiFidelity DNA Polymerase, which incorporates a hot-start activation to its exonuclease activity, providing reliable and sensitive results, in contrast to enzymes from other suppliers (Figures 3 and 4). In addition, this enzyme also adds an A over- hang during the final extension step allowing direct UA- or TA-cloning. T a q D N A P o l y m e r a s e M M H o t S t a r T a q P l u s H o t S t a r T a q S u p p l i e r T I * S u p p l i e r T I * S u p p l i e r R M 10 1 10 1 10 1 10 1 10 1 ng Primer– – dimers Q I A G E N S u p p l i e r S S u p p l i e r T I * S u p p l i e r T I * S u p p l i e r R Figure 2. Highest specificity with HotStarTaq Plus Polymerase. PCR was carried out using QIAGEN HotStarTaq Plus, HotStarTaq and Taq DNA Polymerases and three hot-start PCR enzymes from the indicated suppliers. Parallel reactions were performed following the suppliers’ recommendations, using 50 ng human genomic DNA. A 1.5 kb fragment of the human CFTR gene was amplified in 35 PCR cycles. M: markers. Figure 3. The importance of optimized high-fidelity systems for sensitive and the reliable PCR. High-fidelity PCR was performed using HotStar HiFidelity DNA Polymerase Kit (QIAGEN) and the four high-fidelity PCR enzymes from the indicated suppliers. A 2.3 kb fragment of the human IL9R gene was amplified from the indicated amounts of genomic DNA in 40 PCR cycles. M: marker. The HotStar HiFidelity DNA Polymerase Kit provided highly sensitive results using 1 ng template. * Two different enzymes from Supplier TI . M 101101101101101 ng

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