DNA polymerases

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The key to PCR is the DNA polymerase that synthesizes new DNA strands. General characteristics of DNA polymerases include: • Thermostability • Processivity • Speed • Fidelity, and • Specificity Some DNA polymerases can withstand high temperatures longer than others over the course of PCR cycling. This is often described as the enzyme’s thermostability or half-life at a specific temperature. All DNA polymerases have 5-prime to 3-prime polymerase activity to synthesize new DNA strands. The number of nucleotides the DNA polymerase can incorporate without dissociating from the template defines its processivity. Processivity generally depends on the enzyme’s affinity for the DNA template during amplification. High processivity often contributes to the DNA polymerase’s speed, as well as resistance to inhibitors. The higher the processivity of a DNA polymerase, the faster it can synthesize the new strands. Fast enzymes are especially beneficial in amplifying long templates. Some DNA polymerases also have 3-prime to 5-prime exonuclease activity to remove mismatched nucleotides. This is also called proofreading activity. Proofreading DNA polymerases are used for high-fidelity PCR because they make fewer mistakes during DNA amplification. DNA polymerases without 3-prime to 5-prime exonuclease activity are known as non-proofreading or low-fidelity PCR enzymes. They are mainly used for standard PCR. One common issue encountered in PCR is low specificity or background amplification. PCR specificity can be improved by modifying DNA polymerases to be inactive at room temperature during reaction setup, but activated at a higher temperature at the start of PCR cycling. This approach is commonly known as hot-start PCR.

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