What is high-fidelity PCR?


"For downstream applications like cloning, mutation analysis, and sequencing, PCR sequence accuracy is critical. You can achieve it, with high-fidelity DNA polymerases, and feel confident in your experimental results. That accuracy is referred to as fidelity—how accurate a DNA polymerase can replicate target sequences without. Fidelity is determined by the polymerase’s accurate reading of template sequences and preferential binding of correct nucleotides during synthesis. In addition, high-fidelity polymerases have a proofreading capability called 3′ to 5′ exonuclease activity. It recognizes misincorporated nucleotides and replaces them with correct ones. Fidelity is often expressed as a relative value in comparison to Taq DNA polymerase. This is determined by measuring error rates in nucleotide incorporation, using methods like: colony-screening assays, Sanger sequencing, and next-generation sequencing. When you're comparing fidelity between different polymerases, look for values measured using the same method and reaction conditions. In this example, the fidelity of the DNA polymerases was measured by next-generation sequencing. It's a highly sensitive method. Notice how Invitrogen Platinum SuperFi II DNA polymerase shows more than 300 times higher fidelity than Taq DNA polymerase. Consequently, for downstream applications like cloning and sequencing, PCR amplicons of Platinum SuperFi II DNA polymerase would contain extremely low errors. In summary, you can have the utmost confidence in PCR sequence accuracy. Simply choose a DNA polymerase with the highest fidelity."

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