Sequence Errors in PCR Product (PCR (Polymerase Chain Reaction))

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Sequencing of the PCR product reveals mutations, insertions, deletions, or other sequence variations compared to the original template DNA. Common causes: 1) Low fidelity polymerase lacking 3' to 5' exonuclease proofreading activity 2) Excessive number of PCR cycles allowing error accumulation 3) Unbalanced dNTP concentrations (deviation from equimolar 200 µM each) 4) Excessive Mg²⁺ concentration reducing polymerase fidelity 5) Template DNA damaged by UV exposure or degradation 6) Toxic sequences causing mutations during bacterial propagation Solutions: 1) Use high-fidelity polymerase such as Q5® (NEB #M0491) or Phusion™ (NEB #M0530) with proofreading activity 2) Reduce number of PCR cycles to minimum required for visible product 3) Prepare fresh equimolar dNTP mix at 200 µM each nucleotide 4) Decrease Mg²⁺ concentration in 0.2–1 mM increments 5) Repair damaged template DNA using PreCR® Repair Mix (NEB #M0309) 6) Limit UV exposure time during gel analysis; clone toxic sequences into non-expression or low-copy vectors