Sequence Errors Within PCR Product Body (PCR (Invitrogen Guide))

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Sequencing reveals point mutations, insertions, or deletions within the amplified fragment that are not present in original template. Common causes: 1) Low-fidelity DNA polymerase lacking proofreading activity 2) Excess Mg2+ concentration favoring nucleotide misincorporation 3) Unbalanced dNTP concentrations (non-equimolar dATP, dCTP, dGTP, dTTP) increasing error rate 4) High number of PCR cycles increasing mismatched nucleotide incorporation 5) UV damage from short-wavelength (254–312 nm) light box exposure Solutions: 1) Use DNA polymerases with exceptionally high fidelity for cloning, sequencing, and mutagenesis applications 2) Review and reduce Mg2+ concentration to prevent misincorporation 3) Ensure equimolar concentrations (typically 200 µM each) of dATP, dCTP, dGTP, and dTTP 4) Reduce cycle number without lowering yield; increase template input to avoid excessive cycles 5) Use long-wavelength UV (360 nm) light box with minimal illumination time; alternatively use longer-wavelength excitation dyes 6) Sequence both DNA strands with duplicate samples to verify results