Sequence Errors at PCR Product Termini (PCR (Invitrogen Guide))

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Sequencing shows errors, truncations, or unexpected sequences specifically at the 5′ or 3′ ends of amplified products. Common causes: 1) Primer design with direct repeats causing sequence misalignment at product ends 2) Low primer quality with non-full-length oligos truncated at 5′ ends 3) Contaminating nucleases or exonucleases degrading product termini 4) UV damage from gel visualization Solutions: 1) Avoid direct repeats within primer sequences to prevent misalignment at PCR product ends 2) Order PCR primers with purification to remove truncated non-full-length oligos 3) Use molecular-grade, nuclease-free reagents; set up reactions on ice to minimize exonuclease activity 4) Use long-wavelength UV (360 nm) with limited exposure or longer-wavelength excitation dyes 5) Sequence both DNA strands with duplicate samples to confirm reliability