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Why do overlapping peaks often occur when sequencing PCR products?
Overlapping peaks (mixed sequences) in PCR product sequencing generally occur for the following reasons:
(1) The PCR template is impure or the PCR primers lack specificity, resulting in amplification products that include fragments of similar length to the target fragment, which cannot be separated even by gel electrophoresis. Sequencing such PCR products results in overlapping peaks.
(2) Structural reasons can cause overlapping peaks in PCR product sequencing. The presence of polyA/G/C/T tracts or other complex structures of unknown origin can lead to overlapping sequencing results.
Why must a sequencing primer bind specifically to the DNA template?
The key to successful sequencing is that the sequencing primer has only one binding site on the DNA template molecule. If the sequencing primer has more than one binding site on the DNA template, it will cause the primer to initiate extension at multiple sites during the sequencing reaction. This will manifest as double peaks or messy peaks on the sequencing chromatogram, making the sequence unreadable.