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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.
What are the requirements for sequencing primers?
General requirements for sequencing primers:
(1) Specific binding to the sequencing template, with no more than 4 base mismatches.
(2) Must not contain mixed bases.
(3) Length: 17–25 bases.
(4) High purity, preferably PAGE purified.
(5) Dissolved in ddH₂O, not TE buffer.