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My understanding is that PCR is carried out until a fluorescent nucleotide halts replication. The segments of DNA are fed through the capillary tube based on size, sifting through the segments from smallest to largest; subsequently, the sequence is read from one end to the other.

I'm confused as to how we know that each nucleotide in the sequence has been accounted for, or if a nucleotide has been counted twice. If two fragments of the same size, ending with the same ddNTP, pass through the capillary tube at slightly different times, how do we know if that specific nucleotide hasn't been double-counted?

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In chain-termination sequencing, a population of molecules is detected as opposed to a single one. The readout looks like this:

enter image description here [ http://seqcore.brcf.med.umich.edu/doc/dnaseq/trouble/badseq.html ]

The peaks represent intensity of the four different fluorophores at the detector. Every fragment that is terminated at the same spot will have the same fluorophore and the same length. Because capillary gel electrophoresis separates molecules by size, these fragments will pass through at approximately the same time. "Approximately the same time" is why you see broad-ish and overlapping peaks on the readout as opposed to sharp and distinct lines.

Ideally, the readout will appear like the left half of the image, where there's minimal overlap between peaks. However, often you will see overlapping peaks, as in the right half of the image. The link above explains many reasons for why this occurs. Whether or not a peak is correctly assigned is determined by Phred scores, which is a statistical analysis of the shape and resolution of each peak.

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    $\begingroup$ I would like to also add that this is precisely why we use a capillary tube. The small surface area makes it ideal for resolution of small things like nucleotides. That is the whole idea behind chromatography. Trying to separate things that may only be modestly different in size (or charge) to separate. The more resolution something has, the farther each of those peaks will be from each other, and the sharper each peak will be, i.e. a very sharp line instead of a broad peak. $\endgroup$
    – jwillis0720
    Commented Dec 25, 2014 at 8:18
  • $\begingroup$ how do we know if a certain nucleotide hasn't been accounted for? (e.g., what if a fragment that terminated at nucleotide #70 was not produced?) $\endgroup$
    – Alzeon
    Commented Dec 25, 2014 at 23:36
  • $\begingroup$ @Alzeon Statistically, that seems unlikely given that there is billions of DNA molecules in a sequencing reaction. $\endgroup$
    – canadianer
    Commented Dec 26, 2014 at 0:16

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