Because this is done in real-time where the x-axis of the graph is time, the first 3 nucleotides dispensed (deoxythymidine triphosphate [dTTP], deoxyguanosine triphosphate [dGTP], and dCTP) do not elongate the growing strand because the first complementary nucleotide is dATP. Thus, there are no peaks on the pyrogram for these dispensed dNTPs (abbreviated T, G, and C in the graph) because they could not be incorporated into the growing strand.
The optimized dispensation sequence generates a cleaner pyrogram with an easily identifiable mutant peak at the second dATP dispensed (Figure 5, A).
The subsequent addition of dATP brings the molecules back in phase because the polymerase replicating the wild-type allele does not advance, whereas the one for the mutant advances by the one nucleotide.
The first nucleotide dispensed, dATP, is not incorporated into either nascent strand, whereas the next one, dGTP, is incorporated twice on the strand for the wild-type allele and only once on the strand for the mutant allele, causing the polymerase molecules to go out of phase.