Sequence signatures identify different markers in a mixed DNA solution. (A) Sequences of the six genetic markers pCaVIT-1 to -6. The blue A on the left is the last common adenine shared between all pCa-VIT1-6 plasmids upstream of the polymorphic region. Adenine residues marked in green are theoretically expected to yield discriminating peaks; ther A residues are indicated in black. The expected position (in bp) of each base from the fluorescent sequencing primer is indicated at the top. (B) Experimentally observed positions of discriminating A-peaks (named VIT1.1 to VIT6.2 at the top) on sequence traces (not shown) from individually sequenced markers. Sequences of all six markers are slightly distorted in order to match the actual position of A peaks observed on individual electropherograms. The red A residues were each confirmed to yield a discriminating peak. Though not theoretically predicted to do so, the red-boxed As proved to yield discriminating peaks experimentally. The remaining green As failed to produce discriminating-peaks. Sequencing reactions of pCa-VIT1-6 were repeated three times, and the observed position of all peaks was highly reproducible: +/- 0.2 bp among repeats. At the bottom, the number in blue is the average position of the last A common to all sequences upstream of the differential markers. The numbers in red are the average positions of the observed discriminating peaks. (C) Single-letter Sequencing electropherogram from a mixed DNA solution containing all 6 markers. A mix of all 6 pCaVIT1-6 plasmids in equal amounts was used as a template for PCR and subsequent single-letter sequencing. All discriminating-peaks defined in B are indicated by arrows on the electropherogram. The shaded blue peak corresponds to the last common A. The scale at the top indicates the nucleotide position relative to that of the sequencing primer. The scale on the left is used for scoring the height of the peaks in arbitrary units provided by the STRand program. The two purple peaks correspond to molecular weight markers. # These peaks are artefacts. ‡ Though appearing as discriminating, these peaks actually overlap small artefactual peaks observed on at least one electropherogram from individually sequenced markers (not shown). They are thus not used further.