Effect of template complexity on the allele-specific extension reaction on primer arrays. (A)Average signal intensity ratios and signal intensities, obtained when seven mutations amplified in a single 7-plex PCR (indicated by 7), were genotyped directly and at increased complexity in the presence of 22 additional (indicated as 7 + 22 = 29) and 99 additional (106) PCR fragments in similar molar amounts. A mixture containing all the other PCR fragments (99*), except the 7-plex PCR product, was included as control for the template-independent extension or cross reaction with nonspecific targets. The average signal intensity ratio (reflecting specific vs. misincorporation of CY5-dNTPs) and signal intensity from quadruplicate genotyping reactions were given the value 1 for the 7-plex PCR product. The signal intensity ratios and signal intensities for the complex mixtures are given in relation to this value. The standard deviations were calculated for the overall change in signal ratio and signal intensity for the seven analyzed mutant sites. (B) Agarose gel image of the mixtures of PCR products analyzed in the experiment described in A.
