Fluorescent hot-stop PCR.

Accuracy of the quantitation of the relative abundance of 2 DNA species by the newly developed fluorescent hot-stop PCR was verified by performing a number of PCR amplifications using different molar ratios of pT-Aα-wt and pT-Aα-Arg149stop plasmids as template. Labeled PCR products were subjected to allele-specific digestion withMaeIII and quantitated on an Abi-310 Genetic Analyzer. Areas of fluorescence peaks corresponding to the mutant and wild-type restriction fragments were measured by GeneScan Analysis software 3.1. (A) GeneScan Analysis windows showing fluorescence peaks corresponding to wild-type (280 nt) and Arg149stop mutant (309 nt) single-stranded fragments. The x-axis represents GeneScan data points and the y-axis represents fluorescence units (FUs). (B) Results of the semiquantitative analysis. The reported values correspond to the peak areas, setting the more abundant mRNA species of each experiment equal to 100%.