High resolution melting analysis of RB1 exons 21, 22 and 23. (A) To detect heterozygous sequence changes in RB1, constructs were created that encode either exon 21, 22 or 23 with or without single base pair substitutions. Equal quantities of these constructs were mixed, PCR amplified, and subjected to melting analysis alongside human genomic DNA samples. (B) Each DNA sample was analyzed in duplicate and the differences in melting profiles are plotted as a normalized difference plot. Representative difference plots for each exon are shown. In each case the pair of heterozygous plasmid controls (shown in red) is readily distinguishable from the homozygous human samples (shown in blue) by the Gene Scanning software as it assigns the colours.