Enzymatic mismatch cleavage and fluorescence detection of potato polymorphisms. IRDye 700 image shown for optimization of input genomic DNA for PCR amplification (A). DNA amounts of 25, 10, 4, 1.5, 0.5, 0.25, 0.1, 0.04, 0.03, 0.015, 0.025 and 0.01 ng were tested in PCR reactions with fluorescently labeled primers (listed on top of image). After PCR, samples were denatured and annealed to form heteroduplexes between polymorphic amplicons. Mismatches were cleaved by incubating samples with a crude celery extract containing the CEL I enzyme. Samples were resolved by denaturing polyacrylamide gel electrophoresis and visualized with the LI-COR DNA analyzer. Bands represent nucleotide polymorphisms. Molecular weights estimated using the GelBuddy program  are listed to the left of the image. Polymorphisms were also detected in pooled samples. At each DNA concentration, two samples of the Draja (Dr), Sponta (S) and Diamond (D) genotypes were tested (Panel B, image from lanes marked with a horizontal box in panel A). True polymorphisms produce a band in the IRDye700 image channel and a band in the IRDye800 image channel whose molecular weights sum to the weight of the full length PCR product. The high background and low band resolution observed in sample Draja1 is indicative of genomic DNA degradation.