OPTIMIZED PCR LABELING IN MUTATIONAL AND MICROSATELLITE ANALYSIS

To optimize the labeling and visualization olf PCR products we tested different variables, including deoxynucleotide concentration and ratio , dilution of labeled product, number of PCR cycles, and use of oneste p or nested labeling protocols. Labeling was achieved using a fixed am ount of labeled dATP, whose relative specific activity was varied by a dding increasing amounts of cold dATP. Optimal PCR-labeling intensity was reached at dATP concentrations between 0.9 and 7.0 mu mol/L, with a peak at 1.8 mu mol/L. This concentration corresponded to an optimal ratio between the increase in specific activity and the decrease in DN A yield. Nucleotide imbalances >1:2 were not advantageous. Mutational analysis by single-strand conformational polymorphism (SSCP) was used to validate PCR-labeling protocols. The limiting nucleotide concentrat ions did not affect SSCP. Clear SSCP patterns were obtained using DNA templates of different sizes derived from several genes. SSCP patterns obtained using one-step or nested PCR-labeling protocols were equival ent and were visualized after overnight exposure, using [alpha(35)S]dA TP as the label. Dilutions of labeled products ranging between 1:10 an d 1:2.5 influenced SSCP patterns, and the lowest dilution tested produ ced better-defined and more-intense signals. Optimized SSCP conditions allowed the detection of novel and previously characterized nucleotid e variants. Clear microsatellite typing was also obtained using optimi zed protocols and [alpha(35)S]dATP as the label.