EVALUATION OF RUNNING CONDITIONS FOR SSCP ANALYSIS - APPLICATION OF SSCP FOR DETECTION OF POINT MUTATIONS IN THE LDL-RECEPTOR CONE

We have performed analyses of single-strand conformation polymorphisms (SSCP) of the promoter region and the translated parts of the 18 exon s of the low-density lipoprotein receptor (LDLR) gene. DNA from 20 unr elated familial hypercholesterolemia (FH) patients was studied. Four d ifferent running conditions were used for the nondenaturing gel electr ophoresis to systematically evaluate how differences in the running co nditions affect the sensitivity of the assay. These conditions were 15 W, 40 W and 50 W in the absence of glycerol, and 50 W in the presence of 10% glycerol. SSCP analyses of the 18 PCR fragments for the 20 sub jects revealed a total of 46 genotypes at 15 W, 45 at 50 W, 42 at 40 W , and 41 at 50 W with 10% glycerol. A total of 53 different genotypes were observed when the results of the four conditions were considered together. Assuming that the four conditions together detected 100% of the different genotypes, the sensitivity of the four individual condit ions ranged between 87% (15 W) and 77% (50 W with 10% glycerol). There were marked differences among the different running conditions to det ect abnormal SSCP patterns of individual exons. Therefore, different c onditions should be used for the different exons of the LDLR gene.