OPTIMIZATION AND PROPERTIES OF A UHG FOR GENOTYPING OF HEMOGLOBIN-S AND HEMOGLOBIN-C

The use of universal heteroduplex generators (UHG) as an effective mea ns of screening for specific mutations has been previously reported. H ere, we report the optimisation of a UHG system used for the rapid and simple detection of sickle cell hemoglobinopathies, HbS and HbC. The test involves heteroduplex formation between between polymerase chain reaction (PCR) amplified beta-globin gene first exon sequences, and a UHG. The UHG is a synthetic DNA molecule homologous to HbA but which c ontains a small deletion adjacent to the HbS and HbC mutation sites in codons 5 and 6. Heteroduplexes are resolved on nondenaturing polyacry lamide minigels and are diagnostic of HbS and HbC in homozygous and he terozygous individuals. A blind trial of UHG genotyping involving elev en previously sequenced DNAs showed complete concordance between metho ds. In addition, we identi fled a characteristic heteroduplex banding pattern for the H2H silent mutation (CAC --> CAT) in codon 2. (C) 1995 Wiley-Liss, Inc.