Optimisation and properties of a UHG for genotyping of hemoglobins S and C

The use of universal heteroduplex generators (UHG) as an effective means of screening for specific mutations has been previously reported. Here, 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 contains a small deletion adjacent to the HbS and HbC mutation sites in codons 5 and 6. Heteroduplexes are resolved on nondenaturing polyacrylamide minigels and are diagnostic of HbS and HbC in homozygous and heterozygous individuals. A blind trial of UHG genotyping involving eleven previously sequenced DNAs showed complete concordance between methods. In addition, we identified a characteristic heteroduplex banding pattern for the H2H silent mutation (CAC-->CAT) in codon 2.