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.
Wood
N
N
University of Bristol Department of Transplantation Sciences, Bristol Homoeopathic Hospital, Cotham, England.
Standen
G
G
Old
J
J
Bidwell
J
J
D000755:Anemia, Sickle Cell / Q000175:diagnosis / Q000235:genetics; D001483:Base Sequence; D002986:Clinical Trials as Topic; D005091:Exons; D005838:Genotype; D005914:Globins / Q000235:genetics; D006444:Hemoglobin C / Q000235:genetics*; D006451:Hemoglobin, Sickle / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry* / Q000235:genetics*
D006451:Hemoglobin, Sickle; D009692:Nucleic Acid Heteroduplexes; D005914:Globins; D006444:Hemoglobin C