title: Multivariant confirmation of sickle cell disease using a non-radioactive minisequencing reaction.
abstract: A non-radioactive solid-phase minisequencing method for confirmation of abnormal hemoglobin variants causing sickle cell disease has been developed. In this method amplified 5'-biotinylated target sequences containing normal and mutation sites are immobilized onto streptavidin-coated microplates. Detection primers corresponding to target sequences are annealed immediately adjacent to the mutation site and single-step, hapten-labeled nucleotide primer extension reactions are performed. The incorporation of the labeled nucleotide is detected through immunological reaction with an enzyme-labeled anti-hapten conjugate and a substrate. The method enables confirmation of mutations of the beta-globin gene variants (Hbs S, C, E D-Punjab, O-Arab) and the alpha-globin gene variant (Hb G-Philadelphia). The test was evaluated using characterized dried blood spot specimens (n = 100) The advantages of the procedure are easy performance and objectiveness. The non-radioactive minisequencing assay will prove helpful for genotyping in neonatal screening for hemoglobinopathies and in prenatal and pre-implantational diagnostics.
authors.affiliation: Labsystems Research Laboratories, Helsinki, Finland.
authors.forename: T
authors.initials: T
authors.lastname: Tuuminen
authors.forename: H
authors.initials: H
authors.lastname: Ingman
authors.forename: B L
authors.initials: BL
authors.lastname: Therrell
authors.forename: A
authors.initials: A
authors.lastname: Kallio
mesh_terms: D000755:Anemia, Sickle Cell / Q000175:diagnosis* / Q000235:genetics; D003937:Diagnosis, Differential; D004797:Enzyme-Linked Immunosorbent Assay; D005470:Fluorometry; D006455:Hemoglobins, Abnormal / Q000235:genetics*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D016037:Single-Blind Method
chemical_list: D006455:Hemoglobins, Abnormal