title: Reverse dot-blot detection of the African-American beta-thalassemia mutations.
abstract: DNA-based diagnosis of the beta thalassemias provides accuracy to newborn screening genetic counseling, and prenatal diagnosis. However, the use of polymerase chain reaction (PCR)-based methods is challenged by the great number of different-beta-thalassemia mutations that exist even within defined ethnic groups. In this regard, the reverse dot-blot method offers a means of screening for several mutations with a single hybridization reaction. We have applied the reverse dot-blot method to the detection of the beta-thalassemia mutations of African-Americans. We used two biotin-labeled primer pairs in a duplex reaction to amplify and label two beta-globin target DNA fragments that encompass all known African-American beta-thalassemia mutations. The PCR products were denatured and hybridized to polyT-tailed, membrane-fixed, allele-specific probe pairs for the hemoglobin (Hb) S, Hb C, and 14 beta-thalassemia mutations and their corresponding wild-type sequences. Seven common mutations plus Hb S and Hb C were included on one diagnostic strip, and seven less common beta-thalassemia mutations were included on another strip. Carefully controlled, high stringency hybridization allowed accurate distinction of these alleles. Reverse dot-blot diagnosis of the less common beta-thalassemia mutations precludes the need for alternative, more technically challenging methods. This method provides a rapid, accurate method for diagnosis of beta thalassemia among African-Americans and other ethnic groups in which beta thalassemia occurs.
authors.affiliation: Department of Medicine, Chulalongkorn Hospital, Chulalongkorn University, Bangkok, Thailand.
authors.forename: P
authors.initials: P
authors.lastname: Sutcharitchan
authors.forename: R
authors.initials: R
authors.lastname: Saiki
authors.forename: T H
authors.initials: TH
authors.lastname: Huisman
authors.forename: A
authors.initials: A
authors.lastname: Kutlar
authors.forename: V
authors.initials: V
authors.lastname: McKie
authors.forename: H
authors.initials: H
authors.lastname: Erlich
authors.forename: S H
authors.initials: SH
authors.lastname: Embury
mesh_terms: D001483:Base Sequence; D044383:Black People / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005914:Globins / Q000235:genetics*; D006444:Hemoglobin C / Q000235:genetics; D006451:Hemoglobin, Sickle / Q000235:genetics; D006453:Hemoglobinopathies / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D017086:beta-Thalassemia / Q000175:diagnosis* / Q000235:genetics; D001741:Black or African American
chemical_list: D017931:DNA Primers; D006451:Hemoglobin, Sickle; D005914:Globins; D006444:Hemoglobin C