title: Direct detection of beta thalassemic mutations: use of biotin-labelled allele specific probes.
abstract: Mutations at positions beta IVS1-6, beta IVS1-110, and beta 39 of the beta globin gene are responsible for the three most common thalassemic genes in the Mediterranean population. The polymerase chain reaction (PCR) was employed to amplify a 536 base pair segment surrounding this region. Nonradioactive labelling of an oligonucleotide probe, specific for the beta IVS1-110 mutation, was achieved by incorporation of biotin-16-dUTP into a standard 3'-end labelling procedure. This probe was subsequently hybridized with the PCR amplification product and permitted detection of the mutant gene in a homozygous beta thalassemic child by a simple colour detection method using a streptavidin-alkaline phosphatase conjugate and NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) substrate. A known cloned mutant gene was similarly detected. Results could be obtained within 48 hr. These findings suggest that such an approach could provide a rapid and specific means for detection of beta thalassemic mutations without the need for radioactive probes.
authors.affiliation: Thalassemia Laboratory, Department of Pathology, Royal Women's Hospital, Carlton, Australia.
authors.forename: J G
authors.initials: JG
authors.lastname: Hendy
authors.forename: M N
authors.initials: MN
authors.lastname: Cauchi
mesh_terms: D000483:Alleles; D001710:Biotin; D002648:Child; D005819:Genetic Markers / Q000032:analysis; D006720:Homozygote; D006801:Humans; D009154:Mutation; D013789:Thalassemia / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D001710:Biotin