Direct detection of β thalassemic mutations: Use of biotin‐labelled allele specific probes

Mutations at positions βIVSS 1–6 , βIVS 1–110 , and β 39 of the β 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 βIVS 1–110 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 β 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 β thalassemic mutations without the need for radioactive probes.