Direct detection of beta thalassemic mutations: use of biotin-labelled allele specific probes
| Autor: | J. G. Hendy, M. N. Cauchi |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Genetic Markers
Mutation education.field_of_study Base pair Population Homozygote Biotin Hematology Biology medicine.disease_cause Molecular biology law.invention law medicine Humans Thalassemia Globin Beta (finance) Molecular probe education Oligomer restriction Child Polymerase chain reaction Alleles |
| Zdroj: | American journal of hematology. 34(2) |
| ISSN: | 0361-8609 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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