Direct detection of β 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.

Mutational Analysis of Beta-Thalassemia Cases from the Aegean Region of Turkey Using an Allele-Specific Oligonucleotide Hybridization Technique

“…Using PCR, it has been possible to detect beta-thalassemia mutations by a variety of techniques such as dot-blot hybridization with ASO probes, restriction enzyme analysis, chemical cleavage of mismatched duplexes, denaturing gradient gel electrophoresis and allele-specific PCR by color amplification [9, 10, 11, 25, 26, 27, 28]. Over the past 15 years, ASO hybridization has been extensively used as probes to diagnose genetic and infectious diseases.…”

“…This heterogeneity makes molecular diagnosis of this disorder time-consuming despite the availability of the polymerase chain reaction (PCR)-dot blot hybridization techniques [8, 9, 10, 11]. The aim of our study was to determine the point mutations of beta-thalassemia patients from the Aegean region of Turkey by using an allele-specific oligonucleotide (ASO) hybridization technique and to evaluate the efficiency of this technique in our patients.…”

Biophysical Studies of the Translation Initiation Pathway with Immobilized mRNA Analogs

“…End labelling with biotinylated residues -one method for the targeted introduction of biotinylated residues at the 3'-end of an RNA molecule relies on the ability of certain enzymes to append nucleotides to existing RNA sequences. One published method employs poly(A) polymerase to incorporate biotinylated adenine moieties at the 3'end of a transcript previously generated by standard in vitro transcription (14). In contrast to the oligo-capture method, the biotin moieties are covalently attached to the RNA, and the strength of the bond to the solid support thus corresponds to the strength of the biotin-Streptavidin bond, i.e.…”

Usefulness of Determination of Factor VIII Intron 7 Polymorphism by a Non-Radioactive Technique for Carrier Detection of Hemophilia A

“…The samples were heated for 5 min at 95° C, put on ice and spotted on nitrocellulose filters with a Bio-Rad Slot-Blot apparatus. The filters were then hybridized with the oligonucleotide probes 11.9 (5'-GCAAGACACTCTGACATTG-3') and 11.1 (5'-GCAAGACACTCTAACATTG-3') (Applied Biosystems, Warrington, UK), non-radioactively labeled by substitution of a Biotin-lldUTP nucleotide analogue (5). After hybridization with the two 19 bp allele-specific oligonucleotides, filters were washed twice with 2 × SSPE (300 mmol/1 NaCl, 20 mmol/1 NaHP0 4 , 2 mmol/1 EDTA pH 7.4), 0.1% SDS for 10 min at 58° C and 56° C, respectively, and the streptavidin-alkaline phosphatase reaction developed by the DNA detection system (BRL, Uxbridge, England).…”