Vol. 86, pp. 6230–6234, August 1989 | RANDALL K. SAIKI*, P. SEAN WALSH*, COREY H. LEVENSON†, AND HENRY A. ERLICH*
The article describes a method for the simultaneous screening of a sample for all known allelic variants at an amplified locus using sequence-specific oligonucleotide probes. The technique involves immobilizing the probes onto nylon membranes by adding homopolymer tails and covalently binding them with UV irradiation. The target DNA is PCR-amplified and then hybridized to the membrane under stringent conditions. Detection is nonradioactive, involving the binding of streptavidin-horseradish peroxidase to biotinylated DNA, followed by a colorimetric reaction. This method has been applied to HLA-DQA genotyping and the detection of Mediterranean β-thalassemia mutations. The authors demonstrate the feasibility of this approach for analyzing multiple genetic loci and its potential for automating the process.The article describes a method for the simultaneous screening of a sample for all known allelic variants at an amplified locus using sequence-specific oligonucleotide probes. The technique involves immobilizing the probes onto nylon membranes by adding homopolymer tails and covalently binding them with UV irradiation. The target DNA is PCR-amplified and then hybridized to the membrane under stringent conditions. Detection is nonradioactive, involving the binding of streptavidin-horseradish peroxidase to biotinylated DNA, followed by a colorimetric reaction. This method has been applied to HLA-DQA genotyping and the detection of Mediterranean β-thalassemia mutations. The authors demonstrate the feasibility of this approach for analyzing multiple genetic loci and its potential for automating the process.