Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes.

TLDR

DNA mutation detection by hybridization with sequence‑specific probes is facilitated by PCR amplification, yet conventional assays require separate hybridization for each probe. The authors present a method enabling simultaneous screening of all known allelic variants at an amplified locus. They immobilize oligonucleotide probes with homopolymer tails on a nylon membrane via UV cross‑linking, then hybridize biotinylated PCR products under stringent conditions and detect binding non‑radioactively with streptavidin‑HRP and a colorimetric readout. The technique successfully genotyped six HLA‑DQA alleles and detected nine Mediterranean β‑thalassemia mutations.

Abstract

The analysis of DNA for the presence of particular mutations or polymorphisms can be readily accomplished by differential hybridization with sequence-specific oligonucleotide probes. The in vitro DNA amplification technique, the polymerase chain reaction (PCR), has facilitated the use of these probes by greatly increasing the number of copies of target DNA in the sample prior to hybridization. In a conventional assay with immobilized PCR product and labeled oligonucleotide probes, each probe requires a separate hybridization. Here we describe a method by which one can simultaneously screen a sample for all known allelic variants at an amplified locus. In this format, the oligonucleotides are given homopolymer tails with terminal deoxyribonucleotidyltransferase, spotted onto a nylon membrane, and covalently bound by UV irradiation. Due to their long length, the tails are preferentially bound to the nylon, leaving the oligonucleotide probe free to hybridize. The target segment of the DNA sample to be tested is PCR-amplified with biotinylated primers and then hybridized to the membrane containing the immobilized oligonucleotides under stringent conditions. Hybridization is detected nonradioactively by binding of streptavidin-horseradish peroxidase to the biotinylated DNA, followed by a simple colorimetric reaction. This technique has been applied to HLA-DQA genotyping (six types) and to the detection of Mediterranean beta-thalassemia mutations (nine alleles).