Detection of β-Thalassemia Mutations by Aso Hybridization of PCR Amplified DNA With Digoxigenin ddUTP Labeled Oligonucleotides

Abstract

A simple procedure for nonradioactive labeling of oligonucleo tides has recently been developed (1). It consists of 3′ end labeling of oligonucleotides with terminal transferase by incorporation of a single digoxigenin labeled dideoxy uridine triphos phate. We used these oligonucleotides for allele specific oligomer hybridization of polymerase chain reaction amplified DNA, followed by an enzyme-linked immunoassay and subsequent enzyme-catalyzed color reaction. We compared this procedure with the standard radioactive oligonucleotide hybridization technique through the detection of the most common Mediterranean β-thalassemia mutations. This procedure was also used for the confirmation of a new mutation at position -87 (C→A) (2) of the β-globin gene and for the subsequent family analysis.