Detection of beta-thalassemia mutations by ASO hybridization of PCR amplified DNA with digoxigenin ddUTP labeled oligonucleotides.
A simple procedure for nonradioactive labeling of oligonucleotides 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 triphosphate. 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 beta-thalassemia mutations. This procedure was also used for the confirmation of a new mutation at position -87 (C----A) (2) of the beta-globin gene and for the subsequent family analysis.
Efremov
D G
DG
Department of Hematology, Faculty of Medicine, Skopje, Yugoslavia.
Dimovski
A J
AJ
Efremov
G D
GD
D000483:Alleles; D003124:Colorimetry; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D004253:DNA Nucleotidylexotransferase; D054306:Dideoxynucleotides; D004076:Digoxigenin; D004797:Enzyme-Linked Immunosorbent Assay; D005914:Globins / Q000235:genetics*; D006801:Humans; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D013789:Thalassemia / Q000235:genetics*; D014500:Uracil Nucleotides
D054306:Dideoxynucleotides; D009838:Oligodeoxyribonucleotides; D014500:Uracil Nucleotides; C063034:2',3'-dideoxyuridine-5'-triphosphate; D005914:Globins; D004247:DNA; D004253:DNA Nucleotidylexotransferase; D004076:Digoxigenin