Competitive assay to improve the specificity of detection of single-point mutations in alpha 1-antitrypsin deficiency.
Allele-specific oligonucleotides are used widely for the detection of single point mutations in genes. A modification of this assay based on competition has been developed for detection of the Z mutation of alpha 1-antitrypsin (alpha 1-AT). The normal alpha 1-AT allele is referred to as M, and the Z mutation arises from a single base substitution. Amplified DNA products corresponding to homozygous M, heterozygous MZ, and homozygous Z obtained by the polymerase chain reaction were incubated with a twofold molar excess of unlabeled oligonucleotide prior to hybridization with a radiolabeled oligonucleotide. Thus, initial incubation with unlabeled M-specific oligonucleotide was followed by hybridization with radiolabeled Z-specific oligonucleotide, and vice versa. This assay increased the specificity of single-point mutation detection three- to four-fold. Furthermore, specific hybridization was obtained at a lower temperature as a consequence of improving the signal-to-noise ratio.
Gunneberg
A
A
Department of Clinical Chemistry, Southmead Hospital, Westbury-on-Trym, Bristol, UK.
Scobie
G
G
Hayes
K
K
Kalsheker
N
N
D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000032:analysis*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D000515:alpha 1-Antitrypsin / Q000235:genetics*; D019896:alpha 1-Antitrypsin Deficiency
D015345:Oligonucleotide Probes; D000515:alpha 1-Antitrypsin; D004247:DNA