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 alp ha1-antitrypsin (alpha1-AT). The normal alpha1-AT allele is referred t o as M, and the Z mutation arises from a single base substitution. Amp lified DNA products corresponding to homozygous M, heterozygous MZ, an d homozygous Z obtained by the polymerase chain reaction were incubate d with a twofold molar excess of unlabeled oligonucleotide prior to hy bridization with a radiolabeled oligonucleotide. Thus, initial incubat ion with unlabeled M-specific oligonucleotide was followed by hybridiz ation with radiolabeled Z-specific oligonucleotide, and vice versa. Th is 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-noi se ratio.