Screening for defined cystic fibrosis mutations by solid-phase minisequencing.
We have developed a rapid method for the quantitative detection of point mutations and deletions. In this minisequencing method, enzymatically amplified DNA, 5'-biotinylated in one strand, is bound to a solid phase and denatured. A detection primer, constructed to end immediately before the mutation, is annealed to the immobilized single-stranded template and elongated with a single, labeled deoxynucleoside residue. We have applied the solid-phase minisequencing method to the detection of the major mutation, delta F508, causing cystic fibrosis (CF). In the presence of the allele with the delta F508 mutation, [3H]dTTP is incorporated; with the nonmutated allele, [3H]dCTP is incorporated. Thus, samples from heterozygous individuals allow the incorporation of both labels. The method was evaluated by analyzing 59 coded DNA specimens collected from 20 Finnish CF patients and their parents. The ratio of [3H]C to [3H]T gave unambiguously the allele combination. The solid-phase minisequencing method was also applicable to the analysis of three CF mutations simultaneously, i.e., delta F508, G542X, and G551D. We conclude that the microtiter-plate-based minisequencing test is an accurate method for the screening of defined sequence alterations in the CF gene.
Jalanko
A
A
Orion Corp., Orion Pharmaceutica, Biotechnology, Helsinki, Finland.
Kere
J
J
Savilahti
E
E
Schwartz
M
M
Syvänen
A C
AC
Ranki
M
M
Söderlund
H
H
D001483:Base Sequence; D002872:Chromosome Deletion; D003550:Cystic Fibrosis / Q000235:genetics*; D003570:Cytidine Triphosphate / Q000378:metabolism; D004247:DNA / Q000737:chemistry*; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D013942:Thymine Nucleotides / Q000378:metabolism
D013942:Thymine Nucleotides; D003570:Cytidine Triphosphate; D004247:DNA; C024157:thymidine 5'-triphosphate