Polymorphism analysis and gene detection by minisequencing on an array of gel-immobilized primers.
Two procedures, multibase and multiprimer, have been developed for single nucleotide extension of primers immobilized within polyacrylamide gel pads on a microchip. In the multibase assay, a primer is next to a polymorphic nucleotide; the nucleotide is identified by the specificity with which the primer incorporates fluorescently labeled dideoxyribo-nucleoside triphosphates. In the multiprimer assay, several primers containing different 3'-terminal nucleotides overlapping the variable nucleotide in DNA are used. The polymorphic nucleotide is identified according to the primer that is extended. The methods were compared for diagnosis of beta-thalassemia mutations. Isothermal amplification of the fluorescent signal was achieved by performing both assays at elevated temperature. Anthrax toxin genes were identified in a model system using this amplification method.
Dubiley
S
S
Joint Human Genome Program, Engelhardt Institute of Molecular Biology, 32 Vavilov Str., Moscow 117984, Russia.
Kirillov
E
E
Mirzabekov
A
A
D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers; D005453:Fluorescence; D005914:Globins / Q000235:genetics; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA; D017086:beta-Thalassemia / Q000235:genetics
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D017931:DNA Primers; C030325:anthrax toxin; D005914:Globins