Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports.
A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene.
Guo
Z
Z
Department of Chemistry, University of Wisconsin, Madison 53706-1396.
Guilfoyle
R A
RA
Thiel
A J
AJ
Wang
R
R
Smith
L M
LM
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D003432:Cross-Linking Reagents; D005091:Exons / Q000235:genetics; D019793:Fluorescein; D005452:Fluoresceins; D005898:Glass; D006801:Humans; D007091:Image Processing, Computer-Assisted; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000302:isolation & purification; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic
D003432:Cross-Linking Reagents; D005452:Fluoresceins; D015345:Oligonucleotide Probes; D014442:Monophenol Monooxygenase; D019793:Fluorescein