Detection of single base alterations in genomic DNA by solid phase polymerase chain reaction on oligonucleotide microarrays.
DNA microarray technology holds significant promise for human DNA diagnostics. A number of technical approaches directed at the parallel identification of mutations or single nucleotide polymorphisms make use of polymerase-based specificity, like minisequencing or allele-specific primer elongation. These techniques, however, require separate laborious sample amplification, preparation, and purification steps, making large-scale analyses time and cost consuming. Here, we address this challenge by applying an experimental setup using simultaneous solid and liquid phase PCR on polyethyleneimine-coated glass slides, a novel microarray support allowing on-chip amplification reactions with exquisite specificity. A gene-specific oligonucleotide tiling array contains covalently attached allele-specific primers which interrogate single nucleotide positions within a genomic region of interest. During a thermal cycling reaction amplification products remain covalently bound to the solid support and can be visualized and analyzed by the incorporation of fluorescent dyes. Using the described procedure we unequivocally defined the presence of point mutations in the human tumor suppressor gene p53 directly from a natural DNA source. This semi-multiplex solid phase amplification format allowed the rapid and correct identification of 20 nucleotide positions from minute amounts of human genomic DNA. Our results suggest that this approach might constitute a vital component of future integrated DNA chip devices used in gene analysis.
Huber
M
M
VBC-Genomics Bioscience Research GmbH, 1030 Vienna, Austria.
Losert
D
D
Hiller
R
R
Harwanegg
C
C
Mueller
M W
MW
Schmidt
W M
WM
D001482:Base Composition / Q000235:genetics; D003110:Colonic Neoplasms / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics*; D016158:Genes, p53 / Q000235:genetics; D016678:Genome; D006801:Humans; D020125:Mutation, Missense / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm