Single-nucleotide polymorphism analysis by hybridization protection assay on solid support.
The clinical need for high-throughput typing methods of single-nucleotide polymorphisms (SNPs) has been increasing. Conventional methods do not perform well enough in terms of speed and accuracy to process a large number of samples, as in clinical testing. We report a new DNA microarray method that uses hybridization protection assay (HPA) by acridinium-ester-labeled DNA probes. Probes were immobilized on the bottom of streptavidin-coated microtiter plates by streptavidin-biotin binding. We studied aldehyde dehydrogenase 2 (ALDH2) genotyping using two probes, discriminating A/G polymorphism. We also designed four probes to type the Alzheimer's disease-related gene ApoE, which has three genotypes (ApoE2, 3, and 4) determined by two SNP loci (C/T polymorphism). SNP analysis of the ALDH2 gene or the ApoE gene from human genome samples by solid-phase HPA was successful. Unlike other methods, the microarray by HPA does not require a washing step and can be completed within 30min. It also has advantages in discriminating one-base mismatch in targets. These characteristics make it a good candidate for practical SNP analysis of disease-related genes or drug-metabolizing enzymes in large numbers of samples.
Chugai Diagnostics Science, Tokyo, Japan. gotossm@chugai-pharm.co.jp
Susumu
S
Goto
Aki
A
Takahashi
Keiichi
K
Kamisango
Koichi
K
Matsubara
D000166:Acridines / Q000737:chemistry / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000097:blood / Q000235:genetics*; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D001057:Apolipoproteins E / Q000097:blood / Q000235:genetics*; D019921:Biotinylation; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D015342:DNA Probes; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results
D000166:Acridines; D001057:Apolipoproteins E; D017931:DNA Primers; D015342:DNA Probes; D004247:DNA; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial