Allele-specific oligonucleotide hybridization (ASOH), also known as dot-blot analysis, is used for genotyping of highly polymorphic regions of DNA. ASOH can be thought of as a variation of the Southern blot, in that patient DNA amplified by PCR is bound to a membrane and hybridized with labeled allele-specific oligonucleotide probes.23 Reverse dot-blot analysis differs from ASOH in that unlabeled allele-specific oligonucleotide probes are spotted onto different membrane locations and hybridized with labeled PCR amplicons.
For ASOH, the PCR products are denatured and a small amount of denatured (single stranded) amplicon is spotted onto a nylon or nitrocellulose membrane. The amplicon is permanently bound to the membrane by baking under vacuum or UV cross-linking. Amplicons from different specimens can be spotted at different locations to interrogate the genotype of multiple specimens simultaneously. Duplicate membranes are made for each probe type. Each membrane is hybridized with two different labeled oligonucleotide probes (one complementary to the mutant sequence and another to the normal sequence of the same DNA region). The membranes are washed to remove non-specifically bound probe. Samples that hybridize strongly to only one probe indicate homozygosity for the normal or mutant allele; those that hybridize with both probes are heterozygous. The oligonucleotide probes are labeled and detected by radioactivity (often avoided in clinical molecular laboratories), fluorescence, colorimetry, chemilumi-nescence, or mass spectrometry. One criticism of ASOH is the potentially ambiguous discrimination of a positive signal. Optimization of the assay and the use of both positive and negative controls help to define and score ASOH results.
Example of Application of ASOH 1. Low-resolution HLA typing
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