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Molecular Basis of Heredity: Part 4. Gene Identification and Tests

Author(s): Raye L. Alford, PhD

Allele Specific Oligonucleotide (ASO) Analysis

ASO (allele specific oligonucleotide) hybridization begins with PCR. Once amplified, the target DNA is hybridized (affixed) to a nylon membrane or another solid substrate in spots (without prior electrophoresis). Oligonucleotide probes specific for normal and mutant sequences are radioactively, chemiluminescently, or fluorescently labeled and hybridized to the substrate-bound target DNA. Competitive binding between mutant and normal probes permits probe binding dependent upon the sequence of the target DNA.

ASO is very effective and reliable and is used for many DNA tests where only one or a few mutations cause the disease. The development of 96- and 384- tray formats permit automation and the simultaneous analysis of many specimens, making ASO useful for high throughput diagnostic testing. ASO effectively identifies sought after mutations, but unlike sequencing, cannot typically detect mutations not directly tested. Another common problem with ASO is with polymorphic sequences that occur near the sequence variation that is being sought. If these polymorphisms interfere with probe binding, false positive or false negative results can be obtained. In these cases, special consideration must be given to probe design to avoid errors in interpretation of ASO test results.

The image on the slide shows the ASO analysis of a mutation in the CFTR gene associated with cystic fibrosis (CF) in a family with one affected child and a fetus of unknown genotype. In this example, the parents are both carriers of a CF-associated allele. On ASO analysis, they show a signal with both the normal and mutant probes, consistent with their carrier status. The child, who is affected by CF, shows an ASO signal only for the mutant probe, indicating inheritance of two copies of the CF allele, one from each parent. The fetus also has inherited a CF-associated allele from each parent and does not show a signal from the normal probe. This fetus is predicted to be affected with CF. The column labeled X represents the negative control.

There are a number of variations on allele specific methods, all based on the same principle: primers and probes only bind to complementary DNA sequences. This feature of DNA hybridization is exploited to identify normal and mutant alleles in patient specimens.