The human disease sickle cell anemia is caused by a genetic mutation in the codon for the sixth amino acid of the blood protein beta-hemoglobin. The normal DNA sequence G-A-G codes for the amino acid glutamate, while the mutation changes the middle adenine to a thymine, leading to the sequence G-T-G (G-U-G in the mRNA). This altered sequence substitutes a valine into the final protein, distorting its structure.
To test for the presence of the mutation in a DNA sample, an ASO probe would be synthesized to be complementary to the altered sequence, here labeled as "S". As a control, another ASO would be synthesized for the normal sequence "A". Each ASO is fully complementary to its target sequence (and will bind strongly), but has a single mismatch against its non-target allele (leading to weaker interaction). The first diagram shows how the "S" probe is fully complementary to the "S" target (top), but is partially mismatched against the "A" target (bottom).
A segment of the beta-hemoglobin genes in the sample DNA(s) would be amplified by PCR, and the resulting products applied to duplicate support membranes as Dot blots. The sample's DNA strands are separated with alkali, and each ASO probe is applied to a different blot. After hybridization, a washing protocol is used which can discriminate between the fully complementary and the mismatched hybrids. The mismatched ASOs are washed off of the blots, while the matched ASOs (and their labels) remain.
In the second diagram, six samples of amplified DNA have been applied to each of the two blots. Detection of the ASO label that remains after washing allows a direct reading of the genotype of the samples, each with two copies of the beta-hemoglobin gene. Samples 1 and 4 only have the normal "A" allele, while samples 3 and 5 have both the "A" and "S" alleles (and are therefore heterozygous carriers of this recessive mutation). Samples 2 and 6 have only the "S" allele, and would be affected by the disease. The small amount of cross hybridization shown is typical, and is considered in the process of interpreting the final results.