Allele-specific oligonucleotide

These probes can usually be designed to detect a difference of as little as 1 base in the target's genetic sequence, a basic ability in the assay of single-nucleotide polymorphisms (SNPs), important in genotype analysis and the Human Genome Project.

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.

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 RFLP assay was briefly adapted to the use of oligonucleotide probes,[3] but this technique was quickly supplanted by ASO analysis of polymerase chain reaction (PCR) amplified DNA.

However, the simplicity and versatility of the combined PCR/ASO method has led to its continued use, including with non-radioactive labels, and in a "reverse dot blot" format where the ASO probes are bound to the membrane and the amplified sample DNA is used for hybridization.

The use of synthetic oligonucleotides as specific probes for genetic sequence variations was pioneered by R. Bruce Wallace, working at the City of Hope National Medical Center in Duarte, California.

In 1979 Wallace and his coworkers reported the use of ASO probes to detect variations in a single-stranded bacterial virus,[4] and later applied the technique to cloned human genes.

In 1983[5] and 1985[2] Wallace's lab reported the detection of the mutation for sickle cell anemia in samples of whole genomic DNA, although this application was hampered by the small amount of label that could be carried by the ASO.