Allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) is an alternative method for mutation detection.

The advantage of ASO-PCR is that it is a fast, simple and non-radioactive method.

Increasingly, many single base pair substitutions are being discovered that lead to inherited diseases, predisposition to genetic disorders and cancer.

The ability to amplify specific DNA sequences by polymerase chain reaction (PCR) has made it possible to quickly and accurately diagnose many inherited diseases.

Prior to the use of PCR, point mutations were identified using cloning and direct sequencing.

Also Southern blotting and hybridisation with labelled oligonucleotide probes centred on the mutation site or digestion with restriction endonucleases.

These methods have been greatly improved by PCR, which allows amplification of DNA fragments containing polymorphic sites from minute amounts of DNA.

However, these techniques tend to be time-consuming, complex, require the use of a radioactive marker and, in the case of restriction endonuclease detection, are only applicable when the mutation alters a known excision site.

An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA material, complementary to the sequence of a variable target DNA.

It acts as a screening for the presence of the target in a Southern blot assay or, more commonly, in the simpler dot blot assay. It is a common tool used in Genetic Testing, forensic, and Molecular Biology research.

An ASO is typically an oligonucleotide 15-21 nucleotide bases in length. It is designed (and used) in a way that makes it specific to a single version, or allele, of the DNA being tested.

The length of the ASO, which strand is chosen and the conditions by which it is bound to (and washed from) the target DNA all play a role in its specificity.

These probes can generally be designed to detect a difference of as little as 1 base in the target gene sequence, a core capability in the single nucleotide polymorphism (SNP) assay, important in genotyping and Human Genome Project analyses.

To be detected after it has bound to its target, the ASO must be tagged with a radioactive, enzymatic or fluorescent tag.

ASO technology harnesses to detect a base pair difference (cytosine versus thymine) to measure methylation at a specific CpG site.

Allele-specific oligonucleotide polymerase chain reaction (ASO PCR) is an alternative method for mutation detection in which only the perfectly matched oligonucleotide can act as a primer for amplification. The advantage of ASO-PCR is that it is a fast, simple and non-radioactive method.

ASO-PCR, also known as amplification refractory mutation system (ARMS) was first described for the detection of mutations in the α1-antitrypsin gene.

It has since been adopted in the study of several genes, including prenatal diagnosis of cystic fibrosis, apolipoprotein E polymorphisms and point mutations in the ras oncogene.

In this technique, oligonucleotide primers are designed to be complementary to the normal (wild-type) or mutant sequence, and both are used together with a common primer.

Because DNA polymerase lacks 3′ exonuclease activity, it is unable to repair a single-base mismatch between primer and template at the 3′ end of DNA primers.

Therefore, if oligonucleotide primers are designed to contain mismatches near or at the 3′ end, the primer will extend or not depending on which alternative single base polymorphisms are present in the target sequence.

Therefore, under the appropriate stringent conditions, only target DNA exactly complementary to the primer will be amplified.

A simple protocol for allele-specific nucleotide PCR is shown below.

The allele-specific polymerase chain reaction (ASPCR) is an application of the polymerase chain reaction (PCR) that allows the direct detection of any point mutation in human DNA by analysis of PCR products on an agarose or polyacrylamide gel stained with ethidium bromide.

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

In 1979, Wallace and his co-workers reported the use of ASO probes to detect variations in a single-stranded bacterial virus, and subsequently applied the technique to clone human genes.

In 1983 and 1985 Wallace’s laboratory reported mutation detection for sickle cell anaemia in “whole genomic DNA” samples, although this application was hampered by the small number of tags that could be carried by ASO.

Fortunately PCR, a method to greatly amplify a specific segment of DNA, was also reported in 1985.

In less than a year PCR had been paired with ASO analysis. This combination solved the problem of ASO labelling, since the amount of target DNA could be expanded over a pre-existing one.

Also, the specificity of the PCR process itself could be added to that of the ASO probes, reducing the problem of spurious binding of the ASO to non-target sequences.

The combination was specific enough that it could be used in a simple dot blot fashion, avoiding the laborious and inefficient southern white/black blot method.

New DNA sequencing of a series of fish identifies : a single nucleotide polymorphism (SNP) corresponding to two alleles (A and B) of a single gene (top, middle).

Two PCR primers are constructed that are specific for SNP alleles A and B, respectively.

These allele-specific oligonucleotides (ASOs) are paired with an anchor primer for a DNA sequence common to all fish (above, right).

The progress of the PCR is monitored in “real time” and a successful reaction indicates the presence of the corresponding SNP allele.

A fish farmer wants to know whether some of the fish (males) used in a mass spawning experiment contribute disproportionately to the gene pool of the next generation.

She selects two males that are homozygous for SNP A and B, respectively, and one unmarked female (above, left).

The three fish can spawn at random; 48 larvae are selected (bottom left).

ASO A and B tests are run simultaneously on all larvae in a 96-well plate format, in alternating rows (bottom, right).

Here, only 8 of the 48 larvae have the B allele, indicating a five-fold reproductive advantage of the A parent.

A fisheries manager wants to know the relevant abundance of fish eggs of two different species of similar size found in the plankton.

She identifies a region in which the two species differ by a single SNP, producing alleles A and B, respectively (above, left).

A- and B-specific ASOs are constructed (top right).

DNA is extracted from each of the 48 eggs (bottom left), and the A and B ASOs are run on each egg simultaneously in a 96-well plate format, in alternating rows (bottom right).

Here, only 8 of the 48 larvae have the B allele, indicating a five-fold abundance of the A species relative to the B species.

This “RT-PCR ASO” method is an alternative to the traditional requirement for electrophoretic assays, and enables rapid, large-scale screening of larval populations and cohorts.

Studencki AB, Conner BJ, Impraim CC, Teplitz RL y Wallace RB “Discrimination between human beta A, beta S and beta C globin genes using allele-specific oligonucleotide hybridisation probes”. Am J Hum Genet vol. 37 (1), págs. 42-51 (1985).
^ a B Saiki, RK; Scharf S; Faloona F; Mullis KB; Horn GT; Erlich HA; Arnheim N (20 de diciembre de 1985). “Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for the diagnosis of sickle cell disease.”. Ciencias. 230 (4732): 1350–4. Bibcode:1985 Sci … 230.1350S. doi:10.1126 / science.2999980. PMID 2999980. Archivado desde el original el 19 de diciembre de 2008.
^ Wallace, RB; Shaffer, J; Murphy, RF; Bonner, J; Hirose, T; Itakura, K (1979). “Hibridación de oligodesoxirribonucleótidos sintéticos con ADN Phi-X 174: el efecto del desajuste de un solo par de bases”. Investigación de ácidos nucleicos. 6 (11): 3543–3558. doi:10.1093 / nar / 6.11.3543. PMC 327955. PMID 158748.
^ Conner BJ, Reyes AA, Morin C, Itakura K, Teplitz RL y Wallace RB “Detección del alelo de la beta S-globina de células falciformes por hibridación con oligonucleótidos sintéticos”. Proc Natl Acad Sci USA. vol. 80 (1), págs. 278-282 (1983).
^ Saiki RK, Bugawan TL, Horn GT, Mullis KB y Erlich HE “Análisis de ADN de beta-globina y HLA-DQ amplificado enzimáticamente con sondas de oligonucleótidos específicos de alelo” Nature vol. 324 (6093) págs. 163-166 (1986).