PCR-based Allele-Specific Oligonucleotide (ASO) test:
differential male success in breeding

          DNA re-sequencing of a series of fish identifies a Single Nucleotide Polymorphism (SNP) corresponding to two alleles (A & B) of a single gene (top, middle). Two PCR primers are constructed that are specific for SNP alleles A & B, respectively. These allele-specific oligonucleotides (ASOs) are paired with an anchor primer for a DNA sequence common to all fish (top, right). Progress of the PCR is monitored in "real time", and a successful reaction indicates the presence of the corresponding SNP allele.

          Aquaculture application: An aquaculturist wishes to know if some of the (male) fish used in a mass-spawning experiment contribute disproportionately to the gene pool of the next generation.  She selects two males that are homozygous for SNPs A & B, respectively and an unmarked female fish (top, left). The three fish are allowed to spawn at random; 48 larvae are selected (bottom left). The A & B ASO tests are both run simultaneously on all larvae in an 96-well plate format, in alternate rows (bottom, right). Here, only 8 of the 48 larvae have the B allele, indicating a five-fold reproductive advantage of the A parent.

          Biodiversity application: A fisheries manager wishes to know the relevant abundance of fish eggs from two different species with similar size that occur in the plankton. She identifies a region in which the two species differ by a single SNP, producing alleles A & B, respectively (top, left). A- & B-specific ASOs are constructed (top right).  DNA is extracted from each of 48 eggs (bottom left), and the A & B ASO tests are run on each  egg simultaneously in a 96-well plate format, in alternate rows (bottom, right). Here, only 8 of the 48 larvae have the B allele, indicating a five-fold abundance of species A relative to species B.

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