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.