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Sample collection, DNA preparation,
and PCR (Polymerase Chain Reaction) by the Gene Conservation
Laboratory
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| DNA analysis of fish and shellfish tissue
is a powerful tool used by the department for management
of Alaska's fishery resources. Genetic markers are used to identify
appropriate population units (discrete stocks) for management and
can be used to identify individuals of particular stocks in mixed-stock
fisheries. The ability to identify stock origins can also assist
the enforcement of conservation closures. In addition to providing
population tags, genetic variability itself is important for the
survival of a population. |
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 There are many methods used
to capture and collect fish and shellfish for DNA analysis. Beach
seines, gill nets and hook and line are often used to collect salmon
off the spawning beds. Specimens are also collected from fishing
boats and from shore based processors, after the catch has been
landed. |
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Tissue samples for DNA analysis are typically taken from live or recently dead
fish. Fin tissue is often used because sampling is relatively fast, logistically simple, and is non-lethal. Fin clips from a population may be placed in separate vials (one sample per vial) of in a single common bottle and preserved in ethanol. Tissue samples collected for allozyme analysis (heart, liver, muscle, eye) can also be used for DNA analysis, increasing the amount of genetic information from each sample. |
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 DNA is extracted from the various tissue samples using Qiagen’s DNeasy® 96 Blood & Tissue Genomic DNA Purification Kits. The final step elutes purified DNA in AE buffer which is ready for PCR. Extractions are performed in sets of 95 individual samples contained in a 96-well plate with the last well left vacant as a No Template Control (NTC.) |
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Four 96-well plates of Extracted DNA are transferred to multiple replicates of
384-well plates for high throughput needs using a Biomek® FX. Automation
reduces the risks of loading errors between replicates. |
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 Polymerase chain reaction
(PCR) preparation: combining DNA sample with oligonucleotide primers,
deoxynucleotide triphosphates, and the thermostable Taq DNA polymerase
in a suitable buffer. PCR's are prepared under a plexiglass enclosed
hood with positive airflow to minimize risk of sample contamination. |
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 Loading PCR samples onto thermal cyclers where repetitive heating and cooling of the prepared mixture takes place for several hours until the desired amount of amplification of the DNA is achieved. |
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to Techniques
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On to SNPs |
