Alaska Department of Fish and Game
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Gene Conservation Laboratory
Sample collection, DNA preparation, and Polymerase Chain Reaction (PCR)
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.
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.
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.
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.)
The Biomek™ liquid handling robot is used to pipette whole plates of DNA and assay reagent into our genotyping microarrays. The automated process helps reduce human error, precision, and chip preparation time where the accuracy necessary is at the sub microliter level.
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.
Microsatellite 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. These samples will be run with the ABI 3730 capillary sequencer for allele size.
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.