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Basic Steps in a Forensic DNA Analysis

    DNA is present in a variety of living or once-living sources, including hair, feathers, and scales, blood, tissue or body fluid samples, as well as food items such as fresh meat, fish, or shellfish, or dried, canned, smoked, or otherwise preserved food, etc. The small size of many forensic samples and the very small quantity of DNA present means that it is first necessary to increased the number of DNA copies present.

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"DNA Xeroxing" by the PCR

     The polymerase chain reaction (PCR) is a method of 'DNA xeroxing' that amplifies the small amount of a particular DNA sequence present in a sample to usable quantities. The PCR experiment includes a DNA source, a pair of short DNA primers that flank the DNA region to be amplified, a heat-resistant DNA copying enzyme (Taq), and a pool of the four DNA building-block (dNTPs). The reaction is carried out in a computer-regulated heating block (a thermal cycler). PCR includes three stages: the reaction is first heated to melt the DNA strands, cooled so that the primers can find the DNA targets and stick to them, and finally heated again to allow the Taq enzyme to copy the DNA. Each PCR cycle doubles the amount of the DNA of interest: ten doublings produce 1,000 copies, and 30 cycles yields 230 copies for a billion-fold amplification. This produces a sufficient quantity of the gene region of interest for DNA sequencing.

 

"False Colour" automated DNA sequencing

     After PCR, a sequencing reaction is set up that synthesizes multiple copies of both DNA strands by incorporating a set of fluorescently-labeled DNA bases, A C G & T, into the new DNA [left]. The DNA sequence of each strand can then be read as the successive 'rungs' in the sequencing 'ladder'. The gel image on the automated DNA sequencer [middle] builds a "false colour" composite representation of 24 different DNA sequences. In the magnified view at right, each lane shows a different DNA sequence as a "ladder". For example, in the fourth lane, the first few bases will be read as ATTTGAATTC . Terra Nova Genomics uses instruments that can sequence 96 reactions simultaneously.

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DNA sequence Chromatogram

     Each channel is scanned by the computer from bottom to top [white line in the 7th channel]. The scan is converted to a chromatogram, in which each band corresponds to a coloured peak: the order of peaks gives the DNA sequence.
 
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Detection of DNA sequence variation ("SNPs")

     Differences among DNA sequences of individuals and species arise from mutations that alter the four-letter code.  Such single-letter changes are called single-nucleotide polymorphisms (SNPs). The top window shows an 80-letter stretch of DNA from eight Atlantic Cod. The black dots flag two SNP positions where individuals differ.  As seen in the lower window, most individuals have the sequence
CATAG: individuals BS02 & BS11 share a CT SNP variant at the third position (TATAG); individual BS03 has a unique AG SNP at the sixth position (CATGG).  Identification of such genetic differences permits inferences about relationships among individuals within species (genealogy), in time and space (phylogeography), and the evolutionary relationships (phylogeny) of related species.
 
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"The Case of the Falsified Fillets"
DNA Forensics for Commercial Food Fisheries

The species identity of fish products that has been skinned, cleaned, filleted, cooked, frozen, dried, and / or salted can be difficult to determine. Questions may arise as to the origin of a commercial product: Is the catch in a boat's hold properly reported? Is the label on a package is accurate. A product may be physically altered to pass as another, for example by bleaching of fillets. Although physical identification may be impossible once skin and scales are gone, DNA survives processing in sufficient quantities to provide a reliable test.

In the forensic test shown here, the identity of salt-cured fish fillets in a comme
rcial batch of “Cod” was questioned. Terra Nova Genomics compared the DNA sequences from fillet to a data base of known cod, pollock, and hake species. The analysis produces a "family tree", which s hows that each of the four fillets belongs to a different commercial species: walleye or Alaska pollock, Atlantic cod, Pacific cod, and pollock or saithe.

Forensic Fish Samples


"The Case of the Scurrilous Scallops"
Species-Specific Oligonucleotides (SSOs) as a forensic DNA test

      In some forensic cases, the cost of direct sequence analysis of large numbers of individuals is prohibitive. The case illustrates the development of a novel industrial DNA forensic method by Terra Nova Genomics.

     The DNA sequences of a particular gene (COI) are known for many species of scallops, including two found in the Northwest Atlantic, Sea Scallops (Placopecten) and Icelandic Scallops (Chlamys). We combined an PCR primer (an "oligo") for a DNA sequence identical in both species (ScallopR2) with oligos specific for either species (PmaCOIF1 and CisCOIF2). The sizes of the PCR amplification product expected from either species are different, longer in Placopecten and shorter in Chlamys. Use of species-specific oligonucleotides (SSOs) thus identifies species directly from the sizes of the PCR fragments, without the need for sequencing. The same procedure can be adapted for any species
 
     A fisherman had a hold full of scallops that he claimed were from the open fishery for Icelandic Scallops, but which DFO Enforcement officers suspected they from the closed fishery for Sea Scallops. Since a small proportion of bycatch from the closed fishery might be considered acceptable, the legal question was: What fraction of the total catch was from the prohibited species? We extracted DNA from more than 900 individual scallops and applied the SSO test to each. Of the 80 scallops in the test below, all but eight (blue arrows) show the larger DNA fragment, which indicates that 90% are Sea Scallops, the closed fishery.  In the complete series from two vessels, almost two-thirds were Sea Scallops. This evidence resulted in conviction, confiscation of the vessel, and heavy fine.
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DNA Microarrays: the ArkChip for multi-species fisheries and forensics

     A DNA re-sequencing microarray allows a sequence of up to 500,000 ACGTs to be determined in a single experiment. The microrarray is designed from a known reference sequence for the species of interest, and allows the same sequence to be read repeatedly from additional individuals. This is particularly well-suited to population genomics, where the collection of sequences from multiple individuals is called "iterative sequencing".

      The example shows a complete 15,452bp human mtDNA genome sequence arranged on microarray pattern. Each nucleotide position is represented in a vertical block of 4 cells in 5 rows (ACGT + a blank). In each block, the cell with the strongest DNA binding identifies the base present at that position. In the magnified insert view, the sequence of bases in each of four blocks is easily read by computer as the left-to-right order of successive brightest squares.

     In an important biotechnological breakthrough, Terra Nova Genomics has shown that it is possible to use a multi-species ArkChip microarray to sequence the mtDNA genomes from several different species simultaneously, without interference ("cross-talk") among species. The larger figure shows a larger microarray that includes reference genomes from seven species, on which complete mtDNA genome sequences have been sequenced from four species (Atlantic Cod, Atlantic Wolffish, Newfoundland Caribou, and Harp Seal). Because the design, manufacture, and processing of the microarray is a fixed cost, the added cost of sequencing multiple species is limited to extraction and amplification of DNA.

     Terra Nova Genomics is planning a second-generation ArkChip 2.0 that will include ~20 different species. This converts academic research to an industrial tool for large-scale, long term monitoring of multiple commercial fishery stocks. Such species can also serve as valuable bioindicators for potential environmental damage from oil spill. Finally, because mtDNA genome sequences of individuals within species are unique, it allows forensic identification and discrimination of individuals within species.

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