DNA sequencing-based identification is becoming more common in routine laboratory work and species identification. The DNA based identification tools exploit diversity among DNA sequences to identify organisms and employ DNA sequences as ‘barcodes’. The advantages of using DNA barcoding in forensic science and conservation genetics are that the same method is used across multiple taxonomic groups, and it also allows the identification of parts or modified products of organisms that do not display specific morphological characters. The most common molecular method of species identification is the sequencing of regions in mitochondrial DNA (mtDNA). The Cytochrome b (Cyt b) gene, cytochrome oxidase I (COI) gene, 12s rRNA and 16s rRNA region and the hypervariable or control region (HV) found within the D-loop region of the mitochondria has been commonly used as DNA barcodes in the field of forensic genetics, species identification, population genetics, phylogenetic studies and conservation genetics
Cytochrome b has both variable and conserved regions and is a widely used locus. It has the widest taxonomic representation in GenBank at National Centre for Biotechnology Information (NCBI) database. The DNA Barcode of Life initiative proposed the use of COI gene locus for barcoding to develop a widely accepted simple diagnostic tool based on strong taxonomic knowledge that is collated in the DNA barcode reference library published in the Consortium for the Barcode of Life (CBOL).
Wildlife DNA Technology
Wildlife crime is a global problem and it involves four major categories: (1) illegal killing or poaching; (2) possessing and modifying; (3) trading, shipping or moving; and (4) inflicting cruelty to wildlife. Illegal wildlife trade is a multi-billion-dollar industry that is threatening the world’s biodiversity. There is an increasing demand for species identification in the fields of international trade and forensics. Identification of wildlife parts and products is always a challenging task. In many criminal cases, morphological and microscopic examination is sufficient for the identification of such specimens upon the availability of reference data. But many samples received in a forensic laboratory are naturally degraded, old, modified or carved, and diagnostic morphological features may be absent. In such conditions where traditional techniques are inadequate, DNA based identification methods may be useful for the proper identification of samples. DNA based identification methods are routinely being used in forensics for human identification using short tandem repeats (STRs), mitochondrial sequences of hypervariable regions (HVs) and single nucleotide polymorphisms (SNPs). The basic set up and the technicality is very similar and wildlife forensic genetics also applies methods used in human identification, taxonomy and phylogenetics for purposes related to wildlife crime. Different studies using cytochrome b and cytochrome oxidase I indicate that there are enough regions of variability for the primers to distinguish between different species.
There are a number of different extraction methods for different types of samples. By using these extracted DNA templates, the target region is amplified by a polymerase chain reaction (PCR). The universal primers allow for the amplification of species-specific fragments of DNA which can be sequenced and compared to a reference sample.
Application and Challenges
The application of molecular identification techniques has gained popularity in every field of biological sciences after the widespread development and application of PCR. The biological samples in minor quantities can be useful for the identification of species with high degrees of specificity and reliability. The development and standardization of various DNA isolation techniques has made it possible to apply such techniques to forensic genetics. The locus used in species testing usually has very little intraspecific variation, and commonly all members of the same species will produce the same results. Various universal primers are available for DNA barcoding and many of them have proven useful for the identification of biological specimens to species level and even subspecies level. The use of mitochondrial DNA regions has proven effective and applicable for species identification. Although a minute quantity of any sample is deemed sufficient for DNA barcoding, some highly degraded samples may not generate sufficient sequence data while some other samples may even produce mixed sequences as a result of contamination. The most common application has been species identification, but increasingly DNA is being applied to analyze the geographic location of origin or to prove or disprove claims that a listed species has been captive bred as opposed to its having been taken from a wild population. For DNA barcoding application to forensics, the available database of taxonomic reference sequences, is currently very limiting. Free access to the open database of DNA sequences kept in the NCBI and other thirdparty databases is quite invaluable for wildlife forensic science. It provides the preliminary identification of any new or unknown DNA sequence within seconds. The disadvantage is that these databases are not wellregulated and hence errors or contaminated sequences can lead to misidentifications requiring laboratories to maintain an in-house database for legal purposes.
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The Author: Nirajan Thapa Kshetry is a Senior Scientific Officer and Unit Chief, DNA Unit, National Forensic Science Laboratory, Nepal.