Validation of the barcoding gene COI for use in forensic genetic species identification

The application of forensics to wildlife crime investigation routinely involves genetic species identification based on DNA sequence similarity. This work can be hindered by a lack of authenticated reference DNA sequence data resulting in weak matches between evidence and reference samples. The introduction of DNA barcoding has highlighted the expanding use of the mtDNA gene, cytochrome c oxidase I (COI), as a genetic marker for species identification. Here, we assess the COI gene for use in forensic analysis following published human validation guidelines. Validation experiments investigated reproducibility, heteroplasmy, mixed DNA, DNA template concentration, chemical treatments, substrate variation, environmental conditions and thermocycling parameters. Sequence similarity searches using both GenBank BLASTn and BOLD search engines indicated that the COI gene consistently identifies species where authenticated reference sequence data exists. Where misidentification occurred the cause was attributable to either erroneous reference sequences from published data, or lack of primer specificity. Although amplification failure was observed under certain sample treatments, there was no evidence of environmentally induced sequence mutation in those sequences that were generated. A simulated case study compared the performance of COI and cytochrome b mtDNA genes. Findings are discussed in relation to the utility of the COI gene in forensic species identification.     

A molecular genetic approach for species identification of mammals and sex determination of birds in a forensic case of poaching from South Korea

DNA-based analysis was performed using partial mitochondrial cytochrome b genes of five mammalian specimens and Chromo-Helicase-DNA-binding (CHD) genes of five pheasants to determine whether specimens were from illegally hunted animals. Mammalian specimens were identified as being those of horse, roe deer, and cow through gene amplification using cytb981f and cytb981r primer set and sequencing. CHD genes were revealed to be those of three male and two female pheasants through polymerase chain reaction amplification. Because hunting of roe deers and female pheasants is prohibited in Korea, these results provided forensic evidences of illegal wild animal hunting.