Co-amplification of human specific and conserved region in cytochrome b gene confirms human bloodstains
Keywords:
Human bloodstains, Cytochrome b gene, Duplex PCR, Forensic scienceAbstract
The objective of this study was to determine human bloodstains by duplex PCR analysis of cytochrome b gene (Cyt b). Two regions of Cyt b consisting of conserved and human specific regions were the targets of interest. The primers specific to these regions were designed and tested with DNA extracted from human and animal (dog, cat, pig, chicken and cow) blood samples. A PCR product of Cyt b conserved region (~309 bp) was found in both human and animal blood samples while a DNA fragment of human specific region (~412 bp) was seen only in human blood sample. Duplex PCR analysis was then carried out. Human blood could be discriminated from animal blood by the number of amplified fragment, two for human and one for animal blood samples. The duplex PCR analyses of mixed human and animal bloodstains were also investigated. Duplex PCR could determine human DNA even when the quantity of human blood was 100 fold less than that of animal blood. The effects of temperature and age of bloodstains on human blood determination were then examined. Human bloodstain samples were kept at different temperatures (25, 35, 45 and 55°C) for different periods of time (1, 7, 14 and 30 days). The results demonstrated that duplex PCR analysis is able to determine human bloodstains in all experimented conditions.References
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