Species identifications

Samples subjected to mtDNA typing are not necessarily derived from humans, but usually this does not impose much trouble because the sequencing of HV-I and HV-II not only provides individual identification but also confirms whether the sample is of human origin. However, when amplification of HV-I and HV-II fails, species identification of DNA extracted from a sample may be useful in order to ascertain whether failure in amplification is due to the sample being non-human in origin (e.g. plants and animals) or whether it resulted from other causes (e.g. an insufficient amount of DNA and/or decomposition).

Cytochrome b (cyt b) is one component of the electron transport (respiratory chain) enzyme complexes involved in oxidative phosphorylation. In many types of species, including plants and animals, the cyt b gene exists in mtDNA (Figure 8.1). In view of the endosymbiosis hypothesis, the cyt b gene escaped incorporation by the nucleus and accumulated mutations over evolutionary time, leading to the present cyt b gene sequence differences among species. In other words, cyt b genes are species-specific and have been used for species determination in phylogenetic and forensic investigations (Kocher et al., 1989; Parson et al., 2000). In these investigations, a portion of the cyt b gene is amplified using 'universal' primers and then sequenced. The universal primers were carefully designed to hybridize to highly conserved sites in order to allow the amplification of mtDNA across a broad range of species, yet sequence differentiation in a region sandwiched between the hybridization sites is sufficiently large to allow the discrimination of species.

The sequencing approach is very efficient because DNA samples from different species can be positively identified. However, confirmation of human samples is likely to be useful in routine forensic cases. Matsuda et al., (2005) developed a PCR-based method in which DNA extracted from samples was amplified using 'human-specific' primers and then subjected to gel electrophoresis. The human-specific primers were designed to hybridize to human cyt b gene sites, which differed from those of the chimpanzee by 26% (Figure 8.3). The results of this method were determined simply by the presence (positive results) or the absence of a visible band (negative results), with no bands observed in DNA of animals, including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) (Figure 8.4). Thus, samples producing a single band can be reasonably interpreted as being of human origin. Samples producing no visible bands, however, are inconclusive. In such cases, the employment of other cyt b gene primers as a positive control, and if necessary, the subsequent sequence analysis may achieve conclusive results (Parson et al., 2000).

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