While domestic cat and dog evidence may be the most frequently encountered types of animal DNA found at a crime scene, there are certain instances when the identification of the species of origin and subsequent analysis of other animal DNA may be useful. Species identification could be particularly useful in cases such as poaching, illegal trade, and violations of the Endangered Species Act. Classical molecular methods for species identification have been mainly based on immunological testing, including precipitation and agglutination reactions.^42-44 However, molecular genetics-based approaches for the identification of species of origin have been developed, including the use of the mtDNA-containing cytochrome b (cyt b) gene and rRNA genes.^45-52

The most common molecular genetic approaches to species identification involve analysis of the mitochondrial DNA cytochrome b gene.  Amplification of the cyt b gene with primers flanking a large portion (981 bp out of 1140 bp) of the sequence and digestion of the resulting amplimer with restriction enzymes, such as ALU 1 and NCO 1, can be used to produce species specific RFLP (restriction fragment length polymorphism) fragments of various lengths.⁵³ Alternative RFLP methods that use smaller non-contiguous amplimers from within the cyt b gene (464 bp and 358 bp) have been developed specifically for forensic use.^49-51

Alternatively, direct Sanger sequencing of portions of the cyt b gene provides a more definitive, albeit more costly and time consuming, species identification strategy.^46-48,50

Ribosomal RNA genes and other gene markers such as TP53 have also been reported as being useful for species identification.^45,47 In a comparison study of β-actin, TP53, cyt b, mtDNA D-loop, and 28S rRNA amplimers, the best species resolution was obtained using the TP53 gene.⁴⁷ Sequencing of a short hypervariable 20 nucleotide fragment of the 12S rRNA gene by pyrosequencing appears to be capable of species identification.⁴⁵

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