Routine Forensic Use of the Mitochondrial 12S Ribosomal RNA Gene for Species Identification

Abstract

Since July 2004, Mitotyping Technologies has been amplifying and sequencing a approximately 150 base pair fragment of mitochondrial DNA (mtDNA) that codes for 12S ribosomal RNA, to identify the species origin of nonhuman casework samples. The approximately 100 base pair sequence product is searched at http://www.ncbi.nlm.nih.gov/BLAST and the species match is reported. The use of this assay has halved the number of samples for which no mtDNA results are obtained and is especially useful on hairs and degraded samples. The availability of species determination may aid forensic investigators in opening or closing off lines of inquiry where a highly probative but challenging sample has been collected.

Forensic species identification based on size variation of mitochondrial DNA hypervariable regions

“…Nucleotide sequences of mtDNA-HV should be interpreted with caution because of the risk of heteroplasmy and/or contamination [26,34]. However, identification by the comparison of animal mtDNA-HV sequences is very important for forensic investigation since shed hairs from domestic animals, especially dogs and cats, are frequently recovered from crime scenes and can be used as evidence [11,35]. By morphological observations alone, it is impossible to distinguish between individuals of a species [36].…”

“…Consequently, sequencing [1-3, 5, 6, 8-12, 14, 16], restriction fragment length polymorphism analysis [4,15], and species-specific polymerase chain reaction (PCR) [7,13] are indispensable for species discrimination. Due to the high copy number of mtDNA (∼100 to >1,000 copies/cell), these analytical methods are highly sensitive and reliable and are very useful for poor quality samples such as old bloodstains, animal hairs, bird feathers, and bone particles [2,3,5,7,[11][12][13]. Another advantage of these assays is that a BLAST search can be used to determine the species of a sample, even if there is no reference sample [2,3,5,11,12].…”

“…Due to the high copy number of mtDNA (∼100 to >1,000 copies/cell), these analytical methods are highly sensitive and reliable and are very useful for poor quality samples such as old bloodstains, animal hairs, bird feathers, and bone particles [2,3,5,7,[11][12][13]. Another advantage of these assays is that a BLAST search can be used to determine the species of a sample, even if there is no reference sample [2,3,5,11,12]. Recently, to distinguish mammal species from mixtures, a multiplex assay was developed based on different sizes of PCR products using lots of species-specific primers designed on the CYB gene [17].…”

“…The cytochrome b (CYB) [1][2][3][4][5][6][7][8], cytochrome oxidase I [9], and 12S [10][11][12] and 16S ribosomal RNA (rRNA) genes [12,13] in mitochondrial DNA (mtDNA) have been widely used to discriminate among species. D-loops have also been targeted for which universal primers amplified similar sized fragments among all species [14][15][16].…”

Bite through the tent

“…Analysis of mitochondrial deoxyribunucleic acid (mtDNA) is the method of choice due to a higher success rate processing low copy number or degraded DNA samples as well as to its arrangement of conserved and specific regions. For species determination, the mitochondrial cytochrome b (cytb) [2,3] and 12S ribosomal ribunucleic acid (12S rRNA) [4][5][6] genes proved useful together with cytochrome I oxidase and 16S ribosomal RNA genes [1,7]. The most common approach in sporadic forensic species determination is Sanger sequencing.…”