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When a highly probative crime scene sample gives “no results” for the standard human mitochondrial DNA or STR assays, what can be done? One question remains unanswered: is this a highly degraded human sample with unrecoverable DNA, or a non-human sample? The hair in the victim’s hand, the hair adhering to the undercarriage of a vehicle in a hit-and-run accident, and the hair in the back of a suspect’s pickup truck are all evidence samples that may shed light on what happened in a particular crime. For skeletal remains, unless anatomically recognizable bones are recovered from a burial site, the species of origin may be indeterminate, even though a missing person was believed to have been buried nearby. Extremely fragmented and degraded bones can also present an analytical challenge.
Animals whose DNA has been observed in laboratory casework.
For many cases, determination that a highly probative hair or bone believed to be human is actually from another species can be helpful, opening or closing off lines of inquiry in the case investigation. The general but unfortunate trend of reduced training of and availability of experienced hair examiners sometimes results in the submission of non-human or indeterminable hairs to the laboratory for DNA analysis. Figure 1 shows a range of variation in hair appearance found in fur-bearing species; although hair microscopy may be recommended prior to DNA analysis, this type of examination is sometimes beyond the practical scope of the individual laboratory or even the greater laboratory system.
Figure 1: Photomicrographs of hair shafts, for different species of mammals. Left to right: human, human, cat, deer, and dog.
Several useful DNA sequencing approaches to species determination have been published, all of which use conserved mitochondrial DNA regions such as cytochrome b and ribosomal DNA for primer annealing. Simplicity, ease of validation, and utility on degraded samples are critical in a forensic context. Our laboratory routinely applies PCR and sequencing of a short fragment of mtDNA that codes for mitochondrial 12S ribosomal RNA (12S rRNA; human mtDNA rCRS nucleotide positions 650-1603) to forensic casework.1 The advantage of this assay, beyond the obvious one of using the naturally abundant mtDNA genome, is that the amplicon size is approximately 150 base pairs (bp), therefore rendering it useful for all but the most degraded samples. In this respect, it is similar to the mini-primer set approach used by many labs for degraded but abundant control region mtDNA. The region that is amplified corresponds to nucleotide positions 1095-1198 in humans and the primers correspond to nucleotide positions 1071-1094 and 1199-1221 per the revised Cambridge Reference Sequence numbering scheme. Figure 2 shows the highly conserved primer regions (in green) and the high nucleotide species diversity present between these primers within this portion of the 12S gene. In addition, an online resource exists for search and identification of the many thousands of organisms with homology to this region, thereby making species identification very straightforward. A February 2007 search of the http://www.ncbi.nlm.nih.gov/BLAST site showed that using the search term “12S ribosomal” returned 34,809 entries, using the search term “12S ribosomal + vertebrata” returned 23,717 entries, and using the search term “12S ribosomal + mammalia” returned 7,619 entries. Although some of these entries are duplicates within species, these results indicate that the 12S gene is substantially represented in the database.
Figure 2: Schematic of nucleotide diversity measurements across the 12S gene.
Figure 3: Strategy applied to all forensic casework leading to eventual analysis of species identification if necessary.
A streamlined and routine strategy to identify problematic (degraded or non-human) samples, which can proceed to 12S species analysis if necessary, is shown in Figure 3. Hairs, bones, teeth, and tissue samples are extracted and amplified according to standard mitochondrial DNA methods. A set of human-specific primers is first used to attempt amplification of the second half of hypervariable region 1 (16162-16400). If that amplification fails, human specific mini-primer sets are used to attempt amplification of positions 16131-16218 and 16209-16356 to assess whether degraded human mtDNA is present. If these amplifications fail also, species identification is undertaken. A 10-15 ul aliquot of DNA extraction product is amplified with 12S forward (12SF: 5’-ACTGGGATTAGATACCCCACTATG-3’) and 12S reverse (12SR: 5’-ATCGATTATAGAACAGGCTCCTC-3’) primers for 12 minutes at 96oC followed by 40 cycles of denaturation at 95oC for 15 seconds, annealing at 53oC for 30 seconds, and extension at 72oC for 45 seconds. The extraction reagent blank, a known human DNA (positive control) and a negative PCR control are amplified in parallel. A portion of the purified amplification product is cycle-sequenced with the same primers used for amplification. After analysis/editing, the consensus sequence is copied and pasted into the nucleotide-nucleotide BLAST function at the Web site http://www.ncbi.nlm.nih.gov/BLAST (Entrez, National Center of Biotechnology Information public databases). Following the prompts generates a report showing the degree of sequence homology with organisms in the database. The most likely species match can then be reported.
Between validation in June 2004 and June 2011, species identification using the 12S assay was used at Mitotyping Technologies in 20 cases on a total of 36 samples (Table 1), all but three of them hairs. In almost all these cases, the samples were submitted without having had the benefit of experienced microscopic examination, or a non-human origin was suspected based on sample morphology, and a species confirmation was desired. In many cases, the hair was small or fragmented, meaning that hair microscopy was more difficult for an inexperienced examiner.
|1||1.2 cm hair||Canis familiaris (dog)|
|1||0.9 cm hair||Sus scrofu (feral pig)|
|1||2 cm hair||Procyon lotor (raccoon)|
|2||6 cm hair||Canis familiaris (dog)|
|2||8 cm hair||Canis familiaris (dog)|
|3||4.5 cm hair||Canis familiaris (dog)|
|3||2 cm hair||Canis familiaris (dog)|
|3||4 cm hair||Canis familiaris (dog)|
|4||4 cm hair||Felis catus (cat)|
|5||2.5 cm hair||Canis familiaris (dog)|
|6||3 cm hair||Capra hircus (goat)|
|7||0.6 cm hair||Ovis aries (sheep)|
|8||2 cm hair||Canis familiaris (dog)|
|9||clump tissue||Rattus norvegicus (rat)|
|9||clump tissue||Rattus norvegicus (rat)|
|10||2 cm hair||Canis familiaris (dog)|
|11||1.3 cm hair||Canis familiaris (dog)|
|12||10.8 cm hair||Bos grunniens (yak)|
|13||4 cm hair||Canis familiaris (dog)|
|13||8 cm hair||Canis familiaris (dog)|
|13||1.8 cm hair||Canis familiaris (dog)|
|14||1.5 cm hair||Canis familiaris (dog)|
|15||2 mm hair||Canis familiaris (dog)|
|15||1 mm hair||Canis familiaris (dog)|
|15||2 mm hair||Canis familiaris (dog)|
|16||1 cm hair||Canis familiaris (dog)|
|16||3.6 cm hair||Felis catus (cat)|
|17||2.5 cm hair||Canis familiaris (dog)|
|18||2 cm hair||Ovis aries (sheep)|
|18||1.5 cm hair||Canis familiaris (dog)|
|18||2 cm hair||Ovis aries (sheep)|
|18||2 cm hair||Marmota sp. (marmot)|
|18||1.1 cm hair||Ovis aries (sheep)|
|19||bone||Gallus gallus (chicken)|
|20||2 cm hair||Canis familiaris (dog)|
Figure 4: Yak,
In Case 12, beauty salon owners wished to know if their hairpiece supplier was providing them with guaranteed human hair as promised. A microscopic hair examination done by an outside consultant revealed the hair to be animal hair, and the 12S assay showed the species to be Bos grunniens (yak). Upon further investigation, we learned that yaks are common sources of human-appearing hairs that are frequently incorporated into hair weaves and wigs in the beauty industry.
In Case 9, investigators re-opened an unsolved 50-year-old homicide in which tissue had been collected from an automobile undercarriage. The tissue was sent for mitochondrial DNA analysis and comparison to a maternal relative of the missing victim. Attempts to amplify HV1 and HV2 with standard and mini-primer sets were unsuccessful. When the 12S assay was performed, the tissue was determined to belong to Rattus norvegicus (common rat). This discovery resulted in steering the investigation away from homicide by vehicle.
Other cases turned up hairs from domestic animals such as dog, cat, sheep, and goat as well as from wild animals such as raccoon and feral pig. Since the majority of our casework involves hairs, and most of the 12S assays have been applied to hairs, by definition a physical characteristic of mammals, this assay is highly useful. For a laboratory already performing DNA sequencing, the addition of a 12S assay requires no change in general sequencing or data editing routines.
Of interest might be the extent of 12S homology within and between species, or whether there would be a likelihood of spurious matches. While it would be difficult to investigate systematically the homologies for all possible species, the 100 most homologous species returns for the Canis familiaris search were examined. All 100% homologies (N=41) were either Canis familiaris (domestic dog), Canis latrans (coyote), or Canis lupus (wolf), and there were other Canis entries at 99% (N=7), 98% (N=1), 97% (N=1) and 94% (N=1) homologies. There was no intromission of non-Canis species into this cluster, but species falling with the range of 94% to 91% homology included the bushbuck, bat, red fox, giant eland, weasel, giraffe, musk deer, spotted linsang, seal, and cow. Keeping in mind that most forensic cases will not involve animals like seals or bats, it appears that there can be a high degree of confidence in those species returned in the BLASTn search. Although there is no guarantee that a recovered animal hair or tissue sequence will find a perfect BLAST match, we note that the profiles of most common North American mammals are present in the database and the assay as designed is sufficient for general forensic species identification.
Investigation of primate homologies within the NCBI database revealed that Pan troglodytes (chimpanzee) shares a 98% homology with the human 12S region used here, while Gorilla gorilla and Macaca mulatta (rhesus macaque) share 97% and 90% homologies with humans, respectively. Therefore, it is unlikely that a non-human primate hair could be easily confused with a human hair using this system.
We previously reported that 7.2% of our hair samples gave no profile due to either a non-human origin or absence of recoverable mtDNA due to degradation.2 Since application of the 12S assay, the frequency of no-result hairs has dropped to 4.5%. A test yielding no results is now most likely due to the complete absence of mtDNA due to degradation rather than to a non-human source. The size of the 12S amplicon, 150 bp, is the same as the average size of the mini-primer set amplicons used for the control region, meaning it has the same efficacy in recovery of degraded mtDNA for non-human samples as the mini-primer set approach does for human samples.
This method can be applied to any kind of biological material such as hair, tissue, bone, blood, or saliva. Once the species origin of a sample is identified, a species-specific STR or mitochondrial DNA control region analysis, if available, could be applied to the remaining DNA extraction product for further animal individualization. In addition to aiding in identification of non-human sources of samples in the mainstream criminal justice system, this method has broad applications in forensic wildlife studies, including poaching and the sale of endangered species.
- Melton T and Holland C. Routine use of the mitochondrial 12S ribosomal RNA gene for species identification. J Forensic Sci 2007:52:1305-1307.
- Melton T, Nelson K. Forensic mitochondrial DNA analysis of 691 casework hairs. J Forensic Sci 2005;50:73-80.
Terry Melton, Ph.D., is a Forensic Examiner and the Laboratory Director of Mitotyping Technologies in State College, Pennsylvania, a private laboratory that provides DNA analysis services to law enforcement, attorneys, and other members of the criminal justice community. Terry is a Fellow of the American Academy of Forensic Sciences and a member of the editorial board of the Journal of Forensic Sciences. Mitotyping Technologies, 2565 Park Center Boulevard, Suite 200, State College, PA 16801, firstname.lastname@example.org, www.mitotyping.com