Diagnostic models to predict nuclear DNA and mitochondrial DNA recovery from incinerated teeth.

Teeth are frequently used for human identification from burnt remains, as the structure of a tooth is resilient against heat exposure. The intricate composition of hydroxyapatite (HA) mineral and collagen in teeth favours DNA preservation compared to soft tissues. Regardless of the durability, the integrity of the DNA structure in teeth can still be disrupted when exposed to heat. Poor DNA quality can negatively affect the success of DNA analysis towards human identification. The process of isolating DNA from biological samples is arduous and costly. Thus, an informative pre-screening method that could aid in selecting samples that can potentially yield amplifiable DNA would be of excellent value. A multiple linear regression model to predict the DNA content in incinerated pig teeth was developed based on the colourimetry, HA crystallite size and quantified nuclear and mitochondrial DNA. The chromaticity a* was found to be a significant predictor of the regression model. This study outlines a method to predict the viability of extracting nuclear and mitochondrial DNA from pig teeth that were exposed to a wide range of temperatures (27 to 1000 °C) with high accuracy (99.5-99.7%).

Freeze-drying improves DNA yield from teeth.

The common method of preparing teeth prior to DNA extraction involves cleaning, decontamination, drying and pulverisation. Moisture in post-mortem teeth can promote bacterial growth and hydrolytic damage that could contribute to DNA degradation, whilst also possibly reducing the efficiency of sample pulverisation and DNA release. Here we compared DNA extraction from pig teeth, with- and without freeze-drying, to examine the impact of removing moisture on DNA yield. Quantitative real-time polymerase chain reaction (qPCR) was used to quantify an 83 bp mitochondrial DNA fragment and two nuclear DNA fragments of 82 bp and 150 bp. The comparative results showed that sample preparation with freeze-drying resulted in a higher DNA yield without compromising the DNA quality. This study highlights the advantage of incorporating a freeze-drying to improve the DNA yield and minimising the loss of DNA during sample preparation of teeth.

The development of a tool to predict temperature-exposure of incinerated teeth using colourimetric and hydroxyapatite crystal size data.

This study presents a novel tool to predict temperature-exposure of incinerated pig teeth as a proxy for understanding impacts of fire on human teeth. Previous studies on the estimation of temperature-exposure of skeletal elements have been limited to that of heat-exposed bone. This predictive tool was developed using a multinomial regression model of colourimetric and hydroxyapatite crystal size variables using data obtained from unheated pig teeth and teeth incinerated at 300 °C, 600 °C, 800 °C and 1000 °C. An additional variable based on the observed appearance of the tooth was included in the tool. This enables the tooth to be classified as definitely burnt (600 °C-1000 °C) or uncertain (27 °C/300 °C). As a result, the model predicting the temperature-exposure of the incinerated teeth had an accuracy of 95%. This tool is a holistic, robust and reliable approach to estimate temperature of heat-exposed pig teeth, with high accuracy, and may act as a valuable proxy to estimate heat exposure for human teeth in forensic casework.

Integrating spectrophotometric and XRD analyses in the investigation of burned dental remains.

Heat alters colour and crystallinity of teeth by destruction of the organic content and inducing hydroxyapatite crystal growth. The colour and crystallite changes can be quantified using spectrophotometric and x-ray diffraction analyses, however these analyses are not commonly used in combination to evaluate burned dental remains. In this study, thirty-nine teeth were incinerated at 300-1000 °C for 15 and 30 min and then measured using a spectrophotometer and an x-ray diffractometer. Response variables used were lightness, L*, and chromaticity a* and b* and luminance (whiteness and yellowness) for colour, and crystal size for crystallinity. Statistical analysis to determine the attribution of these variables revealed yellowness and crystal size were significantly affected by temperature (p < 0.05), whilst duration of heat-exposure showed no significant effect. This study suggests the inclusion of both spectrophotometric and x-ray diffraction in investigating thermal-heated teeth is useful to accurately estimate the temperature teeth are exposed to.

An internationally standardized species identification test for use on suspected seized rhinoceros horn in the illegal wildlife trade.

Rhinoceros (rhino) numbers have dwindled substantially over the past century. As a result, three of the five species are now considered to be critically endangered, one species is vulnerable and one species is near-threatened. Poaching has increased dramatically over the past decade due to a growing demand for rhino horn products, primarily in Asia. Improved wildlife forensic techniques, such as validated tests for species identification of seized horns, are critical to aid current enforcement and prosecution efforts and provide a deterrent to future rhino horn trafficking. Here, we present an internationally standardized species identification test based on a 230 base pair cytochrome-b region. This test improves on previous nested PCR protocols and can be used for the discrimination of samples with <20pg of template DNA, thus suitable for DNA extracted from horn products. The assay was designed to amplify water buffalo samples, a common 'rhino horn' substitute, but to exclude human DNA, a common contaminant. Phylogenetic analyses using this partial cytochrome-b region resolved the five extant rhino species. Testing successfully returned a sequence and correct identification for all of the known rhino horn samples and vouchered rhino samples from museum and zoo collections, and provided species level identification for 47 out of 52 unknown samples from seizures. Validation and standardization was carried out across five different laboratories, in four different countries, demonstrating it to be an effective and reproducible test, robust to inter laboratory variation in equipment and consumables (such as PCR reagents). This is one of the first species identification tests to be internationally standardized to produce data for evidential proceedings and the first published validated test for rhinos, one of the flagship species groups of the illegal wildlife trade and for which forensic tools are urgently required. This study serves as a model for how species identification tests should be standardized and disseminated for wildlife forensic testing.

The complete mitochondrial genome of Axis porcinus (Mammalia: Cervidae) from Victoria, Australia, using MiSeq sequencing.

The mitochondrial genome of the hog deer (Axis porcinus) was sequenced using an Illumina MiSeq. The assembled genome consists of 16,351 bp, and shared a 99.8% similarity to the published chital deer (Axis axis) genome, suggesting that they belong to the same species. Further research is ongoing to understand why these mitochondrial genomes are highly similar.

Molecular identification of python species: development and validation of a novel assay for forensic investigations.

Python snake species are often encountered in illegal activities and the question of species identity can be pertinent to such criminal investigations. Morphological identification of species of pythons can be confounded by many issues and molecular examination by DNA analysis can provide an alternative and objective means of identification. Our paper reports on the development and validation of a PCR primer pair that amplifies a segment of the mitochondrial cytochrome b gene that has been suggested previously as a good candidate locus for differentiating python species. We used this DNA region to perform species identification of pythons, even when the template DNA was of poor quality, as might be the case with forensic evidentiary items. Validation tests are presented to demonstrate the characteristics of the assay. Tests involved the cross-species amplification of this marker in non-target species, minimum amount of DNA template required, effects of degradation on product amplification and a blind trial to simulate a casework scenario that provided 100% correct identity. Our results demonstrate that this assay performs reliably and robustly on pythons and can be applied directly to forensic investigations where the presence of a species of python is in question.

Identification multiplex assay of 19 terrestrial mammal species present in New Zealand.

An identification assay has been developed that allows accurate detection of 19 of the most common terrestrial mammals present in New Zealand (cow, red deer, goat, dog, horse, hedgehog, cat, tammar wallaby, mouse, weasel, ferret, stoat, sheep, rabbit, Pacific rat, Norway rat, ship rat, pig, and brushtail possum). This technique utilizes species-specific primers that, combined in a multiplex PCR, target small fragments of the mitochondrial cytochrome b gene. Each species, except hedgehog, produces two distinctive species-specific fragments, making the assay self-confirmatory and enabling the identification of multiple species simultaneously in DNA mixtures. The multiplex assay detects as little as 100 copies of mitochondrial DNA, which makes it a very reliable tool for degraded and trace samples. Reliability, accuracy, reproducibility, and sensitivity tests to validate the technique were performed. The technique featured here enabled a prompt response in a predation specific event, but can also be useful for wildlife management and conservation, pest incursions detection, forensic, and industrial purposes in a very simple and cost-effective manner.

Reconstructing mammalian phylogenies: a detailed comparison of the cytochrome B and cytochrome oxidase subunit I mitochondrial genes.

The phylogeny and taxonomy of mammalian species were originally based upon shared or derived morphological characteristics. However, genetic analyses have more recently played an increasingly important role in confirming existing or establishing often radically different mammalian groupings and phylogenies. The two most commonly used genetic loci in species identification are the cytochrome oxidase I gene (COI) and the cytochrome b gene (cyt b). For the first time this study provides a detailed comparison of the effectiveness of these two loci in reconstructing the phylogeny of mammals at different levels of the taxonomic hierarchy in order to provide a basis for standardizing methodologies in the future. Interspecific and intraspecific variation is assessed and for the first time, to our knowledge, statistical confidence is applied to sequence comparisons. Comparison of the DNA sequences of 217 mammalian species reveals that cyt b more accurately reconstructs their phylogeny and known relationships between species based on other molecular and morphological analyses at Super Order, Order, Family and generic levels. Cyt b correctly assigned 95.85% of mammal species to Super Order, 94.31% to Order and 98.16% to Family compared to 78.34%, 93.36% and 96.93% respectively for COI. Cyt b also gives better resolution when separating species based on sequence data. Using a Kimura 2-parameter p-distance (x100) threshold of 1.5-2.5, cyt b gives a better resolution for separating species with a lower false positive rate and higher positive predictive value than those of COI.

A technique for the quantification of human and non-human mammalian mitochondrial DNA copy number in forensic and other mixtures.

The number of mitochondria per cell varies by cell type and the number of mitochondrial DNA (mtDNA) genomes varies per mitochondrion. Biological samples from unknown species are encountered frequently in forensic science investigations and are often contaminated with human mtDNA making analysis difficult. Currently, no techniques to quantify non-human mtDNA are available. We report on a method to accurately quantify, sensitive to 100 copies (1.7fg), mtDNA from human and non-human sources when present as a mixture. The test developed uses the cytochrome b (cytb) and the ribosomal 12S genes on the mitochondrial genome. Universal and human specific fragments of similar size are amplified and quantified using SYBR Green. We validate the test with 24 human samples and 27 non-human mammalian samples. The human fraction of a sample can then be subtracted from the universal fraction for an accurate estimation of non-human mtDNA copy number.

A multiplex assay to identify 18 European mammal species from mixtures using the mitochondrial cytochrome b gene.

A novel species-specific multiplex to identify 18 common European mammalian species (badger, cat, cow, dog, donkey, fox, goat, guinea pig, harvest mouse, hedgehog, horse, house mouse, human, pig, rabbit, rat, red deer and sheep), many of which are often associated with forensic investigations, has been developed. The assay is based on the mitochondrial cytochrome b gene, which is commonly used in species identification and phylogeny studies. Areas of homology and variation were identified and were used to create universal and species-specific primers. The species-specific primers were designed such that they will only react with the species for which they were designed. Two primer sets were designed for each species making the test self-confirmatory. All primer sets produced the expected results. The multiplex was balanced at template concentration of 40 000 copies (approximately 1.36 pg). Validation was accomplished by analysing the same sample ten times to determine run variation and several samples for each species to determine between-sample variation. Twenty-eight additional mammalian species were reacted with the multiplex. The multiplex provides, for the first time, a definitive method for identification of species in a forensic context.