Discrimination between human and animal DNA: application of a duplex polymerase chain reaction to forensic identification

Identification of a report's species is one of the basic analyses in forensic laboratories. The authors report the case of 6 bone fragments recovered in a wooded area, which were not attributable to 1 animal species on the basis of morphologic examination. The aim of this study was to develop a duplex polymerase chain reaction (PCR) to discriminate human and animal origin of bone fragments. The method is based on the PCR amplification of cytochrome b and a 16S ribosomal mitochondrial DNA fragment, which has never been tested up to now. Our protocol combines a single-round PCR with direct visualization of amplicons in agarose gel, without sequencing analysis of the PCR products. The presence of a single band (359 bp) indicates a nonhuman origin of the sample, whereas 2 bands (157 and 359 bp) indicate a human biologic sample.This method revealed to be useful for forensic purposes because the 16S ribosomal mitochondrial DNA is a small human-specific fragment that is easily amplifiable even with degraded DNA from biologic materials such as old bones.     

Criteria of personal identification by the hyoid bone

We studied 16 sizes of the hyoid bone (HB) from 158 corpses of known gender, Russians, who had died at the age over 15 years by the K. Miller et al program (1998) and 205 back projection electroroentgenograms of the neck of patients of the traumatological hospital in the town of Kirov. HB skeletization was made by the enzymatic drug enzyme. SPSS package and multidimentional statistics (regression and discriminant analyses) were used for data processing. It was established that HB is an informative object of forensic medical personal identification allowing determination of the body length and gender as well as anatomical, x-ray and osteometric individual features. Precision of human body length diagnosis by HB is 6.71-7.26 cm. Therefore, this method should be used in combination with the diagnosis by long tubular bones. Diagnosis of human gender by osteometric signs of HB based on one-dimentional discriminant analysis is feasible in 80-85%, based on canonical discriminant analysis--in 91.1% cases. The method of anatomic, osteometric and roentgenological assessment of HB individuality can be used in personal identification by intravital x-ray of cervical organs and in traumatic defects of HB. The methods may be applied in forensic medical practice in expert examination of body fragments or isolated bone fragments.     

Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region

Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog, goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic specimens.     

The effects of skeletal preparation techniques on DNA from human and non-human bone

The forensic pathologist increasingly relies on the forensic anthropologist to be the consulting expert in human identification. Likewise, if identification is not possible from visual inspection of skeletal remains, the forensic biologist may be called upon to conduct DNA analysis. The possibility of downstream DNA testing needs to be considered when skeletal preparation techniques are employed to deflesh human remains, as they have the potential to strongly impact genetic analyses and subsequent identification. In this study, three cleaning techniques, boiling bone in water, in bleach, and in powdered detergent/sodium carbonate, were tested for their effect on nuclear and mtDNA recovery from a variety of human and non-human bones. A statistically significant reduction in DNA yields occurred in non-human bones cleaned with bleach, and DNA degradation was apparent electrophoretically. The human bones also showed much lower yields from bleach cleaning, while the detergent/carbonate method allowed the largest segments of DNA to be amplified, indicating it may have a less degradative effect on bone DNA than either of the other cleaning processes.     

Fungal DNA challenge in human STR typing of bone samples

The present study focuses on possible cross-reaction of fungal DNA with human STR primers that may affect subsequent forensic DNA analysis of forensic samples. Specificity of human STR markers namely HUMAMEL, HUMCSF1PO, D8S306, HUMTH01, HUMvWA, HUMFES/FPS, HUMF13A01, HUMDHFRP2, HUMFGA and HUMTPOX was tested using DNA of 24 different filamentous fungal isolates obtained from exhumed bone samples. The specificity of these ten STR markers for human DNA was demonstrated. Presence of non-human DNA in five bone samples analyzed did not alter scoring of detected alleles. Notably, amplification was inhibited in the presence of a high proportion of fungal DNA compared to human DNA (1000 ng: 1 ng) in DNA mixture experiments. The results of the present study underscore the importance of carefully analyzing the presence of non-human biological contaminants that may affect DNA typing of environmentally challenged forensic samples to avoid spurious data interpretation.     

The 2000-2001 GEP-ISFG Collaborative Exercise on mtDNA: assessing the cause of unsuccessful mtDNA PCR amplification of hair shaft samples

We report the results of Spanish and Portuguese working group (GEP) of International Society of Forensic Genetics (ISFG) Collaborative Exercise 2001-2002 on mitochondrial DNA (mtDNA) analysis. 64 laboratories from Spain, Portugal and several Latin-American countries participated in this quality control exercise. Five samples were sent to the participating laboratories, four blood stains (M1-M4) and a sample (M5) consisting of two hair shaft fragments. M4 was non-human (Felis catus) in origin; therefore, the capacity of the labs to identify the biological source of this sample was an integral part of the exercise. Some labs detected the non-human origin of M4 by carrying out immuno-diffussion techniques using antihuman serum, whereas others identified the specific animal origin by testing the sample against a set of animal antibodies or by means of the analysis of mtDNA regions (Cyt-b, 12S, and 16S genes). The results of the other three human blood stains (M1-M3) improved in relation to the last Collaborative Exercises but those related to hairs yielded a low rate of success which clearly contrasts with previous results. As a consequence of this, some labs performed additional analysis showing that the origin of this low efficiency was not the presence of inhibitors, but the low quantity of DNA present in these specific hair samples and the degradation.As a general conclusion the results emphasize the need of external proficiency testing as part of the accreditation procedure for the labs performing mtDNA analysis in forensic casework.     

Implant Bone Integration Importance in Forensic Identification

Odontological identification consists of the comparison of antemortem dental information regarding a missing person with postmortem data from an unidentified corpse or human remains. Usually, the comparison concerns morphologic features that the operator chooses among all the visible characteristics because of inter‐individual uniqueness; for this reason, implants can be of enormous assistance. A case concerning the recovery of a burnt oral implant, connected to a bone fragment, among 2780 charred bone fragments, suspected to have belonged to a victim of homicide, is presented to demonstrate that dental implants and their site of bone integration represent a very precious element for personal forensic identification. Because of their morphological invariability in time and because of their morphologic uniqueness, they were used as evidence to associate unidentified human charred remains to a missing person where DNA analysis failed to do so. The case illustrates the fundamental contribution, not yet described in literature, given by the clinical aspects of tooth replacement with dental implants to a forensic discipline. Clinical practitioners should therefore be aware of the great importance of their work and of dental records in a forensic identification scenario.     

DNA extraction of burnt bone and teeth casework samples using bead-beating homogenization technique

Sample disruption was a necessary step for DNA isolation. Bone and teeth were useful biological sources particular in human remains and advance decomposed bodies. The compact bone and teeth required several preparation steps prior to analyzing process. However, the methods in standard protocol were laborious and time consuming. An alternating pulverization, bead beating homogenizer, was purposed in its effectiveness for forensic casework. (1) Here, we applied this technique to the burnt cracked bone and tooth that recovered from house fire for forensic DNA analysis. After cleansed an external surface, the eight multidirectional motion tissue homogenizer, Precellys® evolution, was utilized to pulverize bone and tooth followed by a DNA extraction and amplification. For detection with a capillary electrophoresis, full profiles of autosomal STRs and Y-chromosomal STRs were recovered from tooth sample but the partial profile STR was demonstrated in bone sample. The new technique in bone homogenization was less time consuming (around 30 s), less exposure to chemical agents (no need of liquid nitrogen), high efficiency, with high-throughput productivity.     

Mitochondrial Genome DNA Analysis of the Domestic Dog: Identifying Informative SNPs Outside of the Control Region*

Abstract: While the mitochondrial control region has proven successful for human forensic evaluations by indicating ethnic origin, domestic dogs (Canis lupus familiaris) of seemingly unrelated breeds often form large groups based on identical control region sequences. In an attempt to break up these large haplotype groups, we have analyzed the remaining c. 15,484 base pairs of the canine mitochondrial genome for 79 dogs and used phylogenetic and population genetic methods to search for additional variability in the form of single nucleotide polymorphisms (SNPs). We have identified 356 SNPs and 65 haplotypes in the remainder of the mitochondrial genome excluding the control region. The exclusion capacity was found to be 0.018. The mitochondrial control region was also evaluated for the same 79 dogs. The signals from the different fragments do not conflict, but instead support one another and provide a larger fragment of DNA that can be analyzed as forensic evidence.     

Case involving differentiation of deer and human bone fragments

In a recent Louisiana forensic anthropology case, it was necessary to attempt species identification of six small bone fragments. The primary concern was whether or not they matched the fractured humerus of a woman killed by two shotgun blasts and then disposed of in the Mississippi River. These tiny fragments were recovered by law enforcement officers inside a jeep pickup and at the gas station where the vehicle had been cleaned. The police suspect claimed that these fragments were from a deer that he had recently killed. The small size of the pieces precluded positive recognition of human versus nonhuman origin based upon gross morphology and cortical thickness. Microscopic examination was possible. This analysis involved comparison of the unknown specimens to reference deer and human thin sections including bone recovered from the woman during autopsy. Examination of the jeep and gas station fragments revealed no plexiform bone, secondary (not primary) osteons, and variability in size and shape of the osteons and Haversian canals. These and other variables identified the bone fragments as human.     

法医骨组织学研究

在实际检案中 ,当现场发现的骨骼残片体积较小时 ,用解剖学观察无法进行骨骼残片的法医鉴定 ,需使用骨组织学的方法进行骨骼残片的个体识别。目前 ,这是法医人类学中一门较活跃的领域 ,即法医骨组织学。法医骨组织学的内容主要包括两个方面 :(1)骨骼残片是否属于人类骨骼 ,或属于何种动物骨骼。这方面的研究包括人类骨骼的组织学特征研究及不同动物的组织学特征研究。 (2 )人类骨骼个体识别的组织学研究。这方面的研究主要包括 ,人类骨骼的组织学特征的年龄判断 ,例如股骨、胫骨、肱骨、锁骨等 ,以及使用骨组织学方法 ,进行人类骨骼的年龄评价的准确性研究。本文对上述内容进行了综述。     

Effects of processing techniques on the forensic DNA analysis of human skeletal remains

Human remains processed by forensic anthropologists may potentially be used for genetic analysis. Therefore, the condition of the deoxyribonucleic acid (DNA) in processed remains may become an issue for future analysis. Processing techniques employed by anthropologists are highly variable and scanning electron microscopy reveals significant alterations to the bone surface depending upon the technique used. Such damage to the bone indicates differences may exist in quality and quantity of DNA extracted. This study assessed how five processing procedures used by major forensic anthropology laboratories around the country affects the amounts of DNA extracted from human rib bones and the subsequent DNA analysis. The DNA was analyzed using the short tandem repeat (STR) locus CSF1PO and amelogenin. The findings indicate processing procedures used by forensic anthropologists do not adversely affect DNA analysis but prolonged exposure to heat during processing may decrease the yield of information from the DNA.     

Analysis of an ancestry using cremated old human remains from the Korean War victims

Analysis of DNA from burnt bone fragment is the very hard work for the human identification in forensic casework. In general, cremated bone with an artificial damage is more difficult to get an intact DNA than a singed one because of the chemical and biological drastic changes such as protein denaturation and destruction. In this study, we pursue the best technical approach for the minimal damage and the contamination of DNA from other factors in the preconditioning and the extraction process based on over 70 years old Korean War victim skeletal that was burnt and buried in Korean Peninsula. First of all, we removed the pollutant and the dust from the burnt bones using dental instruments, and then incubated with EDTA buffer at 25 ℃ to remove inhibitors such as calcium and mineral. In order to compare the DNA preservation ability between a pellet and a supernatant, samples are repeatedly tested to collect washed EDTA buffer several times to separate. Each of isolated materials is secondly cleaned with the organic extraction method using phenol and analyzed mtDNA sequence with the in-house method for the ancestry assay. The better discrimination ability was appeared in the supernatant than the pellet. Nevertheless, many of the forensic geneticists use a powdering method for getting more DNA, we applied EDTA buffer in the preconditioning step to eliminate every contamination. As a result, the contamination factor was efficiently removed and the ancestry was estimated as per the written information. Consequently, cremated bone is identified to belong in the D4 mtDNA haplogroup which is commonly reported in ethnic groups in Asia especially Korea. This is a preliminary study of a human identification over an ancestry analysis to give information against a mass disaster in a future. Through a higher process optimization and better analytical methods toward more remains, which are genetically difficult to analyze, will support to examine the identity of the post cremated remains.     

A duplex real-time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples: implications for quantifying DNA in degraded samples

A duplex real-time qPCR assay was developed for quantifying human nuclear and mitochondrial DNA in forensic samples. The nuclear portion of the assay utilized amplification of a approximately 170-190 bp target sequence that spans the repeat region of the TH01 STR locus, and the mitochondrial portion of the assay utilized amplification of a 69 bp target sequence in the ND1 region. Validation studies, performed on an ABI 7000 SDS instrument using TaqMan detection, demonstrated that both portions of the duplex assay provide suitable quantification sensitivity and precision down to 10-15 copies of each genome of interest and that neither portion shows cross-reactivity to commonly encountered non-human genomes. As part of the validation studies, a series of DNase-degraded samples were quantified using three different methods: the duplex nuclear-mitochondrial qPCR assay, the ABI Quantifiler Human DNA Quantification Kit qPCR assay, which amplifies and detects a 62 bp nuclear target sequence, and slot blot hybridization. For non-degraded and moderately degraded samples in the series, all three methods were suitably accurate for quantifying nuclear DNA to achieve successful STR amplifications to yield complete profiles using the ABI AmpFlSTR Identifiler kit. However, for highly degraded samples, the duplex qPCR assay provided better estimates of nuclear template for STR amplification than did either the commercial qPCR assay, which overestimated the quantity of STR-sized DNA fragments, leading to an increased proportion of undetected alleles at the larger STR loci, or slot blot hybridization, which underestimated the quantity of nuclear DNA, leading to an increased proportion of STR amplification artifacts due to amplification of excess template.     

Craniometric diagnosis of skeletal and somatotype mass

The paper presents diagnostic models for evaluation of ash mass (bone fragments, bone ashes), sex, length and mass of the burned body. Individual data by M. Warren and W. Maples (1997) on 85 adult Americans were used. On the basis of a canonical discriminant analysis and multiple linear regression equations the authors have developed models for diagnosis of ash residue mass as well as the burned body's sex, body length and mass. The above models can be used directly on the place of the criminal body cremation. They do not require determination of anatomic appurtenance of bone fragments and their osteometric examination.     

Difficulties of sex determination from forensic bone degraded DNA: A comparison of three methods

Sex determination is of paramount importance in forensic anthropology. Numerous anthropological methods have been described, including visual assessments and various measurements of bones. Nevertheless, whatever the method used, the percentage of correct classification of a single bone usually varies between 80% and 95%, due to significant intra- and inter-population variations, and sometimes variations coming from secular trends. DNA is increasingly used in a forensic context. But forensic DNA extraction from bone raises several issues, because the samples are very often badly altered and/or in very small quantity. Nuclear DNA is difficult to get from degraded samples, according to low copy number, at least in comparison with mitochondrial DNA. In a forensic context (as in a paeleoanthropological context) DNA sex determination is usually complicated by the weak amount of DNA, the degraded nature of nucleic acids, the presence of enzymatic inhibitors in DNA extracts, the possible faint amplification of Y band and the risk of contamination during either excavation or manipulation of samples.The aim of this work was to compare three methods of DNA sex determination from bones: procedure #1 using a single PCR amplification, procedure #2 using a double PCR amplification, and procedure #3 adding bleaching for decontamination of the bone, instead of simply rubbing the bone. These processes were applied to samples of bones (49 samples coming from 39 individuals) that were in various states of post mortem alteration.The main results are the following. (i) No DNA could be extracted from three skulls (parietal bones, mastoid process), the compact bone of one rib, and the diaphysis of one femur; (ii) there was a contamination in three skulls; and (iii) the Y band did not appear in two male cases, with one of the three procedures (male tibia, procedure #2) and with procedures #2 and #3 (male femur).This study emphasises the main issue while working with altered bones: the impossibility to extract DNA in some cases, and, worth of all, the contamination of the sample or the faint amplification of Y band which leads to a wrong sex answer. Multiple and significant precautions have to be taken to avoid such difficulties.     

Interindividual morphologic variability as reason for misinterpretations in forensic osteology exemplified by a rib

Bone finds make great demands on the examiner. The first priority is to establish the remains as human, because then further analyses have to be performed for identification. Especially for bone fragments it may be extremely difficult to determine whether they are of human or animal origin. In the reported case a bone fragment similar to a human rib was found in a medieval town together with more than 300 non-human bones. As it turned out, a medieval cesspit had been opened during excavation works. Even an mtDNA analysis could not answer the question whether the rib was of human origin. A direct comparison with numerous ribs of humans and mammals showed the great interindividual variability, but in the end it was possible to rule out a human origin and to identify the fragment as the third left rib of a pig.     

Histological Determination of the Human Origin of Bone Fragments

Abstract:  A frequently encountered task in the forensic scenario is verification of the human origin of severely degraded fragments of bone. In these cases histological methods which consider osteon size and morphology can prove to be useful. The authors in the present study verify the applicability of published algorithms to flat and subadult bones from human, dog, cat, cow, rabbit, sheep, pig, chicken, quail, and turkey samples. Metric analysis was performed on 2031 Haversian canals. Analyses carried out on human samples confirmed a success rate of around 70% on long adult bones; however the percentage of wrong answers was particularly high in the case of newborns and older subadults as well as on flat bones in general. Results therefore suggest that such regression equations should be limited only to bone fragments from long adult bones.     

An extremely sensitive species-specific ARMs PCR test for the presence of tiger bone DNA

The survival of the tiger (Panthera tigris) is seriously threatened by poaching to provide raw materials for Traditional Chinese Medicines (TCMs). Most highly prized are the tiger's bones, which are used in combination with other animal and plant derivatives in pills and plasters for the treatment of rheumatism and other ailments. Hundreds of patent remedies have been produced which claim to contain tiger bone, but proof of its presence is needed, if legislation prohibiting the trade in endangered species is to be enforced. A highly sensitive tiger-specific real-time PCR assay has been developed to address this problem. Using primers specific to the tiger mitochondrial cytochrome b gene, successful amplification has been reliably achieved from blood, hair and bone as well as from a range of TCMs spiked with 0.5% tiger bone. Although capable of detecting fewer than 10 substrate molecules, the seven varieties of TCM pills and plasters tested showed no detectable trace of tiger DNA before spiking. Furthermore, sequencing several "tiger bone" fragments seized from TCM shops has shown that they actually originated from cattle and pigs. The potential effects of traditional bone preparation methods, evidence that much lower concentrations are used than alleged on TCM packaging, and substitution of bones from other species all suggest a low likelihood of detecting tiger DNA in patent medicines. Despite this, the basic methods have been thoroughly proven and can be readily applied to derivatives from other CITES protected species providing a rapid and highly sensitive forensic test for species of origin. Potential applications to the monitoring of wild populations are demonstrated by the successful identification of shed hairs and faecal samples.     

Identification of mammal species using species-specific DNA pyrosequencing

In forensic casework it is highly relevant to be able to deduce the species origin of an unknown biological sample. For such a purpose we have designed and developed an assay for species identification based on DNA sequencing of two short mitochondrial DNA amplicons. In short, partial 12S rRNA and partial 16S rRNA fragments (approximately 100bp) are amplified by PCR followed by direct sequencing using pyrosequencing technique. Due to properties of the chosen targets, the same PCR conditions and primers were used irrespective of the true species of an unknown sample. A total of 28 different mammals present in the European fauna were sequenced both for the partial 12S rRNA and the partial 16S rRNA sequences for accuracy verification. Together the two sequences showed to have a high divergence factor, discriminating almost all mammals. Furthermore, the human reference nucleotide sequences were always at least nine nucleotides different compared to the other sequenced species both at the partial 12S rRNA and the partial 16S rRNA sequences.