陈旧骨骼DNA提取及性别鉴定

用本室建立的方法对3～15年的陈旧骨骼进行DNA提取，并用X、Y同源引物扩增AInelogenin基因X、Y特异性片段，所有检材均得出正确结论，表明该方法快速、灵敏、可靠，适用于陈旧骨骼性别鉴定。     

Testing the validity of metacarpal use in sex assessment of human skeletal remains

To assess the potential of employing metacarpals in assessing sex of human skeletal remains, previous investigators have generated regression equations (Scheuer & Elkington, 1995) and linear discriminant functions (Falsetti, 1995; Stojanowski, 1999) based upon measurements from metacarpals. Results have varied in overall accuracy and which metacarpal produces the greatest accuracy. Using a contemporary sample, this study seeks to evaluate the validity of using metacarpals to assign sex by testing methodologies of previous studies. Measurements defined by previous authors were repeated on metacarpals from 23 adult cadavers and data were subjected to regression equations and linear discriminant analysis according to previous methodologies. Accuracy in sex determination from methods of Scheuer & Elkington (1993) and Falsetti (1995) were lower than originally reported while accuracy from methods of Stojanowski (1999) were higher than previously reported. These results suggest that the use of metacarpals in sex determination may be limited and should be applied cautiously.     

Simple and highly effective DNA extraction methods from old skeletal remains using silica columns

The recovery of DNA data from old skeletal remains is often difficult due to degraded and very low yield of extracted DNA and the presence of PCR inhibitors. Herein, we compared several silica-based DNA extraction methods from artificially degraded DNA, DNA with PCR inhibitors and DNA from old skeletal remains using quantitative real-time PCR. We present a modified large-scale silica-based extraction combined with complete demineralization, that enables maximum DNA recovery and efficient elimination of PCR inhibitors. This is performed with high concentration of EDTA solution for demineralization of bone powder followed by QIAamp^® spin columns and buffers from the QIAquick^® PCR purification kit. We have successfully used this modified technique to perform STR analysis for 55-year-old skeletal remains. The results of this study will contribute to solve the forensic cases dealing with skeletal remains.     

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.     

Comparison of two methods for isolating DNA from human skeletal remains for STR analysis

Abstract: The quality and efficiency of a standard organic DNA isolation method and a silica‐based method using the QIAGEN Blood Maxi Kit were compared to obtain human DNA and short tandem repeats (STRs) profiles from 39 exhumed bone samples for paternity testing. DNA samples were quantified by real‐time PCR, and STR profiles were obtained using the AmpFlSTR^® Identifiler^® PCR amplification kit. Overall, the silica‐based method recovered less DNA ranging from 0 to 147.7 ng/g (average 7.57 ng/g, median = 1.3 ng/g) than did the organic method ranging from 0 to 605 ng/g (average 44.27 ng/g, median = 5.8 ng/g). Complete profiles (16/16 loci tested) were obtained from 37/39 samples (95%) using the organic method and from 9/39 samples (23%) with the silica‐based method. Compared with a standard organic DNA isolation method, our results indicate that the published silica‐based method does not improve neither the quality nor the quantity of DNA for STR profiling.     

The talus: sex assessment of prehistoric New Zealand Polynesian skeletal remains

Prehistoric Polynesian skeletal remains are frequently being recovered in New Zealand due to the increasing pace of urbanisation. Since such material must often be reinterred quickly, it is important that the sex of individuals be determined from the remains in a relatively short time. For this purpose, discriminant function analysis was utilised for sex determination of prehistoric adult New Zealand Polynesian tali (24 males and 27 females). Five measurements were taken and subjected to Statistical Package for the Social Sciences (SPSS) discriminant function analysis. For the discriminant functions derived, accuracy of sex determination ranged from 85.1 to 93.3%. Reduction in error over random assignment by sex ranged from 70 to 87%.     

The calcaneus: sex assessment of prehistoric New Zealand Polynesian skeletal remains

The increasing pace of urbanisation has meant that prehistoric Polynesian skeletal remains are frequently being recovered in New Zealand. Since such material must often be reinterred quickly, it is important that the sex of individuals be determined from the remains in a relatively short time. For this purpose, discriminant function analysis was utilised for sex determination of prehistoric adult New Zealand Polynesian calcanei (26 male and 22 female). Five measurements were taken and subjected to Statistical Package for the Social Sciences (SPSS) discriminant function analysis. For the discriminant functions derived, accuracy of sex determination ranged from 88.4 to 93.5%. Reduction in error over random assignment by sex ranged from 77 to 87%.     

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Bones and teeth often represent the only sources of DNA available for identifying human remains. DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium. Because of the extensive mineralisation, the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction (PCR) inhibitors. Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform. To improve the efficiency of DNA extraction from skeletal remains, the present study focuses on a modification to these already available protocols. In this study, different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol, a supplementary protocol, and a modified protocol. The modified approach included a decalcification step, whereas the Qiagen protocols worked directly on non-decalcified powder. In all three procedures, 150 mg samples were used for DNA extraction. We evaluated the quantity of DNA recovered from samples, the presence of any PCR inhibitors co-extracted, the level of DNA degradation, the quality of short tandem repeat (STR) profiles, and the reproducibility of the modified procedure. When compared with the other protocols, the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors. Additionally, the STR profiles were reliable and of high quality. In our opinion, the decalcification step increases DNA recovery by softening tissues, which allows lysis solutions to act more effectively. Furthermore, the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols. These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples, such as bones and teeth.

Key points

• Bones and teeth often represent the only sources of DNA for identifying human remains.
• The choice of an efficient DNA extraction procedure is important for maximizing DNA recovery and removing PCR inhibitors.
• This study focuses on modifications to the previously available Qiagen-based protocols.
• The modified protocol enabled the best recovery of DNA, and both quality and quantity were superior to those of the previously available Qiagen-based protocols.
• The STR profiles obtained from samples extracted using the modified protocol were reliable and of high quality.

     

Determining the sex of human remains through cranial morphology

Sex determination is the keystone of a biological profile, yet few qualitative methods of cranial sex determination have been tested. This analysis examines the accuracy and precision of 17 morphological features of the skull commonly used to determine the sex of unknown skeletal remains. The sample consists of 46 identified skulls from the 19th century St. Thomas' Anglican Church Cemetery in Belleville, Canada. Nasal aperature, zygomatic extension, malar size/rugosity, and supraorbital ridge proved the most useful; of secondary value are chin form and nuchal crest; mastoid size is of tertiary consideration; nasal size and mandibular symphysis/ramus size rank fourth; forehead shape ranks fifth; and palate size/shape are sixth. Skull size/architecture provides an internal standard to assess the relative sizes of other traits. This research is a necessary step in establishing the credibility of morphological sex determination with respect to the Daubert and Mohan criteria for admissibility in a court of law.     

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.     

The femoral head: sex assessment of prehistoric New Zealand Polynesian skeletal remains

Prehistoric Polynesian skeletal remains are frequently being recovered in New Zealand due to the increasing pace of urbanisation. Since such material must often be reinterred quickly, it is important that the sex of individuals be determined from the remains in a relatively short time. For this purpose, discriminant function analysis was utilised for sex determination of prehistoric adult New Zealand Polynesian femora (47 male and 44 female). Three measurements of the femoral head were taken and subjected to Statistical Package for the Social Sciences (SPSS) discriminant function analysis. For the discriminant functions derived, accuracy of sex determination ranged from 80.9% to 82.4%. Reduction in error over random assignment by sex ranged from 62% to 65%.     

The accuracy of sex identification in European skeletal remains using the phenice characters

Three documented European skeletal series were examined to assess the accuracy and reliability of the pubic variables described by Phenice for correctly identifying the sex of adult human skeletal remains. The accuracy and objectivity of these variables, as reported by Phenice, Kelley, Sutherland and Suchey, and Lovell, could not be confirmed on this European material. In general, the subpubic concavity feature, when used alone, proved to be the most reliable variable for sex identification. In this study, the level of correct sex identification that could be achieved using the Phenice variables was shown to be significantly affected by the previous experience of the observer.     

Analysis of pathological and non-pathological human skeletal remains by FT-IR spectroscopy

In this study, we report the chemical analyses of various non-pathological, tuberculosis and syphilis infected bone samples from different burial environments by Fourier transform infrared spectroscopy (FT-IR), in the framework of a general study of diagenesis. Dating human skeletal remains is one of the most important and yet unreliable aspects of forensic anthropology. In this paper, a new method has been suggested, using the crystallinity index and carbonate-phosphate index as a means of distinction between recent and archaeological, anthropological bone samples. Pathological bone samples were analyzed with the same method to see if changes in crystallinity interfere with the process of dating.     

DNA extraction: an anthropologic aspect of bone remains from sixth- to seventh-century ad bone remains

In the archeological site of the early Christian Episcopal complex of Saint Peter, in Canosa di Puglia (Bari, Italy), during the operations of archaeological excavations, tombs were discovered. They were dated between the sixth and seventh centuries ad with carbon 14 methodology. Five skeletons were found in the 5 tombs: 28A: male individual, 43 years old. The height was 170 cm; the biomass was 65.7 kg. The analysis of the bones indicated several noteworthy pathologies, such as a number of hypoplasia lines of the enamel, the presence of Schmorl hernias on the first 2 lumbar vertebrae, and the outcome of subacromial impingement syndrome. 28E was a male individual, with a biologic age of death of between 44 and 60 years. The height was 177 cm. He had a posttraumatic fracture callus of the medial third of the clavicle, with an oblique fracture rima. 29B was a female individual, 44-49 years old. The height was 158.8 cm; the biomass was 64.8 kg. There was Wells bursitis on the ischial tuberosity on both sides. 29E was a male individual, 45-50 years old. The height was 169.47 cm; the biomass was 70.8 kg. The third and the fourth vertebrae showed Baastrup syndrome (compression of the vertebral spine). There were radiologic signs of deformity on the higher edge of the acetabula and results of frequent sprains of the ankles. 31A was a male individual, 47-54 years old. The height was 178.65 cm; the biomass was 81 kg. The vertebral index showed a heavy overloading in the thoracic lumbar region. There were bony formations under the periosteum on both on the higher and medium facets of the first metatarsus and on the higher and lateral facets of the fifth metatarsus on both sides. As the topography indicates, these small ossifications coincided with the contact points between the back of the foot and parts of the upper shoe. From the osseous remains, in particular from the teeth (central incisors), the DNA was extracted and typed to identify potential family ties among all the subjects. The extraction technique used came from the DNA Promega technique, partially modified by the authors. Stay times of the sample in the extraction buffer were increased and were increased the polymerase chain reaction (PCR) cycles.     

Identification of the skeletal remains of Josef Mengele by DNA analysis.

There has been considerable controversy over the identity of the skeletal remains exhumed in Brazil in 1985 and believed to be those of Dr Josef Mengele, the Auschwitz 'Angel of Death'. Bone DNA analysis was therefore conducted in an attempt to provide independent evidence of identity. Trace amounts of highly degraded human DNA were successfully extracted from the shaft of the femur. Despite the presence of a potent inhibitor of DNA amplification, microsatellite alleles could be reproducibly amplified from the femur DNA. Comparison of the femur DNA with DNA from Josef Mengele's son and wife revealed a bone genotype across 10 different loci fully compatible with paternity of Mengele's son. Less than 1 in 1800 Caucasian individuals unrelated to Mengele's son would by chance show full paternal inclusion. DNA analysis therefore provides very strong independent evidence that the remains exhumed from Brazil are indeed those of Josef Mengele.     

Extraction and amplification of authentic DNA from ancient human remains

A total of 36 ancient human remains from 12 individuals (three tooth/bone samples each) and one sample each of three individuals from the newest time was typed in a blind study using the amelogenin sex test. Prior to this molecular sex determination the sex of the individual was determined morphologically. The success rate of the amelogenin amplifications was >90%. For every individual an unambiguous molecular sex typing result was obtained. Furthermore, the morphological and molecular sex determinations were in accordance with each other, giving evidence for the authenticity and ancient origin of the polymerase chain reaction (PCR) amplifications.     

The sciatic notch/acetabular index as a discriminator of sex in European skeletal remains

The sciatic notch/acetabular index and its rule of thumb application was tested on two European skeletal series of documented sex. The results showed poor consistency between documented and estimated sex. Of the two variables comprising the index, it appeared that sciatic notch width was a particularly poor discriminator of sex. It is suggested that this may be due to differences in the relationship between sciatic notch form and body size in different ethnic groups.     

DNA typing of human remains found in damp environments

DNA typing is often used to determine identity from human remains. The environment to which the material has been exposed, however, is crucial for the success of the investigation. Damp conditions in particular can cause a rapid degradation of DNA, even in bone and teeth, and thus reduce the chances of successful typing. Here, we present the results of investigations performed on four skulls that had been lying in a damp environment for periods ranging from almost 1 year to about 45 years. In none of these cases was DNA typing successful on bone or, where present, on teeth. Where remnants of brain tissue were used, however, complete STR typing was possible in one case, partial STR typing in another, and mtDNA sequencing could be carried out in three cases. These findings suggest that brain tissue is relatively resistant to putrefaction in damp environments and, unlike bone, appears to exhibit a certain degree of protection against DNA degradation.