Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains.

The application of deoxyribonucleic acid (DNA) typing methods for the potential identification of unknown human remains was investigated. DNA was isolated from compact bone tissue from badly decomposed bodies and from known and unknown human remains, using a decalcification and ion wash procedure. Restriction fragment length polymorphism (RFLP) analysis of variable number of tandem repeats (VNTR) loci yielded results in some cases, but more often the DNA was too degraded to produce RFLP patterns. No RFLP profiles could be obtained from putrefied soft tissues. However, DNA extracted from compact bone tissue of human remains up to eleven years old was successfully amplified using the polymerase chain reaction (PCR) for the VNTR loci D1S80, D17S5, COL2A1, and APO B, as well as the HLA-DQ alpha locus. This is especially significant, since PCR results were obtained from those samples whose DNA had been degraded substantially and had yielded no RFLP patterns. All DNA types determined from the compact bone tissue from decomposed bodies whose identification had been established first by other means (and whose parents or offspring were available for typing) demonstrated mendelian inheritance of the alleles of the loci analyzed. These results suggest that amplification and typing of DNA extracted from compact bone of human remains could be useful in establishing the identity of a person, as well as in excluding possible false identifications.     

Identification of male bloodstains by dot hybridization of human Y chromosome-specific deoxyribonucleic acid (DNA) probe

The sex determination of bloodstains was performed using a human Y chromosome-specific (DNA) fragment of 1.9-kb length as a hybridization probe. The DNA samples were taken from 1- and 4-week-old bloodstains of males and females, respectively. Strong signals with male DNA were observed by Y-probe, while faint signals with female DNA were detected. In addition, clear signals were observed in the extract samples from male bloodstains (16-week-old) on paper. Dot hybridization of the Y-probe would be widely applicable to studies on sex determination of medicolegal materials such as blood, bloodstains, teeth, and cadaverous parts.     

Parentage determination on aborted fetal material through deoxyribonucleic acid (DNA) profiling

After a rape, women who are pregnant often elect to abort the fetus. The authors describe ten cases in which deoxyribonucleic acid (DNA) typing was performed on the aborted fetal material to provide evidence of the genetic constitution of the suspect. The problems encountered with this new technique are discussed.     

Cocaine in decomposed human remains.

From March 1988 through March 1990, at the Philadelphia Medical Examiner's Office toxicology laboratory, samples from 77 decomposed human bodies were tested for the presence of cocaine, employing gas chromatography/mass spectrometry (GC-MS). The material analyzed included decomposed soft tissue, bloody decomposition fluid, mummified tissue, maggots, and beetle feces. Twenty-two cases (28.6%) were positive for cocaine, many of these cases in states of advanced decomposition. These findings indicate the usefulness of testing decomposed tissue for cocaine in all cases where its presence is suspected. This is contrary to what might be expected, since cocaine is generally labile and rapidly broken down by both enzymatic and nonenzymatic mechanisms.     

Identification of decomposed human remains from radiographic comparisons of an unusual foot deformity

A case of positive identification from decomposed human remains using an unusual foot deformity is presented. Scrutiny of the decedent revealed foot deformities, which upon examination, prompted further inquiry. Radiographic comparisons and defleshing each foot established bilateral talipes equinovarus (TEV, clubfoot). Positive identification was based upon unique skeletal features present in the radiographs.     

Big game species identification by deoxyribonucleic acid (DNA) probes.

Species identification is important in many big game forensic science cases but cannot always be accomplished because of the lack of adequate techniques. The authors have developed deoxyribonucleic acid (DNA) probes for elk, deer, and antelope by isolating highly repeated satellite sequences. These DNA probes distinguish among deer, elk, and antelope, although not between different species of deer. Because of the high number of sequence copies per genome, these probes are extremely sensitive, requiring less than 10 ng of total genomic DNA. The developmental protocol for these probes is relatively simple and is applicable to many other species.     

Individual identification from semen by the deoxyribonucleic acid (DNA) fingerprint technique

For individual identification from semen, the deoxyribonucleic acid (DNA) fingerprint technique was used. In a blind trial, we succeeded in determining the semen donors among several volunteers comparing the DNA fingerprints of the blood and semen samples, respectively. Thereafter, we examined semen in a condom left beside a naked female dead body. The DNA fingerprint of the semen was recognized to be identical to that of the blood from a suspected man arrested later. This is the first report that the DNA fingerprint technique was practically used in a criminal investigation in Japan.     

Repetitive deoxyribonucleic acid (DNA) and human genome variation--a concise review relevant to forensic biology

The various classes of human repetitive deoxyribonucleic acid (DNA) are described, with particular emphasis being given to their variation in the human genome. The significance of this information to forensic science is discussed.     

Anthropological and radiographic comparison of vertebrae for identification of decomposed human remains

This case study demonstrates the importance of involving an anthropologist in forensic situations with decomposed remains. Anthropological consultation was used in conjunction with the comparison of antemortem and postmortem radiographs to establish positive identification of unknown, decomposed remains. The remains had no traditional identifying features such as fingerprints or dental. Through anthropological analysis, it was determined the decedent was male, between 20 and 23 years at time of death and c. 5'2' tall. This information allowed for a presumptive identification and a request for antemortem radiographs. The missing person was identified comparing the spinous processes of the cervical and thoracic vertebrae between ante- and postmortem radiographs.     

Identification of remains by sequencing of mitochondrial DNA control region

The maternity of two newborns who were murdered and abandoned >5 and 10 years were analyzed by amplification and direct sequencing of mitochondrial DNA (mtDNA) control regions. Sequences of two hypervariable segments from each femur bone sample and the blood of the putative mother showed four mutations in hypervariable region I and two mutations in addition to two nucleotide insertions in hypervariable region II compared with the reference sequence, and all sequences were identical. The genotype of these individuals is found to be relatively rare in the Japanese population, and it was strongly suggested that both sets of newborn remains really were children of the putative mother. Sexes of the remains were determined to be female and male by amplifying a segment of the X-Y homologous gene, amelogenin. These results demonstrate that sequencing of mtDNA is a useful tool for genetic identification of aged and decomposed materials.     

Using accumulated degree-days to estimate the postmortem interval from decomposed human remains

Forensic anthropologists often rely on the state of decomposition to estimate the postmortem interval (PMI) in a human remains case. The state of decomposition can provide much information about the PMI, especially when decomposition is treated as a semi-continuous variable and used in conjunction with accumulated-degree-days (ADD). This preliminary study demonstrates a supplemental method of determining the PMI based on scoring decomposition using a point-based system and taking into account temperatures in which the remains were exposed. This project was designed to examine the ways that forensic anthropologists could improve their PMI estimates based on decomposition by using a more quantitative approach. A total of 68 human remains cases with a known date of death were scored for decomposition and a regression equation was calculated to predict ADD from decomposition score. ADD accounts for approximately 80% of the variation in decomposition. This study indicates that decomposition is best modeled as dependent on accumulated temperature, not just time.     

Personal identification from human remains by mitochondrial DNA sequencing

The authors report four cases in which severely damaged human remains were identified by mitochondrial DNA (mtDNA) sequencing. Degraded DNA was extracted from highly adipoceratous tissues using the phenol-chloroform method and polymerase chain reaction amplified for sequencing of two hypervariable regions, hypervariable region 1 and hypervariable region 2, of mitochondrial DNA. They also sequenced these regions of blood samples that were obtained from the presumptive mother or sister of the human remains. The sequencing results were compared with each other and with the Anderson's sequence. It was concluded from the sequence data that a lower part of a body in case 1 and some organs in case 2 were from the same woman, and a human head in case 3 and a female body in case 4 were from the relative of a presumptive mother and a sister, respectively.     

Case report: Identification of skeletal remains using short-amplicon marker analysis of severely degraded DNA extracted from a decomposed and charred femur

Applying two extraction protocols to isolate DNA from a charred femur recovered after a major forest fire, a range of established and recently developed forensic marker sets that included mini-STRs and SNPs were used to type the sample and confirm identity by comparison to a claimed daughter of the deceased. Identification of the remains suggested that the individual had been dead for 10 years and the DNA was therefore likely to be severely degraded from the combined effects of decomposition and exposure to very high temperatures. We used new marker sets specifically developed to analyze degraded DNA comprising both reduced-length amplicon STR sets and autosomal SNP multiplexes, giving an opportunity to assess the ability of each approach to successfully type highly degraded material from a challenging case. The results also suggest a modified ancient DNA extraction procedure offers improved typing success from degraded skeletal material.     

Sensitive and specific quantification of human genomic deoxyribonucleic acid (DNA) in forensic science specimens: casework examples.

We describe the forensic science application of a method for quantification of human genomic deoxyribonucleic acid (DNA). The two cases cited in this report involve DNA samples extracted from skin tissue and bloodstained clothing recovered from different crime scenes. High-molecular-weight DNA was recovered from both specimens, and the concentrations of these DNAs were estimated to be approximately 0.5 microgram/microL by ethidium bromide/agarose gel electrophoresis. Using the human-specific DNA probe p17H8 (locus D17Z1) to quantify the amount of human genomic DNA in these samples, it is shown that less than 1% of the DNA isolated from the skin tissue is of human origin and that the DNA isolated from the bloodstained clothing is effectively devoid of human DNA sequences. These case examples illustrate the need to quantify not only the total amount of DNA recovered from forensic casework material, but also the proportion of the DNA that is of human origin.     

Sex identification in fresh blood and dried bloodstains by a nonisotopic deoxyribonucleic acid (DNA) analyzing technique

Deoxyribonucleic acid (DNA) specimens were prepared from blood or bloodstain extracts, and the content of a Y-chromosome specific DNA fragment was investigated by the Southern hybridization method using a nonisotopic staining technique. Thus obtained patterns of male DNA showed a clear band, whereas broad stains with some faint bands appeared on the patterns of DNA from both sexes. This method is expected to be a new powerful mean of forensic medical examination.     

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.     

The autodegradation of deoxyribonucleic acid (DNA) in human rib bone and its relationship to the time interval since death

This research explored the feasibility of using the degradation rate of deoxyribonucleic acid (DNA) in human rib bone to determine the time interval since death. Postmortem human rib samples were surface sterilized and incubated under sterile conditions in either high or low humidity conditions at room temperature for a period of weeks. At selected times, portions of the bone were cut away, and the DNA from these samples was extracted and subjected to strand separating gel electrophoresis. The DNAs in the gels were transferred to a nylon membrane, preserving their relative positions as in the gel, and probed with radioactive total genomic human DNA. Autoradiograms produced were scanned and digitized. When the samples were incubated under identical conditions, the degradation rate of DNA in samples from different individuals appeared very similar. The DNA degradation rate may vary with temperature and humidity more than it varies between individuals.     

"Sexing" deoxyribonucleic acid (DNA) on DNA fingerprint gel: an internal control for DNA fingerprint evidence

Deoxyribonucleic acid (DNA) isolated from male and female fresh blood samples was processed exactly as for routine DNA fingerprint analysis; that is, the DNA was digested with particular restriction endonucleases and fractionated by agarose gel electrophoresis. Ultraviolet (UV) visualization of ethidium-bromide (EtBr)-stained gels revealed a sex-specific banding pattern, which depended only on the restriction enzyme used. By means of this test, which is based on direct detection of particular sex-specific restriction fragments in human DNA digests, the authors succeeded in determining the sex of DNA obtained from biological specimens recovered as criminal evidence in rape cases. The data obtained demonstrate that direct sexing of DNA on DNA fingerprint gel appears to be useful as an intermediate control step in DNA fingerprinting analysis used for the purpose of assailant identification.     

Characterization of deoxyribonucleic acid (DNA) obtained from teeth subjected to various environmental conditions.

This study was designed to determine the effects of various environmental factors on the deoxyribonucleic acid (DNA) obtained from dental pulp. Extracted teeth were subjected to the following conditions: varying pH (3,7,10); temperature (4 degrees C, 25 degrees C, 37 degrees C, incineration); humidity (20%, 66%, 98%); various types of soil (sand, potting soil, garden soil); seawater; burying the teeth outdoors, and aging (one week to six months). In addition, teeth that had been extracted and held at room temperature for 16 and 19 years were also examined. Following isolation of DNA, the samples were analyzed on yield gels to determine the concentration and integrity of the recovered DNA. Restriction digestion with Pst I was followed by electrophoresis of the generated fragments, Southern transfer to nylon membranes, and hybridization to both human and bacterial probes. It was determined that, aside from soil, the environmental conditions examined did not affect the ability to obtain high-molecular-weight human DNA from dental pulp. Restriction fragment length polymorphic (RFLP) analysis of selected samples was performed. Dental pulp patterns were compared with bloodstain exemplars, revealing matching patterns, although an increase in band-shifting was observed with extended exposure to elevated temperatures.     

Analysis of restriction fragment length polymorphisms in deoxyribonucleic acid (DNA) recovered from dried bloodstains

Deoxyribonucleic acid (DNA) was recovered from dried bloodstains aged up to three years and shown to be of high molecular weight. DNA was digested with restriction endonucleases and fractionated by agarose gel electrophoresis. Following transfer to a filter, DNA was hybridized with two different radioactively labeled recombinant probes which recognize highly polymorphic regions in human DNA. The autoradiographic pattern observed was not altered by sample age, and the size of the alleles was consistent with those observed in the general population. Therefore, DNA of high molecular weight prepared from dried blood samples can be used for identification.