Victor W. Weedn

Mitochondrial DNA Sequence Analysis of Human Skeletal Remains: Identification of Remains from the Vietnam War

Deoxyribonucleic acid (DNA) sequence analysis of the control region of the mitochondrial DNA (mtDNA) genome was used to identify human skeletal remains returned to the United States government by the Vietnamese government in 1984. The postmortem interval was thought to be 24 years at the time of testing, and the remains presumed to be an American service member. DNA typing methods using nuclear genomic DNA, HLA-DQ alpha and the variable number of tandem repeat (VNTR) locus D1S80, were unsuccessful using the polymerase chain reaction (PCR). Amplification of a portion of the mtDNA control region was performed, and the resulting PCR product subjected to DNA sequence analysis. The DNA sequence generated from the skeletal remains was identical to the maternal reference sequence, as well as the sequence generated from two siblings (sisters). The sequence was unique when compared to more than 650 DNA sequences found both in the literature and provided by personal communications. The individual sequence polymorphisms were present in only 23 of the more than 1300 nucleotide positions analyzed. These results support the observation that in cases where conventional DNA typing is unavailable, mtDNA sequencing can be used for human remains identification.

Extraction, Evaluation, and Amplification of DNA from Decalcified and Undecalcified United States Civil War Bone

Deoxyribonucleic acid (DNA) was extracted from documented skeletal specimens of U.S. Civil War soldiers to determine the need for decalcification prior to extraction. The polymerase chain reaction (PCR) was performed to determine if the calcification state had an effect on the ability to amplify the extracts and to determine how successful amplification would be with these aged specimens. Bone samples were pulverized to a fine powder and divided into two sets. One set of samples was decalcified and the other set left undecalcified. Both sets were extracted using an organic procedure. The results demonstrate that decalcification is not a necessary step in the extraction process and that the yield of DNA is generally two times greater when decalcification is omitted. Furthermore, the calcification state had no effect on the ability to perform the PCR. Although the extracted DNA was very degraded, a 410 base pair (bp) segment of the mitochondrial DNA (mtDNA) control region was amplified. These results suggest that DNA can be extracted and amplified from 125 year old bone without decalcification, which may assist in the identity of modern and historic forensic specimens.

A systematic approach to the sampling of dental DNA

As investigations into the forensic aspects of DNA analysis continue, the human tooth will play a dual role in identification. Dentin and enamel provide a protective enclosure for genomic and mitochondrial DNA as well as providing the basis for radiographic, biochemical, and ultrastructural forensic studies. The purpose of this investigation is to establish technical guidelines, based on histology and experimental evidence, for the management and sampling of dental DNA. The anatomic location of dental DNA is discussed with emphasis on the conservation of tooth structure during sampling. Ten pairs of maxillary right and left third molars were sampled for DNA following storage for 18 weeks at ambient temperature and humidity. Right third molars were crushed, whereas the left third molars were sectioned conservatively prior to sampling the DNA. The quantity and quality of human DNA obtained from each tooth was compared, as well as the radiographic appearance of remaining hard tissue and the overall simplicity of each approach. DNA typing was performed, both sequence and length based analyses, comparing teeth from the same individual and teeth from different donors. The results of this study suggest that the odontologist will maximize the dental DNA yield by crushing the entire specimen but that substantial yields of human DNA can be obtained by using a conservative technique that preserves the tooth structure. In addition, the method of sampling does not affect the ability to perform DNA typing analyses.

Electrophoretic separations of polymerase chain reaction-amplified DNA fragments in DNA typing using a capillary electrophoresis- laser induced fluorescence system

Abstract Analysis of polymerase chain reaction (PCR)-amplified DNA fragments for human identification requires high-resolution separation and efficient detection of amplified alleles. Capillary electrophoresis (CE) with its speed, automation, high resolution and efficiency shows promise for analysing the amplified DNA fragments. CE with UV detection, however, suffers from lack of detector sensitivity owing to the limited detection path length of the capillary. By the use of intercalating dyes (TOTO and YOYO) a laser-induced fluorescence (LIF) detection system can provide much greater sensitivity for detecting DNA fragments. Femtogram quantities of dsDNA (φX174 HaeIII restriction digest mixture) per nanoliter of injected volume have been detected. Application of CE-LIF to analysis of PCR-amplified DNA fragments from three different genetic loci (apolipoprotein B, VNTR locus D1S80, mitochondrial DNA) is shown here. Further, the resolving power of a polymer-network capillary separation system is compared to that of a capillary-gel separation system.

Rapid PCR for Identity Testing Using a Battery-Powered Miniature Thermal Cycler

A microfabricated, battery-powered thermal cycler was implemented in PCR-based DNA typing for human identification. HLA DQ alpha and an STR triplex were PCR amplified using a device known as the Miniature Analytical Thermal Cycling Instrument (MATCI). The extremely efficient heating properties of the MATCI enabled thermal cycling to be completed in as little as 21 min. In addition, the feasibility of using the real-time fluorescent detection system of the MATCI was demonstrated. The successful application of this portable, prototype device to forensic identity testing is a significant milestone towards the eventual development of a completely integrated DNA testing instrument that would also incorporate sample preparation and allele detection.

Retinal Hemorrhage in an Infant After Cardiopulmonary Resuscitation

Unexplained retinal hemorrhages in infants are usually indicative of child abuse. We present the case of an infant with retinal hemorrhages following cardiopulmonary resuscitation, who had not been abused. Cardiopulmonary resuscitation should be added to the list of causes of retinal hemorrhages in infants and children.

Review of: Forensic Pathology Reviews: Volume 1

Forensic Pathology Reviews is a new (April 2004) and ambitious series published by Humana Press and edited by Michael Tsokos, MD of the Institute of Legal Medicine at the University of Hamburg. It is a part of an explosive growth in forensic publications that is difficult not to link to the recent interest from the spate of television shows or forensics. However, the introduction to the series correctly refers to a significant expansion of scientific progress in the forensic sciences and states: “The Forensics Pathology Reviews knowledge on special topics in the field, focusing closely on the dynamic and rapidly growing evolution of medical science and law.” The introduction further states that the series is intended to take a problem-oriented approach and provide comprehensive and well documented reviews of the international literature with insights into new diagnostic techniques.

Stud guns revisited: report of a suicide and literature review.

Stud guns (powder-actuated fastening tools) are a commonly used construction tool. Accidental injuries and fatalities are no longer frequent, presumably because of current safety features and practices. A case of an intentional fatal wound (suicide) is described. A literature review of stud gun injuries is also presented.

Short tandem repeat loci: Application to forensic and human remains identification

The short tandem repeat (STR) locus ACTBP2 (common name SE33) was analyzed for its potential use in forensic and human remains identification. PCR amplification conditions were determined, and an allele-specific ladder was generated so that discrete alleles could be scored. The allele frequency distributions were determined for both Caucasian and Black populations. The frequency data meets Hardy-Weinberg expectations, and the allele distributions were similar from one racial group to another and between ethnic groups. SE33 analysis was subsequently used to confirm the identification of human remains for the Office of the Armed Forces Medical Examiners.

National Association of Medical Examiners Position Paper: Medical Examiner, Coroner, and Forensic Pathologist Independence:

Objective Forensic pathologists play a vital role in the justice system in matters concerning questions of death. Science as applied in the justice system should be objective and neutral. Since the goals of medical examiners and coroners are to determine the cause and manner of death for certification and public health functions (goals different and distinct from the missions of law enforcement agencies), it is important that medicolegal death investigation be independent. Accurate investigation, examination, reporting, and testimony by forensic pathologists are important to determine the cause and manner of death of individuals who die under sudden, unexpected, or violent circumstances. These cases can become the focus of political or legal pressure by individuals or offices seeking to influence the pathologists findings. This pressure, even if seemingly unsuccessful in an individual case, can introduce error, bias, and corruption into the medicolegal investigation process. This paper reinforces the principle that medical examiners, coroners and forensic pathologists should be allowed to perform medicolegal investigations free of these influences. Participants The ad hoc Committee on Medical Examiner Independence of the National Association of Medical Examiners (NAME), a self-selected volunteer committee, developed this position paper. The findings are based on surveys of the NAME membership regarding members’ experience with, and reaction to, outside influence. Evidence Surveys of NAME members revealed that medical examiner independence was important to most members. Over 70% of survey respondents had been subjected to pressures to influence their findings, and many had suffered negative consequences for resisting those influences. In a separate study, over 30% of respondents indicated that fear of litigation affected their diagnostic decision-making. In 2009, the National Research Council of the National Academies published recommendations to strengthen the forensic sciences; they specifically recommended that medical examiner and coroner offices should be independent from law enforcement agencies and prosecutors’ offices. Consensus Process This position paper represents the consensus of the ad hoc Committee on Medical Examiner Independence, submitted to the Executive Committee and Board of Directors of NAME, and subject to comment and review by the membership. Conclusions It is the position of NAME that forensic pathologists working in or for medical examiner or coroner offices or as private consultants should be permitted to objectively pursue and report the facts and their opinions of those cases which they are investigating independent of political influences from other agencies within their respective jurisdictions and independent of the threat of litigation.