Joseph E. Warren, Arthur V. Eisenberg, Ph.D., John V. Planz, Ph.D., Robert Benjamin, Ph.D., and Phillip
Hartman, Ph.D.
Tarrant County Medical Examiner’s Office, Fort Worth, TX and Department of Biology,
University of North Texas, Denton, TX

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Analysis of Mitochondrial DNA (mtDNA) has proven to be a highly useful tool for the study of human identification and individualization. Its high copy number per cell, material mode of inheritance in humans and high degree of polymorphism found in the DNA sequences of Hypervariable Region 1 (HV1) and Hypervariable Region 2 (HV2) of the D-loop region in the mitochondrial genome are three of the main reasons why this is so. Furthermore, the relative small size of the base pair sequences that are of interest (approximately 341 base pairs for HV1 and 267 base pairs for HV2) make them good candidates for DNA amplification via the Polymerase Chain Reaction (PCR) technique.

The area of study in which mtDNA has proven to be most useful has been the analysis of very old human remains, such as skeletal material, mummified tissue, hair and teeth. The fields of Population Genetics, Evolutionary Biology, Anthropology, Paleontology and History have recently begun to benefit from this analytical method. Forensic Biology, Forensic Anthropology, Forensic Odontology, and Forensic Pathology can also be helped by this method, especially in the analysis of unidentified human remains. If a scientist has access to any source of genetic material from a material relative of the suspected remains, then a comparison of the DNA sequence from the mtDNA of the living relative, to that of the remains can be highly informative in determining identity. Also, in cases of linking a victim and/or suspect to a crime scene where only limited or old DNA samples are available, such as found in hair or body stains, mtDNA comparisons can be performed.

It has been assumed that mtDNA is very stable. However, most of the samples used in mtDNA studies have consisted of bone and hair. The DNA in these tissues are fairly well protected from environmental exposure. In order to validate a new technique for forensic use, one of the criteria is how a variety of investigator induced environmental conditions can affect the outcome of the test. This is the primary purpose of this study.

Three 10 mL vials of EDTA preserved blood were removed from one human male volunteer. This blood was then either treated with a variety of substances one might encounter at a crime scene (soap, tap water, ethanol, bleach, gasoline, motor oil, etc.,) and then spotted onto a clean cotton cloth, dried and stored at room temperature. Another set of stains were made and subjected to exposure to such environmental conditions as sunlight, burial, heat, humidity, and other conditions. The DNA was then isolated using the organic method developed by the F.B.I., followed by treatment with the Microcon 100 concentrator. Primer sets that are specific for the mtDNA region, HV1 15971-16258 were utilized to PCR amplify the DNA. The resulting amplification products were then assayed by the use of a 2% agarose TBE mini-gel.

Ethidium Bromide was used to stain the DNA and a photograph was taken. If mtDNA amplification occurred, then a band of 287bp should appear when compared to the DNA ladder. The results of these tests will be presented. In general, mtDNA proved to be stable in most, but not all cases.

A brief discussion will also be given on a variety of manual DNA sequencing strategies that laboratories which do not have access to an automatic DNA sequencer can use. Future research will include using these methods to sequence the DNA from the exposed samples to determine if any sequence changes occurred when compared to control sample.