Two sets of studies were performed to evaluate the forensic utility of sequencing human mitochondrial DNA (mtDNA) derived from various tissues and amplified by the polymerase chain reaction (PCR). Sequencing was performed on a Perkin-Elmer/Applied Biosystems Division (PE/ABD) automated DNA sequencer (model 373A). The first set of experiments included typical validation studies that had previously been conducted on forensic DNA markers, such as: chemical contaminant effects on DNA from blood and semen and the effect of typing DNA extracted from body fluid samples deposited on various substrates. A second set of experiments was performed strictly on human hair shafts. These studies included typing mtDNA from hairs that were: (1) from different body areas, (2) chemically treated, (3) from deceased individuals, and (4) deliberately contaminated with various body fluids. The data confirm that PCR-based mtDNA typing by direct automated sequencing is a valid and reliable means of forensic identification.

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Hair samples were typed from three individuals who exhibited length heteroplasmy in the homopolymeric cytosine stretches (C-stretch) in hypervariable region 2 (HV2). The study demonstrated that for different hairs within an individual, the HV2 C-stretch region can vary with respect to the number of cytosines and/or proportion of C-stretch length variants. Length heteroplasmy may occur regardless of the prominent length variant present in this region. Differences in the number of cytosines at the C-stretch region, or a variation in the relative amounts of heteroplasmic length variants, cannot be used to support an interpretation of exclusion.

A family exhibiting heteroplasmy at position 16355 in hypervariable region I of the human mtDNA control region has been identified. This family consists of a mother, daughter, and son. DNA samples extracted from blood stains, buccal swabs, and hairs from these individuals were amplified by PCR and sequenced utilizing fluoresence-labeled dye terminator chemistry in an automated DNA sequencer. In both the daughter and mother, heteroplasmy was observed in DNA extracted from blood stains, buccal swabs, and hairs. In the blood stains, the proportion of cytosine was greater than thymine in both individuals. Buccal swab extracts showed a more balanced contribution from the two nucleotides. Telogenic hair root and hair shaft samples exhibited a wide range of nucleotide contributions at this position, from predominately cytosine in some samples to predominately thymine in others. The apparent stochastic segregation of mitotypes in hair samples is discussed from a forensic viewpoint, and the mechanism of mtDNA heteroplasmy is considered.

The ability to identify individual human hosts based on analyses of blood recovered from the digestive tract of hematophagous arthropods has been a long-term pursuit in both medical and forensic entomology. Blood meal individualization techniques can bring important advancements to studies of vector-borne disease epidemiology. Forensically, these analyses may aid in assailant identification in violent crime cases where blood-feeding insects or their excreta are recovered from victims or at crime scenes. Successful isolation, amplification, and sequencing of human mitochondrial DNA obtained from adult human crab lice fed on human volunteers are reported. Adult lice were removed from recruited volunteers frequenting inner city health clinics. Live lice were killed by freezing and subsequently air dried at ambient temperature. A saliva sample was obtained from each volunteer and served as a DNA reference sample. Volunteers were afforded free, approved pediculosis treatment. Individual lice were subsequently processed using procedures developed for the extraction of mitochondrial DNA from human hair, teeth, and bone. The resulting DNA was amplified by the polymerase chain reaction and sequenced. Our results point to valuable avenues for future entomological research.

The isolation, amplification, and characterization of human DNA from hematophagous (blood feeding) and necrophagous (carrion feeding) arthropods have been advanced significantly by the development of polymerase chain reaction (PCR) DNA sequencing methodologies. Historically, DNA technology has been successfully utilized to identify individual hosts upon which species of hematophagous arthropods have fed. The analysis of hematophagous insects' gut content blood meals has led to major advances in medical entomology and vector-borne disease epidemiology. In the forensic arena, the ability to apply similar techniques to insects recovered from badly decomposed remains has been greatly enhanced through the advent of mitochondrial DNA (mtDNA) techniques. Mitochondrial DNA analyses have been utilized to identify both the human remains upon which fly larvae (maggots) have fed and the species of the larvae themselves. The preliminary work detailed here demonstrates, for the first time, the successful application of mtDNA sequencing techniques to the analysis of necrophagous beetle larvae. A small sample of sap beetle larvae, Omosita spp. (Coleoptera: Nitidulidae), was collected from human skeletal remains during anthropological examination and analyzed for human DNA using mtDNA sequencing. The beetle larvae yielded mtDNA matching that of the host human bone. The results detailed here further demonstrate the robust nature of human mtDNA and the ability to recover valuable mtDNA evidence from forensically important, late decompositional stage insect species.

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