Assessing population diversity in southern Italy has traditionally relied on archaeological and historic evidence. Although informative, these lines of evidence do not establish specific instances of within lifetime mobility, nor track population diversity over time. In order to investigate the population structure of ancient South Italy I sequenced the mitochondrial DNA (mtDNA) from 15 Iron Age (7th – 4th c. BCE) and 30 Roman period (1st – 4th c. BCE) individuals buried at Iron Age Botromagno and Roman period Vagnari, in southern Italy, and analyzed δ18O and 87Sr/86Sr values from a subset of the Vagnari skeletal assemblage. Phylogenetic analysis of 15 Iron Age mtDNAs together with 231 mtDNAs spanning European prehistory suggest that southern Italian Iapygians share close genetic affinities to Neolithic populations from eastern Europe and the Near East. Population pairwise analysis of Iron Age, Roman, and mtDNA datasets spanning the pan-Mediterranean region (n=357), indicate that Roman maternal genetic diversity is more similar to Neolithic and Bronze Age populations from central Europe and the eastern Mediterranean, respectively, than to Iron Age Italians. Genetic distance between population age categories imply moderate mtDNA turnover and constant population size during the Roman conquest of South Italy in the 3rd century BCE. In order to determine the local versus non-local demographic at Vagnari, I measured the 87Sr/86Sr and 18O/16O of composition of 43 molars, and the 87Sr/86Sr composition of an additional 13 molars, and constructed a preliminary 87Sr/86Sr variation map of the Italian peninsula using disparate 87Sr/86Sr datasets. The relationship between 87Sr/86Sr and previously published δ18O data suggest a relatively low proportion of migrants lived at Vagnari (7%). This research is the first to generate whole-mitochondrial DNA sequences from Iron Age and Roman period necropoleis, and demonstrates the ability to gain valuable information from the integration of aDNA, stable isotope, archaeological and historic evidence. / Thesis / Doctor of Philosophy (PhD) / With biochemical information obtained from teeth, this study examines the population structure and geographic origins in two archaeological communities located in southern Italy. Analysis of classical remains has traditionally been the subject of historical and archaeological inquiry. However, new applications evaluate these population changes with integrated stable isotope and ancient DNA techniques. Overall, the biochemical results suggest that the pre-Roman communities harbor deep maternal ancestry originating from eastern Europe and the eastern Mediterannean. These results, when compared to the genetic diversity of Roman and broader Mediterranean populations, indicate that the Romans share closer genetic similarity with ancient Stone and Bronze Age communites from Europe and the eastern Mediterranean, than with the pre-Roman community studied here. Furthermore, tooth chemistry results indicate a predominantly local population buried in the Roman period cemetery.

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Traditionally, biological evidence at a crime scene is collected, packaged, labeled, and then transported to the crime laboratory for DNA analysis. This is a deliberate process requiring exte~ive training and documented protocols. By defirtition, crime scenes are contaminated. When biological evidence has been deposited by an individual, preservation and testing should be conducted as soon as possible. Due to the extremely large volume ofcases submitted to forensic laboratories for analysis, cases must be prioritized which may result in a delay in processing ofbiological materials. The Mobile Molecular™ Laboratory (MML) from MJ Research, Inc.(Watertown, MA) is a mini, portable DNA laboratory which may assist the DNA analyst in achieving quick preliminary test results at a catastrophic crime scene or accident. At crime scenes where the amount ofbiological evidence is astronomical, collection and preliminary DNA testing ofthe samples would· enhance prioritization not only for collection purposes but for subsequent laboratory testing as well. The MML includes the following instrumentation: PTC-150 Minicycler (MJ Research, Watertown, MA), UV transilluminator (Fotodyne, Inc., Hartland~ WI) , FCR10 Polaroid camera (Fotodyne, Inc, Hartland, WI), mini gel box (Fotodyne, Inc., Hartland, WI), power supply (Fotodyne, Inc., Hartland, WI), and microcentrifuge (Denver Instrument Company, Aarvada, CO). The MML has been used on site to analyze isozymes among rare Antarctic mosses, amplify mtDNA sequences copied from whale meat for sale in Japan, and analyze Ebola virus among primates in the Philippines. Subsequently, prototypes ofthe MML have been tested in remote areas ofBrazil, the American Southwest, and the Caribbean. Anin situ crime scene DNA screening procedure has been developed using the MML system to detect Short Tandem Repeat (STR) allele patterns. A rapid extraction procedure, the UltraClean™ Bloodspin kit from MoBio Laboratories, Inc, has been optimized for biological stains and can be completed in approximately 10 minutes. The PTC-150 Minicycler™ completes amplification ofpolymorphic STR loci (System I: Dl6S539, Amelogenin, and Dl3S317 and System II: CSFlPO, Penta E, and O5S818) in approximately 70 minutes. Resultant STR patterns from biological evidence allow for discrimination ofone sample from another. The use ofMML technology at a crime scene has several advantages including allowing DNA evidence to play a vital role in the early investigative time period, providing guidance in the collection ofsamples, and being able to easily prioritize samples in the laboratory. Caveats to MML use include laborious protocols, necessity of having a DNA analyst in the field, and unpredictable environmental conditions.

The primary goal ofthis !esearch project was to determine if sequence variation exists between individuals who possess the same sized HumFGA alleles. A comprehensive set of procedures was developed that were used to isolate the DNA from blood samples, locate the HumFGA allele of interest and subsequently sequence the DNA fragments and determine if sequence variation existed in the flanking regions and/or the repeat region ( e.g., TTTC) for the HumFGA loci. Blood samples were collected from 104 convicted offenders from the Virginia Division of Forensic Science's DNA Data Bank who possessed a HumFGA allele 21, 22, 23, or 24. The samples collected for this study were obtained from individuals from the Caucasian, African American, and Hispanic population groups in order to determine if sequence variation might be observed more predominantly in a particular population group. To eliminate potential errors due to base misincorporation during the sequencing process both the forward and reverse strands ofDNA fragments were sequenced. In addition, to ensure that any sequence variation observed was genuine and not due to base misincorporation, several colonies from a single sample were sequenced. To further examine the possibility of detecting sequence variations at the HumFGA locus, sizing data for all ofthe samples sequenced, including additional samples, totaling 619 samples containing the HumFGA alleles 21 (196), 22 (129), 23 (121), and 24 (173) were examined to look for any sizing trends that may occur. The sizing data collected from 27 polyacrylamide typing gels containing ~amples amplified using the GenePrint® PowerPlexTM 2.1 System Kit (Promega Corporation, Madison, WI) and analyzed on a FMBIO II Fluorescent Image Analysis System (Hitachi Genetic Systems/MiriaBio, Alameda, CA) were examined. The sizing data from samples at a particular allele (i.e., 21), which were analyzed on the same typing gel, were compared across all 27 polyacrylamide typing gels (Tables 3-5 through 3-8). To further evaluate any structural differences that may exist at the HumFGA 21, 22, 23, or 24 alleles, which might cause the DNA fragments at this locus to migrate slightly differently on a polyacrylamide typing gel than other commonly used forensic loci, the HumFGA sizing data were compared to sizing data obtained for the Hum.CSFlPO 11, 12, and 13 alleles. No sequence variation was found in the flanking or repeat regions for any of the samples sequenced. Therefore, if sequence variation for the HumFGA locus does exist, it is rare and will not routinely be observed.

The goal of this project was to reduce processing time for forensic DNA testing without incurring significant added costs and/or the need for new instrumentation, while still generating high quality profiles. This was accomplished by: 1) extraction normalization using the ChargeSwitch® Forensic DNA Purification Kit such that a small range of DNA concentrations was consistently obtained, eliminating the need for sample quantification and dilution; 2) developing fast PCR protocols for STR primer sets using shorter amplification methods, low volume reactions and non-fast thermal cyclers; and 3) developing a quicker 3130xl Genetic Analyzer detection method using an alternative polymer/array length combination. Extraction normalization was achieved through a reduction in bead quantity, thereby forcing an increase in bead binding efficiency. Four products (AmpliTaq Gold® Fast PCR Master Mix, KAPA2G™ Fast Multiplex PCR Kit, SpeedSTAR™ HS DNA Polymerase and Type-it Microsatellite PCR Kit) were evaluated for low volume (3μl) fast PCR on a 384-well Veriti® thermal cycler with the Identifiler primer set. KAPA2G™ was selected for 3μl fast PCR protocols using PowerPlex 16 HS and Identifiler Plus primer sets (42-51min), as well as 5μl and 6μl Identifiler fast reactions on a 9700 thermal cycler (51-60min). Alternative detection (POP-6™/22cm) achieved 24-28min run times, but with decreased resolution as compared to traditional POP-4®/36cm detection for alleles >200bp; however, 1bp resolution was still obtainable for alleles <300bp. These modifications resulted in robust databasing processes with up to a 37% reduction in processing time for buccal swabs and Buccal DNA Collectors™ using the three primer sets evaluated (3μl fast PCR reactions) and generated high quality STR profiles with ≥90% pass rates.

<p>The field of forensic genetics is growing fast and the development and optimisation of more sensitive, faster and more discriminating forensic DNA analysis methods is highly important. In this thesis, an evaluation of the use of novel DNA technologies and the development of specific applications for use in forensic casework investigations are presented.</p><p>In order to maximise the use of valuable limited DNA samples, a fast and user-friendly Real-time PCR quantification assay, of nuclear and mitochondrial DNA copies, was developed. The system is based on the 5’ exonuclease detection assay and was evaluated and successfully used for quantification of a number of different evidence material types commonly found on crime scenes. Furthermore, a system is described that allows both nuclear DNA quantification and sex determination in limited samples, based on intercalation of the SYBR Green dye to double stranded DNA. </p><p>To enable highly sensitive DNA analysis, Pyrosequencing of short stretches of mitochondrial DNA was developed. The system covers both control region and coding region variation, thus providing increased discrimination power for mitochondrial DNA analysis. Finally, due to the lack of optimal assays for quantification of mitochondrial DNA mixture, an alternative use of the Pyrosequencing system was developed. This assay allows precise ratio quantification of mitochondrial DNA in samples showing contribution from more than one individual.</p><p>In conclusion, the development of optimised forensic DNA analysis methods in this thesis provides several novel quantification assays and increased knowledge of typical DNA amounts in various forensic samples. The new, fast and sensitive mitochondrial DNA Pyrosequencing assay was developed and has the potential for increased discrimination power.</p>

The field of forensic genetics is growing fast and the development and optimisation of more sensitive, faster and more discriminating forensic DNA analysis methods is highly important. In this thesis, an evaluation of the use of novel DNA technologies and the development of specific applications for use in forensic casework investigations are presented. In order to maximise the use of valuable limited DNA samples, a fast and user-friendly Real-time PCR quantification assay, of nuclear and mitochondrial DNA copies, was developed. The system is based on the 5’ exonuclease detection assay and was evaluated and successfully used for quantification of a number of different evidence material types commonly found on crime scenes. Furthermore, a system is described that allows both nuclear DNA quantification and sex determination in limited samples, based on intercalation of the SYBR Green dye to double stranded DNA. To enable highly sensitive DNA analysis, Pyrosequencing of short stretches of mitochondrial DNA was developed. The system covers both control region and coding region variation, thus providing increased discrimination power for mitochondrial DNA analysis. Finally, due to the lack of optimal assays for quantification of mitochondrial DNA mixture, an alternative use of the Pyrosequencing system was developed. This assay allows precise ratio quantification of mitochondrial DNA in samples showing contribution from more than one individual. In conclusion, the development of optimised forensic DNA analysis methods in this thesis provides several novel quantification assays and increased knowledge of typical DNA amounts in various forensic samples. The new, fast and sensitive mitochondrial DNA Pyrosequencing assay was developed and has the potential for increased discrimination power.

One electron oxidation of DNA has been studied extensively over the years. When a charge is injected into a DNA duplex, it migrates through the DNA until it reaches a trap. Upon further reactions, damage occurs in this area and strand cleavage can occur. Many works have been performed to see what can affect this damage to DNA. Netropsin is a minor groove binder that can bind to tracts of four to five A:T base pairs. It has been used in the studies within to determine if it can protect DNA against oxidative damage, caused by one-electron oxidation, when it is bound within the minor groove of the DNA. By using a naphthacenedione derivative as a photosensitizer, several DNA duplexes containing netropsin binding sites as well as those without binding sites, were irradiated at 420 nm, analyzed, and visualized to determine its effect on oxidative damage. It has been determined netropsin creates a quenching sphere of an average of 5.8 * 108 Šwhether bound to the DNA or not. Herein we will show netropsin protects DNA against oxidative damage whether it is free in solutions or bound within the minor groove of a DNA duplex.