Social and Behavioral Sciences Commons^™

Evaluation Of Direct-To-Amplification Cell Lysis Techniques For Forensically Relevant Non-Sperm Cells, Rhea Arya

Master of Science in Forensic Science Directed Research Projects

While efforts have been made to reduce the pervasive backlog of sexual assault evidence collection kits, the actual laboratory process remains very time-consuming due to the requirement of a differential lysis step before DNA purification, as well as intricate mixture analysis towards the end of the DNA workflow. Previous work in the Dawson Green laboratory at VCU has developed alternative solutions for differential extractions with sexual assault samples using both an in-tube and microdevice assay. Prior work led to the identification of an alternative sperm lysis method – alkaline lysis using 1M NaOH. However, the current lysis method used for …

Optimization Of Microfluidic Device Methodology For Optical Trapping Epithelial Cells In A Mixture, Brittney N. Hackworth

Master of Science in Forensic Science Directed Research Projects

Evidence containing biological mixtures can make DNA interpretation more difficult due to ambiguous allele calls and artifacts that may be present within electropherograms. Separating out these cell mixtures can be advantageous to the analysts’ analysis methods for identifying STR profiles with higher accuracy. A potential cell separation method that could be implemented in the beginning of the forensic DNA workflow is optical trapping.

This research utilized a 5W ytterbium linearly polarized laser split into two optical traps, paired with a microfluidic device to isolate spermatozoa and vaginal epithelial cells from a 1:1 mixture of vaginal fluid and 1:20 diluted seminal …

Horizontal And Vertical Transmission Of Microbes Associated With Blow Flies Of Forensic Importance, Gabrielle Alisa Burton

Master of Science in Forensic Science Directed Research Projects

Entomological and microbiological evidence can be used in the estimation of postmortem intervals (PMI). This study observes the horizontal and vertical transmission of the microbial community associated with blow flies of forensic importance. Variable region four (V4) of the 16S ribosomal DNA (16S rDNA) was amplified and sequenced from various life stages of blow flies (n=72) using dual-index high throughput sequencing strategy on the MiSeq FGx platform. No significant difference in bacterial community structure was observed between blowfly adults with carrion access and blowfly adults with no carrion access. Dysgonomonas, unclassified Proteobacteria, Escherichia, Ignatzschia, Providencia, …

Comparison Of Cell Counts To Dna Yield In Mock Biological Evidence Samples, Jacob Sewell

Master of Science in Forensic Science Directed Research Projects

The implementation of rapid DNA analysis systems is of interest to forensic DNA casework, as this technology exhibits a sample-to-profile time of approximately two hours with limited handling of samples. This decreases the likelihood of contamination, and can potentially reduce DNA backlogs; however, current rapid DNA analysis systems do not execute one crucial step of the forensic DNA analysis workflow: human DNA quantitation. This step is necessary for DNA profiles to be compliant with the FBI’s Quality Assurance Standard 9.4, allowing DNA profiles to be uploaded and searched within the CODIS database. Coupling traditional qPCR methods with rapid DNA analysis …

Development And Validation Of Microrna Markers For Forensic Body Fluid Identification Using Rt-Qpcr, Jane Goble

Master of Science in Forensic Science Directed Research Projects

Body fluid identification is one of the first and most fundamental steps in the forensic DNA workflow. It allows analysts to determine where a sample might exist on an item of evidence, as well as the type of sample that they are dealing with. However, current serological methods have been documented to produce false or inaccurate results in some cases, and may consume large amounts of sample during processing. Molecular methods, such as the use of microRNA and microbial profiles have been introduced as a means to increase specificity of testing, and have even been combined into a singular assay …

Assessment Of The Acidified Hydrogen Peroxide Method On Uncontrolled Fired Cartridge Case Samples, Kylie S. Richardson

Master of Science in Forensic Science Directed Research Projects

The use of fingerprints to identify individuals in forensic cases has been useful for many years due to the persistence and individuality of the prints. In addition, the presence of firearms in forensic casework is steadily increasing over the years. This study aims to expand upon prior research in the development and recovery of latent prints on fired, brass ammunition using an acidified hydrogen peroxide method. Although latent prints are left on the cartridge casing of ammunition during the handling and loading of ammunition into the firearm, it is hypothesized that the pressure within the barrel of the firearm during …

Development Of A Loop-Mediated Isothermal Amplification (Lamp) Assay For Detection Of Mirnas, Alisha B. Whelan

Master of Science in Forensic Science Directed Research Projects

Body fluid identification serves a vital role in contextualizing biological evidence received in forensic laboratories. While several fluids can be presumptively detected using immunological or catalytic tests, these tests are susceptible to false results and have limited sensitivity. True confirmatory testing is still somewhat limited in forensic laboratories. With the advancement of DNA typing technologies, laboratories can develop DNA profiles from trace amounts of sample, increasing the demand for sensitive and accurate body fluid detection. This project proposes using loop-mediated isothermal amplification (LAMP) as a method to rapidly detect microRNAs (miRNAs) that are differentially expressed in various body fluids with …

Stability And Variation Within Donors Of Mirna Markers For Body Fluid Identification, Kelsey R. Price

Master of Science in Forensic Science Directed Research Projects

Traditional enzymatic tests for body fluid identification are prone to false negatives, false positives, and several body fluids do not have confirmatory or reliable presumptive tests. Therefore, molecular-based tests may be more reliable, such as messenger RNA (mRNA), microbial DNA, or microRNA (miRNA) assays.

miRNAs are small, noncoding RNAs whose main function in-vivo is the regulation of protein expression by selectively suppressing the translation of their corresponding mRNAs. They also lack a poly-A tail, and because of this, their small size, and their association with other molecules such as the RISC protein complex, they are more robust than other RNAs, …

A Quantifiler Trio-Based Hrm Mixture Screening Assay For The Quantstudio 6 Flex Qpcr Platform, Dayanara A. Torres

Master of Science in Forensic Science Directed Research Projects

At present, the forensic DNA workflow is not capable of providing information about the contributor status (single source vs. mixtures) of evidentiary samples prior to end-point analysis. This shortcoming can exacerbate the challenges inherent to mixtures and low-template DNA samples. Provided additional sample information earlier in the workflow, protocols could be implemented to mitigate these challenges. High-resolution melt (HRM) curve analysis is a technique used to detect genetic variation in DNA fragments and in the last decade has been evaluated for use in differentiation of samples by genotype and/or contributor status. To this end, a proof-of-concept HRM assay using the …

Optimization And Comparison Of Methods For Separation Of Spermatozoa From Superabsorbent Polymer-Containing Forensic Evidence, Hannah Wells

Master of Science in Forensic Science Directed Research Projects

Sexual assault is currently one of the most prevalent crimes that affects victims of all ages. Forensic DNA analysts often confirm proof of contact in sexual assault cases through the identification of spermatozoa and subsequent STR profiling. Diapers and other feminine hygiene products, such as maxi pads and ultrathin pads, are types of superabsorbent polymer-containing evidence that complicate the process of DNA analysis due to the trapping of the spermatozoa in the gel-like matrix. In this study, a comparison of methods was performed to determine which extraction technique produces the highest sperm cell and DNA yield and generates a usable …

Impact Of 1,4-Dithiothreitol (Dtt) On Microbial Community Structures Associated With Human Semen Samples, Christian R. Blaise

Master of Science in Forensic Science Directed Research Projects

Body fluid identification (BFID) is one of the most important steps in forensic science as it provides important context to how DNA analysis should be performed on biological samples found at the scene of a crime. Many scientific methods on BFID have been developed over the years with respective benefits and flaws. One emerging method in recent years is microbial signature based BFID. This method uses the same DNA extract that is utilized for human DNA profile generation for microbial analysis. Although DNA is extracted from all body fluid samples without any additional steps, extraction of human DNA from semen …

Powerplex® Fusion 6c System Versus Powerplex® Fusion 5c: A Comparison Of Performance Metrics, Caitlin M. Mccaughan

Master of Science in Forensic Science Directed Research Projects

Following the FBI mandated expansion of the CODIS core loci from 13 to 20, several manufacturers developed short tandem repeat (STR) typing kits in response to the new criteria. One such manufacturer was the Promega Corporation, which released the PowerPlex® Fusion 5C megaplex STR typing kit (Fusion 5C) in 2012. Currently, the Virginia Department of Forensic Science (VDFS) utilizes this amplification kit for both casework and database applications. In 2015, Promega released the PowerPlex® Fusion 6C STR typing kit (Fusion 6C), which contains three additional loci beyond those included in PowerPlex® Fusion 5C, and a sixth color channel. The power …

Creation And Optimization Of An Optical Trapping Microfluidic Device For The Separation Of Mock Forensic Sexual Assault Samples, Mackenzie Lally

Master of Science in Forensic Science Directed Research Projects

Cell mixtures are often seen in forensic samples and commonly involve sexual assault cases where mixtures of sperm cells and vaginal epithelial cells are frequently encountered. This produces challenges in downstream analysis in the form of STR mixture profiles. The only method currently in use in crime laboratories for front-end sperm and epithelial cell separation is differential extraction. This method often results in STR mixture profiles due to carryover into both the male and female fractions and suffers from a wide range of efficiency depending on the laboratory or individual processing the sample. Optical trapping offers an alternative method for …

Evaluation Of Cell Lysis Techniques For Direct Amplification Of Sexual Assault Samples, Sarah Schellhammer

Master of Science in Forensic Science Directed Research Projects

Sexual assault is one of the most common crimes committed today with a delay in sample processing, resulting in the “rape kit backlog”. The traditional method to extract DNA from these samples containing both male and female DNA is a differential extraction; taking hours to process. This study examined a variety of different cell lysis techniques to develop a method which can consistently generate high-quality profiles at a fast rate and with low volumes when compared to the currently used method in our laboratory.

Six lysis methods, in addition to the currently used forensicGEM™ Sperm kit, were performed on …

Evaluation Of Dna Extraction Efficiencies Of Promega’S Dna Iq™ Methods And Casework Extraction Kit For Low Template Samples, Emily M. Anderson

Master of Science in Forensic Science Directed Research Projects

Biological evidence from crime scene samples frequently contain low levels of DNA, such as the most predominant form of evidence, which is DNA deposited by handling objects or “touch evidence”. To maximize the DNA yield recovered from theses challenging samples, forensic laboratories must optimize the extraction methods utilized to isolate and purify DNA for downstream short tandem repeat (STR) amplifications. Currently, the Virginia Department of Forensic Science (VADFS) uses a DNA IQ™ System (DNA IQ) extraction method for isolation of DNA from most forensic samples. This extraction procedure, which combines DNA IQ™ lysis buffer and Dithiothreitol (DTT), has been validated …

Postmortem Interval Estimation Based On Eukaryotic Community Associated With Soil Under Decomposing Porcine Remains, Amanda M. Haase

Master of Science in Forensic Science Directed Research Projects

Recent studies have shown that bacteria associated with soils under a carcass change significantly both temporally and spatially. These studies were done in both laboratory and field conditions, but with limited replications. In addition, limited information exists on temporal and spatial changes in eukaryotic community structure associated with soil under decomposing remains. This study was designed to fill in these gaps and expand on previous studies to improve postmortem interval (PMI) estimation techniques as well as to identify cadaver decomposition islands (CDI) based on eukaryotic community structure associated with soil under vertebrate remains. To accomplish this goal, soil samples were …

Development Of A Quantitative Pcr (Qpcr) Based Method For Studying Temporal Dna Degradation In Waterlogged Bone, Isis Thornton

Master of Science in Forensic Science Directed Research Projects

Human activities are often centered around the presence of water, thus it is not surprising that there are many water-related human deaths. Accumulated degree days (ADD), and other aquatic variables may affect DNA retrieval from waterlogged bone. Calcium and collagen in bone can inhibit the PCR necessary to produce an STR profile; the current solution is a time-consuming organic extraction. While there are examples of research on DNA degradation in terrestrial bone over time, there has been little work done on submerged bone samples and they are usually limited to case studies. The major aim of this study was to …

Expanded Testing Of An Integrated Qpcr Mixture Screening Assay Using High Resolution Melt Curve Analysis And Support Vector Machine Modeling, Andrea L. Williams

Master of Science in Forensic Science Directed Research Projects

In the conventional forensic DNA workflow, the number of contributors in a sample is unknown until the final step of STR analysis. We propose a high-resolution melt curve (HRM) mixture screening assay, which uses support vector machine (SVM) modeling of melt morphologies of D5S818 and D18S51 amplicons integrated into a common qPCR-based human DNA quantification kit, to differentiate between single-source samples (and their genotypes) and mixtures at an earlier stage in the DNA workflow (quantification). Previously, using data generated from whole melt curves, 87.5% of single-source samples and 100% of 1:1 mixture samples (2 contributors) classified accurately.

In this study, …