Keywords

Trace, biological, evidence, tissue, donor, identity, body fluid, micro volume, dna, rna, forensics, forensic biology, touch, profiling, single cell, micromanipulation, few cells, clumps, particles

Abstract

In forensic casework analysis it is necessary to obtain genetic profiles from increasingly smaller amounts of biological material left behind by perpetrators of crime. The ability to obtain profiles from trace biological evidence is demonstrated with so-called ‘touch DNA evidence’ which is perceived to be the result of DNA obtained from shed skin cells transferred from donor to an object or person during physical contact. However, the current method of recovery of trace DNA involves cotton swabs or adhesive tape to sample an area of interest. This “blindswabbing” approach may result in the recovery of biological material from different individuals resulting in admixed DNA profiles which are often difficult to interpret. Profiles recovered from these samples are reported to be from shed skin cells with no biological basis for that determination. A specialized approach for the isolation of single or few cells from ‘touch DNA evidence’ is necessary to improve the analysis and interpretation of recovered profiles. Here we describe the development of optimized and robust micro volume PCR reactions (1-5 μL) to improve the sensitivity and efficiency of ‘touch DNA’ analysis. These methods will permit not only the recovery of the genetic profile of the donor of the biological material, but permit an identification of the tissue source of origin using mRNA profiling. Results showed that the 3.5 uL amplification volume, a fraction of the standard 25 uL amplification volume, was the most ideal volume for the DNA assay, as it had very minimal evaporation with a 50% profile recovery rate at a single cell equivalent input (~5 pg) with reducing amplification volume alone. Findings for RNA showed that by reducing both amplification steps, reverse transcriptase PCR (20 uL) and body fluid multiplex PCR (25 uL), to iv 5 uL, ideal results were obtained with an increase in sensitivity and detection of six different body fluids down to 50 pg. Once optimized at the trace level, the assays were applied to the collection of single and few cells. DNA findings showed that about 40% of a full profile could be recovered from a single buccal cell, with nearly 80% of a full profile recovered from only two cells. RNA findings from collected skin particles of “touched” surfaces showed accurate skin detection down to 25 particles and detection in one clump of particles. The profiles recovered were of high quality and similar results were able to be replicated through subsequent experiments. More studies are currently underway to optimize these developed assays to increase profile recovery at the single cell level. Methods of doing so include comparing different locations on touched surfaces for highest bio-particle recovery and the development of physical characteristics of bio-particles that would provide the most ideal results

Notes

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Graduation Date

2013

Semester

Fall

Advisor

Ballantyne, John

Degree

Master of Science (M.S.)

College

College of Sciences

Department

Chemistry

Degree Program

Forensic Science; Forensic Biochemistry Track

Format

application/pdf

Identifier

CFE0005385

URL

http://purl.fcla.edu/fcla/etd/CFE0005385

Language

English

Release Date

June 2015

Length of Campus-only Access

1 year

Access Status

Masters Thesis (Open Access)

Subjects

Dissertations, Academic -- Sciences, Sciences -- Dissertations, Academic

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