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THE DEVELOPMENT OF A "GENETIC EYEWITNESS" PROFILING SYSTEM FOR LOW TEMPLATE FORENSIC SPECIMENS: IDENTIFICATION OF NOVEL PROTEIN, RNA, DNA BIOMARKERS
Title
THE DEVELOPMENT OF A "GENETIC EYEWITNESS" PROFILING SYSTEM FOR LOW TEMPLATE FORENSIC SPECIMENS: IDENTIFICATION OF NOVEL PROTEIN, RNA, DNA BIOMARKERS.
Creator
Hanson, Erin, Ballantyne, Jack, University of Central Florida
Abstract / Description
In many criminal investigations, valuable information regarding the physical appearance of suspected perpetrators or the time and order of events that transpired are provided by eyewitness accounts. However, the information obtained from eyewitnesses is often constrained by human recollection or subjective accounts and provides a biased description of the perpetrator's appearance or an inaccurate time line of events. Additionally, in numerous situations eyewitness accounts may not be...
Show moreIn many criminal investigations, valuable information regarding the physical appearance of suspected perpetrators or the time and order of events that transpired are provided by eyewitness accounts. However, the information obtained from eyewitnesses is often constrained by human recollection or subjective accounts and provides a biased description of the perpetrator's appearance or an inaccurate time line of events. Additionally, in numerous situations eyewitness accounts may not be available. An increasing reliance therefore is placed on the biological evidence recovered during criminal investigations to act as a silent witness, providing unbiased and scientific information that may aid in the resolution of criminal investigations. While the current capabilities of operational forensic crime laboratories include analytical methods to allow for a determination of the origin of a biological stain and for the recovery of a genetic profile of the donor, the sensitivity of such methods is not always sufficient to accommodate the limited amounts of biological material often recovered in forensic casework, Therefore, it is critical that continual advancements in the analysis of low template samples be made. In this report, we have sought to identify novel protein, RNA and DNA biomarkers that, in combination with enhanced profiling strategies, would allow for a determination of the time since deposition, the body fluid of origin and the genetic profile of the donor ("genetic eyewitness") of forensic low template specimens. First, we have developed a novel strategy for the determination of the time since deposition of dried bloodstains using spectrophotometric analysis of hemoglobin. An examination of the Soret band (lambda max = 414nm) in aged bloodstains has revealed a previously unidentified hypsochromic shift as the age of the stain increases. The extent of this shift permits a distinction to be made between stains that differ in age by only minutes, hours, days and months thus providing the highest resolution of any previously developed method. We also demonstrate that it may be possible to utilize a decline in enzyme activity to determine the age of a forensic biological stain. Second, we demonstrate that the differential expression of a panel of nine miRNAs allows for the identification of the body fluid origin of forensic biological stains using as little as 50pg of total RNA. This is the highest reported sensitivity of any RNA-based approach and this assay has demonstrated a high degree of specificity for each body fluid tested. The final task of this work was to identify novel DNA biomarkers and to develop enhanced profiling strategies to allow for greater sensitivity and reliability in the genetic profiling of low template samples. We demonstrate that the use of laser capture micro-dissection and enhanced amplification strategies resulted in the ability to obtain genetic profiles from as few as 2-5 epithelial cells and 5-10 sperm cells with greater reproducibility than previously reported studies. The use of a novel whole genome amplification method provided the ability to not only increase the quantity of genetic material obtained from micro-dissected cells but also the ability to recover additional genetic information from individual samples using novel DNA biomarkers. The novel biomarkers and profiling strategies described in this report provide the basis for the establishment of a molecular "genetic eyewitness" from low template forensic samples and demonstrate the future potential for routine and reliable analysis of trace amounts of genetic material recovered from low template biological evidence.
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Date Issued
2008
Identifier
CFE0002373, ucf:47785
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0002373
THE USE AND DEVELOPMENT OF LASER MICRODISSECTION TO SEPARATE SPERMATOZOA FROM EPITHELIAL CELLS FOR STR ANALYSIS
Title
THE USE AND DEVELOPMENT OF LASER MICRODISSECTION TO SEPARATE SPERMATOZOA FROM EPITHELIAL CELLS FOR STR ANALYSIS.
Creator
Sanders, Christine, Ballantyne, Jack, University of Central Florida
Abstract / Description
Short Tandem Repeat (STR) analysis has become a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. This report describes the use of laser...
Show moreShort Tandem Repeat (STR) analysis has become a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. This report describes the use of laser microdissection (LMD) for the separation of pure populations of spermatozoa from two-donor cell mixtures. In this study, cell separation was demonstrated by microscopic identification of histologically stained spermatozoa and female buccal cell mixtures, and STR analysis of DNA obtained from the separated sperm cells. Clear profiles of the male donor were obtained with the absence of any additional alleles from the female donor. Five histological stains were evaluated for use with LMD and DNA analysis: hematoxylin/eosin, nuclear fast red/picroindigocarmine, methyl green, Wright's stain, and acridine orange. Hematoxylin/eosin out-performed all other stains however nuclear fast red/picroindigocarmine could be used satisfactorily with STR analysis. In addition, three DNA isolation methods were evaluated for LMD collected cells: QIAamp (Qiagen), microLYSIS (Microzone Ltd.) and Lyse-N-Go (Pierce Chemical Co.). MicroLYSIS performed poorly, yielding low levels of PCR product. Lyse-N-Go extraction was effective for the recovery of DNA from LMD collected sperm cells while QIAamp isolation performed best for the recovery of DNA from LMD collected epithelial cells. LMD is shown to be an effective, low-manipulation separation method that enables the recovery of sperm while excluding epithelial cell DNA.
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Date Issued
2005
Identifier
CFE0000876, ucf:46652
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0000876
The Owl Sensor: a smart nanostructure for single nucleotide variation analysis
Title
The Owl Sensor: a smart nanostructure for single nucleotide variation analysis.
Creator
Karadeema, Rebekah, Kolpashchikov, Dmitry, Chumbimuni Torres, Karin, Harper, James, University of Central Florida
Abstract / Description
Analysis of single nucleotide variations (SNVs) in DNA and RNA sequences is extensively used in healthcare for detection of genetic mutations and analysis of drug resistant pathogens. Here we developed a nucleic acid sensor able to differentiate between a fully matched analyte and one with a SNV in a wide temperature range of 5(&)deg;C-32(&)deg;C. The sensor, dubbed here the 'Owl Sensor' due to the complex's resemblance to owl eyes, utilizes recent developments in DNA nanotechnology and...
Show moreAnalysis of single nucleotide variations (SNVs) in DNA and RNA sequences is extensively used in healthcare for detection of genetic mutations and analysis of drug resistant pathogens. Here we developed a nucleic acid sensor able to differentiate between a fully matched analyte and one with a SNV in a wide temperature range of 5(&)deg;C-32(&)deg;C. The sensor, dubbed here the 'Owl Sensor' due to the complex's resemblance to owl eyes, utilizes recent developments in DNA nanotechnology and synthetic biology to self-assemble a fluorescent DNA nanostructure called a Double Crossover, or DX Tile, capable of differentiating SNVs in a large temperature range, including ambient temperature. In the presence of fully matched nucleic acid analytes, a stable complex is formed with high fluorescent signal; however in the presence of a single base variation in the analyte, unfavourable helicity results in little-to-no observed complex formation. The novelty of the approach is that selectivity of analyte recognition is, at least in part, determined by the structural rigidity of the entire nanostructure rather than by the stability of analyte-probe hybrid, as is the case for conventional hybridization probes. The rigid nanostructure collapses if a minor imperfection, e.g. if a single-base mispairing, is present. Owl Sensor differentiates fully matched analyte from mismatched in a wide temperature range, with mismatched analyte producing only the background fluorescence, selectivity that is hard to achieve by conventional hybridization probes. Owl Sensor therefore promises to add to the toolbox for diagnosis of genetic disorders and infectious diseases at ambient temperatures.
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Date Issued
2016
Identifier
CFE0006691, ucf:51916
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006691