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Title: MITOCHONDRIAL DNA ANALYSIS BY PYROSEQUENCING.
Creator: Hastings, Patsy-Ann Susan, Ballantyne, Jack, University of Central Florida
Abstract / Description: Mitochondrial DNA (deoxyribo nucleic acid) is typically used in forensic casework when small quantities of high molecular weight quality DNA is not expected to be present thus negating the chances of obtaining usable nuclear DNA. Typical samples that utilized mitochondrial DNA analysis are: hair, bones, teeth, ancient remains (samples or remains that are at least 100 years old) or very old samples (samples that are less than 100 but greater than 10 years old). The current method used to...
Show moreMitochondrial DNA (deoxyribo nucleic acid) is typically used in forensic casework when small quantities of high molecular weight quality DNA is not expected to be present thus negating the chances of obtaining usable nuclear DNA. Typical samples that utilized mitochondrial DNA analysis are: hair, bones, teeth, ancient remains (samples or remains that are at least 100 years old) or very old samples (samples that are less than 100 but greater than 10 years old). The current method used to evaluate mitochondrial DNA is Sanger sequencing. Although robust, it is also time consuming and labor intensive, on the other hand pyrosequencing is a nonelectrophoretic, rapid, reliable, and sensitive sequencing method which can be easily automated. Therefore pyrosequencing could enable the widespread use of mitochondrial DNA in forensic casework and reduce the amount of time spent on each sample without compromising quality.The aim of this study is to evaluate the efficacy of pyrosequencing for forensic DNA applications, in particular mitochondrial DNA. Two dispensation orders, cyclic and directed, were examined to determine if there is any effect on the sequence generated. The accuracy of pyrosequencing was evaluated by sequencing samples of known sequence provided by the FBI. The sensitivity of pyrosequencing was evaluated by sequencing samples at different DNA concentrations and inputs. Experiments were conducted to determine the ability of pyrosequencing to detect mixtures and heteroplasmy. Additionally, the ability of pyrosequencing to sequence damaged/degraded DNA was evaluated using blood, semen, and saliva samples that were subjected to three different environmental conditions. A blind study will be conducted to confirm the accuracy of pyrosequencing. Finally, a comparison study will be conducted in which pyrosequencing will be compared to Sanger sequencing.
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Date Issued: 2004
Identifier: CFE0000098, ucf:46116
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0000098

Title: Homologous Pairing Through DNA Driven Harmonics - A Simulation Investigation.
Creator: Calloway, Richard, Proctor, Michael, Kincaid, John, Jaganathan, Balasubramanian, Gerber, Matthew, Chai, Xinqing, University of Central Florida
Abstract / Description: The objective of this research is to determine if a better understanding of the (")molecule of life("), deoxyribonucleic acid or DNA can be obtained through Molecular Dynamics (MD) modeling and simulation (M(&)S) using contemporary MD M(&)S. It is difficult to overstate the significance of the DNA molecule. The now-completed Human Genome Project stands out as the most significant testimony yet to the importance of understanding DNA. The Human Genome Project (HGP) enumerated many areas of...
Show moreThe objective of this research is to determine if a better understanding of the (")molecule of life("), deoxyribonucleic acid or DNA can be obtained through Molecular Dynamics (MD) modeling and simulation (M(&)S) using contemporary MD M(&)S. It is difficult to overstate the significance of the DNA molecule. The now-completed Human Genome Project stands out as the most significant testimony yet to the importance of understanding DNA. The Human Genome Project (HGP) enumerated many areas of application of genomic research including molecular medicine, energy sources, environmental applications, agriculture and livestock breeding to name just a few. (Science, 2008) In addition to the fact that DNA contains the informational blueprints for all life, it also exhibits other remarkable characteristics most of which are either poorly understood or remain complete mysteries. One of those completely mysterious characteristics is the ability of DNA molecules to spontaneously segregate with other DNA molecules of similar sequence. This ability has been observed for years in living organisms and is known as (")homologous pairing.(") It is completely reproducible in a laboratory and defies explanation. What is the underlying mechanism that facilitates long-range attraction between 2 double-helix DNA molecules containing similar nucleotide sequences? The fact that we cannot answer this question indicates we are missing a fundamental piece of information concerning the DNA bio-molecule. The research proposed herein investigated using the Nano-scale Molecular Dynamics NAMD (Phillips et al., 2005) simulator the following hypotheses:H(Simulate Observed Closure NULL) : = Current MD force field models when used to model DNA molecule segments, contain sufficient variable terms and parameters to describe and reproduce directed segregating movement (closure of the segments) as previously observed by the Imperial College team between two Phi X 174 DNA molecules. H(Resonance NULL) : = Current MD force field models when used to model DNA molecule segments in a condensed phased solvent contain sufficient variable terms and parameters to reproduce theorized molecular resonation in the form of frequency content found in water between the segments. H(Harmonized Resonance NULL) : = Current MD force field models of DNA molecule segments in a condensed phase solvent produce theorized molecular resonation in the form of frequency content above and beyond the expected normal frequency levels found in water between the segments. H(Sequence Relationship NULL): = The specific frequencies and amplitudes of the harmonized resonance postulated in H(Harmonized Resonance NULL) are a direct function of DNA nucleotide sequence. H(Resonance Causes Closure NULL) : = Interacting harmonized resonation produces an aggregate force between the 2 macro-molecule segments resulting in simulation of the same directed motion and segment closure as observed by the Imperial College team between two Phi X 174 DNA molecules. After nearly six months of molecular dynamic simulation for H(Simulate Observed Closure NULL) and H(Resonance Causes Closure NULL) no evidence of closure between two similar sequenced DNA segments was found. There exist several contributing factors that potentially affected this result that are described in detail in the Results section. Simulations investigating H( Resonance NULL), H(Harmonized Resonance NULL) and the emergent hypothesis H(Sequence Relationship NULL) on the other hand, revealed a rich selection of periodic pressure variation occurring in the solvent between simulated DNA molecules. About 20% of the power in Fourier coefficients returned by Fast Fourier Transforms performed on the pressure data was characterized as statistically significant and was located in less than 2% of the coefficients by count. This unexpected result occurred consistently in 5 different system configurations with considerable system-to-system variation in both frequency and magnitude. After careful analysis given the extent of our experiments the data was found to be in support of H( Resonance NULL), and H(Harmonized Resonance NULL) . Regarding the emergent hypothesis H(Sequence Relationship NULL), further analysis was done on the aggregate data set looking for correlation between nucleotide sequence and frequency/magnitude. Some of the results may be related to sequence but were insufficient to prove it. Overall the conflicting results were inconclusive so the hypothesis was neither accepted nor rejected. Of particular interest to future researchers it was noted that the computational simulations performed herein were NOT able to reproduce what we know actually happens in a laboratory environment. DNA segregation known to occur in-vitro during the Imperial College investigation did not occur in our simulation. Until this discrepancy is resolved MM simulation should not as yet be considered a suitable tool for further investigation of Homologous Chromosome Pairing. In Chapter 5 specific follow on research is described in priority of need addressing several new questions.
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Date Issued: 2011
Identifier: CFE0004472, ucf:49302
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004472

Title: Strategies for Enhanced Genetic Analysis of Trace DNA from Touch DNA Evidence and Household Dust.
Creator: Farash, Katherine, Ballantyne, John, Kolpashchikov, Dmitry, Bridge, Candice, University of Central Florida
Abstract / Description: In forensic casework it is often necessary to obtain genetic profiles from crime scene samples that contain increasingly smaller amounts of genetic material, called Low Template DNA (LTDNA). Two examples of LTDNA sources are touch DNA evidence and dust bunnies. Touch DNA refers to DNA that is left behind through casual contact of a donor with an object or another person. Touch DNA can be used to prove a suspect was present at a crime scene. Dust bunnies, or dust conglomerates, typically...
Show moreIn forensic casework it is often necessary to obtain genetic profiles from crime scene samples that contain increasingly smaller amounts of genetic material, called Low Template DNA (LTDNA). Two examples of LTDNA sources are touch DNA evidence and dust bunnies. Touch DNA refers to DNA that is left behind through casual contact of a donor with an object or another person. Touch DNA can be used to prove a suspect was present at a crime scene. Dust bunnies, or dust conglomerates, typically contain trapped shed skin cells of anyone in the vicinity along with fibers, dirt, hair, and other trace materials. Dust specimens are a potential source of forensic evidence that has been widely underutilized in the forensic community. This is unfortunate because a dust bunny could not only be used to associate a person or crime scene (-) through trace materials such as fibers (-) but also to positively identify (-) through a DNA profile. For example, if a dust specimen is found on a piece of evidence suspected of being moved from its original location, for instance as a body that is too heavy to carry and therefore collects dust while being dragged, then it could be used to link a suspect to a crime scene.Standard methods for obtaining and analyzing touch DNA have been established, but the techniques are not ideal. First, by nature, the 'blind-swabbing' technique, which involves cotton swabs or adhesive tape being applied to an area of interest, can artificially create mixtures of biological material that was originally spatially separated. Second, because the amount of DNA present is typically very low, standard analysis methods may not be sensitive enough to produce probative profiles. In the case of mixtures, the minor component's DNA may go undetected. Dust specimens contain degraded genetic material that has been accumulating for an unknown amount of time. Additionally, dust is usually a conglomeration of genetic material from multiple donors so a mixed profile, if any, is likely to be recovered if standard analysis methods are used.In order to overcome these obstacles presented by LTDNA, a micro-manipulation and combined cell lysis/direct PCR amplification technique has been developed that is sensitive enough to obtain full or probative STR profiles from single or clumped bio-particles collected from touch DNA and dust evidence. Sources of touch DNA evidence such as worn clothing items, touched objects, and skin/skin mixtures are easily sampled using an adhesive material on a microscope slide. Dust specimens can be dispersed onto an adhesive material as well. Targeted bio-particles are then (")picked(") with a water-soluble adhesive on a tungsten needle and deposited into a micro-volume STR amplification mix. Individual selection and analysis of isolated bio-particles reduces the chance of mixed profile recovery. To aid in the release of genetic material present in the bio-particles, a lysis mix containing a thermostable proteinase is then added to the sample. Samples are then analyzed using standard capillary electrophoresis (CE) methods.In addition to identifying the donor source of these LTDNA sources, it would be beneficial to a criminal investigation to identify the tissue source of the biological material as well. While it is widely speculated that the material originates from shed skin cells, there is little confirmatory evidence proving this assertion. Knowledge of the nature of the evidence could be vital to prevent its misinterpretation during the investigation and prosecution of a crime. Here tissue specific mRNA biomarkers have been evaluated for their use in tissue source determination using a highly sensitive High Resolution Melt (HRM) temperature assay that detects the selectively amplified targets based on their melt temperatures.Using the enhanced genetic analysis technique described above, DNA profile recovery has been markedly enhanced in sources of Touch DNA evidence and dust specimens compared to standard methods. Additionally, the molecular-based characterization method could potentially provide a better understanding of the meaningfulness of the recovered DNA profiles. This optimized strategy provides a method for recovering highly probative data from biological material in low template samples in an efficient and cost effect manner.
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Date Issued: 2015
Identifier: CFE0006033, ucf:50979
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006033

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

Title: EPIGENETIC CONTROL MECHANISMS IN SOMATIC CELLS MEDIATED BY DNA METHYLTRANSFERASE 1.
Creator: Lee, Bongyong, Muller, Mark, University of Central Florida
Abstract / Description: DNA methylation regulates gene expression through a complex network of protein/protein and protein/DNA interactions in chromatin. The maintenance methylase, DNA methyltransferase 1 (DNMT1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications in somatic cells. The mechanistic details that explain how DNMT1 catalytic action is directed in a chromatin setting are not well understood. We hypothesize that post...
Show moreDNA methylation regulates gene expression through a complex network of protein/protein and protein/DNA interactions in chromatin. The maintenance methylase, DNA methyltransferase 1 (DNMT1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications in somatic cells. The mechanistic details that explain how DNMT1 catalytic action is directed in a chromatin setting are not well understood. We hypothesize that post translational modifications and a variety of protein-protein interactions processes are key regulatory elements that set the methylation of CpG elements essential for normal growth behavior in somatic cells. These fundamental processes can be disrupted by DNA damage leading to inappropriate gene silencing and loss of growth control in somatic cells. First, we show that DNMT1 is post-translationally modified by sumoylation and we have mapped these sumoylation sites by defined mutations. Sumoylated DNMT1 is catalytically active on genomic DNA in vivo and substantially increases the enzymatic activity of DNMT1 both in vitro and in chromatin. These data establish that sumoylation modulates the endogenous activity of a prominent epigenetic maintenance pathway in cells. Second, we investigated novel mechanisms whereby somatic cells can erase then reset DNA methylation events in somatic cells. In this study, the relationship between DNA damage and gene silencing was explored. To this end, we generated a HeLa cell line containing a specialized GFP reporter cassette (DRGFP) containing two mutated GFP genes and a unique I-SceI restriction endonuclease site. These cells do not express GFP. A unique double strand break is then delivered by transfecting in the gene for I-SceI. About 4% of the cells produced a functional GFP by gene conversion and homologous recombination (HR); however roughly half of the GFP recombinants expressed the gene poorly and this was attributed to gene silencing. Silencing of the GFP expressing cell clones was due to DNA methylation and could be reversed using a drug that inhibits global methylation (5-aza-2'-deoxycytidine). Approximately half of the repaired genes were heavily methylated, and half were hypomethylated. That is, a key intermediate methylation state after HR repair is hemimethylated DNA, defined as methylation limited to one strand. Evidence is given that DNMT1 is acting as a de novo methylase at the HR repair patches in cells. Moreover, the DNA damage inducible protein, GADD45, interacts specifically with the catalytic domain of DNMT1 and GADD45 binds with extremely high affinity to hemimethylated DNA sites. Thus, GADD45 is a key regulatory element in silencing of HR repaired DNA segments and appears to inhibit the activity of DNMT1. Consistent with these results, we found that GADD45 increased the expression of recombinant GFP following HR repair, further suggesting its role in orchestrating strand specific DNA methylation by DNMT1. Since these experiments were performed in live cells, there is strong physiological relevance. We propose that DS DNA damage and the resulting HR process involves precise, strand selected DNA methylation mediated by the prominent methylase enzyme, DNMT1. Moreover, DS DNA break repair through HR and gene conversion, may potentially erase and reset DNA methylation patterns and therefore alter the expression of repaired genes. The overall process is tightly regulated by the DNA damage inducible protein GADD45, which may coordinate strand specific methylation by recruiting DNMT1 to HR repair templates. The ability of GADD45 to modulate DNMT1 catalytic activity may explain its role as a passive mediator of demethylation that has been reported by other groups. The overall process of silencing post DNA repair is a strong evolutionary force that may predispose cells to malignant transformation.
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Date Issued: 2009
Identifier: CFE0002872, ucf:48026
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0002872

Title: A Cytoplasmic-Replicating RNA Virus Sensitizes Cancer Cells to DNA Modifying Agents.
Creator: Fox, Candace, Parks, Griffith, Copik, Alicja, Khaled, Annette, Zervos, Antonis, University of Central Florida
Abstract / Description: The Parainfluenza virus 5 (PIV5) mutant P/V-CPI- is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burden in mouse model systems. Here we show that P/V-CPI- infection of human laryngeal cancer HEp-2 cells resulted in the majority of the cells dying, but unexpectedly, a population of cells emerged as P/V-CPI- persistently infected (PI) cells. P/V-CPI- PI cells had elevated levels of basal caspase activation, and viability was highly...
Show moreThe Parainfluenza virus 5 (PIV5) mutant P/V-CPI- is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burden in mouse model systems. Here we show that P/V-CPI- infection of human laryngeal cancer HEp-2 cells resulted in the majority of the cells dying, but unexpectedly, a population of cells emerged as P/V-CPI- persistently infected (PI) cells. P/V-CPI- PI cells had elevated levels of basal caspase activation, and viability was highly dependent on activity of cellular inhibitors of apoptosis, such as Survivin. In challenge experiments with external inducers of apoptosis, the PI cells were highly sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in the phosphorylation cascade controlling DNA damage signaling pathways, as well as translocation of damage-specific DNA binding protein 1 (DDB1) to the nucleus. Similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI-, as well as acute infections with WT PIV5. Based on this finding, we tested the hypothesis that histone deacetylase (HDAC) inhibitors would also act with P/V-CPI- infection to enhance cancer cell killing. Using human lung and laryngeal cancer cell lines, 10 HDAC inhibitors were tested for their effect on viability of P/V-CPI- infected cells. HDAC inhibitors such as scriptaid enhanced caspase-3/7, -8 and -9 activity induced by P/V-CPI- and overall cell toxicity. Scriptaid treatment also enhanced the spread of P/V-CPI- through a population of cancer cells and suppressed interferon-beta induction through blocking phosphorylation and nuclear translocation of Interferon Regulatory Factor 3 (IRF-3). These results support a therapeutic approach of combining parainfluenza infection and chemotherapy, but also raise questions on the mechanism by which a cytoplasmic-replicating RNA virus can alter cellular DNA damage responses.
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Date Issued: 2019
Identifier: CFE0007803, ucf:52355
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007803

Title: THE BIOCHEMICAL REACTIONS OF DRY STATE DNA.
Creator: Marrone, April, Ballantyne, John, University of Central Florida
Abstract / Description: The biochemistry of dry state DNA is of interest to the fields of forensics, ancient DNA, and DNA storage. The exact chemical nature of the degradation of the DNA molecule in the dry state has not been studied prior. If determined what chemical changes the DNA molecule undergoes, to what degree and in what time frame then protocols can be implemented to bypass the impact of this damage or to repair it when necessary. It is suspected that similar reactions occur to the dry state DNA molecule...
Show moreThe biochemistry of dry state DNA is of interest to the fields of forensics, ancient DNA, and DNA storage. The exact chemical nature of the degradation of the DNA molecule in the dry state has not been studied prior. If determined what chemical changes the DNA molecule undergoes, to what degree and in what time frame then protocols can be implemented to bypass the impact of this damage or to repair it when necessary. It is suspected that similar reactions occur to the dry state DNA molecule as does to the hydrated molecule. It cannot be assumed, however that these types of chemical processes occur to the same extent and at the same rates. In general the generic process of hydrolysis encompasses two important reactions, that of deamination and of base loss from the 2'-deoxyribose backbone. Base loss is believed to ultimately lead to chain scission. It is also suspect that reactive oxygen species (ROS) have an important role in the chemistry associated with DNA. Species such as hydroxyl radicals (OH•) and singlet oxygen (1O2) can lead to strand scissions and chemically modified bases. Throughout this project various techniques were used to determine damage to DNA and its molecular constituents under conditions leading to hydrolytic and oxidative damage. Novel techniques used in this study include ion-pairing chromatography and denaturing HPLC (DHPLC) to measure glycosidic bond cleavage and strand breaks. The extent to which the macromolecule haemoglobin (Hb) can lead to oxidative damage of DNA in dried blood stains by acting as a Fenton chemistry catalyst was evaluated. Additionally the enzymatic activity of the extracellular nuclease from Alteromonas espejiana, BAL 31 was studied as it pertains to the degradation of single-stranded short homopolymeric oligonucleotides. This study serves as the basis for future, more in depth experimentation into the more specific nature of dry state DNA biochemistry. It was found that to a large extent the same degradation reactions (base hydrolysis, base modifications, and strand breaks) do occur in the dry state as in the hydrated state when heat and UV radiation are used as energy sources. Reaction rates indicate that base hydrolysis and deamination occur much more slowly, yet have the same energies of activation in both states. Single strand breaks of dry state duplex DNA occur with a half life of 24 ± 2 days and appears to occur in a mechanistic manner which could be of interest when attempting to repair such damage. In addition, base loss alone does not correlate with the extent of single strand breaks detected. Thermodynamic data can lead to the conclusion that DNA degradation in both dry and hydrated states is not a spontaneous process. It is also concluded that though the Hb molecule undergoes oxidative changes over time, these changes do not impact its ability to become a more aggressive Fenton reagent. However, the presence of Hb in the vicinity of DNA does create the opportunity for OH• induced damage to the deoxyribose sugar, and most likely the DNA bases themselves. This study also reveals that the general purpose BAL 31 nuclease commonly used in molecular genetics exhibits a hithertofore non-characterized degree of substrate specificity with respect to single-stranded DNA oligomers. Specifically, BAL 31 nuclease activity was found to be affected by the presence of guanine in ssDNA oligomers.
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Date Issued: 2009
Identifier: CFE0002547, ucf:47663
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0002547

Title: ASSESSMENT AND IN VITRO REPAIR OF DAMAGED DNA TEMPLATES FROM FORENSIC STAINS.
Creator: Hall, Ashley, Ballantyne, Jack, University of Central Florida
Abstract / Description: DNA extracted from biological stains is often intractable to analysis. This may due to a number of factors including a low copy number (LCN) of starting molecules, the presence of soluble inhibitors or damaged DNA templates. Remedies may be available to the forensic scientist to deal with LCN templates and soluble inhibitors but none presently exist for damaged DNA. In fact, only recently has the biochemical nature, the extent of DNA damage in physiological stains and the point at which the...
Show moreDNA extracted from biological stains is often intractable to analysis. This may due to a number of factors including a low copy number (LCN) of starting molecules, the presence of soluble inhibitors or damaged DNA templates. Remedies may be available to the forensic scientist to deal with LCN templates and soluble inhibitors but none presently exist for damaged DNA. In fact, only recently has the biochemical nature, the extent of DNA damage in physiological stains and the point at which the damage inflicted upon a particular sample precludes the ability to obtain a genetic profile for purposes of identification been examined. The primary aims of this work were first to ascertain the types of DNA damage encountered in forensically relevant stains, correlating the occurrence this damage with the partial or total loss of a genotype, and then to attempt the repair of the damage by means of in vitro DNA repair systems. The initial focus of the work was the detection of damage caused by exogenous, environmental sources, primarily UV irradiation, but also factors such as heat, humidity and microorganism growth. Results showed that the primary causes of the damage that resulted in profile loss were strand breaks, both single and double stranded, as well as modifications to the DNA structure that inhibited its amplification. Armed with this knowledge, the next focus was the repair of the damage by means of in vitro DNA systems. Efforts have been concentrated on single strand break/gap repair and translesion synthesis assays. By modifying the assays and employing various combinations of the systems, a genetic signature has been recovered from previously intractable samples. Additionally, the effects that various storage conditions have on the DNA in physiological stains stored in a laboratory were examined. The optimal long term storage conditions for biological evidence has been a matter of debate in the forensic community for some time. But, no comprehensive study had previously been undertaken to describe the effects of dehydration and temperature on degradation and the ability to obtain a genetic profile on bloodstains kept in different types of storage media at a range of temperatures. To examine this, bloodstains were either allowed to dry overnight or placed in the storage medium while still wet and were stored at room temperature, 4oC or 30oC for up to four years. Results showed that specimens dehydrated prior to storage were very stable, and these bloodstains showed no degradation or loss of a genetic profile for up to four years.
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Date Issued: 2005
Identifier: CFE0000878, ucf:46647
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0000878

Title: A TRIPARTILE BIOSENSOR FOR REAL-TIME SNPS DETECTION IN DNA HAIRPIN MOTIF.
Creator: Nguyen, Camha, Kolpashchikov, Dmitry, University of Central Florida
Abstract / Description: The hybridization between two complementary strands of nucleic acid is the basis for a number of applications in DNA and RNA analysis, including in vivo RNA monitoring, microarrays, SNPs detection, and so on. The short oligonucleotide probes form Watson/Crick base pairs (A-T and G-C) with the analyzed nucleic acid. Molecular beacon (MB) probe is one of the most advantageous tools for nucleic acid analysis in real-time. A traditional MB probe consists of a DNA strand folded in hairpin motif...
Show moreThe hybridization between two complementary strands of nucleic acid is the basis for a number of applications in DNA and RNA analysis, including in vivo RNA monitoring, microarrays, SNPs detection, and so on. The short oligonucleotide probes form Watson/Crick base pairs (A-T and G-C) with the analyzed nucleic acid. Molecular beacon (MB) probe is one of the most advantageous tools for nucleic acid analysis in real-time. A traditional MB probe consists of a DNA strand folded in hairpin motif with a fluorophore attached to the 5'end and a quencher attached to the 3' end. The loop segment is complementary to the analytes. Upon hybridization to a complementary single-stranded nucleic acid, MB probe switches to the elongated conformation, which separates the fluorophore from the quencher, resulting in high fluorescence signal. However, DNA or RNA folded in hairpin motifs are difficult to analyze by a conventional MB probes. Inefficient formation of the duplex between the secondary analyte and the MB probe results in low or undetectable fluorescent signal. In this project, we developed a tripartite probe consisting of one MB probe and two adaptor strands to genotype single nucleotide polymorphism (SNPs) in DNA hairpin motifs in real-time fluorescent assays. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to an MB probe. One adaptor strand hybridizes to the analyte and unwinds its secondary structure, and the other strand forms stable complex only with the fully complementary analyte sequence. The tri-component probe promises to simplify nucleic acid analysis at ambient temperatures in such application as in vivo RNA monitoring and isothermal detection of specific DNA/RNA targets.
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Date Issued: 2011
Identifier: CFH0003768, ucf:44722
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0003768

Title: A major double strand repair pathway and cancer-associated circulating proteins are effecters of epigenetic revision.
Creator: Allen, Brittany, Masternak, Michal, Khaled, Annette, Zhao, Jihe, Muller, Mark, Siddiqi, Shadab, University of Central Florida
Abstract / Description: DNA methylation is a vital epigenetic process that acts as a major control mechanism for gene expression. In addition to its essential role in many normal cellular processes, it is also implicated in a wide variety of disease states and processes including cancer. Along with genetic mutations, aberrant DNA methylation patterns, specifically the inappropriate DNA methylation or demethylation of CpG residues, may activate oncogenes or suppress tumor suppressor genes, respectively. These changes...
Show moreDNA methylation is a vital epigenetic process that acts as a major control mechanism for gene expression. In addition to its essential role in many normal cellular processes, it is also implicated in a wide variety of disease states and processes including cancer. Along with genetic mutations, aberrant DNA methylation patterns, specifically the inappropriate DNA methylation or demethylation of CpG residues, may activate oncogenes or suppress tumor suppressor genes, respectively. These changes can generate or facilitate the progression of tumorigenesis and tend to accumulate throughout the development of cancer. Although they play such a major role in cancer and in other diseases, it remains unclear what causes these epigenetic revisions to occur. This dissertation will focus on uncovering mechanisms that are sources of epigenetic revision, specifically as they relate to cancer. Due to rapid cell division and increased DNA damage, cells are increasingly dependent on DNA repair as they continue on a path of tumorigenic progression. We hypothesize that DNA repair, specifically the repair of DNA double strand breaks (DSB) by Non-Homologous End Joining (NHEJ) may play a role in inappropriate epigenetic revision. Using a GFP reporter system inserted into the genome of HeLa cells, we are able to induce targeted DNA damage that enables the cells, after successfully undergoing NHEJ repair, to express WT GFP. These GFP+ cells were segregated into two expression classes, one with robust expression (Bright) and the other with reduced expression (Dim). Using a DNA hypomethylating drug (AzadC) we were able to demonstrate that the different GFP expression levels was due to differential methylation statuses of CpGs in regions on either side of the break site. Deep sequencing analysis of this area in sorted Bright and Dim populations revealed a collection of different epi-alleles that display patterns of DNA methylation following repair by NHEJ. These patterns differ between Bright and Dim cells which are hypo- and hypermethylated, respectively, and between the post-repair populations and the original, uncut cells. These data suggest that NHEJ repair facilitates a rewrite of the methylation landscape in repaired genes, elucidating one potential source for the altered methylation patterns seen in cancer cells.The Dim cells generated during this study are known to have a hypermethylated GFP gene that is correlated with reduced expression, allowing it to be used as a screening tool for hypomethylating agents. We used this tool to screen the blood serum of patients with head and neck squamous cell carcinoma (HNSCC). We found that the serum from HNSCC patients, but not from healthy individuals, contains some factor that causes hypomethylation in exposed cells. Further, we were able to identify this factor as a protein capable of effecting changes in DNA methylation, gene expression, and miRNA levels in the treated Dim cells. The novel concept presented in this study has immense implications on the study of cancer progression as it evidences circulating proteins, presumably released by cancer cells, which are able to effect gene expression in cells that are distal to the location of the cancer. Further, the fact that these proteins are in circulation makes them a potential target for use in diagnostics. Changes in DNA methylation play a major role in the development of cancer and understanding the mechanisms by which this occurs could provide new therapeutic targets for preventing this process from contributing to tumorigenesis. This dissertation presents potential sources of epigenetic revision in cancer and thus provides answers to a major question that has yet to be answered in the area of cancer research.
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Date Issued: 2017
Identifier: CFE0006555, ucf:51333
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006555

Title: LITERATURE REVIEW ON THE USE OF NUCLEIC ACID-BASED LOGIC GATES FOR THE DETECTION OF HUMAN DISEASES.
Creator: Blanco Martinez, Enrique J, Kolpashchikov, Dmitry, University of Central Florida
Abstract / Description: Conventional methods for diagnosis of human disease are, at times, limited in different regards including time requirement, either experimental or data processing, sensitivity, and selectivity. It is then that a Point of Care Criteria, which considers the true utility and usefulness of the device, is employed to propose new diagnostic devices capable of overcoming the aforementioned shortcomings of conventional tools. Nucleic acid, characterized for its predictable base-pairing nature, is...
Show moreConventional methods for diagnosis of human disease are, at times, limited in different regards including time requirement, either experimental or data processing, sensitivity, and selectivity. It is then that a Point of Care Criteria, which considers the true utility and usefulness of the device, is employed to propose new diagnostic devices capable of overcoming the aforementioned shortcomings of conventional tools. Nucleic acid, characterized for its predictable base-pairing nature, is considered to be a highly-selective, yet greatly modifiable device. Its behavior is then described through Boolean Logic, where "true" or "false" outputs are mathematically described as "1" and "0", respectively. This mathematical approach is then referred to as Logic Gates, where outputs can be predicted based on satisfied environmental conditions. The mechanisms, capable of exhibiting Logic Gate behavior, are described.
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Date Issued: 2017
Identifier: CFH2000256, ucf:46006
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000256

Title: APTAMERIC SENSORS: IN VITRO SELECTION OF DNA THAT BINDS BROMOCRESOL PURPLE.
Creator: Miller, Derek B, Kolpshchikov, Dmitry, University of Central Florida
Abstract / Description: Aptamers being used as sensors is an emerging field that has capabilities of being tomorrow's diagnostic tools. As aptameric sensors have become more popular, their visualization systems have been limited. The majority of today's aptameric sensors require expensive machinery such as a fluorometer in order to visualize results. We propose a system that will cut the need for instrumentation and be detected via the naked eye. With the selection of an aptamer to bind the pH indicating dye...
Show moreAptamers being used as sensors is an emerging field that has capabilities of being tomorrow's diagnostic tools. As aptameric sensors have become more popular, their visualization systems have been limited. The majority of today's aptameric sensors require expensive machinery such as a fluorometer in order to visualize results. We propose a system that will cut the need for instrumentation and be detected via the naked eye. With the selection of an aptamer to bind the pH indicating dye bromocresol purple (BCP) this may be achieved. When rendered active, the binding towards BCP will facilitate a color change from yellow to purple or vice versa. Previous studies have shown albumin contains the ability to facilitate this role and we now intend to use a DNA aptamer to achieve this as well. The BCP aptamer has the potential to serve as a signaling domain to any already selected aptamer thus making it a universal tool for both research and diagnostic measures. We have found that an alternative structure-switching systematic evolution of ligands by exponential enrichment (SELEX) method which left the dye unaltered was not sufficient for selecting an aptamer. We believe that a classical SELEX will enable us to select an aptamer that may be used to accomplish this role as a universal visual detector.
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Date Issued: 2016
Identifier: CFH2000112, ucf:45951
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000112

Title: ANALYSIS OF MITOCHONDRIAL DNA CODING REGION SNPS BY PYROSEQUENCING.
Creator: Parker, Kyle, Ballantyne, Jack, University of Central Florida
Abstract / Description: To date, the use of mitochondrial DNA in forensic analysis has relied on the presence of variations in the control region to differentiate between samples. One problem that this analysis has shown is the occurrence of common Haplogroup H haplotypes or identical sequences. Thus, there is a need to enhance the distinguishing power of this type of analysis. One option has been to investigate the mitochondrial coding region for polymorphisms that could differentiate between samples with identical...
Show moreTo date, the use of mitochondrial DNA in forensic analysis has relied on the presence of variations in the control region to differentiate between samples. One problem that this analysis has shown is the occurrence of common Haplogroup H haplotypes or identical sequences. Thus, there is a need to enhance the distinguishing power of this type of analysis. One option has been to investigate the mitochondrial coding region for polymorphisms that could differentiate between samples with identical control region haplotypes. The goal of this study has been to identify polymorphic coding region sites for development in a Pyrosequencing assay that would effectively enhance the discriminatory power of mitochondrial DNA analysis. With this goal in mind, five duplexes have been successfully developed and tested, utilizing the ten polymorphic sites that had been selected, with most sites being specific to Caucasians. Validation studies were performed to test the durability of the assay. The specificity of the assay to primate and non-primate species was determined to be limited to primate species only. Sample variations, including mixtures, dilutions and environmental exposure, were utilized to assess the sensitivity of the Pyrosequencing method. It was found that a minimum initial DNA input of 10fg was necessary for reliable results. The Pyrosequencing assay was able to detect mixtures at a 1:1 ratio and environmental samples exposed to the elements from up to 1 week for blood and 6 weeks for semen. Samples designed to simulate typical casework materials were analyzed and found to provide for consistent results, including trace fingerprints and digested hair shafts. These validation results provide the conclusion that this assay is suitable for use in forensic casework and demonstrate that the mitochondrial coding region provides a viable alternative to hypervariable region analysis.
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Date Issued: 2007
Identifier: CFE0001562, ucf:47132
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0001562

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

Title: ANALYSIS OF THE REPAIR OF TOPOISOMERASE II DNA DAMAGE.
Creator: Goldstein, Eric, Muller, Mark, University of Central Florida
Abstract / Description: A large number of anti-cancer chemotherapeutics target DNA topoisomerases. Etoposide is a specific topoisomerase II poison which causes reversible double strand DNA breaks. The focus of this project is to analyze the repair of DNA damage induced by etoposide.. Double strand DNA break repair is mediated by through either non-homologous end joining (NHEJ) or homologous recombination. NHEJ repairs through direct ligation of a double stranded break while homologous recombination utilizes a...
Show moreA large number of anti-cancer chemotherapeutics target DNA topoisomerases. Etoposide is a specific topoisomerase II poison which causes reversible double strand DNA breaks. The focus of this project is to analyze the repair of DNA damage induced by etoposide.. Double strand DNA break repair is mediated by through either non-homologous end joining (NHEJ) or homologous recombination. NHEJ repairs through direct ligation of a double stranded break while homologous recombination utilizes a homologous template to recover the wild type sequence. A reporter cassette, RYDR-GFP, has been stably integrated into HeLa cells. This reporter contains an ultra-high affinity topoisomerase II cleavage site (RY) placed in the middle of a mutant GFP sequence. Flanking this sequence is a corresponding stretch of wild type GFP that is used as template to repair the break and restore gene function yielding GFP positive cells. Titrations with etoposide have shown that a logarithmic increase in drug concentration yields a corresponding increase in repair through homologous recombination (HR). This result demonstrates that topoisomerase II mediated damage is efficiently repaired by the process of HR. To examine NHEJ repair, a doxycycline inducible, stably integrated NHEJ HeLa cell reporter cassette was also evaluated. The data indicates that repair of topoisomerase II mediated DNA damage occurs more efficiently through the HR pathway. Collectively, the data suggests that tumor cells proficient in HR repair may effectively elude treatment by topoisomerase II targeting drugs.
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Date Issued: 2011
Identifier: CFH0003785, ucf:44767
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0003785

Title: GOLD (III) MACROCYCLES ARE DNA INTERCALATORS THAT INHIBIT TOPOISOMERASE I AND II.
Creator: Fagenson, Alexander, Muller, Mark, University of Central Florida
Abstract / Description: Human Topoisomerase IB (TOP1) and Topoisomerase IIα (TOP2α) are essential nuclear enzymes that control DNA topology during DNA replication, gene transcription and cell division. These enzymes carry out their catalytic function by making transient single-strand (type I) or double-strand (type II) breaks in the DNA. In vivo, these complexes are short-lived but can be exploited by anti-cancer drugs to mechanistically kill cancer cells. Two general classes of compounds can kill cancer cells...
Show moreHuman Topoisomerase IB (TOP1) and Topoisomerase IIα (TOP2α) are essential nuclear enzymes that control DNA topology during DNA replication, gene transcription and cell division. These enzymes carry out their catalytic function by making transient single-strand (type I) or double-strand (type II) breaks in the DNA. In vivo, these complexes are short-lived but can be exploited by anti-cancer drugs to mechanistically kill cancer cells. Two general classes of compounds can kill cancer cells through a topo-targeted mechanism. Interfacial Poisons (IFPs) act at the enzyme-DNA interface to inhibit the religation reaction, resulting in the accumulation of DNA double-stand breaks (DSBs) in the genomic setting. Catalytic Inhibitor Compounds (CICs) act by interfering with other steps of the catalytic cycles such as DNA/protein binding or the cleavage reaction. In this work we identify new Au3+ macrocyclic gold complexes that act as CICs of both TOP1 and TOP2α. The complexes exhibit square planar geometry with an aromatic system that allows for DNA intercalation with binding affinities in the low micromolar range. A cytotoxicity screen across 60 human cancer cell lines performed by the National Cancer Institute (NCI, USA) reveals significant anti-tumor potential. Our lead compound (butyl gold(III) macrocycle, cmpd 3.) is currently undergoing further studies in animal models at the NCI. In vitro assays with purified DNA and enzyme reveal the Au3+ ion to be the quintessential switch that allows for DNA intercalation and subsequent inhibition of TOP1 and TOP2α.
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Date Issued: 2012
Identifier: CFH0004161, ucf:44823
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004161

Title: Conformations and Dynamics of Semi-Flexible Polymers.
Creator: Huang, Aiqun, Bhattacharya, Aniket, Kokoouline, Viatcheslav, Tatulian, Suren, Campiglia, Andres, University of Central Florida
Abstract / Description: In this dissertation, we investigate the conformations, transverse fluctuations and dynamics of two-dimensional (2D) semi-flexible polymers both in the bulk and under channel confinement. We present unified scaling relations in regard to various quantities of interest for a broad range of combinations of chain length and chain stiffness using Langevin dynamics simulation. We also present a three-dimensional (3D) heterogeneous semi-flexible chain model for a double stranded DNA (dsDNA). Our...
Show moreIn this dissertation, we investigate the conformations, transverse fluctuations and dynamics of two-dimensional (2D) semi-flexible polymers both in the bulk and under channel confinement. We present unified scaling relations in regard to various quantities of interest for a broad range of combinations of chain length and chain stiffness using Langevin dynamics simulation. We also present a three-dimensional (3D) heterogeneous semi-flexible chain model for a double stranded DNA (dsDNA). Our model not only confirms the established findings for homogeneous dsDNA, but also predicts new physical phenomenon for heterogeneous dsDNA. The problems studied in this dissertation are relevant to analysis of the conformations and dynamics of biopolymers (such as DNA) in living organisms, and also offer insights for developing devices which operate on the single-molecule level.In particular, we present a unified description for the dynamics of building-blocks (monomers) of a semi-flexible chain. We consider the full range of flexibility from the case where the chain is fully flexible (no stiffness at all) to the case where the chain behaves like a rod (infinite stiffness). Our theory predicts qualitatively different sub-diffusive regimes for the monomer dynamics originating from the chain stiffness by studying the mean square displacement (MSD) of the monomers before the chain dynamics become purely diffusive.For the conformations in the bulk, we present results confirmed and agreed by two completely different models of semi-flexible polymers, with one of which is the bead-spring model (studied by Langevin dynamics) in the continuum space, the other (studied by Monte Carlo) is a self-avoiding walk chain on the square lattice, where only discrete bond angles are possible. We point out the universal features of chain conformations and fluctuations which are independent of the models.For the conformations under channel confinement, we discover qualitatively different conformations and dynamics of the chain as a function of the channel width and chain stiffness, and show how globule like shapes ((")de Gennes blobs(")) for more flexible chains continuously go over to shapes in the form of deflections from the wall ((")Odijk limit(")) for more stiff chains. We provide theoretical arguments how these regimes occur and interpolate among each other as one varies different parameters of the model. We also demonstrate the effect of physical dimensions (either 2D or 3D) on these regimes and argue that since in 2D the excluded volume (EV) effect is more severe compared to 3D, certain regimes do not exist in 2D.Finally, we study a model of a dsDNA , where both base-pairing and base-stacking interactions are accounted for albeit at a low computational cost compared to the other existing models. Our model correctly recovers the stiffness for dsDNA and ssDNA at different temperatures. Under most conditions of interest, a dsDNA can locally denature and form bubbles due to thermal fluctuations. At a critical temperature, a dsDNA undergoes a phase transition, in which the two strands of dsDNA completely melt to two single strands (two ssDNA). By considering EV interactions and calculating the bubble size distribution, recent studies have shown that this denaturation process is a first order transition. We show that for a homogeneous dsDNA made of only AT or GC pairs, our simulation results agree with the previous conclusion of first order transition, however, for sequences of periodic AT and GC regions, when the periodic size is relatively large compared to the sequence length, we show that the bubble size distribution exhibits peaks expressing the sequence pattern, and more importantly, the denaturation is no longer a first order transition.All these studies reported in the dissertation are relevant to the physics of living systems.
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Date Issued: 2016
Identifier: CFE0006464, ucf:51429
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006464

Title: Methodological Improvements in the mRNA Profiling Assays for Incorporation into DNA Casework Workflows.
Creator: Volk, Paris, Ballantyne, John, Gerasimova, Yulia, Baudelet, Matthieu, University of Central Florida
Abstract / Description: Currently, DNA profiling is the gold standard to identify an individual. However, determining body fluid origin is important in criminal investigations, offering additional information surrounding the circumstances of a crime. However, crime labs can only definitively identify blood and semen and presumptively saliva using techniques that consume time and sample and do not simultaneously identify all forensically relevant body fluids. This causes many crime labs to want to bypass body fluid...
Show moreCurrently, DNA profiling is the gold standard to identify an individual. However, determining body fluid origin is important in criminal investigations, offering additional information surrounding the circumstances of a crime. However, crime labs can only definitively identify blood and semen and presumptively saliva using techniques that consume time and sample and do not simultaneously identify all forensically relevant body fluids. This causes many crime labs to want to bypass body fluid identification altogether. Therefore, advances into more definitive molecular-based body fluid methods are necessary. One such technique is mRNA profiling because it provides a highly sensitive and specific approach to definitively identifying all relevant body fluids in parallel. Although advancements have been made, improvements to mRNA profiling methodologies still need to be researched such as 1) possible mRNA recovery from established DNA workflows and 2) possible integration of mRNA profiling into an upfront male DNA screening assay for triaging sexual-assault evidence likely to contain male DNA and reduce/eliminate a significant bottleneck in the standard DNA workflow of microscopic sperm identification. This study was designed to address these two issues by evaluating a novel way to recover RNA, for body fluid identification, from the waste fractions of a PrepFiler(TM) DNA extraction, and from the DNA extracts directly. Next, this study aimed to provide a relatively quick molecular-based approach for screening sexual-assault evidence. It involves extraction of RNA using the Dynabeads(TM) mRNA DIRECT(TM) Kit, while saving the extraction waste fractions for downstream male-DNA quantitation and STR profiling. The RNA is then used in a rapid and sensitive 1-step combined reverse transcription-HRM assay to positively detect the presence of sperm. Both non-conventional co-extraction methods successfully addressed current body fluid identification challenges and allowed for easy integration into existing workflows when single sourced, mixture and mock casework samples were analyzed.
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Date Issued: 2019
Identifier: CFE0007551, ucf:52627
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007551

Title: IN VITRO SELECTION OF DNA APTAMERS AGAINST PROSTATE CANCER PEPTIDE BIOMARKERS.
Creator: Kuguoglu, Elif, Kolpashchikov, Dmitry, University of Central Florida
Abstract / Description: This project is aimed toward finding DNA aptamers against prostate cancer peptide antigens. DNA aptamers can function to find and indicate the presence of certain molecules in a specimen. These aptamers will be obtained through the process of evolutionary selection, a specific process called SELEX which stands for Systemic Evolution of Ligands by Experimental Enrichment. By conducting several rounds of SELEX, a DNA aptamer will be selected to bind to a known peptide antigen. A biotinylated...
Show moreThis project is aimed toward finding DNA aptamers against prostate cancer peptide antigens. DNA aptamers can function to find and indicate the presence of certain molecules in a specimen. These aptamers will be obtained through the process of evolutionary selection, a specific process called SELEX which stands for Systemic Evolution of Ligands by Experimental Enrichment. By conducting several rounds of SELEX, a DNA aptamer will be selected to bind to a known peptide antigen. A biotinylated column will be utilized to stabilize a random library of DNA aptamers, and those peptides that bind to certain aptamers will cause a conformational change leading to the elution of those specific DNA aptamers. This SELEX process will be conducted again on the eluted aptamers to further select for strong binding DNA aptamers. The DNA aptamers that are obtained can further on be sequenced or used for prostate cancer research studies. Another possible usage of aptamers is to diagnose and determine the stage of various different cancer types. Our prediction is that this research will produce a DNA aptamer that will bind to a specific prostate cancer peptide antigen.
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Date Issued: 2014
Identifier: CFH0004671, ucf:45294
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004671

Title: THE USE OF PYROSEQUENCING FORTHE ANALYSIS OF Y CHROMOSOME SINGLE NUCLEOTIDE POLYMORPHISMS.
Creator: Fletcher, Jeremy Charles, Ballantyne, Jack, University of Central Florida
Abstract / Description: The potential value of the Y chromosome for forensic applications has been recognized for some time with the current work dedicated to Short Tandem Repeat analysis and Single Nucleotide Polymorphism (SNP) discovery. This study examined the ability of two different SNP analysis methods to determine if they could be utilized in forensic applications and ultimately be developed into an established system for Y chromosome SNP analysis. This study examined two principle SNP analysis systems:...
Show moreThe potential value of the Y chromosome for forensic applications has been recognized for some time with the current work dedicated to Short Tandem Repeat analysis and Single Nucleotide Polymorphism (SNP) discovery. This study examined the ability of two different SNP analysis methods to determine if they could be utilized in forensic applications and ultimately be developed into an established system for Y chromosome SNP analysis. This study examined two principle SNP analysis systems: single base extension and Pyrosequencing. Pyrosequencing was determined to be superior to single base extension, due to the wealth of information provided with sequencing and the flexibility of designing primers for analysis. Using Pyrosequencing, 50 Y chromosome loci were examined and the minimum loci required for maximum diversity for the development of a Y chromosome SNP analysis system were chosen. Thirteen loci were selected based on their ability to discriminate 60 different individuals from three different racial groups into 15 different haplogroups. The Y chromosome SNP analysis system developed utilized nested PCR for the amplification of all 13 loci. Then they were sequenced as groups, ranging from one to three loci, in a single reaction. The Y chromosome SNP analysis system developed here has the potential for forensic application since it has shown to be successful in the analysis of blood, buccal swabs, semen, and saliva, works with as little as 5 pg of starting DNA material, and will amplify only male DNA in the presence of male/female mixtures in which the female portion of the sample overwhelmed the male portion 30,000 to 1.
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Date Issued: 2004
Identifier: CFE0000071, ucf:46089
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0000071

DNA (10)	+ -
Forensic Science (4)	+ -
Biosensors (2)	+ -
DNA damage (2)	+ -
Pyrosequencing (2)	+ -

RNA (2)	+ -
SELEX (2)	+ -
SNP (2)	+ -
forensic (2)	+ -
phylogeography (2)	+ -
4-way junction (1)	+ -
Anti cancer compounds (1)	+ -
Aptamer (1)	+ -
BAL 31 nuclease (1)	+ -
Biological (1)	+ -
Body Fluid (1)	+ -
Body Fluid Identification (1)	+ -
Body fluid identification (1)	+ -
Brazil (1)	+ -
Bromocresol Purple (1)	+ -
Brownian Dynamics (1)	+ -
Caribbean (1)	+ -
Cell Separation (1)	+ -
Chemotherapy (1)	+ -
Clumps (1)	+ -
Co-extraction (1)	+ -
Coding Region (1)	+ -
Colorimetric Assay (1)	+ -
DNA Analysis (1)	+ -
DNA Motifs (1)	+ -

ETD Collection (22)	+ -
Honors Collection (7)	+ -

University of Central Florida (29)	+ -
Kolpashchikov, Dmitry (8)	+ -
Ballantyne, Jack (7)	+ -
Ballantyne, John (5)	+ -
Muller, Mark (5)	+ -

Hoffman, Eric (2)	+ -
Khaled, Annette (2)	+ -
Zhao, Jihe (2)	+ -
Allen, Brittany (1)	+ -
Baudelet, Matthieu (1)	+ -
Bhattacharya, Aniket (1)	+ -
Blanco Martinez, Enrique J (1)	+ -
Bridge, Candice (1)	+ -
Calloway, Richard (1)	+ -
Campiglia, Andres (1)	+ -
Chai, Xinqing (1)	+ -
Chakrabarti, Debopam (1)	+ -
Chumbimuni Torres, Karin (1)	+ -
Cook, Geoffrey (1)	+ -
Copik, Alicja (1)	+ -
Cox, Amanda (1)	+ -
Cyril Sagayaraj, Vidusha (1)	+ -
Ding, Jun (1)	+ -
Doonan, Terry (1)	+ -
Fagenson, Alexander (1)	+ -
Farash, Katherine (1)	+ -
Ferreira, Carlos (1)	+ -
Fletcher, Jeremy Charles (1)	+ -
Fox, Candace (1)	+ -
Gerasimova, Yulia (1)	+ -

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