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- Title
- SPLIT DEOXYRIBOZYME PROBE FOR EFFICIENT DETECTION OF HIGHLY STRUCTURED RNA TARGETS.
- Creator
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Solarez, Sheila Raquel, Gerasimova, Yulia, De Bekker, Charissa, University of Central Florida
- Abstract / Description
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Transfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information and as regulators for gene expression; uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell. Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis.[1] tRNAs have a highly stable structure that can present a...
Show moreTransfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information and as regulators for gene expression; uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell. Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis.[1] tRNAs have a highly stable structure that can present a challenge for their detection using conventional techniques.[2] To enable signal amplification and lower detection limits, a split probe - split deoxyribozyme (sDz or BiDz) probe, which uses a double-labeled fluorogenic substrate as a reporter - has been introduced. In this project we developed an assay based on sDz probe to detect yeast tRNA[Phe] as a proof-of-principle highly structured target. An sDz probe was designed specific to tRNA[phe] that could efficiently unwind stable secondary and tertiary structure of the target RNA thereby providing an efficient tool for tRNA detection.[3] The efficiency of the developed sDz probe was compared with a currently used state-of-the-art hybridization probe - molecular beacon probe. The results obtained in the project further demonstrate the power of sDz probes for the detection of highly structured RNA analytes. The split probes show signal amplification capabilities in detection of structured analytes, which will benefit diagnostics, fundamental molecular biology research and therapeutic fields.
Show less - Date Issued
- 2018
- Identifier
- CFH2000311, ucf:45728
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000311
- Title
- SPLIT PROBE DETECTION OF THE INFLUENZA A VIRUS FOR IMPROVED DIAGNOSTICS IN A POINT OF CARE SYSTEM.
- Creator
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Yishay, Tamar, Gerasimova, Yulia, Harper, James, University of Central Florida
- Abstract / Description
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A group of Influenza viruses, RNA containing viruses of the Orthomyxoviridae family, consists of Influenza virus types A-D and has been known to cause the Flu, a respiratory illness associated with numerous detrimental symptoms that can lead to death. Influenza A virus (IAV) is constantly changing and is capable of causing pandemics. Currently used diagnostic methods include virus culturing, immunoassays including rapid influenza detection tests (RIDTs), and molecular assays including those...
Show moreA group of Influenza viruses, RNA containing viruses of the Orthomyxoviridae family, consists of Influenza virus types A-D and has been known to cause the Flu, a respiratory illness associated with numerous detrimental symptoms that can lead to death. Influenza A virus (IAV) is constantly changing and is capable of causing pandemics. Currently used diagnostic methods include virus culturing, immunoassays including rapid influenza detection tests (RIDTs), and molecular assays including those based on RT-PCR. Most of the methods can be only performed in the certified diagnostic laboratories equipped with sophisticated instrumentation and/or special biosafety facilities. The results using these methods are not available on a timely basis. RIDTs provide response within 15 minutes but are unable to differentiate between the IAV subtypes. New diagnostic technique, which allows reliable detection of the influenza virus infection and virus genotyping at point-of-care setting, are needed to prevent the spread of the virus and the occurrence of a pandemic. In this project, we propose to use split G-quadruplex (G4) peroxidase probes targeting a fragment of the IAV genome amplified using an isothermal RNA amplification reaction for the detection of IAV infection and virus genotyping. The probes selectively report the virus RNA target with a color change, which can be read by the naked eye. They are capable of differentiating the targets containing as little as a single-nucleotide variation in their sequences. This study aims to optimize the probes, test their selectivity, and calculate the detection limit.
Show less - Date Issued
- 2019
- Identifier
- CFH2000533, ucf:45639
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000533
- Title
- Methodological Improvements in the mRNA Profiling Assays for Incorporation into DNA Casework Workflows.
- Creator
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Volk, Paris, Ballantyne, John, Gerasimova, Yulia, Baudelet, Matthieu, University of Central Florida
- Abstract / Description
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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.
Show less - Date Issued
- 2019
- Identifier
- CFE0007551, ucf:52627
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007551