Matching Items (4)

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Single cell RT-qPCR based ocean environmental sensing device development

Description

This thesis research focuses on developing a single-cell gene expression analysis method for marine diatom Thalassiosira pseudonana and constructing a chip level tool to realize the single cell RT-qPCR analysis.

This thesis research focuses on developing a single-cell gene expression analysis method for marine diatom Thalassiosira pseudonana and constructing a chip level tool to realize the single cell RT-qPCR analysis. This chip will serve as a conceptual foundation for future deployable ocean monitoring systems. T. pseudonana, which is a common surface water microorganism, was detected in the deep ocean as confirmed by phylogenetic and microbial community functional studies. Six-fold copy number differences between 23S rRNA and 23S rDNA were observed by RT-qPCR, demonstrating the moderate functional activity of detected photosynthetic microbes in the deep ocean including T. pseudonana. Because of the ubiquity of T. pseudonana, it is a good candidate for an early warning system for ocean environmental perturbation monitoring. This early warning system will depend on identifying outlier gene expression at the single-cell level. An early warning system based on single-cell analysis is expected to detect environmental perturbations earlier than population level analysis which can only be observed after a whole community has reacted. Preliminary work using tube-based, two-step RT-qPCR revealed for the first time, gene expression heterogeneity of T. pseudonana under different nutrient conditions. Heterogeneity was revealed by different gene expression activity for individual cells under the same conditions. This single cell analysis showed a skewed, lognormal distribution and helped to find outlier cells. The results indicate that the geometric average becomes more important and representative of the whole population than the arithmetic average. This is in contrast with population level analysis which is limited to arithmetic averages only and highlights the value of single cell analysis. In order to develop a deployable sensor in the ocean, a chip level device was constructed. The chip contains surface-adhering droplets, defined by hydrophilic patterning, that serve as real-time PCR reaction chambers when they are immersed in oil. The chip had demonstrated sensitivities at the single cell level for both DNA and RNA. The successful rate of these chip-based reactions was around 85%. The sensitivity of the chip was equivalent to published microfluidic devices with complicated designs and protocols, but the production process of the chip was simple and the materials were all easily accessible in conventional environmental and/or biology laboratories. On-chip tests provided heterogeneity information about the whole population and were validated by comparing with conventional tube based methods and by p-values analysis. The power of chip-based single-cell analyses were mainly between 65-90% which were acceptable and can be further increased by higher throughput devices. With this chip and single-cell analysis approaches, a new paradigm for robust early warning systems of ocean environmental perturbation is possible.

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Date Created
  • 2013

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Design, analytics and quality assurance for emerging personalized clinical diagnostics based on next-gen sequencing

Description

Major advancements in biology and medicine have been realized during recent decades, including massively parallel sequencing, which allows researchers to collect millions or billions of short reads from a DNA

Major advancements in biology and medicine have been realized during recent decades, including massively parallel sequencing, which allows researchers to collect millions or billions of short reads from a DNA or RNA sample. This capability opens the door to a renaissance in personalized medicine if effectively deployed. Three projects that address major and necessary advancements in massively parallel sequencing are included in this dissertation. The first study involves a pair of algorithms to verify patient identity based on single nucleotide polymorphisms (SNPs). In brief, we developed a method that allows de novo construction of sample relationships, e.g., which ones are from the same individuals and which are from different individuals. We also developed a method to confirm the hypothesis that a tumor came from a known individual. The second study derives an algorithm to multiplex multiple Polymerase Chain Reaction (PCR) reactions, while minimizing interference between reactions that compromise results. PCR is a powerful technique that amplifies pre-determined regions of DNA and is often used to selectively amplify DNA and RNA targets that are destined for sequencing. It is highly desirable to multiplex reactions to save on reagent and assay setup costs as well as equalize the effect of minor handling issues across gene targets. Our solution involves a binary integer program that minimizes events that are likely to cause interference between PCR reactions. The third study involves design and analysis methods required to analyze gene expression and copy number results against a reference range in a clinical setting for guiding patient treatments. Our goal is to determine which events are present in a given tumor specimen. These events may be mutation, DNA copy number or RNA expression. All three techniques are being used in major research and diagnostic projects for their intended purpose at the time of writing this manuscript. The SNP matching solution has been selected by The Cancer Genome Atlas to determine sample identity. Paradigm Diagnostics, Viomics and International Genomics Consortium utilize the PCR multiplexing technique to multiplex various types of PCR reactions on multi-million dollar projects. The reference range-based normalization method is used by Paradigm Diagnostics to analyze results from every patient.

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Date Created
  • 2014

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Mass spectrometric and molecular analyses of biological agents in environmental compartments

Description

This thesis discusses the use of mass spectrometry and polymerase chain reaction (PCR), among other methods, to detect biomarkers of microorganisms in the environment. These methods can be used to

This thesis discusses the use of mass spectrometry and polymerase chain reaction (PCR), among other methods, to detect biomarkers of microorganisms in the environment. These methods can be used to detect bacteria involved in the degradation of environmental pollutants (bioremediation) or various single-celled pathogens, including those posing potential threats as bioterrorism agents. The first chapter introduces the hurdles in detecting in diverse environmental compartments in which they could be found, a select list of single-celled pathogens representing known or potential bioterrorism agents. These hurdles take the form of substances that interfere either directly or indirectly with the detection method. In the case of mass spectrometry-based detection, many of these substances (interferences) can be removed via effective sample pretreatment. Chapters 2 through 4 highlight specific methods developed to detect bioremediation or bioterrorism agents in environmental matrices. These methods are qualitative mass spectrometry, quantitative PCR, and quantitative mass spectrometry, respectively. The targeted organisms in these methods include several bioremediation agents, e.g. Pseudomonas putida F1 and Sphingomonas wittichii RW1, and bioterrorism agents, e.g. norovirus and Cryptosporidium parvum. In Chapter 2, I identify using qualitative mass spectrometry, biomarkers for three bacterial species involved in bioremediation. In Chapter 3, I report on a new quantitative PCR method suitable for monitoring of a key gene in yet another bioremediation agent, Sphingomonas wittichii RW1; furthermore, I apply this method to track the efficacy of bioremediation in bioaugmented environmental microcosms. In Chapter 4, I report on the development of new quantitative mass spectrometry methods for two organisms, S. wittichii RW1 and Cryptosporidium parvum, and evaluate two previously published methods for their applicability to the analysis of complex environmental samples. In Chapter 5, I review state-of-the-art methods for the detection of emerging biological contaminants, specifically viruses, in environmental samples. While this summary deals exclusively with viral pathogens, the advantages and remaining challenges identified are also applicable to all single-celled organisms in environmental settings. The suggestions I make at the end of this chapter are expected to be valid not only for future needs for emerging viruses but also for bacteria, eukaryotic pathogens, and prions. In general, it is advisable to continue the trend towards quantification and to standardize methods to facilitate comparison of results between studies.

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Created

Date Created
  • 2012

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Low power, high throughput continuous flow PCR instruments for environmental applications

Description

Continuous monitoring in the adequate temporal and spatial scale is necessary for a better understanding of environmental variations. But field deployments of molecular biological analysis platforms in that scale are

Continuous monitoring in the adequate temporal and spatial scale is necessary for a better understanding of environmental variations. But field deployments of molecular biological analysis platforms in that scale are currently hindered because of issues with power, throughput and automation. Currently, such analysis is performed by the collection of large sample volumes from over a wide area and transporting them to laboratory testing facilities, which fail to provide any real-time information. This dissertation evaluates the systems currently utilized for in-situ field analyses and the issues hampering the successful deployment of such bioanalytial instruments for environmental applications. The design and development of high throughput, low power, and autonomous Polymerase Chain Reaction (PCR) instruments, amenable for portable field operations capable of providing quantitative results is presented here as part of this dissertation. A number of novel innovations have been reported here as part of this work in microfluidic design, PCR thermocycler design, optical design and systems integration. Emulsion microfluidics in conjunction with fluorinated oils and Teflon tubing have been used for the fluidic module that reduces cross-contamination eliminating the need for disposable components or constant cleaning. A cylindrical heater has been designed with the tubing wrapped around fixed temperature zones enabling continuous operation. Fluorescence excitation and detection have been achieved by using a light emitting diode (LED) as the excitation source and a photomultiplier tube (PMT) as the detector. Real-time quantitative PCR results were obtained by using multi-channel fluorescence excitation and detection using LED, optical fibers and a 64-channel multi-anode PMT for measuring continuous real-time fluorescence. The instrument was evaluated by comparing the results obtained with those obtained from a commercial instrument and found to be comparable. To further improve the design and enhance its field portability, this dissertation also presents a framework for the instrumentation necessary for a portable digital PCR platform to achieve higher throughputs with lower power. Both systems were designed such that it can easily couple with any upstream platform capable of providing nucleic acid for analysis using standard fluidic connections. Consequently, these instruments can be used not only in environmental applications, but portable diagnostics applications as well.

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Agent

Created

Date Created
  • 2013