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Al-hashimi, Sora. "Kvantifiering med digital droplet polymerase chain reaction av gyrA-genen med och utan mutationen S83L." Thesis, Örebro universitet, Institutionen för hälsovetenskaper, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-92989.
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Den vanligaste cancerformen hos män är prostatacancer, med 10 000 nya dödsfall årligen. Vid prostatacancerdiagnostik utförs prostata biopsi. För att minska risken för komplikationer i samband med biopsi ges i Sverige en singeldos av antibiotikapreparatet Ciprofloxacin. Andelen bakterier som är resistenta mot ciprofloxacin har ökat. För detektion av genmutationer som orsakar antibiotikaresistens kan droplet digital PCR (ddPCR) användas. Det är en metod som ger en absolut kvantifiering av antalet DNA-sekvenser. Den är baserad på vatten olja-emulsionsdroppsystem. Syftet med denna studie var att optimera och validera en digital droplet PCR för att detektera och kvantifiera mutationen S83L i gyrA genen från faecesprover, samt att jämföra digital droplet resultat från studieprover med odlingsresultat från resistensbestämning och ration mellan S83L allelen och vildtyps allelen i prover tagna före och efter biopsi. För att optimera och validera metoden användes prover tagna före och efter biopsi från nio patienter som genomgått en transrektal prostatabiopsi och fått en dos antibiotika profylax i samband med ingreppet. Den optimala annealingtemperaturen bedömdes vara 60°C och den optimala primer- och probekoncentrationen bestämdes till 1,2 µM respektive 0,4µM. Dessa koncentrationer gav lägst antal falskt positiva droppar. Den minsta detektionsnivån för S83L gyrA (EC40) var 160 kopior/ml och för vildtyps gyrA (EC108) var det 78 kopior/ml. Resultatet från valideringen visade att både vildtyps gyrA och S83L gyrA kunde detekteras och kvantifieras i rektalprover från samtliga patienter.The most common form of cancer in men is prostate cancer, with 10,000 new deaths annually. In prostate cancer diagnosis, prostate biopsy is performed. To reduce the risk of complications in connection with biopsy, a single dose of the antibiotic drug Ciprofloxacin is given in Sweden. The proportion of bacteria that are resistant to ciprofloxacin has increased. For the detection of gene mutations that cause antibiotic resistance, droplet digital PCR (ddPCR) can be used. It is a method that provides an absolute quantification of a DNA sequence in a sample. It is based on water oil emulsion drop system. The purpose of this study was to optimize and validate a digital droplet PCR to detect and quantify the S83L mutation in the gyrA gene from faecal samples and to compare digital droplet results from study samples with culture results from resistance determination and the ration between the S83L allele and the wild-type allele in samples taken before and after biopsy. To validate the method, samples taken before and after biopsy were used from nine patients who had undergone a transrectal prostate biopsy and received a dose of ciprofloxacin or trimethoprim in connection with the procedure. The optimal annealing temperature was determined to be 60 °C and the optimal primer and probe concentrations were determined to be 1.2 µM and 0.4µM, respectively. These concentrations gave the lowest number of false positive droplets. The minimum detection level for S83L gyrA (EC40) was 160 copies/ml and for wild-type gyrA (EC108) it was 78 copies/ml. The results showed that both wild-type gyrA and S83L gyrA could be detected and quantified in rectal samples from all nine patients.
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Poleti, Marcelo Lupion. "Análise morfométrica, radiográfica e molecular do processo de reparo alveolar após a terapia fotodinâmica antimicrobiana em ratos Wistar." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/25/25132/tde-03072009-105031/.
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Introdução: Com o aumento da resistência bacteriana à antibioticoterapia tornou-se necessário o desenvolvimento de técnicas antimicrobianas alternativas. Assim, na busca de novas metodologias contra microrganismos, estudos usando a Terapia Fotodinâmica (TFD) antimicrobiana tem sido realizados. Objetivo: Realizar análise morfométrica, radiográfica e molecular do processo de reparo alveolar após a TFD antimicrobiana em ratos Wistar. Material e Métodos: Foram utilizados 85 ratos, divididos nos seguintes grupos: GRUPO C: Animais com alvéolo não tratado (grupo controle); GRUPO S+L: Animais com alvéolo preenchido com soro fisiológico e tratado apenas com aplicação do Laser de baixa intensidade (660 nm), com fluência de 50 J/cm2; GRUPO ATO: Animais com alvéolo tratado apenas com aplicação tópica de azul de toluidina - O (ATO) na concentração de 100 µg/mL e GRUPO ATO+L: Animais com alvéolo tratado com aplicação tópica de azul de toluidina O (ATO) na concentração de 100 µg/mL + aplicação de Laser de baixa intensidade (660 nm), com fluência de 50 J/cm2. Os animais foram sacrificados aos 6, 15 e 28 dias pós-operatório. O mapeamento térmico foi realizado em um animal para confirmar a ausência de efeito térmico do Laser sobre o local irradiado. Foram realizadas as análises microscópicas: qualitativa e quantitativa de tecido conjuntivo, tecido ósseo, coágulo sanguíneo, vaso sanguíneo, infiltrado inflamatório e espaço vazio; radiográfica: por meio do valor de pixel e dimensão fractal e molecular: quantitativa da expressão de genes envolvidos no processo de reparo: colágeno tipo I (COL-I), fator de crescimento do endotélio vascular (VEGF), runt-related transcription factor 2 (RUNX2), osteocalcina (OCN) e fosfatase alcalina (ALP), por meio da PCR Real Time. Resultados e Conclusão: Os resultados demonstraram que a TFD antimicrobiano não atuou negativamente na evolução do processo de reparo alveolar; a análise radiográfica por meio dos valores de pixel pode ser usada como indicador para avaliar a neoformação óssea no processo de reparo alveolar em ratos; a expressão de VEGF pode ser usada como marcador molecular para a angiogênese no processo de reparo alveolar em ratos e a expressão de fosfatase alcalina pode ser usada como marcador molecular nos estágios iniciais do metabolismo ósseo no processo de reparo alveolar em ratos.Introduction: Antibiotics resistance made the development of antimicrobial alternative techniques necessary. Consequently, others alternatives such as antimicrobial photodynamic therapy (PDT) have been studied. Objective: The objectives were to perform morphometric, radiographic and molecular analyses of alveolar repair process in rats after antimicrobial photodynamic therapy. Methods: Eighty-five rats were used in this study, divided according to the following groups: C: untreated socket; S+L: socket treatment with physiologic saline solution and low intensity laser therapy (660nm - 50J/cm2); ATO: socket treatment with topic application of toluidine blue-O (100 µg/mL) and ATO+L: socket treatment with topic application of toluidine blue-O (100µg/ml) and low intensity laser therapy (660nm - 50J/cm2). The animals were sacrificed at a postoperative period of 6, 15 and 28 days. Thermal variation was carried out in an animal to confirm the absence of Laser thermal effect on irradiation area. Quantitative and qualitative microscopic analyses were performed to evaluate the connective tissue, bone tissue, blood clot, blood vessel, inflammatory infiltrate and empty space. Fractal and Pixel radiographic analysis were performed. A quantitative analysis was performed using a RealTimePCR to evaluate the genes expression involved Collagen Type I (COL-I), vascular endothelial growth factor (VEGF), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), alkaline phosphatase (ALP), in the alveolar repair process. Results and Conclusions: Based in the results, it can be concluded that antimicrobial photodynamic therapy did not act negatively in the socket bone repair evaluation. Pixel values analysis could be used as indicators to evaluate the dry socket bone neoformation. The VEGF molecular marker could be used as angiogenesis indicator to evaluate the dry socket bone neoformation as well as alkaline phosphatase as a molecular marker in the early stage of bone neoformation.
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Feugeas, Olivier. "Pcr (polymerase chain reaction) et vih." Lille 2, 1990. http://www.theses.fr/1990LIL2M264.
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Verhaegen, Monique Elise. "Novel approaches in quantitative polymerase chain reaction." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0021/MQ52489.pdf.
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Chiou, Jeffrey Tsungshuan. "A novel capillary polymerase chain reaction machine." Thesis, Massachusetts Institute of Technology, 2001. http://hdl.handle.net/1721.1/8864.
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Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2001.Includes bibliographical references (p. 254-268).
I built a novel prototype capillary polymerase chain reaction machine. The purpose was to perform a single reaction as fast as possible with a reaction volume - 100 nl. The PCR mix is in the form of a 1 /1 droplet that moves between three heat zones inside of a 1 mm I.D. capillary filled with mineral oil via pneumatic actuation. A laser beam waveguides down the capillary until it strikes the drop, at which point it scatters. The scatter is picked up by a series of photodiodes to provide position feedback. Due to the efficient heat transfer arrangement, the drop can transition between different temperature steps in -2 seconds, which includes both drop motion and temperature equilibration. It was extensively tested in both 10-cycle and 30-cycle PCR, including nearly 200 successful 30-cycle runs. The 30-cycle PCR was typically 74% (as high as 78%) efficient, and took only 23 minutes. This compares well with existing machines in the literature.
by Jeffrey Tsungshuan Chiou.
Ph.D.
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Clackson, Timothy Piers. "Antibody engineering using the polymerase chain reaction." Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316695.
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Lantz, Pär-G. "PCR-based detection of microorganisms in complex biological samples." Lund : Dept. of Applied Microbiology, Lund University, 1998. http://catalog.hathitrust.org/api/volumes/oclc/39178906.html.
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Linley, M. "The detection of polymerase inhibiting lesions using the polymerase arrest polymerase chain reaction assay." Thesis, Swansea University, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.637924.
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There is a constant need to determine the genotoxic potential of the agents to which the human population is exposed. The stringent testing of new products is legislatively controlled and dependent on the accumulation of sufficient scientific data to allow an analysis of the risk. It is important to predetermine any risks in the workplace prior to the presentation of disease and to provide factual public information on personal exposure e.g. the risks associated with UV light. Various experimental assays have been developed to assess the genotoxicity, mutagenicity and mcarcinogenicity of given physical and chemical agents. The Polymerase Arrest- Polymerase Chain Reaction (PA-PCR) assay was employed to investigate the genotoxic effects (DNA adducts, DAN strand breaks and DNA crosslinking) of various physical and chemical agents on naked isolated DNA. The assay was modified to provide two adapted methods, which increased the sensitivity of the assay to report DNA damage at significantly lowered exposure levels. The ability of the PA-PCR assay to perform as an initial screening process for genotoxic activity was assessed and determined.
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Nebbali, M. "Human gene mapping using the polymerase chain reaction." Thesis, University of Nottingham, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317395.
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Borges, Pinto Lais Izabel. "Alu-polymerase chain reaction genomic fingerprinting in neuroblastoma." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366679.
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Erill, Sagalés Ivan. "High-speed Polymerase chain reaction in CMOS-compatible chips." Doctoral thesis, Universitat Autònoma de Barcelona, 2002. http://hdl.handle.net/10803/3031.
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En la última década del siglo XX, el campo de los microsistemas para análisis total (µ-TAS) y, más concretamente, el de los DNA-chips ha adquirido una importancia preponderante en el ámbito de los microsistemas. En gran parte, el creciente interés por estos dispositivos se debe a las substanciales mejoras que prometen: análisis más rápidos, baratos y automatizados, pero también es debido a la posibilidad de implementar técnicas analíticas antes impensables (e.g. chips de hibridación). En el caso particular de los DNA-chips, se han desarrollado prototipos funcionales para PCR, LCR, electroforesis en gel, di-electroforesis, hibridación y varias combinaciones de estas técnicas, al tiempo que los chips de hibridación masiva (mayoritariamente basados en arrayers) han llegado a convertirse en un éxito comercial. Aun así, y aunque se ha llevado a cabo mucho trabajo en estos años, es necesaria todavía mucha investigación para afrontar algunos de los principales retos de los DNA-chips. En el transcurso de esta tesis doctoral, se ha llevado a cabo el desarrollo un proceso tecnológico común para la fabricación de DNA-chips multifunción (i.e. sistemas versátiles basados en PCR y electroforesis), poniendo un especial énfasis en la compatibilidad con los procesos CMOS estándar, a fin de conseguir desarrollar prototipos proto-industriales. Como demostrador de esta puesta a punto tecnológica, se han diseñado, fabricado y testado chips de PCR, y la PCR en chips ha sido optimizada con respecto a materiales de fabricación, metodologías de inserción/extracción, composición bioquímica de la mix de PCR, diferentes configuraciones de calentadores/sensores (Peltier/termopares vs. resistencias integradas) y la cinética de la reacción.
In the last decade of the twentieth century, the fields of µ-TAS and, more specifically, DNA-chips have acquired increasing importance in the microsystems arena. The main reason for this surge of interest lies in the advantages these new devices seek to bring forth: faster, cheaper and completely automated analyses, and also in the outbreak of novel analytical techniques (e.g. hybridization chips). In the particular case of DNA-chips, functional prototypes have been demonstrated for PCR, LCR, gel electrophoresis, di-electrophoresis, hybridization and various combinations of these techniques, whilst hybridization chips (mainly arrayer chips) have become a successful market application. But, even though a considerable amount of work has been carried out in these few years, much research is still required to address fundamental problems of DNA-chips.
In this doctoral work, a common-ground technological setup for the production of multifunction DNA-chips (i.e. PCR plus electrophoresis systems) has been laid down, placing strong emphasis in its compatibility with standard CMOS processes in order to produce proto-industrial prototypes. As a demonstrator of this technological setup, PCR-chips have been designed, manufactured and tested, and the chip PCR reaction has been optimized with respect to surface materials, insertion and extraction methods, biochemical mix composition, heater/sensor setups (Peltier/thermocouple vs. thin-film driven systems) and reaction kinetics.
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Aydin, Gamze. "Detection Of Genetically Modified Maize Via Polymerase Chain Reaction." Master's thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/3/12605495/index.pdf.
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In recent years, foods produced by genetic engineering technology have been on the world food market. The biosafety aspects, regulations, and labelling of these foods are still contentious issues in most countries. It is necessary to have approval for the use of GMOs in the production of food. Thus, detection and quantification of GMOs play crucial role for developing regulations on GM foods.
In this study, raw and processed maize samples were analysed for genetic modification using a DNA based detection method, the Polymerase Chain Reaction. Ten raw food and 18 processed maize food including maize flour, starch, corn flakes, maize chips were collected from different markets located in different places in Turkey. The samples were examined for the presence of genetic elements located in the majority of transgenic crops such as NOS terminator, CaMV 35S promoter, kanamycin resistance (KanR) gene, using
conventional PCR with oligonucleotide sets targeting to novel genes. Furthermore screening was conducted via Real-Time PCR assay for NOS terminator and 35S
promoter. For confirming the presence of Bt11 maize lines event specific primers were utilised. Quantification of Bt11 maize lines were performed via Real-Time PCR.
The result indicates that foreign genetic elements were found in all analysed raw material. In six out of 10 raw material, presence of Bt11 gene were identified. GMO detection was also possible for maize flour and starch, however in processed material as corn starch, corn flakes, corn chips and pop corn, transgenes were not detected.
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Ballagi-Pordány, András. "Application of polymerase chain reaction (PCR) in veterinary virology /." Uppsala : Sveriges lantbruksuniv, 1995. http://epsilon.slu.se/avh/1995/91-576-4997-9.gif.
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Woolford, Alison Jane. "Use of polymerase chain reaction for detection of mycobacteria." Thesis, University of Surrey, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308497.
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Gurram, Neil (Neil K. ). "A mathematical model of polymerase chain reaction induced stutter." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/106012.
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Thesis: M. Eng., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2016.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (page 48).
This is a thesis on understanding stutter present in capillary electropherogram readouts as this methodology forms the basis of current DNA fingerprinting. The readouts come from taking samples of various initial template masses of DNA from different individuals, applying polymerase chain reaction (PCR) to the samples, and then running the amplified copies through capillary electrophoresis to produce a readout of peak heights corresponding to alleles on various loci. The alleles correspond to the number of repeats of microsatellites that are usually two to six base pairs in length called short tandem repeats (STRs); the number of repeats of various STRs defines a person's DNA fingerprint. This process introduces artifacts in measurement. Of particular interest in this thesis is stutter, the phenomenon where amplicons with fewer or greater number of STR repeats than the true allele count are generated as an artifact of the PCR. It is of interest to understand the source and nature for this stutter distribution for small starting masses, as it has ramifications on the ability to accurately determine a match between a DNA sample and a crime scene sample. Understanding the stutter distribution in this thesis is achieved through data analysis, probabilistic modeling, and statistics. We find that a mathematical model that combines stochastic effects from PCR with fluorescent noise explains the most significant features of the observed phenomena.
by Neil Gurram.
M. Eng.
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Lewis, Monte. "Thermal cycling design alternatives for the polymerase chain reaction." College Park, Md. : University of Maryland, 2005. http://hdl.handle.net/1903/3061.
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Thesis (M.S.) -- University of Maryland, College Park, 2005.Thesis research directed by: Dept. of Mechanical Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Way, Jaw-Shiow Chu. "Specific detection of Salmonella by multiplex polymerase chain reaction." Diss., The University of Arizona, 1993. http://hdl.handle.net/10150/186207.
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This study evaluates the use of multiplex polymerase chain reaction (PCR) technology for detection of Salmonella species in pure cultures and also in environmental samples. Three sets of oligonucleotide primers were used in the PCR assay: PhoP primers specific to a 299 bp region in the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, E. coli and Citrobacter species, served as a presumptive indicator of enteric bacteria. Subsequently Hin and H-1i primers, which targeted a 236 bp region of the hin/H2 gene, and a 173 bp region of the H-1-i flagellar gene in Salmonella typhimurium respectively, were used for specific detection of salmonellae. Specificity and sensitivity of PCR amplified products were evaluated by ethidium bromide (EtBr) staining or gene probe analysis. Under optimal PCR conditions described in this study, Salmonella species can be specifically detected by these 3 primer sets. The sensitivity of detection in terms of whole cells was a 10⁻⁵ dilution (approximately 10³ cells) of boiled late log phase cultured cells (detection by EtBr) after 25 cycles of PCR and 10⁻⁸ dilution (approximately 10° cells) after a 50 cycle 'double PCR' protocol. A similar level of sensitivity was observed using a gene probe analysis (³²P end-labeling) to detect the PCR amplified products. The multiplex PCR products observed from known environmental isolates allowed identification of several isolates as Salmonella species. These results agreed with conventional analyses. The efficacy of this PCR technology for environmental applications was also evaluated by testing the primers on soil and environmental water samples. All unseeded soil samples showed no PCR amplified products when detected by EtBr staining after 25 and 50 cycles PCR. Only one pond surface water sample resulted in a positive PCR result. This water sample was also positive when tested by conventional methodologies. Seeded soil and well water samples all resulted in PCR products being observed from 25 or 50 cycles. These results indicate that the primers have the potential to specifically detect Salmonella species in environmental samples.
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Muwonge, Abubaker. "Detection Of Genetically Modified Potatoes By The Polymerase Chain Reaction." Master's thesis, METU, 2005. http://etd.lib.metu.edu.tr/upload/12605783/index.pdf.
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Quite a number of important crops have been genetically modified with genes for agronomically important traits, such as insect and viral resistance.
As the numbers of genetically modified foods continue to increase on the market, the need for rapid development of GMO detection methods is indispensable.
This study was carried out to detect if genetically modified potatoes exist on food market in Turkey. Thirty samples from different places were collected. Using a DNA based PCR method, potato samples were examined for the presence of 35S promoter, Nos terminator, neomycin phosphotransferase (nptII) genes, and synthetic cry3A gene which is the general transgene in all approved Newleaf transgenic potato lines.
The experimental design of this study was to detect Newleaf insect resistant lines. In 11 samples at least one genetic element was detected. Sample R from Ankara has shown to be belonging to Newleaf insect resistant lines. Since 35S promoter was not detected in samples M3, 14 and F1, it is proposed that they are belonging to Newleaf virus and insect resistant lines (Newleaf plus or Newleaf Y). Although Nos terminator was not detected in samples H2, Z2 and D, cry3A fragments amplified in those samples have been verified that they are from the synthetic cry3A regions of Newleaf lines.
The detected synthetic cry3A gene in GM potatoes was amplified by specific primers, which cannot amplify Bacillus thuringiensis tenebrionis natural cry3A gene. In addition, the authenticity of the synthetic cry3A PCR products were confirmed by both sequencing and restriction digestions.
Our results showed that genetically modified Newleaf potatoes exist in food market in Turkey. Further studies by accredited laboratories are strongly recommended.
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White, Adam. "Development and application of microfluidic single-cell polymerase chain reaction." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/55734.
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Methods for single-cell analysis are critical to revealing cell-to-cell variability in biological systems, such as during development or onset of disease, where the characteristics of heterogeneity and minority cell populations are obscured by population-averaged measurements. Analysis of individual cells has been limited due to challenges associated with small amounts of starting material, combined with the cost and throughput required to examine large numbers of cells. Microfluidic approaches are well suited to single-cell analysis, providing increased sensitivity, economy of scale, and automation. This thesis presents the development and application of microfluidic technology for single cell gene expression analysis. The foundational contribution of this work is an integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run. This device executes all steps of single cell processing including cell capture, cell lysis, reverse transcription, and quantitative PCR. This device is further expanded upon by integrating the single cell and nucleic acid processing capabilities with final measurement of cDNA by high-density digital PCR. The direct quantification of single molecules by digital PCR has advantages over RT-qPCR in the measurement of low abundance transcripts, as well as obviating the need for relative abundance measurements or calibration standards. This technology is demonstrated in over 5,000 individual cell measurements of mRNA, microRNA, and single nucleotide variant detection in a variety of cell types. Finally, this technology is applied to study the performance of lipid nanoparticles in delivery of RNA, and manipulation of gene expression in cells. The microfluidic integration of cell and nucleic acid processing established in this thesis permits analysis of hundreds of single cells in parallel, while improving work flow and reducing technical variation compared to samples prepared in microliter volumes. Ultimately, this advances the tools available for precisely measuring transcripts in single cells, and has application in research and clinical settings.Science, Faculty of
Graduate
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Tsang, Tsui-ying Stella, and 曾璀瑩. "Application of quantitative polymerase chain reaction in the diagnosisof thalassaemia." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B45010948.
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Gamma, Torres Rafael Enrique. "Application of polymerase chain reaction to rhinovirus detection and analysis." Thesis, University of Essex, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293589.
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Koc, Yasemin. "Optimization of continuous flow polymerase chain reaction with microfluidic reactors." Thesis, Imperial College London, 2008. http://hdl.handle.net/10044/1/8184.
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The polymerase chain reaction (PCR) is an enzyme catalyzed technique, used to amplify the number of copies of a specific region of DNA. This technique can be used to identify, with high-probability, disease-causing viruses and/or bacteria, the identity of a deceased person, or a criminal suspect. Even though PCR has had a tremendous impact in clinical diagnostics, medical sciences and forensics, the technique presents several drawbacks. For example, the costs associated with each reaction are high and the reaction is prone to contamination due to its inherent efficiency and high sensitivity. By employing microfluidic systems to perform PCR these advantages can be circumvented. This thesis addresses implementation issues that adversely affect PCR in microdevices and aims to improve the efficiency of the reaction by introducing novel materials and methods to existing protocols. Molecule-surface-interactions and temperature control/determination are the main focus within this work. Microchannels and microreactors are characterized by extremely high surface-to-volume ratios. This dictates that surfaces play a dominant role in defining the efficiency of PCR (and other synthetic processes) through increased molecule-surface interactions. In a multicomponent reaction system where the concentration of several components needs to be maintained the situation is particularly complicated. For example, inhibition of PCR is commonly observed due to polymerase adsorption on channel walls. Within this work a number of different surface treatments have been investigated with a view to minimizing adsorption effects on microfluidic channels. In addition, novel studies introducing the use of superhydrophobic coatings on microfluidic channels are presented. Specifically superhydrophobic surfaces exhibiting contact angles in excess of 1500 have been created by growing copper oxide and zinc oxide nanoneedles and silica-sol gel micropores on microfluidic channels. Such surfaces utilize additional surface roughness to promote hydrophobicity. Aqueous solutions in contact with superhydrophobic surfaces are suspended by bridging-type wetting, and therefore the fraction of the surface in contact with the aqueous layer is significantly lower than for a flat surface. An additional difficulty associated with PCR on microscale is the detennination and control of temperature. When perfonning PCR, the ability to accurately control system temperatures is especially important since both primer annealing to single-stranded DNA and the catalytic extension of this primer to form the complementary strand will only proceed in an efficient manner within relatively narrow temperature ranges. It is therefore imperative to be able to accurately monitor the temperature distributions in such microfluidic channels. In this thesis, fluorescence lifetime imaging (FLIM) is used as a novel method to directly quantify temperature within microchannel environments. The approach, which includes the use of multiphoton excitation to achieve optical sectioning, allows for high spatial and temporal resolution, operates over a wide temperature range and can be used to rapidly quantify local temperatures with high precision.
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Allmann, Michael. "Applications of the polymerase chain reaction (PCR) in food chemistry /." [S.l.] : [s.n.], 1994. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Mello, Richard. "IDENTIFICATION OF DQ ALPHA POLYMORPHISM USING THE POLYMERASE CHAIN REACTION." VCU Scholars Compass, 1991. https://scholarscompass.vcu.edu/etd/5213.
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This was a study of detection systems for DQ alpha HLA polymorphism that could be exploited for the demonstration of simulated chimerism. Polymorphic segments of DO alpha DNA were amplified by the polymerase chain reaction (PCR). Simulated chimerism was represented by a mixture of minor and major component DNA. The goal was to detect minor component DNA in the presence of major component DNA utilizing various laboratory techniques. Techniques studied included probe strip typing with the AmpliType HLA-DO Alpha test kit, allele-specific amplification, polyacrylamide gel electrophoresis, restriction enzymes, and Southern transfer combined with a peroxidase detection system.
The AmpliType HLA-DO Alpha test kit had a detection sensitivity of at least 0.2%. This is much better than the 10% detection sensitivity in non-PCR techniques. When the 3.0 DC alpha type was mixed as the minor component with undiluted 1.1 DQ alpha type, the detection sensitivity for the 3.0 DC alpha type increased to a detection level of 0.1%.
The allele-specific primers were able to specifically amplify the minor component DNA in the presence of major component DNA. Major component DNA did not amplify and thus did not compete with the minor component DNA for Taq polymerase. The allele-specific primers provided an overall detection sensitivity of 0.2%.
Background interference prevented detection of minor component bands on both polyacrylamide gels stained with ethidium bromide and on Southern blots reacted with the peroxidase detection system.
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Sonmezalp, C. Zeynep. "Detection Of Genetically Modified Insect Resistant Tomato Via Polymerase Chain Reaction." Master's thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/3/12605493/index.pdf.
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Tomato, which is one of the most important component of human diet, has been genetically modified to develop some properties like delayed ripening and insect resistance. In order to give a choice to the consumer, it is necessary to detect and label GM foods.
This study was carried out to detect genetically modified tomato samples purchased from different food markets of Turkey. PCR method was used to detect genetically modified insect resistant tomatoes. The DNAs of collected samples were isolated according to CTAB DNA extraction protocol and the amplification capacity of isolated samples were checked with patatin specific control PCR. Screening tests of tomatoes were done by targeting 35S promoter, Nos terminator and NptII kanamycin resistance gene with four primer sets. It was aimed to detect Cry1A and Cry1Ac genes with three PCR systems, in order to identify insect resistant samples.
From twenty-eight samples, twenty-two gave positive amplification signal in NptII specific PCR system and results were confirmed with sequence analysis. Additionally, we observed seventeen and ten DNA fragments with Cry1A-F/Cry1A-R and Cry1Ac-F/Cry1Ac-R primer sets respectively, it is necessary to confirm these results with DNA sequencing.
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Duman, Zeynep. "Polymerase Chain Reaction (pcr) For Detection Of Borrelia Burgdorferi Sensu Lato." Master's thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/2/12609199/index.pdf.
Full textAbstract:
The present study aimed detection of a human pathogen B. bugdorferi sensu lato species in suspected Lyme borreliosis (LB) patients in Turkey by PCR analysis and supportive serologic tests. The 152 clinical samples (140 serum and blood, 10 cerebrospinal fluid (CSF), 1 synovial fluid, 1 skin biopsy specimens) from 140 patients sent from 22 different cities of Turkey to The Spirochetal Diseases Diagnosis Laboratory of Central Veterinary Control and Research Institute were analysed. Serum samples were subjected to ELISA with a commercial kit and all of the blood, CSF, synovial fluid and skin biopsy samples were examined by PCR. In PCR analysis two primer sets targeting the ospA gene located on the plasmid and ribosomal 23S rRNA gene of B. burgdorferi sensu lato were used. The results indicated that 32,1% (45 of 140) seropositivity was detectable by ELISA. Our results support that there is a risk of acquiring LB in different regions of Turkey. Although considerable positive detections were recorded using serologic tests,none of the specimens were positive in PCR analysis. Further studies on PCR
based methods for detection of B. burgdorferi sensu lato in patients with a high clinical probability of LB apparently may require that the specimen should be taken in the early phases and before the administration of any therapeutic agent.
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Tsang, Tsui-ying Stella. "Application of quantitative polymerase chain reaction in the diagnosis of thalassaemia /." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36433895.
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Ros, Bascuñana Carlos. "Diagnostic application of the polymerase chain reaction (PCR) in veterinary microbiology /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 1997. http://epsilon.slu.se/avh/1997/91-576-5247-3.gif.
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Walker, Ken R. "Rapid detection of Listeria monocytogenes in salad by polymerase chain reaction." Auburn, Ala, 2005. http://repo.lib.auburn.edu/2005%20Summer/master's/WALKER_KEN_28.pdf.
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Phaneuf, Christopher. "Infrared laser-mediated polymerase chain reaction in a polymer microfluidic device." Diss., Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/53068.
Full textAbstract:
The ability to rapidly, sensitively, and accurately detect the presence of a pathogen is a vital capability for first responders in the assessment and treatment of scenarios such as disease outbreak and bioterrorism. Nucleic acid tests such as the polymerase chain reaction (PCR) are supplanting traditional techniques due to the improved speed, specificity, sensitivity, and simplicity. Still, amplification by PCR is often the bottleneck when processing genetic samples. Conventional PCR machines are bulky, slow, and consume large reagent volumes and an affordable, compact, efficient, easy-to-use alternative has yet to emerge. In this work, a microfluidic PCR platform was developed consisting of a low-cost, multi-chamber polymer microchip and a laser-mediated thermocycler capable of independent thermal control of each reaction chamber. Innovations in polymer microchip modeling, fabrication, and characterization yielded a low-cost solution for sample handling. A simple optical system featuring an infrared laser diode and solenoid-driven optical shutter was combined with a microfluidic temperature measurement system utilizing embedded thermocouples to achieve rapid thermocycling capable of multiplexed temperature control. We validated the instrument with sensitive amplifications of multiple viral targets simultaneously. This technology is a breakthrough in practical microfluidic PCR instrumentation, providing the foundation for a paradigm shift in low-cost, high-throughput genetic diagnostics.
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Tully, Gillian. "DNA profiling for forensic identification : evaluation of polymerase chain reaction methods." Thesis, Cardiff University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264882.
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Diss, Timothy Charles. "The polymerase chain reaction in the characterisation and diagnosis of lymphomas." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338653.
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Organji, Sameer R. A. "Detection of #Beta#-lactam resistant Streptococcus pneumoniae by polymerase chain reaction." Thesis, University of Bristol, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297824.
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Sim, Steven Poh Chuen. "An integrated chip-based device for droplet-flow polymerase chain reaction." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/56111.
Full textAbstract:
The polymerase chain reaction (PCR) is an important in-vitro technique in molecular biology for amplifying trace quantities of deoxyribonucleic acid (DNA). PCR is carried out by mixing the DNA molecules to be amplified with primers, polymerase enzymes and deoxynucleotide triphosphates (dNTPs) in a suitable buffer solution. A conventional thermal-cycler is then used to cycle the PCR mixture between multiple temperatures for denaturation, annealing and extension. Bench-top thermal cyclers have large thermal masses and use large sample volumes, leading to overly long cycling times, excessive energy and material consumption, inhomogeneity in the reaction environment, and an inability to handle large numbers of small volume aliquots. Microfluidic technologies overcome many of the limitations of bench-top thermal cyclers, providing a more controlled approach to PCR. Droplet-flow is one of the most promising microfluidic methods for carrying out PCR. The droplet-flow approach uses small water-in-oil droplets for compartmentalisation of the PCR reaction mixture, with each droplet behaving like an individual reaction chamber. By flowing the droplets over different temperatures for denaturation, annealing and extension, rapid thermal cycling can be achieved, greatly reducing the reaction time relative to bench-top thermal cyclers. The use of an oil phase to encapsulate the aqueous PCR mixture as droplets also prevents unwanted surface interactions and flow dispersion that can adversely affect the PCR yield. Here we describe an integrated microfluidic device for carrying out droplet-flow PCR. Instead of using multiple temperature zones to thermally-cycle the flowing droplets, the device used an on-chip radial temperature gradient. The droplets passed through microchannels arranged in a spoke-like geometry, causing them to pass backwards and forwards along the radial temperature gradient and so undergo the repeated thermal cycling required for PCR. The device reported here builds on an earlier plastic microfluidic PCR device (by Schaerli et al.) in which the radial temperature gradient was generated using a bulky external heater and a thermoelectric cooler, together with heat sinks and fans. In the silicon- and glass-based device reported here, integrated heaters, temperature sensors and air gaps (for passive cooling) were used to generate the temperature gradient, leading to significant miniaturisation of the device. The dimensions of the complete device assembly were 6.0 cm x 5.0 cm x 2.0 cm compared to 25.0 cm x 25.0 cm x 25.0 cm for the device by Schaerli et al. Despite the small size of the device, the achievable temperature gradient on the chip was sizeable. For instance, when the central heater was set to 92.0 °C, the temperature at the periphery was ~60.0 °C, corresponding to a temperature difference of ~32.0 °C – easily sufficient for PCR applications. Using chemical modification, the hydrophilic walls of the microchannel were rendered hydrophobic. An on-chip T-junction or flow-focusing junction was subsequently used to merge the oil and aqueous streams to generate the PCR-containing water-in-oil droplets. A PCR recipe was optimised on a bench-top thermal cycle. With this recipe, droplet-flow PCR was conducted on the PCR device by flowing the generated droplets up and down the radial temperature gradient to induce thermal cycling. Gel electrophoresis analysis of the collected droplets from the device showed the presence of the PCR product, confirming the ability of the integrated device to conduct droplet-flow PCR. By varying the central temperature of the PCR device and the flow rate of the droplets, the yield of the PCR product could be tuned. By serially diluting the concentration of the DNA molecules, it was found that the PCR device was able to amplify concentrations as low as 0.01 pM to a level detectable by gel electrophoresis. When coupled to a laser-induced fluorescence detection system, the emission from the PCR mixture in the water-in-oil droplets could be successfully detected for each PCR cycle. The increase in the fluorescence over successive PCR cycles once again verified the feasibility of carrying out droplet-flow PCR on the integrated device.
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Liao, Yu-Hua. "Polymerase chain reaction based cloning of acetate kinase in propionibacterium acidipropionici." The Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1072778140.
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Holladay, Ervin Blair. "Viral gene detection in oral neoplasms using the polymerase chain reaction." The Ohio State University, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=osu1345129906.
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Holladay, E. Blair. "Viral gene detection in oral neoplasms using the polymerase chain reaction /." The Ohio State University, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487694702781994.
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Liao, Yu-Hua. "Polymerase chain reaction based cloning of acetate kinase in Propionibacterium acidipropionici /." Connect to this title online, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1072778140.
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Liu, Tingting. "Electrokinetic Real-Time Polymerase Chain Reaction Toward Point-Of-Care Diagnosis." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/579083.
Full textAbstract:
Rapid diagnosis of infectious disease and timely initiation of proper clinical antibiotic treatment is the determinant in obtaining the optimal clinical outcomes and reducing emergences of multidrug-resistant organisms. In particular, acute infections require the detection to be accomplished in limited time with high sensitivity due to the low concentration of organisms causing the infections. Real-time Polymerase Chain Reaction can provide quantitative identification of specific genetic materials and has revolutionized clinical microbiology laboratory diagnosis. It is becoming a standard for infectious disease detection. However, most real-time PCR instruments on the market are bulky, fragile and costly due to their delicate optical components, which restricted their use to point-of-care application. Modern microfluidic and sensing technology provide a transition from benchtop real-time PCR to miniaturizable, robust, and portable real-time PCR devices to achieve rapid, low-cost, and efficient point-of-care diagnosis. In this work, an innovative electrokinetic PCR (EK-PCR) platform that combines AC electrothermal flow (ACEF) and Joule heating induced temperature gradient to implement thermal cycling for DNA amplification is discussed. In addition, in situ electrochemical sensing is incorporated in the EK-PCR chamber for real-time monitoring of the DNA concentration toward quantification of the initial copies of the DNA template. EK-PCR can improve the energy efficiency with minimized total thermal mass and remain high amplification efficiency. More importantly, it represents a highly integrated strategy for portable point-of-care devices.
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Liu, Pang-I., and 劉邦奕. "Design and Fabrication of Microfluidic Devices for Digital Polymerase Chain Reaction." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/n4zt38.
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Mota, Ana Catarina Candeias. "Real-time droplet monitoring for digital Polymerase Chain Reaction in microfluidic chip." Master's thesis, 2020. http://hdl.handle.net/10362/117487.
Full textAbstract:
Current cancer diagnosis techniques are often dependent on the collection of tumour
tissue, involving invasive processes for the patient. Circulating Tumour DNA (ctDNA)
emerges as an alternative resource for cancer detection and monitoring, that can be har vested from simple blood samples. Digital Polymerase Chain Reaction (dPCR) is a fast
and sensitive technique for DNA amplification, suitable for low DNA concentrations such
as ctDNA. Advances in microfluidics allow the partition of PCR samples into droplets
based in water-in-oil emulsions, so that PCR amplification occurs within each droplet. In
this way, the PCR reaction is a well controlled process with a low probability of contami nation and allowing a high throughput analysis.
The aimed of this work was to develop droplet-based microfluidic device for application
to dPCR technique coupled with real-time droplet monitoring. This work focused on the
design and fabrication of a microfluidic device capable of producing a large number of
uniform droplets with volumes in the nanoliter range and constant frequency. For this,
a polydimethylsiloxane (PDMS) droplet generator device was developed, through photo
and soft-lithography techniques, and tested with several oil/water flow rates ratios. Then,
the droplets generated were characterized in terms of droplet size, velocity and frequency
through the implementation of a powerful open-source software for real-time analysis.
Several tests on different devices were carried out to evaluate the device reproducibility.
Finally, the droplet generator was incorporated with a serpentine design, allowing the
PCR cycles to occur in continuous flow.
The results revealed that was possible to generate droplets with radius between 22-99 µm
and a coefficient of variation bellow 10%. The correspondents volumes ranged between
90 pL-4.18 nL. Moreover, the velocities obtained situated between 0.05 mm/s-7.62 mm/s
with droplet generating frequency of 2-50 Hz.
Regarding to the droplet monitoring, the results of the workflows developed revealed
similarity with the results obtained trough a widely used software for this purposes, with
the advantage of allowing real-time analysis for a larger sample of results.As técnicas actuais usadas no diagnóstico de cancro, geralmente dependem da recolha de tecido tumoral, envolvendo processos invasivos para o paciente. O DNA tumoral circu lante (ctDNA) surge como alternativa para a detecção e monitorização do cancro, podendo ser extraído através de amostras de sangue. A reação em cadeia da polimerase de modo digital (dPCR) é uma técnica rápida e sensível para amplificação de DNA, adequado para baixas concentrações de DNA, como o ctDNA. Os avanços na microfluídica permitem a partição das amostras de PCR em gotas com base em emulsões de água em óleo, de modo que a amplificação por PCR ocorra dentro de cada gota. Deste modo, a reação de PCR é um processo bem controlado com baixa probabilidade de contaminação, permitindo uma análise de alto rendimento. Este trabalho teve como objetivo o desenho e a fabricação de um dispositivo de micro fluídica capaz de produzir um elevado número de gotas uniformes, cujos volumes se encontram na gama dos nanolitros, com frequência constante. Para tal, foi desenvolvido um dispositivo para geração de gotas em polidimetilsiloxano (PDMS), através de técnicas de fotolitografia e litografia suave, tendo sido testado com diversas taxas de fluxos entre óleo / água. Posteriormente, as gotas geradas foram caracterizadas em relação ao seu ta manho, velocidade e frequência através do software de análise de vídeo Bonsai. Diversos testes em diferentes dispositivos foram realizados de modo a avaliar a reprodutibilidade do dispositivo. Por último, o gerador de gotas foi incorporado com desenho da serpentina, permitindo que os ciclos de PCR ocorram em fluxo contínuo. Os estudos realizados revelaram que foi possível gerar gotas com raios entre 22-99 µm, e coeficiente de variação inferior a 10%. Os volumes correspondentes variaram entre 90 pL 4.18 nL. Além disso, as velocidades obtidas situaram-se entre 0.05 mm/s-7.62 mm/s com frequência de geração de gotas de 2-50 Hz. Relativamente à monitorização das gotas, os resultados dos workflows desenvolvidos revelaram similaridade com os resultados obtidos através de um software amplamente utilizado para estes fins, com a vantagem de permitir a análise em tempo real para uma amostra maior de resultados.
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Chen, An-Te, and 陳安得. "DNA Extraction and Real-time Polymerase Chain Reaction on Digital Microfluidic Chip." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/77633924624998769116.
Full textAbstract:
碩士國立臺灣大學
機械工程學研究所
105
This thesis reports the implementation of DNA extraction and real-time PCR (qPCR) on a digital microfluidic (DMF) device. We aim to develop a personalized point-of-care device for molecular diagnosis from human whole blood and commercial reagents kits driven by electrowetting-on-dielectric (EWOD) on a DMF device for DNA extraction and qPCR. The results from on-chip DNA extraction protocols were validated and quantified. In comparison wiht the traditional DNA extraction procedures in tubes, our on-chip extraction starting from a 100 nL whole blood obtained 52.8 % DNA concenttion at 4.45 ng/µL, required 25 % reaction time (from 120 min to 30 min), consumed 0.05 % volume of the entire reagents (from 4222
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Chang, Yi-Hsien, and 張儀賢. "Digital Microfluidic Chips with Low Operation Voltages for Polymerase Chain Reaction pplications." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/53803224655745668820.
Full textAbstract:
碩士國立成功大學
工程科學系碩博士班
93
This study designs and fabricates a digital microfluidic platform with a low operation voltage by utilizing electro-wetting-on-dielectric (EWOD) effect. All the microfluidic movements are realized bet ween two parallel plates on this platform. The digital microfluidics chips (DMC) could make liquids digitalized, allowing for the transportation of droplets without moving micromechanical components in the device. Most fluidic operations such as sample transporting, creation, cutting and merging can be carried out on the chips using discrete droplets. The DMC does not require pumps, mixers and valves as in traditional microfluidic systems. The droplet motion can be manipulated by using surface tension gradient generated by EWOD, which could control of the wettability of liquids on dielectric thin film surfaces while electric potential is applied. This study tested several high K materials such as BST (Ba0.5Sr0.5TiO3), Ta2O5, Si3N4, Parylene and silicon-dioxide (SiO2). By using the Young-Lippmann equation, reducing the operating voltage of EWOD could be realized. Meanwhile, the hysteresis effect of contact angles between the droplet and the Teflon surface could be reduced by filling silicone oil into the parallel open channel. Therefore, the four fundamental microfluidics operations, transport, creation, cutting, and merging, can be performed with a lower voltage of 12 V. Polymerase chain reaction (PCR) is a popular technology in molecular biology. A micro temperature sensor and micro heaters have been integrated into the EWOD chip to allow for PCR operation, which is dependent on three precise temperature steps. Utilizing the characteristics of a temperature with a constant TCR (Temperature Coefficient of Resistance), this study designed a closed-loop temperature controller. The micro temperature sensor, micro heaters, and smaller reagent volume allow for a heating rate of 38.0 degrees per second and a cooling rate of 7.9 degrees per second, thus enabling for faster completion of the PCR cycles. The micro temperature sensor, micro heaters are controlled by a commercial programmable chip (AT90S8535). This chip successfully achieved 25 thermo cycles PCR for the genes of Dengue II Virus cDNA in 55 minutes. Utilizing micro-electro-mechanical-system (MEMS) fabrication technology, the digital microfluidics operation system and micro temperature controller could be successfully integrated into a cheap and biocompatible glass substrate to allow automated rapid infectious disease detection. By using EWOD effect, the chip could make liquids digitalized, permitting droplets to be quickly and precisely transported and mixed without any moving components. This study also designed a new hydrophilic-hydrophobic interface to induce the surface tension gradient to transport the mixed droplets into the reaction chamber. The Dengue II Virus cDNA polymerase chain reaction was also performed on the developed chip which was used to detect microorganism genes by amplifying the target sequence (511bps) with PCR. This study confirmed the feasibility of the PCR with the digital microfluidics operation. The integrated DMC has several advantages for infectious disease detection including (1) low voltage operation DMC, (2) 70% less sample and reagent consumption (3) 50% shorter detection time due to faster thermal cycling, and (4) high accuracy and reproducibility due to a simple and reliable fabrication process. The study successfully applied the concept of Digital Micro Fluidics to the Lab-on-a-chip. The developed microchip will be of great benefit in the future genetic analysis and infectious disease detection.
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Chiang, Tsai-Jung, and 蔣采蓉. "Polymerase Chain Reaction of DNA by Joule Heating on an EWOD-Based Digital Microfluidic Platform." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/52766787872652536373.
Full textAbstract:
碩士國立交通大學
材料科學與工程學系奈米科技碩博士班
99
We investigated joule heating to treat a small amount of biochemical sample driven by electrowetting-on-dielectric (EWOD) on a digital microfluidic platform. For the decreased reagent volume and reduced device size, the heating and cooling rates are thus increased. With the heating and driving abilities, we utilize this platform to realize polymerase chain reaction of DNA with fast heating and cooling rates. On the contrary, conventional equipments usually give lower heating and cooling rates because large amounts of biochemical samples and reagents are required. The reported digital microfluidic system with the joule heating ability would provide an approach to solve the heating/cooling rate issues by reducing the reaction volume from conventional 50 ?愮 to 3 ?愮 at a heating rate of 2.46 oC/s and a cooling rate of 6.94 oC/s The system consumes a much lower power 8.5 x 10-3 W than that consumed in traditional PCR machines. In the experiment, the external voltage at low frequency (1 kHz) can drive droplet by EWOD without temperature change. At high frequency (higher than 100 kHz), the AC electric field heats the droplet and causes an 80 oC temperature change to the temperature of 105.6 oC. The temperature of the tested droplet rises when the external voltage increases. In addition, the heating is also frequency-related. Therefore, this system can control the temperature and position of the droplet for realizing the polymerase chain reaction- by tuning the amplitude and frequency of the applied voltage.
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"An investigation into the determination of relative chromosome dosage by digital PCR." 2009. http://library.cuhk.edu.hk/record=b5896560.
Full textAbstract:
Chan, Ka Ying.Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 133-150).
Abstract also in Chinese.
ABSTRACT --- p.i
摘要 --- p.iii
ACKNOWLEDGEMENTS --- p.iv
CONTRIBUTORS --- p.vi
TABLE OF CONTENTS --- p.vii
LIST OF TABLES --- p.x
LIST OF FIGURES --- p.xi
LIST OF ABBREVIATIONS --- p.xiii
Chapter SECTION I: --- BACKGROUND --- p.1
Chapter CHAPTER 1: --- PRENATAL DIAGNOSIS OF FETAL TRISOMY 21 --- p.2
Chapter 1.1 --- Down syndrome --- p.2
Chapter 1.2 --- Current methods of prenatal diagnosis of fetal trisomy 21 --- p.3
Chapter 1.2.1 --- Non-invasive procedures --- p.3
Chapter 1.2.2 --- Invasive procedures --- p.5
Chapter 1.3 --- Alternative methods for the prenatal diagnosis of fetal trisomy 21 --- p.7
Chapter CHAPTER 2: --- CELL-FREE FETAL NUCLEIC ACIDS IN MATERNAL PLASMA --- p.13
Chapter 2.1 --- Circulating fetal cells --- p.15
Chapter 2.2 --- Circulating cell-free fetal nucleic acids --- p.15
Chapter 2.3 --- Diagnostic applications of cell-free fetal nucleic acids in maternal plasma --- p.17
Chapter 2.4 --- Digital relative chromosome dosage approach --- p.20
Chapter 2.5 --- Validation of digital RCD approach on artificial DNA mixtures --- p.22
Chapter SECTION II --- : MATERIALS AND METHODS --- p.25
Chapter CHAPTER 3: --- QUANTITATIVE ANALYSIS OF NUCLEIC ACIDS --- p.26
Chapter 3.1 --- Subject recruitment and sample collection --- p.26
Chapter 3.2 --- Sample processing --- p.26
Chapter 3.3 --- Nucleic acid extraction --- p.27
Chapter 3.3.1 --- Extraction of DNA from placental tissues --- p.27
Chapter 3.3.2 --- Extraction of DNA from maternal blood cells --- p.27
Chapter 3.4 --- Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) --- p.28
Chapter 3.5 --- Paralogous sequence assays optimisation workflow --- p.31
Chapter 3.5.1 --- Monoplex paralogous sequence assays --- p.31
Chapter 3.5.2 --- Multiplex paralogous sequence assay --- p.38
Chapter 3.6 --- Digital PCR --- p.42
Chapter 3.6.1 --- Principle --- p.42
Chapter 3.6.2 --- Digital multiplex paralogous sequence assay --- p.42
Chapter 3.7 --- Statistical analysis --- p.46
Chapter 3.7.1 --- Disease classification of samples --- p.46
Chapter 3.7.2 --- Poisson distribution --- p.46
Chapter 3.7.3 --- Data analysis --- p.48
Chapter 3.7.4 --- Sequential probability ratio test (SPRT) analysis --- p.49
Chapter SECTION III: --- ASSAY DEVELOPMENT --- p.53
Chapter CHAPTER 4: --- TESTING OF ASSAY SPECIFICITY WITH CORIELL CELL LINES --- p.54
Chapter 4.1 --- Coriell cell lines --- p.54
Chapter 4.2 --- Specificity of initial PCR primers --- p.56
Chapter 4.2.1 --- Principle --- p.56
Chapter 4.2.2 --- Materials and methods --- p.56
Chapter 4.2.3 --- Results --- p.60
Chapter 4.2.4 --- Conclusion --- p.63
Chapter 4.3 --- Specificity of the iPLEX® Gold extension primers --- p.63
Chapter 4.3.1 --- Principle --- p.63
Chapter 4.3.2 --- Materials and methods --- p.64
Chapter 4.3.3 --- Results --- p.65
Chapter 4.4 --- Further analysis on the specificity of PV2107a initial PCR primers --- p.67
Chapter 4.5 --- Conclusion --- p.71
Chapter CHAPTER 5: --- ASSAY OPTIMISATION --- p.72
Chapter 5.1 --- Introduction --- p.72
Chapter 5.2 --- Optimisation of initial PCRs with AmpliTaq Gold® DNA polymerase followed by homogeneous MassEXTEN´DёØ (hME) assays (Sequenom) --- p.72
Chapter 5.2.1 --- Optimisation of initial PCR reactions --- p.72
Chapter 5.2.2 --- Principle of homogeneous MassEXTEN´DёØ assays (Sequenom)… --- p.75
Chapter 5.2.3 --- Homogeneous MassEXTEN´DёØ assays (Sequenom) on euploid and T21 samples --- p.76
Chapter 5.3 --- Assay selection by iPLEX® Gold single base primer extension reactions (Sequenom) --- p.82
Chapter 5.4 --- Optimisation of multiplex PCR with AmpliTaq Gold® DNA polymerase --- p.88
Chapter 5.5 --- Optimisation of multiplex iPLEX® Gold single base primer extension reaction --- p.93
Chapter 5.6 --- Single molecule detection test for the multiplex paralogous sequence assays … --- p.103
Chapter SECTION IV: --- ANALYSIS OF CLINICAL SAMPLES --- p.107
Chapter CHAPTER 6: --- DISEASE CLASSIFICATION OF EUPLOID AND TRISOMY SAMPLES WITH MULTIPLEX PARALOGOUS SEQUENCE ASSAY --- p.108
Chapter 6.1 --- Introduction --- p.108
Chapter 6.2 --- Materials and methods --- p.109
Chapter 6.2.1 --- Sample collection --- p.109
Chapter 6.2.2 --- Experimental design --- p.110
Chapter 6.3 --- Results --- p.111
Chapter 6.4 --- Discussion --- p.114
Chapter SECTION V: --- CONCLUDING REMARKS --- p.122
Chapter CHAPTER 7: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.123
Chapter 7.1 --- Conclusion --- p.123
Chapter 7.2 --- Future perspectives --- p.124
Appendix 1 --- p.126
Appendix II --- p.127
REFERENCE --- p.133
46
Su, Ying-Tu, and 蘇英圖. "Application of MicroEmusification：Emulsion Polymerase Chain Reaction." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/51940901593932821213.
Full textAbstract:
碩士國立臺灣海洋大學
機械與機電工程學系
97
In this thesis, soft lithography technique was used to produce a micro emulsion chip. This chip can handle tiny volume of DNA solution for polymerase chain reation (ePCR). The DNA solution is dispersed in the emulsified droplets in a continuous oil phase, and the droplets become reaction space for PCR. As a result, the consistency of the multiple types of DNA templates existing in the original reaction space can be improved by emulsification process. Also, it will reduce competitive sequence interference and improve DNA magnification. Differing from usual micro emulsification chips, this study focuses on the technical problems involved in dealing with tiny and expensive DNA solution in order to avoid non-uniform droplets in the transient of droplet generation. The main ideas are as follows: (1) Let water first go through transient process and produce water droplets; (2) Use water and bubble to place DNA solution in between, and pass it through emulsification hydrodynamic focusing channel; (3) Use pneumatic membrane valves control the stop and flow of water and DNAsolution. Characterization of the emulsification chips includes droplet sizes, uniformity of droplets and hot-resistance of water-in-oil droplets. The smallest droplet diameter is less than 10 μm and the coefficient of variation is about 2%. The resulting electrophoretograms of both single or multiple templates’ PCR demonstrate that ePCR can improve the precision of amplified DNA segments. It is suggested from the experiment results that ePCR was proven to be efficient and could be used in further genetic research Key word: ePCR, DNA template, transient process, tiny-volume micro-emulsification
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Gi, Kao Li, and 高麗姬. "ABO Genotyping for Mutiplex Polymerase Chain Reaction." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/82466547226309854097.
Full textAbstract:
碩士中央警察大學
刑事警察研究所
87
Abstract ABO blood group is clinically the most important blood group system in transfusion medicine including fours common different phenotypes . It has been a valuable tool in transfusion medicine , physical anthropology , disputed parentage testing , human identification , and in forensic analysis . Historically , forensic and clinical labatories utilize serological techniques to identify ABO blood type . The testing of forensic samples for ABO types using serological techniques has several drawbacks . We has simplified the identification of ABO types by taking advantage of previouly reported ABO DNA sequence difference . The use of single strand conformation polymorphism（SSCP）can separate sequence polymorphism than differ by only one base . We has investigated in 260 Chinese donors by multiplexed Polymerase Chain Reaction . In this study the ABO alleles from exon 6 , producing a 212/213 bp fragment , and exon 7, which produces a fragment of 272 bp , were separated by non-denaturing polyacrylamide gels . The two exons were amplified in a single reaction that produces similar quantities of DNA for both exons . However , in 260 SSCP patterens we can clustered 9 into different distinct and discernible groups . A concordance rate of 98.5 ﹪（256/260 samples）observed between the actural genotype and the serologically-based predicted genotype . These results indicate that the assay provides a rapid , accurate , and simple method for ABO genotyping that serves as a useful supplement to standard serological ABO typing .
48
Chen, Jhao-Rong, and 陳昭榮. "Micro Rayleigh-Bénard polymerase chain reaction system." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/05888212591754965876.
Full textAbstract:
碩士國立清華大學
工程與系統科學系
93
Polymerase chain reaction (PCR) is a molecular biological method for the in vitro amplification of nucleic acid molecule. In this thesis, the research object was to design a micro PCR system which involved a Rayleigh-Bénard convection PCR chip, measurement circuits, and temperature control circuits. Rayleigh-Bénard convection PCR chip was easy to be fabricated, and the sample solution in it can transit its temperature immediately. Thus, the faster the speed of flow is, the higher heating and cooling rate is. Rayleigh-Bénard convection PCR chip can be divided into two parts. First part is a chip with micro heaters and micro temperature sensors. Second part is PDMS reaction chamber designed by Rayleigh-Bénard convection theory. Temperature control system is designed to keep temperature on the top and bottom chips by Pulse width modulation control. Analog temperature signal is precisely measured by circuits. Then the signal is processed by an 8051 single chip, and the output power of micro heaters is controlled to adjust the temperature of top and down plates. Finally, agarose gel electrophoresis is used to verify the practicability of Rayleigh-Bénard convection PCR system. By comparing with the PCR experiments done by the commercial PCR machine and our chips, our chips can plenty decrease the reaction time. And By comparing with the simulation and PCR experiments of different designed sizes, users can use setted parameters and CFD results to do optimal designs and then decrease the total reaction time in the future.
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Tao, Yo-Shen, and 刁宥升. "Design Of Polymerase Chain Reaction System Controller." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/76827215729605694932.
Full textAbstract:
碩士國立暨南國際大學
電機工程學系
90
DNA analysis is a crucial process for biotechnology. Polymerase chain reaction (PCR) is an imperative step for performing DNA analysis. PCR can proliferate a small quantity of DNA sample to a large quantity enough for DNA analysis in a short time. Our goal is to develop a PCR system chip by integrating standard CMOS process and MEMS process. In this thesis, an 8-bit 4-level pipelined programmable micro-controller for temperature control during PCR process is designed by HDL description and implemented by using field programmable gate array (FPGA). The micro-controller includes modules of data path and control unit. Verilog hardware description language is used to model the modules. The individual module is functionally verified by Max Plus II of Altera and integrated into an 8-bit micro-controller. The micro-controller is realized by a FPGA chip, and then micro program for the temperature control of PCR is downloaded into the ROM in FPGA for system test. The micro-controller proposed in this thesis is very simple and easy to implement. It is suitable for general low speed applications. In order to easily integrate the micro-controller into other applications, we will try to parameterize its design variables for adjusting its hardware structure in future works.
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Gao, Li-Ji, and 高麗姬. "ABO Genotyping for Multiplex Polymerase Chain Reaction." Thesis, 1998. http://ndltd.ncl.edu.tw/handle/7433nn.
Full textAbstract:
碩士中央警察大學
刑事警察研究所
87
ABO blood group is clinically the most important blood group system in transfusion medicine including fours common different phenotypes . It has been a valuable tool in transfusion medicine , physical anthropology , disputed parentage testing , human identification , and in forensic analysis . Historically , forensic and clinical labatories utilize serological techniques to identify ABO blood type . The testing of forensic samples for ABO types using serological techniques has several drawbacks . We has simplified the identification of ABO types by taking advantage ofpreviouly reported ABO DNA sequence difference . The use of single strand conformation polymorphism ( SSCP ) can separate sequence polymorphism than differ by only one base . We has investigated in 260 Chinese donors by multiplexed Polymerase Chain Reaction . In this study the ABO alleles from exon 6 , producing a 212/213 bp fragment , and exon 7, which produces a fragment of 272 bp, were separated by non-denaturing polyacrylamide gels. The two exons were amplified in a single reaction that produces similar quantities of DNA for both exons . However , in 260 SSCP patterens we can clustered 9 into different distinct and discernible groups . A concordance rate of 98.5 % (256/260 samples) observed between the actural genotype and the serologically-based predicted genotype . These results indicate that the assay provides a rapid , accurate , and simple method for ABO genotyping that serves as a useful supplement to standard serological ABO typing.