Microfabricated thermal cyclers for nucleic acid amplification by using polymerase chain reaction (PCR) have been demonstrated by several groups over the last decade, with improved cycling speed and smaller volumes when compared to conventional bench-top cyclers. However, high fabrication costs coupled with difficulties in temperature sensing and control remain impediments to commercialization. In this study we have used a silicon-glass device that takes advantage of the high thermal conductivity of silicon but at the same time utilizes minimum number of fabrication steps to make it suitable for disposable applications. The thermal cycler is based on noncontact induction heating developed in this group. The microchip reaction kinetics is studied for the first time in-situ during PCR, using a real-time fluorescence block that is capable of data acquisition every 0.7 s from the microchip. The fluorescence information from SYBR green I dye is used to optimize microchip amplification reactions and confirm the product by melting curve analysis. We have also developed a novel non-contact temperature sensing technique using SYBR green fluorescence that can be used for miniaturized PCR devices. The thesis is organized into the following chapters. In chapter 1 we introduce the basic biology ideas that are required to understand DNA amplification. DNA based analysis requires amplification of low initial concentrations to above detectable limits using a technique known as polymerase chain reaction (PCR). In this process, the sample is cycled through three thermal steps for 3040 times to produce multiple copies of DNA. In microchip PCR, conventional polypropylene tubes using 2050 µL volume are replaced by miniaturized devices using ~1 µL sample volumes. The device response improves in terms of ramp rate and total analysis time due to the small volume and smart design of the materials. In this chapter we summarize some of the issues important for miniaturized PCR devices and compare them with commercial tube PCR systems. In chapter 2 we describe the induction heating technique that was developed by our group for miniaturized devices. Induction heating is a noncontact heating technique unlike resistive heating which has been commonly used for microchip PCR. Though resistive heating is very efficient in terms of heat transfer efficiency, it is not suitable for disposable devices and requires multi-step microfabrication. Other non-contact heating techniques such as hot air and IR heating require larger size arrangements that are not suitable for miniaturized devices. The heating was verified by using a thermocouple soldered at the back of the secondary plate that was also used for feedback to the comparator circuit for control. The simple on-off circuit was able to control within ±0.1 ◦C with heating and cooling ramp rates of 25 ◦C/s and 2.5 ◦C/s respectively. In this chapter, we also describe the design and fabrication of the silicon-glass microchip fabricated in our lab. We have used silicon-glass hybrid device for PCR in which glass with a 2 mm drilled hole is anodically bonded to an oxidized silicon surface. The hole formed the static reservoir for 3 µL volume of amplification solution. During PCR, the solution needs to be cycled to high temperature of ~95 ◦C. Hence it was necessary to seal the tiny droplet of liquid against evaporation at this temperature. The devices after being filled by sample were covered by 4 µL of mineral oil to serve as an evaporation barrier. It was easy to recover the whole sample after amplification for further testing. Chapter 3 describes the development of a fluorescent block for SYBR green I dye (SG) used for real-time monitoring of the amplification. The block contains a blue LED for excitation, a dichroic beamsplitter, and silicon photodiode along with filters and focusing optics. Signal levels being weak, we incorporated lock-in detection technique. A TTL at 190 Hz was used to pulse the excitation source and detect the emission at the same frequency using a commercial lock-in amplifier. The block was first characterized using a commercial thermal cycler and polypropylene tubes with different dilution of initial template copy number, and the results crosschecked with agarose gel electrophoresis. Performing continuous monitoring every 0.7s within cycles, we discovered interesting features during extension which have not been studied previously. During the constant temperature extension step, the fluorescence shows a rise and then saturates until the temperature is cycled to the next set point. We have confirmed the same behavior in single cycle extension control experiments and established its connection with polymerase extension activity. We were thus able to extract the activity rate for two different kinds of polymerase in-situ during PCR. By monitoring PCR reactions with different fixed extension times, we were able to determine the optimum conditions for tube PCR. Chapter 4 implements the ideas of fluorescence monitoring from tube that was explained in the previous chapter for the silicon-glass microchip. Since the microchip uses parameters such as sample volume, ramp rates, stay time etc. which are different from tube PCR, we performed several initial test experiments to establish key capabilities such as low volume detection, 3 µL amplification, surface passivation of silicon-glass etc. The same fluorescence block was used to obtain DNA melting point information by continuously monitoring ds-DNA with SG while the temperature is ramped slowly (melting curve analysis). Depending on ds-DNA present, the fluorescence gives a melting temperature (TM ), which was used to calibrate the mix temperature with respect to the thermocouple sensor. After successfully calibrating the microchip, we confirmed complete chip PCR in silicon-glass devices using induction heater. The continuous monitoring of chip PCR gave similar curves as obtained previously for tubes except that the signal level was lower in silicon devices. Extension fluorescence information was used to find an optimum temperature for microchip that shows a maximum activity rate. Similarly the reaction time was optimized in-situ during PCR by using continuous fluorescence data in a feedback experiment. The commercial lock-in amplifier was also replaced by a homemade circuit to successfully pickup fluorescence signal from the microchip during melting curve analysis. In chapter 5, we describe a novel technique to sense the temperature from the microchip without touching the sample volume. Usually the temperature is monitored by a sensor spatially separated from the mix and it has always been challenging to measure the exact temperature accurately. Most of the sensors are not biocompatible and too large in size to be placed inside the small volume of liquid. We have developed a protocol that involves SG fluorescence with addition of excess sensor DNA to the amplification solution. The sensor DNA added into the mix is non specific to the primer used for amplification of the template. It therefore does not participate in the amplification and its number remains unchanged throughout the 3040 cycles of PCR. If the amount of sensor DNA is titrated accurately, it will saturate the fluorescence envelope which then shows very reproducible thermal response with cycling. We have used this thermal response of the fluorescence for feedback as a temperature sensor. The fluorescence feedback was shown to produce identical amount of product in comparison to thermocouple feedback. The product can also be verified by melting curve analysis if the sensor DNA is chosen carefully depending on the product. In this chapter we also discuss some preliminary experiments with smart devices that will use dye based temperature sensor and control along with fluorescence based amplification monitoring. Chapter 6 summarizes the thesis and discusses some of the future areas which can be explored in the field of microchip PCR devices.

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M.Sc.(Med.), Faculty of Health Sciences, University of the Witwatersrand, 2009 / Community-acquired pneumonia (CAP) is a major cause of morbidity and mortality worldwide. Knowledge of the predominant agents associated with CAP locally is essential, as it represents the basis for empiric antibiotic treatment. The objective of this study was to establish polymerase chain reaction (PCR)-based methods that could be used to identify CAP pathogens. Real-time PCR assays were developed to detect 10 viral and 5 non-viral pathogens as well as 2 internal controls using SYBR Green I and TaqMan probes, in singleplex and multiplex reactions. Six multiplex assays, with sensitivities of 1-10 copies/μl, were successfully developed to simultaneously detect 12 organisms. These reactions were used to test a limited number of patient and simulated samples. Data from the real-time PCR methods compared favourably to those from commercially available conventional PCR kits. These detection methods could be used to complement each other in prevalence studies and in selected diagnostic applications.

Eva Tsui Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 124-140). / SUMMARY / ACKNOWLEDGEMENT / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter CHAPTER II --- LITERATURE REVIEWS --- p.5 / Chapter 2.1 --- Anatomy and Histology of Uterine Cervix --- p.6 / Chapter 2.2 --- Premalignant and Malignant Lesions of Uterine Cervix --- p.9 / Chapter 2.2.1 --- Incidence --- p.9 / Chapter 2.2.2 --- Etiological Factors --- p.10 / Chapter 2.2.3 --- Classification and Histopathology --- p.13 / Chapter A. --- Condyloma --- p.13 / Chapter B. --- Cervical Intraepithelial Neoplasia (CIN) --- p.16 / Chapter C. --- Microinvasive Squamous Cell Carcinoma --- p.19 / Chapter D. --- Invasive Squamous Cell Carcinoma --- p.22 / Chapter E. --- Adenosquamous Carcinoma --- p.25 / Chapter F. --- Adenocarcinoma --- p.26 / Chapter 2.3 --- Human Papillomavirus (HPV) --- p.28 / Chapter 2.3.1 --- Organization of the Viral Genome --- p.28 / Chapter 2.3.2 --- Classification --- p.35 / Chapter 2.4 --- The Impact of Human Papillomavirus (HPV)in Premalignant and Malignant Lesions of Uterine Cervix --- p.35 / Chapter 2.5 --- Cervical Screening --- p.40 / Chapter 2.5.1 --- Cervical Smear --- p.40 / Chapter 2.5.2 --- Cervicography --- p.41 / Chapter 2.5.3 --- HPV Detection --- p.43 / Chapter CHAPTER III --- MATERIALS AND METHODS --- p.54 / Chapter 3.1 --- Study Population --- p.55 / Chapter 3.2 --- Specimen Collection --- p.56 / Chapter 3.3 --- Extraction of Genomic DNA --- p.56 / Chapter 3.4 --- Polymerase Chain Reaction (PCR) --- p.60 / Chapter 3.4.1 --- Oligonucleotide Primers and Positive Controls --- p.60 / Chapter 3.4.2 --- Polymerase Chain Reaction Procedure --- p.62 / Chapter A. --- PCR Optimization --- p.62 / Chapter B. --- Multiplex PCR --- p.63 / Chapter C. --- Single Primer PCR --- p.64 / Chapter 3.4.3 --- Gel Electrophoresis --- p.65 / Chapter 3.5 --- Southern-blot and Hybridization --- p.69 / Chapter 3.5.1 --- Southern-blot --- p.69 / Chapter 3.5.2 --- Nucleic Acid Hybridization --- p.71 / Chapter A. --- Oligonucleotide Probes --- p.71 / Chapter B. --- Radioactive Labelling of Probes --- p.73 / Chapter C. --- Hybridization --- p.74 / Chapter D. --- Removal of Labelled Probes --- p.77 / Chapter CHAPTER IV --- RESULTS --- p.78 / Chapter 4.1 --- Cytological Findings of Cervical Scrapes --- p.79 / Chapter 4.1.1 --- Colposcopy Group --- p.79 / Chapter 4.1.2 --- General Gynaecology Group --- p.81 / Chapter 4.1.3 --- Antenatal Group --- p.81 / Chapter 4.2 --- PCR Optimization --- p.84 / Chapter 4.3 --- Detection of HPV DNA by Multiplex PCR --- p.86 / Chapter 4.3.1 --- Colposcopy Group --- p.86 / Chapter 4.3.2 --- General Gynaecology Group --- p.89 / Chapter 4.3.3 --- Antenatal Group --- p.91 / Chapter 4.4 --- Detection of HPV DNA by Southern-blot Hybridization --- p.91 / Chapter 4.5 --- Detection of HPV DNA by Single Primer PCR --- p.104 / Chapter CHAPTER V --- DISCUSSION --- p.110 / Chapter CHAPTER VI --- CONCLUSION --- p.121 / REFERENCES --- p.124

by Graham Neil Thomas. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 167-191). / Abstract --- p.i / Contents --- p.iii / List of tables --- p.viii / List of figures --- p.x / Abbreviations --- p.xi / Acknowledgements --- p.xiii / Chapter Chapter 1 - --- Introduction / Chapter 1.1 --- Introduction to the genus rotavirus --- p.1 / Chapter 1.2 --- General characteristics of rotavirus --- p.3 / Chapter 1.3 --- Clinical and epidemiological characteristics of rotaviral infections --- p.4 / Chapter 1.3.1 --- Clinical features of rotavirus infection --- p.4 / Chapter 1.3.2 --- Nosocomial rotavirus infection --- p.4 / Chapter 1.3.3 --- Morbidity and Mortality of rotavirus diarrhoea --- p.5 / Chapter 1.3.3.1 --- Seasonal distribution of rotavirus in temperate regions … --- p.5 / Chapter 1.3.3.2 --- Rotavirus infections in developing countries --- p.6 / Chapter 1.3.3.3 --- Rotavirus infections in developed countries --- p.6 / Chapter 1.3.4 --- Host Resistance to rotavirus infection --- p.7 / Chapter 1.3.5 --- Pathogenesis --- p.9 / Chapter 1.4 --- Vaccine development in rotavirus prevention --- p.10 / Chapter 1.4.1 --- Attenuated HRV as candidate vaccine strains --- p.11 / Chapter 1.4.2 --- Animal RV candidate vaccine strains (Jennerian approach)..… --- p.11 / Chapter 1.4.3 --- Intra- and interspecies reassortants vaccine strains --- p.12 / Chapter 1.4.4 --- Passive immunisation --- p.13 / Chapter 1.5 --- Laboratory diagnosis of rotavirus infections --- p.14 / Chapter 1.5.1 --- Detection of rotavirus --- p.14 / Chapter 1.5.2 --- Negative stain electron microscopy (EM) --- p.15 / Chapter 1.5.3 --- Immunological assays for the detection of rotavirus antigens --- p.15 / Chapter 1.5.4 --- Polyacrylamide gel electrophoresis (PAGE) of RV RNA --- p.16 / Chapter 1.5.5 --- Nucleic acid probe hybridisation assays --- p.17 / Chapter 1.6 --- Antigenic classification of rotaviruses --- p.17 / Chapter 1.6.1 --- Rotavirus groups --- p.17 / Chapter 1.6.2 --- Rotavirus subgroups --- p.18 / Chapter 1.6.3 --- Rotavirus serotypes --- p.19 / Chapter 1.6.4 --- Rotavirus genogroups --- p.21 / Chapter 1.7 --- Molecular biology of rotavirus --- p.21 / Chapter 1.7.1 --- Rotavirus genomic organisation --- p.21 / Chapter 1.7.2 --- Gene coding assignments --- p.22 / Chapter 1.7.3 --- Genome and protein structure of rotavirus VP7 --- p.22 / Chapter 1.8 --- Reverse transcriptase-Polymerase chain reaction (RT-PCR) for the genotyping of rotavirus --- p.32 / Chapter 1.8.1 --- Prevention of contamination in RT-PCR --- p.34 / Chapter 1.9 --- Objectives of the study --- p.36 / Chapter Chapter 2 - --- Methods / Chapter 2.1 --- Collection of specimens --- p.38 / Chapter 2.2 --- Standard rotavirus strains --- p.38 / Chapter 2.3 --- Tissue culture techniques --- p.39 / Chapter 2.3.1 --- Growth of MA104 cell line --- p.39 / Chapter 2.3.2 --- Subculturing of MA104 cell line --- p.40 / Chapter 2.3.3 --- Virus propagation and isolation --- p.40 / Chapter 2.3.4 --- Harvesting and purification of viral particles --- p.41 / Chapter 2.4 --- Rotavirus electropherotyping by PAGE --- p.42 / Chapter 2.4.1 --- RNA extraction --- p.42 / Chapter 2.4.2 --- Polyacrylamide gel electrophoresis (PAGE) --- p.43 / Chapter 2.4.3 --- Silver staining of RNA in polyacrylamide gels --- p.43 / Chapter 2.5 --- Enzyme immunoassays in rotavirus typing --- p.44 / Chapter 2.5.1 --- Preparation of monoclonal antibodies (mAb) from hybridoma cell lines --- p.44 / Chapter 2.5.1.1 --- Growth of hybridoma cell lines --- p.44 / Chapter 2.5.1.2 --- Preparation of the ascitic fluid - monoclonal Abs --- p.45 / Chapter 2.5.2 --- Confirmation of mAb activity by immunofluorescence (IF)..… --- p.46 / Chapter 2.5.2.1 --- Preparation of virus-infected cells --- p.46 / Chapter 2.5.2.2 --- Confirmation of the serotype specificity of the mAb by immunofluorescence microscopy --- p.47 / Chapter 2.5.3 --- Polyclonal hyperimmune antisera against rotavirus --- p.48 / Chapter 2.5.4 --- Immunoglobulin purification --- p.48 / Chapter 2.5.5 --- Monoclonal antibody-based serotyping and subgrouping EIA --- p.49 / Chapter 2.6 --- Reverse transcription-Polymerase Chain Reaction genotyping of rotavirus (RT-PCR) --- p.53 / Chapter 2.6.1 --- Primers used in RT-PCR --- p.53 / Chapter 2.6.1.1 --- Preparation of oligonucleotide primers for RV genotyping --- p.53 / Chapter 2.6.1.2 --- Detachment of the oligonucleotide from the column --- p.54 / Chapter 2.6.1.3 --- Purification of the oligonucleotides --- p.55 / Chapter 2.6.1.4 --- Confirmation of oligonucleotide synthesis --- p.58 / Chapter 2.6.2 --- Preparation of specimens --- p.59 / Chapter 2.6.3 --- Reverse transcription of genomic RNA template and PCR…… --- p.64 / Chapter 2.6.4 --- PCR genotyping using full-length cDNA template --- p.64 / Chapter 2.6.5 --- Product identification --- p.65 / Chapter Chapter 3 - --- Results / Chapter 3.1 --- Epidemiology of rotavirus infections in Hong Kong --- p.70 / Chapter 3.2 --- RT-PCR genotyping of rotavirus --- p.74 / Chapter 3.3 --- Seasonal distribution of rotavirus genotypes --- p.76 / Chapter 3.4 --- Comparison of RT-PCR genotyping of the VP7 gene with mEIA…… --- p.79 / Chapter 3.5 --- Relationship between electropherotyping and genotyping by RT-PCR --- p.91 / Chapter 3.6 --- Atypical rotavirus strains --- p.92 / Chapter 3.7 --- Specimens exhibiting multiple genotype specificities --- p.93 / Chapter 3.8 --- HRV RT-PCR genotype primers --- p.95 / Chapter Chapter 4 - --- Discussion / Chapter 4.1 --- Epidemiology of rotavirus infections in Hong Kong --- p.101 / Chapter 4.2 --- RT-PCR genotyping of rotavirus --- p.103 / Chapter 4.2.1 --- Modifications to methodology --- p.104 / Chapter 4.2.2 --- Rotavirus genotypes --- p.107 / Chapter 4.3 --- Comparison of RT-PCR genotyping with mEIA typing --- p.111 / Chapter 4.4 --- Relationship between electropherotyping and RT-PCR genotyping --- p.113 / Chapter 4.5. --- Rotavirus genotype distribution in Hong Kong --- p.115 / Chapter 4.6 --- Specimens containing atypical rotavirus strains --- p.119 / Chapter 4.7 --- Stool specimens exhibiting multiple rotavirus genotypes identified by RT-PCR --- p.122 / Chapter 4.8 --- Specificity analysis of RT-PCR primers --- p.124 / Chapter 4.8.1 --- 5non-coding region (1-28) - primer BEG9 --- p.125 / Chapter 4.8.2 --- 3non-coding region (1033/6-1062) - primers END9/RVG9. --- p.125 / Chapter 4.8.3 --- Variable region A (165-198) - primer aAT8 (G8) --- p.125 / Chapter 4.8.4 --- Variable region B (309-351) - primer aBT1 (G1) --- p.126 / Chapter 4.8.5 --- Variable region C (408-438) - primer aCT2 (G2) --- p.127 / Chapter 4.8.6 --- Variable region D (477-504) - primer aDT4 (G4) --- p.127 / Chapter 4.8.7 --- Variable region E (672-711) - primer aET3 (G3) --- p.128 / Chapter 4.8.8 --- Variable region F (747-776) - primer aFT9 (G9) --- p.128 / Chapter 4.9 --- Future developments of the study --- p.131 / Appendices / Chapter A1 --- Materials --- p.135 / Chapter A2.1-2.9 --- Distribution of electropherotypes between July 1985 and April 1994 --- p.145 / Chapter A3.1-3.11 --- Individual rotavirus strain electropherotypes --- p.153 / Chapter A4.1-4.8 --- "Comparison of nucleotide sequences for the 3', 5'-non-coding regions and variable regions A to F for the VP7 gene" --- p.158 / Chapter A4.9 --- Rotavirus strains and nucleotide sequence references for the VP7 gene --- p.166 / References

Yu Man Him. / Thesis (M.Sc.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 45-50). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgement --- p.ii / Chapter 1. --- Introduction / The mitochondrion --- p.1 / The mitochondrial genome --- p.3 / Mitochondrial DNA and diseases --- p.7 / Circulating plasma DNA --- p.8 / Project aims --- p.8 / Chapter 2. --- Materials and Methods / Choice of gene quantification system --- p.9 / Real time quantitative PGR --- p.11 / 7700Sequence Detection System --- p.14 / The LightCycler ´ؤ an alternative fast analyzer --- p.15 / Quatntitation of starting copy number using real time PCR --- p.17 / Primer and probe design rules --- p.18 / Hot start PCR technique --- p.19 / Other anti-contamination measures --- p.20 / Test subjects --- p.21 / Sample processing --- p.23 / DNA extraction --- p.24 / Chapter 3. --- System Development / Choice of primer and probe sequences --- p.28 / Optimization of PCR conditions --- p.30 / Imprecision of TaqMan assays --- p.33 / MTRNR2 vs. MTCYB probes --- p.33 / Construction of standard curve --- p.35 / Chapter 4. --- Research Application / The trauma model --- p.37 / Plasma DNA as a marker for trauma severity --- p.37 / Results --- p.38 / Disussion --- p.41 / Chapter 5. --- Conclusion --- p.44 / Chapter 6. --- References --- p.45

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 1997. / Includes bibliographical references. Also available on the Internet.