This thesis describes the investigation of quantum dot-doped particle fluorescent technology commercially available for its application to analyte profiling in suspension. The first part of the thesis described the characterisation of the quantum dot-encoded microspheres, QDEMs, developed by Crystalplex (PA, USA). The multiple fluorescence signatures of QDEMs were analysed using microscopy and flow cytometry technology which provided high-content measurements with a single excitation sources and multiple emission wavelength detectors. The sensitivity and stability of the materials was evaluated under typical biomedical conditions encounter in multiple analyte suspension assays. Novel analytical parameters were defined to study QDEM stability and confocal microscopy detection system was used to provide structural and fluorescent imagines of the fluorescent microspheres under various conditions. Composition of the aqueous environment, temperature and physical forces applied to QDEM induced changes in their fluorescent codes and structural properties. Optimal conditions were then defined for the application of the material to biomedical assays. In a second stage, a conjugation method was developed to produce optimised QDEM bioconjugates for the detection of single strand DNA in suspension. The impact of the conjugation buffer, the concentration and the structure of oligonucleotides was evaluated to optimise QDEM bioconjugates. Then, a novel approach was investigated to optimise the hybridisation of ssDNA to QDEM bioconjugates. Experimental design with response surface methodology determined optimum conditions for the hybridisation of oligonucleotides to QDEM surface in suspension array. Finally, the specific hybridisation of ssDNA to QDEM bioconjugates in a small liquid format adapted to single nucleotide polymorphism detection was demonstrated. The work presented here shows the potential of QDEM bioconjugates for suspension array technology and DNA genotyping. Further, this report highlights the challenges that remain for QDEM fluorescent technology to be reliable for biomedical and suspension array applications.

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.

No description available.

Research on human genetic variation has shown that the human genome is not a fixed, invariant framework, but that there can be extensive structural variation. This variation includes copy number variation (CNV), which can lead to changes in DNA dosage contributing significantly to variation between individual human genomes and heritable traits. Human beta-defensins are small, secreted antimicrobial peptides encoded by DEFB genes located in a cluster of at least seven genes on 8p23.1. These genes are highly variable in copy number but accurate measurement of multiallelic copy number variants is challenging, particularly for high copy numbers, and has not been intensively studied until recently. A new PRT-based (Paralogue Ratio Test) triplex assay was developed to accurately measure the multiallelic beta-defensin copy number variation. The Triplex assay was demonstrated to be an accurate and powerful method to measure copy number variation in large case-control association studies. This method was used to study the beta-defensin CNV in psoriasis disease, showing that high beta-defensin copy number is associated with susceptibility to psoriasis in Caucasians. Studying population variation of CNV showed that variation in copy number of beta-defensin is not significantly different across human populations. To understand the evolutionary history of beta-defensin CNV in the primate lineage, the study of CNV at this locus was carried out in great apes. Beta-densin genes are copy variable in human and chimpanzee, but not in gorilla, suggesting that variation in copy number of beta-defensin genes may have arisen in the human-chimpanzee lineage after the divergence with gorilla.

Mitochondrial DNA analysis was undertaken on samples collected from two populations in Peninsular Malaysia, the Modern Malay (102 samples) and Orang Asli (59 samples from Jahai and Kinsiu sub-groups). The hypervariable region 1 (HV1) of the mtDNA control region was amplified and sequenced. Polymorphisms were reported by aligning each sequence to the Cambridge Reference Sequence (CRS). A total of 94 polymorphisms were observed in the Modern Malay samples, which formed 75 different haplotypes. The Orang Asli showed notably lower number of the HV1 region variations, with only 28 polymorphisms and 13 haplotypes observed. Genetic diversity calculated for the Modern Malays and Orang Asli were 0.989 and 0.818, respectively. Probability of random match calculated was 0.0202 for the Modern Malays and 0.1962 for the Orang Asli. The mtDNA coding region variations was examined using RFLP analysis. Combination of both RFLP and HV1 sequence data had placed the Modern Malays into three major Southeast Asian haplogroups, M, B and F. These findings had initially suggested that the Modern Malays shared a common lineage with other populations within this region. Two novel sub-clusters, M21a and R21 were found at a high frequency within the Orang Asli samples. These sub-clusters, which have also been found in other Semang sub-groups appear to be indigenous Semang haplogroups. The limited number of mtDNA haplotypes shared between the Modern Malays and Orang Asli suggested discontinuity of mtDNA between these populations. Even though both populations were believed to be among the earliest populations of Peninsular Malaysia, this result indicates that the Modern Malays were not direct descendants of the Orang Asli. Minisequencing analysis was carried for further interrogation of the mtDNA coding region polymorphisms. Besides mtDNA analysis, the autosomal STR markers were also examined using PowerPlex® 16 system for both populations. These data could provide more information when added to the available STR database for Malaysian populations.

In eukaryotes, recombination plays a critical role in both the production of viable gametes and as a population genetic process. Here, we are interested in studying recombination as it provides insight into a process that has shaped variation. To this end, we study the evolution of cross-over rates in chimpanzees and humans through two experiments. Components of the recombination machinery are well described in yeast and C. elegans, but less so in other species. In humans, cross-over rates vary across physical scales and occur predominantly in narrow ∼2 kb regions called hotspots, where hotspot usage differs considerably between individuals. Differential hotspot usage is associated with specific DNA motifs, and DNA-contacting zinc finger array variants in the transacting PRDM9 H3K4 trimethyltransferase. The precise relationship between DNA motifs, PRDM9 and hotspot activity is not completely understood. Experiment 1. To investigate the importance of PRDM9 motif recognition, which is predicted be different between humans and chimpanzees, and the effect of PRDM9 on the evolution of fine-scale cross-over rates, we sequenced 10 unrelated Pan troglodytes verus (Western chimpanzee) genomes to moderate coverage (∼10×). I validate the approach by demonstrating that fine-scale maps estimated from 10 human genomes of each African and European ancestry recapitulate independently estimated maps. Then I characterise the error modes in sequencing data arising from errors in chemistry, alignment, variant calling, and genotyping. I identify several cryptic error modes missed by state-of-the-art filters and develop methods to counteract them. To guard against genotype error arising from stochastic variation in low to moderate coverage sequencing, I develop methods to incorporate the underlying statistical uncertainty into recombination analyses, evaluate the approaches through simulation (estimated 11% improvement) and empirical assessment (estimated 4% improvement), and discover that the reported genotype uncertainty is poorly calibrated, which limits the approaches. Consequently, a filtering approach was applied to the hard-called chimpanzee genotypes. I estimate recombination rates in chimpanzees through an existing LD-based method. In contrast to humans, there is no increased cross-over localisation around chimpanzee PRDM9 binding predictions, nor motifs consistently associated with activity. Hotspots do not overlap between the two species, indicating that rates evolved rapidly and consistent with PRDM9 localising all hotspots. In contrast, gene pro- moters and CpG islands are common attractors of recombination (2.7-fold increase in rate in chimpanzee, 1.5-fold increase in human), suggesting chromatin state influences hotspot placement but to varying degree in the species. I discuss the potential implications for PRDM9 mechanism. Experiment 2. To enable a more representative characterisation of the spectrum of genome changes occurring in chimpanzee genomes, I analyse data from an extended three generation Western chimpanzee pedigree sequenced at high coverage (∼30×). I use Mendel transmission to filter variants, infer haplotypes, and identify recombination events through a Hidden Markov Model approach. We detect 375 recombination events, of which 3 are double cross-over events. Sex-specific recombination rate estimates in chimpanzees mirror sex differences in humans (N♂/N♀ = 0.58) and have similar levels of total recombination. We resolve recombination events typically at ∼ 856 base-pair resolution. Additionally, analyses of Mendel inconsistencies suggest that extended pedigree sequencing opens the door on studying complex genome changes. These experiments demonstrate the power of comparative analyses, the utility of high throughput sequencing in enabling the study of recombination in almost any species of interest, the challenges in sifting signal from noise in these data, and the need for experimental and algorithmic methods to guard against error.