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1
Glentis, S. "Whole genome amplification for PGD and PND : molecular and a-CGH diagnosis." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18554/.
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Whole genome amplification amplifies the entire genome in a few hours from samples of minimal DNA quantities, even from single cells. This may have many applications, especially in prenatal diagnosis, PGD and PGS. The hypothesis for chapter 3 was: Can multiple displacement amplification (MDA) be used as a universal step prior to molecular analysis for PGD? WGA using MDA (Qiagen) was used on single cells in order to overcome the problem of limited DNA in PGD. MDA allows the diagnosis through haplotyping or a combination of direct and indirect mutation analysis. Different cell types, including buccal cells, lymphocytes, fibroblasts and blastomeres were examined. A modification on the cell lysis buffer was also tested in order to achieve more accurate results. PGD seems to benefit from MDA when multiple tests are performed for direct and indirect analysis. The modified lysis buffer (exclusion of DTT) produced better results than the other lysis buffers and buccal cells do not produce as accurate results as other cell types. The hypothesis was met as the amount of DNA produced by MDA can be used for direct and indirect testing and haplotyping. The hypothesis for chapter 4 was: Is it possible to accurately assess the chromosomes of a single cell by a-CGH? WGA was achieved by MDA and GenomePlex (Sigma) on single lymphocytes, fibroblasts and blastomeres prior to a-CGH analysis. The difficulty of this technique was the high background noise that was produced by WGA that makes interpretation difficult. Different lysis buffers, modifications of the WGA reaction and analysis software were examined for better results. A-CGH slides from different companies and institutions were used. The results showed that GenomePlex produced less background noise compared to MDA but the amplification efficiency of the technique was less reliable. The BlueGnome Cytochip arrays produced the best compared to arrays from any other companies or institutions. More experiments would be necessary to determine if the hypothesis was met as a number of chromosomal abnormalities detected were not always confirmed by other experiments. The hypothesis for chapter 5 was: Can aneuploidy be detected in coelomic fluid using a-CGH? The possibility of using WGA and a-CGH on coelomic fluid was tested as this could be used as an early form of prenatal diagnosis. Coelomic fluid was collected between the 5th and 11th week of pregnancy from women undergoing termination of pregnancy. MDA and GenomePlex were used to amplify the DNA prior to a-CGH analysis. Both genomic (high resolution) and constitutional (low resolution) arrays were tested. The results showed that aneuploidy can be detected by a-CGH. BlueGnome Cytochip slides produced the best results. A triploid sample was detected as normal. The hypothesis was met and even higher resolution could be achieved with the use of GenomePlex and BlueGnome Cytochip arrays. WGA may be very important for downstream genetic tests when the DNA is from very low quality and quantity. Further optimisation of the technique is needed in order to achieve similar results to those of good quality genomic DNA. Arrays from different companies or institutions may produce very different results. In conclusion, the results showed that WGA can benefit PGD and PND, and a-CGH gives great potential to PGS and coelomic fluid diagnosis.
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Jiang, Sheng. "Application of nested PCR, whole genome amplification and comparative genomic hybridisation for single cell genetic analysis." Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366140.
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Anscombe, C. J. "Multiple displacement amplification and whole genome sequencing for the diagnosis of infectious diseases." Thesis, Queen Mary, University of London, 2016. http://qmro.qmul.ac.uk/xmlui/handle/123456789/18409.
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Next-generation sequencing technologies are revolutionising our ability to characterise and investigate infectious diseases. Utilising the power of high throughput sequencing, this study reports, the development of a sensitive, non-PCR based, unbiased amplification method, which allows the rapid and accurate sequencing of multiple microbial pathogens directly from clinical samples. The method employs Φ29 DNA polymerase, a highly efficient enzyme able to produce strand displacement during the polymerisation process with high fidelity. Problems with DNA secondary structure were overcome and the method optimised to produce sufficient DNA to sequence from a single bacterial cell in two hours. Evidence was also found that the enzyme requires at least six bases of single stranded DNA to initiate replication, and is not capable of amplification from nicks. Φ29 multiple displacement amplification was shown to be suitable for a range of GC contents and bacterial cell wall types as well as for viral pathogens. The method was shown to be able to provide relative quantification of mixed cells, and a method for quantification of viruses using a known standard was developed. To complement the novel molecular biology workflow, a data analysis pipeline was developed to allow pathogen identification and characterisation without prior knowledge of input. The use of de novo assemblies for annotation was shown to be equivalent to the use of polished reference genomes. Single cell Φ29 MDA samples had better assembly and annotation than non-amplification controls, a novel finding which, when combined with the very long DNA fragments produced, has interesting implications for a variety of analytical procedures. A sampling process was developed to allow isolation and amplification of pathogens directly from clinical samples, with good concordance shown between this method and traditional testing. The process was tested on a variety of modelled and real clinical samples showing good application to sterile site infections, particularly bacteraemia models. Within these samples multiple bacterial, viral and parasitic pathogens were identified, showing good application across multiple infection types. Emerging pathogens were identified including Onchocerca volvulus within a CSF sample, and Sneathia sanguinegens within an STI sample. Use of Φ29 MDA allows rapid and accurate amplification of whole pathogen genomes. When this is coupled with the sample processing developed here it is possible to detect the presence of pathogens in sterile sites with a sensitivity of a single genome copy.
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Borgström, Erik. "Technologies for Single Cell Genome Analysis." Doctoral thesis, KTH, Genteknologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-181059.
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During the last decade high throughput DNA sequencing of single cells has evolved from an idea to one of the most high profile fields of research. Much of this development has been possible due to the dramatic reduction in costs for massively parallel sequencing. The four papers included in this thesis describe or evaluate technological advancements for high throughput DNA sequencing of single cells and single molecules. As the sequencing technologies improve, more samples are analyzed in parallel. In paper 1, an automated procedure for preparation of samples prior to massively parallel sequencing is presented. The method has been applied to several projects and further development by others has enabled even higher sample throughputs. Amplification of single cell genomes is a prerequisite for sequence analysis. Paper 2 evaluates four commercially available kits for whole genome amplification of single cells. The results show that coverage of the genome differs significantly among the protocols and as expected this has impact on the downstream analysis. In Paper 3, single cell genotyping by exome sequencing is used to confirm the presence of fat cells derived from donated bone marrow within the recipients’ fat tissue. Close to hundred single cells were exome sequenced and a subset was validated by whole genome sequencing. In the last paper, a new method for phasing (i.e. determining the physical connection of variant alleles) is presented. The method barcodes amplicons from single molecules in emulsion droplets. The barcodes can then be used to determine which variants were present on the same original DNA molecule. The method is applied to two variable regions in the bacterial 16S gene in a metagenomic sample. Thus, two of the papers (1 and 4) present development of new methods for increasing the throughput and information content of data from massively parallel sequencing. Paper 2 evaluates and compares currently available methods and in paper 3, a biological question is answered using some of these tools.QC 20160127
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Dillon, Candace. "Assessment of pre-PCR whole genome amplification of single pollen grains using flowering dogwood (Cornus florida)." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1865.
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Studies of gene flow in natural plant populations often focus on either historical or abiotic dispersal methods (e.g. wind, water, gravity), but there is little information available on contemporary, animal-mediated pollen dispersal patterns. Emerging molecular laboratory techniques allow unprecedented insights into spatial patterns of pollen-mediated gene flow. However, to date, technical challenges have limited their widespread application. The genome of a pollen grain can be amplified via whole genome amplification (WGA) prior to traditional amplification via polymerase chain reaction (PCR) to prevent the stochastic effects associated with low copy number amplification. Even still, WGA can suffer from low success rates or poor repeatability. The present study examined the extent to which WGA can be used to aid in understanding insect-mediated pollen flow in Cornus florida (flowering dogwood) within Virginia Commonwealth University’s Inger and Walter Rice Center for Environmental Life Sciences. Initial amplification of DNA isolated from frozen grains was successful, until the pollen had been stored longer than 120 days at -20ºC. After this time point, the PCR targets failed to amplify. The percent success of downstream PCR amplification on fresh pollen grains varied from 20% to 100%, with an average of 62% success. The addition of a common molecular crowder, polyethylene glycol, produced consistent amplification, regardless of input DNA concentration and eliminated the need for triplicate samples. Successful pollination and subsequent reproduction of flowering plants has a substantial ecological and agricultural importance that warrants increased understanding into how insects move pollen across the landscape. Determining the haploid profiles of a single pollen grain will allow scientists to elucidate dispersal patterns of pollen grains and track the movement and efficiency of biotic pollinators.
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Freedman, Benjamin Gordon. "Degenerate oligonucleotide primed amplification of genomic DNA for combinatorial screening libraries and strain enrichment." Diss., Virginia Tech, 2014. http://hdl.handle.net/10919/71346.
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Combinatorial approaches in metabolic engineering can make use of randomized mutations and/or overexpression of randomized DNA fragments. When DNA fragments are obtained from a common genome or metagenome and packaged into the same expression vector, this is referred to as a DNA library. Generating quality DNA libraries that incorporate broad genetic diversity is challenging, despite the availability of published protocols. In response, a novel, efficient, and reproducible technique for creating DNA libraries was created in this research based on whole genome amplification using degenerate oligonucleotide primed PCR (DOP-PCR). The approach can produce DNA libraries from nanograms of a template genome or the metagenome of multiple microbial populations. The DOP-PCR primers contain random bases, and thermodynamics of hairpin formation was used to design primers capable of binding randomly to template DNA for amplification with minimal bias. Next-generation high-throughput sequencing was used to determine the design is capable of amplifying up to 98% of template genomic DNA and consistently out-performed other DOP-PCR primers.
Application of these new DOP-PCR amplified DNA libraries was demonstrated in multiple strain enrichments to isolate genetic library fragments capable of (i) increasing tolerance of E. coli ER2256 to toxic levels of 1-butanol by doubling the growth rate of the culture, (ii) redirecting metabolism to ethanol and pyruvate production (over 250% increase in yield) in Clostridium cellulolyticum when consuming cellobiose, and (iii) enhancing L-arginine production when used in conjunction with a new synthetic gene circuit.Ph. D.
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Lu, Sijia. "Label-Free Optical Imaging of Chromophores and Genome Analysis at the Single Cell Level." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10563.
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Since the emergence of biology as a quantitative science in the past century, a lot of biological discoveries have been driven by milestone technical advances such as X-ray crystallography, fluorescence microscopy and high-throughput sequencing. Fluorescence microscopy is widely used to explore the nanoscale cellular world because of its superb sensitivity and spatial resolution. However, many species (e.g. lipids, small proteins) are non-fluorescent and are difficult to label without disturbing their native functions. In the first part of the dissertation, we explore using three different contrast mechanisms for label-free imaging of these species – absorption and stimulated emission (Chapter 2), heat generation and diffusion (Chapter 3) and nonlinear scattering (Chapter 4). We demonstrate label-free imaging of blood vessels, cytochromes, drugs for photodynamic therapy, and muscle and brain tissues with three dimensional optical sectioning capability. With the rapid development of high throughput genotyping techniques, genome analysis is currently routinely done genome-wide with single nucleotide resolution. However, a large amount of starting materials are often required for whole genome analysis. The dynamic changes in DNA molecules generate intra-sample heterogeneity. Even with the same genome content, different cells often have very different transcriptome profiles in a functional organism. Such intra-sample heterogeneities in the genome and transcriptome are often masked by ensemble analysis. In this second part of the dissertation, we first introduce a whole genome amplification method with high coverage in sequencing single human cells (Chapter 6). We then use the technique to study meiotic recombinations in sperm cells from an individual (Chapter 7). We further develop a technique that enables digital counting of genome fragments and whole genome haplotyping in single cells (Chapter 8). And we introduce our ongoing efforts on single cell transcriptome analysis (Chapter 9). In the end, we introduce our initial effort in exploring the genome accessibility at the single cell level (Chapter 9). Through the development of techniques probing the single cell genome, transcriptome and possibly epigenome, we hope to provide a toolbox for studying biological processes with genome-wide and single cell resolution.Chemistry and Chemical Biology
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Du, Breil de Pontbriand Alexandra. "Cartographie des génomes par HAPPY mapping. Développement d'une amplification "whole genome" et validation sur cartes comparatives homme/chimpanzé/gorille." Rennes 1, 2003. http://www.theses.fr/2003REN10104.
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La comparaison des génomes de grands singes et de l'homme devrait apporter de nouvelles informations concernant l'évolution de l'homme, ces différents genomes restant très proches. Le caryotype humain possède une paire de chromosomes en moins, le chromosome 2 humain étant issu de la fusion télomérique de deux chromosomes ancestraux de singes. Afin de pouvoir mettre en evidence les réarrangements qui ont amené à l'apparition de l'espèce humaine, nous avons décidé d'entreprendre une étude comparative entre l'homme, le chimpanzé et le gorille. Pour y parvenir, nous avons utilisé le HAPPY mapping afin de cartographier plusieurs centaines de marqueurs sur ces trois génomes. Ceci nous a permis d'identifier plusieurs réarrangements chromosomiques, en particulier une inversion et une translocation, et de préciser le point de rupture de syntenie ayant conduit à la formation du chromosome 2 humain. La localisation de ce dernier ayant été confirmée par des expériences de FISH.
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Sandberg, Julia. "Massively parallel analysis of cells and nucleic acids." Doctoral thesis, KTH, Genteknologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-45671.
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Recent proceedings in biotechnology have enabled completely new avenues in life science research to be explored. By allowing increased parallelization an ever-increasing complexity of cell samples or experiments can be investigated in shorter time and at a lower cost. This facilitates for example large-scale efforts to study cell heterogeneity at the single cell level, by analyzing cells in parallel that also can include global genomic analyses. The work presented in this thesis focuses on massively parallel analysis of cells or nucleic acid samples, demonstrating technology developments in the field as well as use of the technology in life sciences. In stem cell research issues such as cell morphology, cell differentiation and effects of reprogramming factors are frequently studied, and to obtain information on cell heterogeneity these experiments are preferably carried out on single cells. In paper I we used a high-density microwell device in silicon and glass for culturing and screening of stem cells. Maintained pluripotency in stem cells from human and mouse was demonstrated in a screening assay by antibody staining and the chip was furthermore used for studying neural differentiation. The chip format allows for low sample volumes and rapid high-throughput analysis of single cells, and is compatible with Fluorescence Activated Cell Sorting (FACS) for precise cell selection. Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences by constantly producing increasing amounts of data from one sequencing run. However, the reagent costs and labor requirements in current massively parallel sequencing protocols are still substantial. In paper II-IV we have focused on flow-sorting techniques for improved sample preparation in bead-based massive sequencing platforms, with the aim of increasing the amount of quality data output, as demonstrated on the Roche/454 platform. In paper II we demonstrate a rapid alternative to the existing shotgun sample titration protocol and also use flow-sorting to enrich for beads that carry amplified template DNA after emulsion PCR, thus obtaining pure samples and with no downstream sacrifice of DNA sequencing quality. This should be seen in comparison to the standard 454-enrichment protocol, which gives rise to varying degrees of sample purity, thus affecting the sequence data output of the sequencing run. Massively parallel sequencing is also useful for deep sequencing of specific PCR-amplified targets in parallel. However, unspecific product formation is a common problem in amplicon sequencing and since these shorter products may be difficult to fully remove by standard procedures such as gel purification, and their presence inevitably reduces the number of target sequence reads that can be obtained in each sequencing run. In paper III a gene-specific fluorescent probe was used for target-specific FACS enrichment to specifically enrich for beads with an amplified target gene on the surface. Through this procedure a nearly three-fold increase in fraction of informative sequences was obtained and with no sequence bias introduced. Barcode labeling of different DNA libraries prior to pooling and emulsion PCR is standard procedure to maximize the number of experiments that can be run in one sequencing lane, while also decreasing the impact of technical noise. However, variation between libraries in quality and GC content affects amplification efficiency, which may result in biased fractions of the different libraries in the sequencing data. In paper IV barcode specific labeling and flow-sorting for normalization of beads with different barcodes on the surface was used in order to weigh the proportion of data obtained from different samples, while also removing mixed beads, and beads with no or poorly amplified product on the surface, hence also resulting in an increased sequence quality. In paper V, cell heterogeneity within a human being is being investigated by low-coverage whole genome sequencing of single cell material. By focusing on the most variable portion of the human genome, polyguanine nucleotide repeat regions, variability between different cells is investigated and highly variable polyguanine repeat loci are identified. By selectively amplifying and sequencing polyguanine nucleotide repeats from single cells for which the phylogenetic relationship is known, we demonstrate that massively parallel sequencing can be used to study cell-cell variation in length of these repeats, based on which a phylogenetic tree can be drawn.QC 20111031
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Gabrieli, A. "STUDIO DI TECNOLOGIE DI AMPLIFICAZIONE E GENOTIPIZZAZIONE DEL GENOMA SU CAMPIONI DI DNA PROVENIENTI DA SANGUE E DA CELLULE DELLA BOCCA PER APPLICAZIONI IN AMBITO EPIDEMIOLOGICO." Doctoral thesis, Università degli Studi di Milano, 2010. http://hdl.handle.net/2434/150115.
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In epidemiological studies the amount of biological material available is a limiting factor. Many studies use DNA as biological sample obtained by venipuncture, but this collection method is invasive especially if donors are children and the elderly. The use of mouth cells can be an alternative source, although you get DNA of poor quality and quantity. To increase the amount of DNA extracted from buccal cells, you can use the "Whole Genome Amplification”. The aim of my PhD project was to develop a method to extract DNA from buccal cells and to study amplification technologies and subsequent genotyping of DNA extracted from blood and buccal cells. The accuracy of WGA was evaluated with different techniques of molecular biology and genotyping: direct sequencing, allelic discrimination assays, microsatellite genotyping and ”genome wide analysis”. Our analysis showed that the WGA can be used to increase the amount of starting biological material, however, it has some limitations, the fact that direct sequencing and analysis with microsatellites in some cases, may cause a loss of 'genetic information’. According to the data found using DNA from buccal cells and WGA, we have genotyped GSTP1 gene polymorphism Ile105/Val105 about 103 people in the Milan area through Real Time. The study of allele frequencies of this polymorphism in the GSTP1 gene is part of a project aiming to determine whether in patients with respiratory diseases there is an interaction between individual genetic predisposition and exposure to a common external agent coming from urban pollution. The genotypic frequencies obtained in our population were not significantly different from those of Tuscany population genotyped for the HapMap project, so our samples will be used as reference for future studies. Furthermore, we showed that both buccal cells and the WGA can be used in epidemiological analysis for genotyping through Real Time PCR. WGA may be a useful way to increase the amount of DNA; DNA extracted from buccal cells can be a valuable resource for genetic studies.
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Forst, Jannine. "Detecting and sequencing Mycobacterium tuberculosis aDNA from archaeological remains." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/detecting-and-sequencing-mycobacterium-tuberculosis-adna-from-archaeological-remains(a806f3a9-8d22-4395-a1ff-a3ffbcb1c8cc).html.
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Tuberculosis has been an important disease throughout human history, shaping countless past populations. The archaeological study of the causative agents of tuberculosis, members of the Mycobacterium tuberculosis Complex (MTBC), is hindered by the non-diagnostic nature of tuberculosis-associated skeletal changes. As such, ancient DNA (aDNA) or palaeogenetic analyses have become an important tool for identifying tuberculosis in past populations. However, due to the age and variable preservation of aDNA, there are often issues with sporadic results and false negatives. The overall aim of the work presented here was to use different methods, including traditional target-specific PCR, to identify and detect tuberculosis aDNA in archaeological remains. The main objectives within this overarching aim were to first test a method called whole genome amplification (WGA), used to non-specifically amplify all the DNA within a sample, and its potential to improve the yield of aDNA from skeletal remains (Chapters 3 and 4). To determine the extent of its impact, WGA was used in a comparative context, where each archaeological sample analysed was separately subjected to two methods of MTBC detection - the traditional targeted PCR method and the same method assisted by the initial application of WGA. The results show that applying WGA before the traditional targeted PCR methodology to detect the presence of MTBC pathogens in skeletal remains is only useful and viable in some cases, likely depending on the age and preservation of the sample. The second objective was to use next generation sequencing to obtain more information on the aDNA composition of certain archaeological samples and answer questions beyond the scope of traditional target-specific PCR techniques (Chapter 5). Although most of the sequencing runs were variably unsuccessful, the composition of two samples, both known to probably contain tuberculosis aDNA, could be analysed. The samples both contained similar amounts of mycobacterial aDNA and varying amounts of both human and even potentially human intestinal flora DNA. Finally, the third objective was to determine if MTBC aDNA could be detected in a rib sample from Private William Braine of the lost Franklin Expedition using standard target-specific PCR (Chapter 6). In this case study, no evidence of tuberculosis ancient DNA was found. The work done through-out highlights the difficulties of ancient DNA research and, in Chapter 4, shows the importance of using more than a single sample to evaluate methods for application in palaeogenetic contexts.
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Jere, Khuzwayo Chidiwa. "Whole genome characterisation and engineering of chimaeric rotavirus-like particles using African rotavirus field strains / Khuzwayo Chidiwa Jere." Thesis, North-West University, 2012. http://hdl.handle.net/10394/8502.
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Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and
RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young
mammals and the need for further development of additional rotavirus vaccines, especially
vaccines effective against regional strains in developing country settings, is increasing. The
design and formulation of new effective multivalent rotavirus vaccines is complicated by the
wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the
propensity of rotaviruses to evolve using mechanisms such as point mutation, genome
segment reassortment, genome segment recombination and interspecies transmission.
Mutations occurring within the primer binding regions targeted by the current commonly
employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel
mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with
online rotavirus genotyping tools will help to understand the complete epidemiology of the
circulating strains which, in turn, is vital for developing intervention measures such as
vaccine and anti-viral therapies.
In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides
that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing,
and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole
genome of rotaviruses. The robustness of this approach was demonstrated in characterising
the complete genetic constellations and evolutionary origin of selected human rotavirus
strains that emerged in the past two decades worldwide, human rotavirus strains frequently
detected in Africa, and the whole genomes of some common strains frequently detected in
bovine species. Most of the characterised strains emerged either through intra- or interspecies
genome segment reassortment processes. The methods used in this study also allowed
determination of the whole consensus genome sequence of multiple rotavirus variants present
in a single stool sample and the elucidation of the evolutionary mechanisms that explained
their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype
rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2)
and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent
amplification of the rotavirus genomes allowed the determination of the consensus nucleotide
sequence for each of the genome segments of the selected study strains directly from stool
sample.
The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and
VP7 of some of the study strains were codon optimised for insect cell expression and used to
generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used
to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs
contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/
ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of
various combinations of VP4 and VP7 were assembled. The outer capsid proteins were
derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or
P[8] genotypes that were directly extracted from human stool faecal specimens. The
structures of these chimaeric RV-VLPs were morphologically evaluated using transmission
electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered
(dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant
rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the
assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs.
These RV-VLPs will be evaluated in future animal studies as potential non-live
rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this
study, namely by using the consensus nucleotide sequence derived from dsRNA extracted
directly from clinical specimens, should speed up vaccine research and development by
bypassing the need to adapt the viruses to tissue culture and circumventing some other
problems associated with cell culture adaptation as well. Thus, it is now possible to generate
RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field
rotavirus strain.Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012
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Adèle-dit-Renseville, Nathalie. "Développement d'une technique d'analyse d'échantillons limites en quantité d'ADN adaptée aux applications médicales et médico-légales." Nantes, 2010. http://www.theses.fr/2010NANT2113.
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Jiao, Xiang. "Somatic Mutations in Breast Cancer Genomes : Discovery and Validation of Breast Cancer Genes." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-182319.
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Breast cancer is the most common cancer in women worldwide. However, the genetic alterations that lead to breast cancer are not fully understood. This thesis aims to identify novel genes of potential mechanistic, diagnostic or therapeutic interest in breast cancers by mutational analysis and whole-genome sequencing. In paper I, sequencing of 36 previously identified candidate genes in 96 breast tumors with patient-matched normal DNA determined the somatic mutation prevalence of these candidate genes and identified additional mutations in Notch, NF-κB, PI3K, and Hedgehog pathways as well as in processes mediating DNA methylation, RNA processing and calcium signaling. In paper II, comparison of massively parallel mate-pair sequencing results of a human genome before and after phi29-mediated multiple displacement amplification (MDA) revealed that MDA introduces structural alteration artifacts, with an emphasis on false positive inversions, and impairs the sensitivity to detect true inversions. Therefore, MDA has limited value in sample preparation for whole-genome sequencing for structural alteration detection. In paper III, massively parallel paired-end sequencing identified gene rearrangements in 15 hormone receptor negative breast cancers. Forty validated rearrangements were predicted to directly affect 30 genes, involved in epigenetic regulation, cell mitosis, signalling transduction and glycolytic flux. RNA interference-based assays revealed the potential roles in cell growth of some affected genes, among which DDX10 was implicated to be involved in apoptosis. In paper IV, a method for statistical evaluation of putative translocations detected by massively parallel paired-end sequencing was proposed. In an application of this method to analyse translocations detected by cancer genome deep paired-end sequencing, 76 putative translocations were classified into four categories, with the majority likely to be caused by mismapping due to repetitive regions. Taken together, this thesis provides insights into genes and pathways mutated in sporadic breast cancer genomes, which broaden our understanding of the genetic basis of breast cancer and may ultimately facilitate the diagnosis and treatment of this disease.
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Rönn, Ann-Charlotte. "Analysis of Nucleotide Variations in Non-human Primates." Doctoral thesis, Uppsala University, Molecular Medicine, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7904.
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Many of our closest relatives, the primates, are endangered and could be extinct in a near future. To increase the knowledge of non-human primate genomes, and at the same time acquire information on our own genomic evolution, studies using high-throughput technologies are applied, which raises the demand for large amounts of high quality DNA.
In study I and II, we evaluated the multiple displacement amplification (MDA) technique, a whole genome amplification method, on a wide range of DNA sources, such as blood, hair and semen, by comparing MDA products to genomic DNA as templates for several commonly used genotyping methods. In general, the genotyping success rate from the MDA products was in concordance with the genomic DNA. The quality of sequences of the mitochondrial control region obtained from MDA products from blood and non-invasively collected semen samples was maintained. However, the readable sequence length was shorter for MDA products.
Few studies have focused on the genetic variation in the nuclear genes of non-human primates. In study III, we discovered 23 new single nucleotide polymorphisms (SNPs) in the Y-chromosome of the chimpanzee. We designed a tag-microarray minisequencing assay for genotyping the SNPs together with 19 SNPs from the literature and 45 SNPs in the mitochondrial DNA. Using the microarray, we were able to analyze the population structure of wild-living chimpanzees.
In study IV, we established 111 diagnostic nucleotide positions for primate genera determination. We used sequence alignments of the nuclear epsilon globin gene and apolipoprotein B gene to identify positions for determination on the infraorder and Catarrhini subfamily level, respectively, and sequence alignments of the mitochondrial 12S rRNA (MT-RNR1) to identify positions to distinguish between genera. We designed a microarray assay for immobilized minisequencing primers for genotyping these positions to aid in the forensic determination of an unknown sample.
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Halilovic, Amina. "SÄKERSTÄLLNING AV SÄLLSYNTA DNA-KONTROLLER MED HELGENOMAMPLIFIERING I KLINISKT SYFTE." Thesis, Malmö högskola, Fakulteten för hälsa och samhälle (HS), 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-24387.
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Vid klinisk enbaspolymorfi (SNP) analys inkluderas DNA-kontroller med kända genotyper i varje analysomgång för att säkerställa riktigheten vad gäller analysresultatet. DNA-kontrollerna har en central roll för resultatens trovärdighet vid genotypningen. Vissa kontrollprover som används är av sällsynt genotyp och kan vara mycket svåra att få tag på. Detta arbete har utförts för att undersöka om det går att erhålla DNA-material från sällsynta genotyper med hjälp av helgenomamplifiering och på så sätt säkerställa en tillgång till dessa. I arbetet testades helgenomamplifiering med hjälp av två olika kit. De helgenomamplifierade produkternas kvantitet och kvalitet analyserades och jämfördes med det ursprungliga DNA:t, med avsikt att redogöra för det mest fördelaktiga kitet för SNP-analys i kliniskt syfte. Båda helgenomamplifierings-kiten påvisade god förmåga att amplifiera genomiskt DNA med hög kvalité. Helgenomamplifierat DNA från det bästa kitet sekvenserades och här var skillnader mellan ursprungligt och helgenomamplifierat DNA marginella. Vid sekvensanalys av ett 464 baspar långt fragment av faktor II genen och 585 baspar långt fragment av ApoE genen på fem helgenomamplifierade DNA-prover påvisades endast en eventuell diskrepans.Clinical single nucleotide polymorphisms (SNP) analysis includes DNA controls with known genotypes in each run to ensure the accuracy of the analysis results. DNA controls have a central role for the credibility of the results in the genotyping process. Some of the used control samples are rare and can be very difficult to obtain. This work was carried out to investigate whether it is possible to obtain DNA from samples with a rare genotype using whole genome amplification and as a result ensure access to these samples. In this work the whole genome amplification method was tested by two different kits. The quantity and quality of the whole genome amplification products were analyzed and compared with the original DNA, with the intention to describe the most advantageous kit for clinical SNP analysis. Both tested kits demonstrated a good ability to amplify genomic DNA with high quality. Whole genome amplified DNA from the best kit was sequenced and the difference between the original DNA and whole genome amplified DNA was negligible. Sequence analysis of 464 base pairs of the factor II gene and 585 base pairs of the ApoE gene in five whole genome amplified DNA samples indicated only one possible discrepancy.
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Muharam, Firman Alamsyah. "Overcoming problems with limiting DNA samples in forensics and clinical diagnostics using multiple displacement amplification." Thesis, Queensland University of Technology, 2006. https://eprints.qut.edu.au/16207/1/Firman_Muharam_Thesis.pdf.
Full textAbstract:
The availability of DNA samples that are of adequate quality and quantity is essential for any genetic analysis. The fields of forensic biology and clinical diagnostic pathology testing often suffer from limited samples that yield insufficient DNA material to allow extensive analysis. This study examined the utility of a recently introduced whole genome amplification method termed Multiple Displacement Amplification (MDA) for amplifying a variety of limited sample types that are commonly encountered in the fields of forensic biology and clinical diagnostics. The MDA reaction, which employs the highly processive bacteriophage φ29 DNA polymerase, was found to generate high molecular weight template DNA suitable for a variety of downstream applications from low copy number DNA samples down to the single genome level. MDA of single cells yielded sufficient DNA for up to 20,000,000 PCR assays, allowing further confirmatory testing on samples of limited quantities or the archiving of precious DNA material for future work. The amplification of
degraded DNA material using MDA identified a requirement for samples of sufficient quality to allow successful synthesis of product DNA templates. Furthermore, the utility of MDA products in comparative genomic hybridisation
(CGH) assays identified the presence of amplification bias. However, this bias was overcome by introducing a novel modification to the MDA protocol. Future directions for this work include investigations into the utility of MDA products in short tandem repeat (STR) assays for human identifications and application of the modified MDA protocol for testing of single cell samples for genetic abnormalities.
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Muharam, Firman Alamsyah. "Overcoming problems with limiting DNA samples in forensics and clinical diagnostics using multiple displacement amplification." Queensland University of Technology, 2006. http://eprints.qut.edu.au/16207/.
Full textAbstract:
The availability of DNA samples that are of adequate quality and quantity is essential for any genetic analysis. The fields of forensic biology and clinical diagnostic pathology testing often suffer from limited samples that yield insufficient DNA material to allow extensive analysis. This study examined the utility of a recently introduced whole genome amplification method termed Multiple Displacement Amplification (MDA) for amplifying a variety of limited sample types that are commonly encountered in the fields of forensic biology and clinical diagnostics. The MDA reaction, which employs the highly processive bacteriophage φ29 DNA polymerase, was found to generate high molecular weight template DNA suitable for a variety of downstream applications from low copy number DNA samples down to the single genome level. MDA of single cells yielded sufficient DNA for up to 20,000,000 PCR assays, allowing further confirmatory testing on samples of limited quantities or the archiving of precious DNA material for future work. The amplification of degraded DNA material using MDA identified a requirement for samples of sufficient quality to allow successful synthesis of product DNA templates. Furthermore, the utility of MDA products in comparative genomic hybridisation (CGH) assays identified the presence of amplification bias. However, this bias was overcome by introducing a novel modification to the MDA protocol. Future directions for this work include investigations into the utility of MDA products in short tandem repeat (STR) assays for human identifications and application of the modified MDA protocol for testing of single cell samples for genetic abnormalities.
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Björnerfeldt, Susanne. "Consequences of the Domestication of Man’s Best Friend, The Dog." Doctoral thesis, Uppsala universitet, Evolutionsbiologi, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7799.
Full textAbstract:
The dog was the first animal to be domesticated and the process started at least 15 000 years ago. Today it is the most morphologically diverse mammal, with a huge variation in size and shape. Dogs have always been useful to humans in several ways, from being a food source, hunting companion, guard, social companion and lately also a model for scientific research. This thesis describes some of the changes that have occurred in the dog’s genome, both during the domestication process and later through breed creation. To give a more comprehensive view, three genetic systems were studied: maternally inherited mitochondrial DNA, paternally inherited Y chromosome and biparental autosomal chromosomes. I also sequenced complete mitochondrial genomes to view the effect new living conditions might have had on dogs’ genes after domestication. Finally, knowledge of the genetic structure in purebred dogs was used to test analytic methods usable in other species or in natural populations where little information is available. The domestication process appears to have caused a relaxation of the selective constraint in the mitochondrial genome, leading to a faster rate of accumulation of nonsynonymous changes in the mitochondrial genes. Later, the process of breed creation resulted in genetically separated breed groups. Breeds are a result from an unequal contribution of males and females with only a few popular sires contributing and a larger amount of dams. However, modern breeder preferences might lead to disruptive selective forces within breeds, which can result in additional fragmentation of breeds. The increase in linkage disequilibrium that this represents increases the value of purebred dogs as model organisms for the identification and mapping of diseases and traits. Purebred dogs’ potential for these kinds of studies will probably increase the more we know about the dog’s genome.
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Darp, Revati A. "Insights into the Role of Oncogenic BRAF in Tetraploidy and Melanoma Initiation." eScholarship@UMMS, 2021. https://escholarship.umassmed.edu/gsbs_diss/1129.
Full textAbstract:
Melanoma, the most lethal form of skin cancer, arises from altered cells in the melanocyte lineage, but the mechanisms by which these cells progress to melanoma are unknown. To understand the early cellular events that contribute to melanoma formation, we examined melanocytes in melanoma-prone zebrafish strains expressing BRAFV600E, the most common oncogenic form of the BRAF kinase that is mutated in nearly 50% of human melanomas. We found that, unlike wild-type melanocytes, melanocytes in transgenic BRAFV600Eanimals were binucleate and tetraploid. Furthermore, melanocytes in p53-deficient transgenic BRAFV600Eanimals exhibited 8N and greater DNA content, suggesting bypass of a p53-dependent arrest that stops cell cycle progression of tetraploid melanocytes. These data implicate tetraploids generated by increased BRAF pathway activity as contributors to melanoma initiation. Previous studies have used artificial means of generating tetraploids, raising the question of how these cells arise during actual tumor development. To gain insight into the mechanism by which BRAFV600E generates binucleate, tetraploid cells, we established an in vitro model by which such cells are generated following BRAFV600E expression. We demonstrate thatBRAFV600E-generated tetraploids arise via cytokinesis failure during mitosis due to reduced activity of the small GTPase RhoA. We also establish that oncogene-induced centrosome amplification in the G1/S phase of the cell cycle and subsequent increase in the activity of the small GTPase Rac1, partially contribute to this phenotype. These data are of significance as recent studies have shown that aneuploid progeny of tetraploid cells can be intermediates in tumor development, and deep sequencing data suggest that at least one third of melanomas and other solid tumors have undergone a whole genome doubling event during their progression. Taken together, our melanoma-prone zebrafish model and in vitro data suggest a role for BRAFV600E-inducedtetraploidy in the genesis of melanomas. To our knowledge, this is the first in vivo model showing spontaneous rise of tetraploid cells that can give rise to tumors. This novel role of the BRAF oncogene may contribute to tumorigenesis in a broader context.
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Ambers, Angie D. "Investigation of Strategies for Improving STR Typing of Degraded and Low Copy DNA from Human Skeletal Remains and Bloodstains." Thesis, University of North Texas, 2014. https://digital.library.unt.edu/ark:/67531/metadc799472/.
Full textAbstract:
Forensic STR analysis is limited by the quality and quantity of DNA. Significant damage or alteration to the molecular structure of DNA by depurination, crosslinking, base modification, and strand breakage can impact typing success. Two methods that could potentially improve STR typing of challenged samples were explored: an in vitro DNA repair assay (PreCR™ Repair Mix) and whole genome amplification. Results with the repair assay showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally-damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR™ assay. The data suggest that the use of PreCR™ in casework should be considered with caution due to the assay’s varied results. As an alternative to repair, whole genome amplification (WGA) was pursued. The DOP-PCR method was selected for WGA because of initial primer design and greater efficacy for amplifying degraded samples. Several modifications of the original DOP-PCR primer were evaluated. These modifications allowed for an overall more robust amplification of damaged DNA from both contemporary and historical skeletal remains compared with that obtained by standard DNA typing and a previously described DOP-PCR method. These new DOP-PCR primers show promise for WGA of degraded DNA.
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Turnbough, Meredith A. "Applications of Molecular Genetics to Human Identity." Thesis, University of North Texas, 2008. https://digital.library.unt.edu/ark:/67531/metadc9730/.
Full textAbstract:
The primary objectives of this project were: 1. to develop improved methods for extraction of DNA from human skeletal remains, 2. to improve STR profiling success of low-copy DNA samples by employing whole genome amplification to amplify the total pool of DNA prior to STR analysis, and 3. to improve STR profiling success of damaged DNA templates by using DNA repair enzymes to reduce the number/severity of lesions that interfere with STR profiling. The data from this study support the following conclusions. Inhibitory compounds must be removed prior to enzymatic amplification; either during bone section pretreatment or by the DNA extraction method. Overall, bleach outperformed UV as a pretreatment and DNA extraction using silica outperformed microconcentration and organic extraction. DNA repair with PreCR A outperformed both whole genome amplification and repair with PreCR T6. Superior DNA extraction results were achieved using the A6 PMB columns (20 ml capacity column with 6 layers of type A glass fiber filter), and DNA repair with PreCR A led to an overall improvement in profile quality in most cases, although whole genome amplification was unsuccessful. Rapid, robust DNA isolation, successful amplification of loci from the sample-derived DNA pool, and an elimination of DNA damage and inhibitors may assist in providing sufficient genetic information from cases that might otherwise lie on the fringe of what is possible to obtain today.
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Raikar, Sanjeev Vencu. "Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression." Phd thesis, Lincoln University. Bio-Protection and Ecology Division, 2007. http://theses.lincoln.ac.nz/public/adt-NZLIU20080214.105406/.
Full textAbstract:
Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression
by Sanjeev V. Raikar
Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus.
Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%).
Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×106 g-1FW was obtained when cell suspensions were used as the tissue source, with enzyme combination A (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots.
The highest protoplast yield (7×106 g-1FW) of L. corniculatus was achieved from cotyledons also with enzyme combination A (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L).
Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm2 for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants.
The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.
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Fredriksson, Mona. "Using Minisequencing Technology for Analysing Genetic Variation in DNA and RNA." Doctoral thesis, Uppsala University, Department of Medical Sciences, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4789.
Full textAbstract:
In this thesis, the four-color fluorescence tag-microarray minisequencing system pioneered by our group was further developed and applied for analysing genetic variation in human DNA and RNA. A SNP marker panel representing different chromosomal regions was established and used for identification of informative SNP markers for monitoring chimerism after stem cell transplantation (SCT). The success of SCT was monitored by measuring the allelic ratios of informative SNPs in follow-up samples from nine patients with leukaemia. The results agreed with data obtained using microsatellite markers. Further the same SNP marker panel was used for evaluation of two whole genome amplification methods, primer extension preamplification (PEP) and multiple displacement amplification (MDA) in comparison with genomic DNA with respect to SNP genotyping success and accuracy in tag-array minisequencing. Identical results were obtained from MDA products and genomic DNA.
The tag-microarray minisequencing system was also established for multiplexed quantification of imbalanced expression of SNP alleles. Two endothelial cell lines and a panel of ten coding SNPs in five genes were used as model system. Six heterozygous SNPs were genotyped in RNA (cDNA) from the cell lines. Comparison of the relative amounts of the SNPs alleles in cDNA to heterozygote SNPs in genomic DNA displayed four SNPs with significant imbalanced expression between the SNP alleles. Finally, the tag-array minisequencing system was modified for detection of splice variants in mRNA from five leukaemia cell lines. A panel of 20 cancer-related genes with 74 alternatively splice variants was screened. Over half of the splice variants were detected in the cell lines, and similar alternative splicing patterns were observed in each cell line. The results were verified by size analysis of the PCR product subjected to the minisequencing primer extension reaction. The data from both methods agreed well, evidencing for a high sensitivity of our system.
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Lovmar, Lovisa. "Methods for Analysis of Disease Associated Genomic Sequence Variation." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4525.
Full text
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Konstantinidis, Michalis. "Preimplantation genetic diagnosis : new methods for the detection of genetic abnormalities in human preimplantation embryos." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:28611f65-7729-4293-9c3f-4fc3f0cc39d7.
Full textAbstract:
Preimplantation genetic diagnosis (PGD) refers to the testing of embryos produced through in vitro fertilization (IVF) in order to identify those unaffected by a specific genetic disorder or chromosomal abnormality. In this study, different methodologies were examined and developed for performance of PGD. Investigation of various whole genome amplification (WGA) methods identified multiple displacement amplification as a reliable method for genotyping single cells. Furthermore, this technology was shown to be compatible with subsequent analysis using single nucleotide polymorphism (SNP) microarrays. Compared to conventional methods used in this study to perform single cell diagnosis (e.g. multiplex PCR), WGA techniques were found to be advantageous since they streamline the development of PGD protocols for couples at high risk of transmitting an inherited disorder and simultaneously offer the possibility of comprehensive chromosome screening (CCS). This study also aimed to develop a widely applicable protocol for accurate typing of the human leukocyte antigen (HLA) region with the purpose of identifying embryos that will be HLA-identical to an existing sibling affected by a disorder that requires haematopoietic stem cell transplantation. Additionally, a novel microarray platform was developed that, apart from accurate CCS, was capable of reliably determining the relative quantity of mitochondrial DNA in polar bodies removed from oocytes and single cells biopsied from embryos. Mitochondria are known to play an important role in oogenesis and preimplantation embryogenesis and their measurement may therefore be of clinical relevance. Moreover, real-time PCR was used for development of protocols for CCS, DNA fingerprinting of sperm samples and embryos and the relative quantitation of telomere length in embryos (since shortened telomeres might be associated with reduced viability). As well as considering the role of genetics in terms of oocyte and embryo viability assessment and the diagnosis of inherited genetic disorders, attention was given to a specific gene (Phospholipase C zeta) of relevance to male infertility. A novel mutation affecting the function of the resulting protein was discovered highlighting the growing importance of DNA sequence variants in the diagnosis and treatment of infertility.
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Raikar, S. V. "Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression." Diss., Lincoln University, 2007. http://hdl.handle.net/10182/301.
Full textAbstract:
Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×10⁶ g⁻¹FW was obtained when cell suspensions were used as the tissue source, with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×10⁶ g⁻¹FW) of L. corniculatus was achieved from cotyledons also with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm² for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.
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Alawi, Mariah. "Optimizing a Selective Whole Genome Amplification (SWGA) Strategy for Clinical Malaria Infections." Thesis, 2019. http://hdl.handle.net/10754/656568.
Full textAbstract:
Plasmodium is a genus well known for causing malaria, a life-threatening infection for many people where malaria is endemic. The blood-borne disease is transmitted by the female Anopheles mosquito. Till date, eight parasite species have been reported to cause malaria in humans that include P. falciparum, P. vivax, P. malariae, P. ovale curtisi, P. ovale wallikeri, P. cynomolgi, P. knowlesi and more recently P. simium. Amongst them, the most genetically understood species is P. falciparum, causing most of the deaths in children from malaria.
Understanding genome variation at the population level of all malaria species is of utmost importance, including clinical cases with very low parasitemia. To achieve this purpose, we need sufficient amounts of parasite DNA material from the pool of host DNA, which always is overrepresented in clinical infections. We utilized a strategy of selective whole genome amplification (SWGA) technology on P. malariae and P. ovale curtisi (two neglected human infecting malaria parasites that often cause mild yet clinically relevant infections with low parasitemia) to efficiently enrich their genomic DNA for high-quality whole genome sequencing. Previous studies on SWGA applied on P. falciparum and P. vivax showed that SWGA could efficiently enrich the amount of starting DNA material from inadequate amounts of parasites directly from clinical samples without separating the host DNA using specifically designed primer sets.
We have successfully designed multiple sets of primers and tested the efficiency of five best primer sets using polymerase chain reaction to enrich the genomes of P. malariae and P. ovale curtisi. The efficiency of primers in enriching the genome was tested on two clinical samples for each of P. malariae and P. ovale curtisi. We were able to enrich the genome of P. malariae with an average of 19-fold (19X) enrichment across both samples. For P. ovale curtisi, we could achieve an enrichment of 3 folds only. Nevertheless, we still obtained a sufficient amount of gDNA to prepare Illumina sequencing libraries and call for SNPs and Indels in a biologically reproducible manner at genome-scale.
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Schoenborn, Veit. "Whole Genome Amplification von Plasma-DNA und Entwicklung eines Ausschlusskriteriums zur Verbesserung der Genotypisierungsqualität." Doctoral thesis, 2008. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-37136.
Full textAbstract:
Plasma- und Serumproben waren in früheren epidemiologischen Studien häufig das einzige biologische Material, das gesammelt und untersucht wurde. Diese Studien besitzen gerade durch ihren sehr langen Untersuchungszeitraum einen riesigen Informationsgehalt und wären ein unbezahlbarer Schatz für genetische Analysen. Oft ist aufgrund damals mangelnder Akquirierung jedoch keine genomische DNA verfügbar. Um die in Plasmaproben in geringer Menge vorkommende DNA verwenden zu können, extrahierten wir die DNA mit Hilfe von magnetischen Partikeln und setzten sie in eine Whole Genome Amplification (WGA) mittels Φ29-DNA-Polymerase ein. Wir stellten 88 Probenpärchen, bestehend aus einer WGA-Plasma-DNA und der korrespondierenden Vollblut-DNA derselben Person, zusammen und genotypisierten bei diesen neun hochpolymorphe Short Tandem Repeats (STR) und 25 SNPs. Die durchschnittliche innerhalb der Probenpaare auftretende Diskordanzrate betrug 3,8% für SNPs sowie 15,9% für STRs. Basierend auf den Ergebnissen der Hälfte der Probenpaare entwickelten wir einen Ausschlussalgorithmus und validierten diesen in der anderen Hälfte der Probenpaare. Mit diesem ist es möglich, zum Einen diejenigen Proben mit einer guten DNA-Qualität herauszufiltern, um Genotypisierungsfehler zu vermeiden, und zum Anderen jene Proben mit insuffizienter DNA-Qualität auszuschließen. Nachdem Proben, die fünf oder mehr homozygote Loci in dem 9-STR-Markerset aufwiesen, ausgeschlossen wurden, resultierte dies in einer Ausschlussrate von 22,7% und senkte die durchschnittliche Diskordanzrate auf 3,92% für STRs bzw. 0,63% für SNPs. Bei SNPs entspricht dieser Wert ungefähr der Fehlerquote, wie er auch bei Genotypisierungen mit Vollblut-DNA in vielen Laboratorien auftritt. Unsere Methode und das Ausschlusskriterium bieten damit neue Möglichkeiten, um zuverlässige DNA aus archivierten Plasmaproben wiederzugewinnen. Dieser Algorithmus ist auch besser geeignet, als nur die eingesetzte DNA-Menge in die WGA-Reaktion als Kriterium zu benützenPlasma and serum samples were often the only biological material collected for earlier epidemiological studies. These studies have a huge informative content, especially due to their long follow-up and would be an invaluable treasure for genetic investigations. However, often no banked DNA is available. To use the small amounts of DNA present in plasma, in a first step, we applied magnetic bead technology to extract this DNA, followed by a whole-genome amplification (WGA) using phi29-polymerase. We assembled 88 sample pairs, each consisting of WGA plasma DNA and the corresponding whole-blood DNA. We genotyped nine highly polymorphic short tandem repeats (STRs) and 23 SNPs in both DNA sources. The average within-pair discordance was 3.8% for SNPs and 15.9% for STR genotypes, respectively. We developed an algorithm based on one-half of the sample pairs and validated on the other one-half to identify the samples with high WGA plasma DNA quality to assure low genotyping error and to exclude plasma DNA samples with insufficient quality: excluding samples showing homozygosity at five or more of the nine STR loci yielded exclusion of 22.7% of all samples and decreased average discordance for STR and SNP markers to 3.92% and 0.63%, respectively. For SNPs, this is very close to the error observed for genomic DNA in many laboratories. Our workflow and sample selection algorithm offers new opportunities to recover reliable DNA from stored plasma material. This algorithm is superior to testing the amount of input DNA
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Lee, Tai-Chun, and 李黛君. "Comparison with Linker adaptor and MDA Two Methodsof Whole Genome Amplification for Single Cell GenomeAmplification Efficiency." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/08385668836849829311.
Full textAbstract:
碩士國立臺灣大學
分子醫學研究所
95
Preimplantation genetic diagnosis (PGD) of single gene disorders relies on PCR-based tests performed on single cells (polar bodies or blastomeres). Single cell PCR protocols are subject to serious difficulties, including contamination, amplification failure, and preferential amplification or the complete absence of one allele (allele dropout, ADO) in heterozygous loci, but it remains the only tool for detecting specific mutation in PGD. The DNA content of single cell was limited. Different whole genome amplification (WGA) techniques have been developed to increase the DNA quantities from clinical samples with limited DNA contents. Therefore, the utilization of genomic amplification methods will be a useful tool in single-cell genetic diagnosis. In this study, the complete genome DNA of all chromosome could be non-specific amplify by two WGA methods, Linker adaptor and the multiple displacement amplification (MDA) method, without the loss of genomic regions or preferential amplification of genomic loci or alleles. The WGA technique was not only increase the original DNA content, but also retained the integrity of whole genome DNA. The two common SNP of β-thalassemia was amplified from the longer or shorter primer set from these amplified genome DNA. DNA sequencing of the PCR product was performed to evaluate the ADO effect during two WGA methods. Total of 222 single cells were picked up and performed the Linker-adaptor and MDA method to amplify the whole genome DNA, respectively. DNA sequencing results of the PCR product from 174 samples found that 91 samples had the complete allele, and 83 samples had the allele drop-out (ADO) effect. The amplification efficiency of linker adaptor and MDA two methods are 90 ％. The ADO rate was 26.67 ％ and 28.89 ％, respectively. These two methods still had higher than 25 ％ of ADO rate. Therefore, the ADO problem was the biggest obstacle for single cell PCR and diagnosis of single-gene disorders.
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Fang, Mei-Ya, and 方美雅. "Establishment of a new strategy for preimplantation genetic diagnosis using whole genome amplification and blastocyst biopsy." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/19165916479697061799.
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碩士國立臺灣大學
分子醫學研究所
99
Preimplantation genetic diagnosis (PGD) is an alternative for prenatal diagnosis. For families with genetic diseases, PGD offers a chance to have an unaffected child, without facing termination of pregnancy. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. The whole genome amplification (WGA) can amplify the limited DNA to process diagnosis. By using WGA, we can improve the accuracy of diagnosis and expand other diagnosis methods for PGD. The main problem of WGA is allele drop out (ADO), which is critical for misdiagnosis and low accuracy. Therefore, we used a different approach to improve the ADO. We compare amplification rate and efficiency in blastomere and trophectoderm biopsy. We can retrieve more cells in the trophectoderm than blastomere, and improved accuracy for PGD. Furthermore, we apply the Rubicon PicoPlex WGA kit for whole genome amplification. Although the ADO is thus reduced, the practicality of Rubicon PicoPlex WGA kit for PGD need to be further evaluated due to the limitation of the new WGA technique. In addition, we successfully improve the ADO by using the LNA probe and PCR clamping. In the end, no matter how we try to improve the examination process, the ADO is still inevitable. Therefore, to establish a high-accuracy method, through the biopsy of blastocyst stage, whole genome amplification, and the double confirmatory platform, are crucial for a high-quality PGD system. Consequently, we could provide a more credible and reliable system for the patients.
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Meehan, Conor J., G. A. Goig, T. A. Kohl, L. Verboven, A. Dippenaar, M. Ezewudo, M. R. Farhat, et al. "Whole genome sequencing of Mycobacterium tuberculosis: current standards and open issues." 2019. http://hdl.handle.net/10454/17276.
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NoWhole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.
European Research Council grant (INTERRUPTB; no. 311725), European Research Council grant (TB-ACCELERATE; no. 638553), Foundation for Innovative New Diagnostics, German Center for Infection Research (DZIF), Deutsche Forschungsgemeinschaft (German Research Foundation) under Germany’s Excellence Strategy (EXC 22167–390884018), FWO Odysseus G0F8316N, US National Institutes of Health BD2K K01 (MRF ES026835), Agence Nationale de la Recherche (ANR-16-CD35-0009)
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Schoenborn, Veit [Verfasser]. "Whole genome amplification von Plasma-DNA und Entwicklung eines Ausschlusskriteriums zur Verbesserung der Genotypisierungsqualität / vorgelegt von Veit Schoenborn." 2009. http://d-nb.info/995554153/34.
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Svensson, Ulrika. "Whole genome amplification of bacterial DNA: Identification of SHV-, LEN- and OPK-genes with SP6 and T7 sequence tagged PCR amplicons." Thesis, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-204697.
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Seifertová, Eva. "Genetické mapování u rodu Xenopus." Doctoral thesis, 2014. http://www.nusl.cz/ntk/nusl-342246.
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The diploid amphibian Xenopus tropicalis represents a significant model organism for studies of early development, genes function and evolution. Such techniques as gynogenesis, injection of morpholino antisense oligonucleotide into fertilized eggs or transgenesis were established. In the recent ten years, many efforts have been made to complete the sequence information. X. tropicalis genome has been sequenced but the completion of its assembly only on the basis of sequence data has been impossible. Therefore, our first work was focused on one of approaches for a genome completing- genetic mapping. First of all, the genetic map of Xenopus tropicalis was established pursuant linkage and physical positions of markers. Since the map contained gaps, we developed a new method for genetic mapping based on the next generation sequencing of laser microdissected arm. Using Illumina next generation sequencing of fifteen copies of a short arm of chromosome 7, we obtained new insights into its genome by localizing previously unmapped genes and scaffolds as well as recognizing mislocalized portions of the genome assembly. This was the first time laser microdissection and sequencing of specific chromosomal regions has been used for the purpose of genome mapping. These data were also used in the evolution study of...