Degenerate-bases: criação de bancos de dados a partir de oligonucleotídeos com bases degeneradas / Degenerate-bases: databases creation from oligonucleotides with degenerated bases

Nas últimas décadas, o uso de oligonucleotídeos desenhados pela Bioinformática tem aumentado consideravelmente devido à criação de novas tecnologias que fazem uso dessas moléculas. Dentro desse contexto, a utilização de oligonucleotídeos contendo bases degeneradas tem sido observada em variações da PCR, sequenciamento, na produção de sondas, aptâmeros entre outras moléculas. Diante desse cenário, torna-se necessária a criação de uma ferramenta capaz de gerar todas as possibilidades que uma sequência com bases degeneradas pode fornecer, tornando possível a criação de um banco de dados local para análises in silico. Este artigo descreve um software desenvolvido para analisar uma sequência de oligonucleotídeos contendo bases degeneradas e gerar, a partir dessa sequência, um arquivo com todas as sequências possíveis com as bases não degeneradas considerando a conversão das bases degeneradas em bases convencionais. O software obteve sucesso nos testes de validação com sequências de comprimentos variados, contendo bases convencionais e degeneradas, o que influencia diretamente o tempo de processamento

Feline Lentivirus: Molecular Analysis and Epidemiology in Southern African Lions

Feline immunodeficiency virus is a retrovirus of domestic cats causing significant lifelong infection. Infection has also been detected in nondomestic species, including African lions. It is endemic in certain populations in east and southern Africa. Infection leads to immunologic dysfunction and immunosuppressive disease in domestic cats; however, little research exists about the pathogenic effects of infection in lions and its epidemiological impact on free-ranging and captive populations. Little is known about the lentivirus in these populations at the molecular and host level. Analysis of the virus from these populations is necessary for development of detection assays that are both sensitive and specific. Whole-blood and serum samples were collected opportunistically from free ranging lions in Kruger National Park, South Africa, and from Hlane Royal National Park, Swaziland. Whole-blood and serum samples were also collected from captive exotic felids in RSA andUS and domestic cats in RSA. A nested polymerase chain reaction assay was performed on all whole-blood samples, and all positive products were sequenced and analyzed phylogenetically. Serum samples were tested for cross-reactive antibodies to domestic feline lentivirus antigens and cross-reactive antibodies to puma lentivirus synthetic envelope peptide antigen. Serum samples were tested for feline haptoglobins and feline alpha-one acid glycoproteins by radial immunodiffusion. This research represents the first epidemiological study of the lion lentivirus among free-ranging lions of Kruger National Park and the first epidemiological study comparing genetic material to antibody-based methods of lentivirus detection on lions in RSA. The polymerase chain reaction assay was successful in amplifying the lion lentivirus from African lions. The conservation management of free-ranging lions must consider the infectious agents to which they are susceptible. No conclusions can be drawn from this investigation with respect to the potential virulence or pathogenic distinctions between viral subtypes, as little is known about the definitive consequences of lion lentivirus infection in African lions. Immunologic studies may uncover potential differences in immune expression among lentivirus-positive and -negative individuals with regard to increased resistance or increased susceptibility to infection or pathogenicity. To this end, lion lentivirus infection status could also be compared to the infection status of bovine tuberculosis

Data Mining Using the Crossing Minimization Paradigm

Our ability and capacity to generate, record and store multi-dimensional, apparently unstructured data is increasing rapidly, while the cost of data storage is going down. The data recorded is not perfect, as noise gets introduced in it from different sources. Some of the basic forms of noise are incorrect recording of values and missing values. The formal study of discovering useful hidden information in the data is called Data Mining. Because of the size, and complexity of the problem, practical data mining problems are best attempted using automatic means. Data Mining can be categorized into two types i.e. supervised learning or classification and unsupervised learning or clustering. Clustering only the records in a database (or data matrix) gives a global view of the data and is called one-way clustering. For a detailed analysis or a local view, biclustering or co-clustering or two-way clustering is required involving the simultaneous clustering of the records and the attributes. In this dissertation, a novel fast and white noise tolerant data mining solution is proposed based on the Crossing Minimization (CM) paradigm; the solution works for one-way as well as two-way clustering for discovering overlapping biclusters. For decades the CM paradigm has traditionally been used for graph drawing and VLSI (Very Large Scale Integration) circuit design for reducing wire length and congestion. The utility of the proposed technique is demonstrated by comparing it with other biclustering techniques using simulated noisy, as well as real data from Agriculture, Biology and other domains. Two other interesting and hard problems also addressed in this dissertation are (i) the Minimum Attribute Subset Selection (MASS) problem and (ii) Bandwidth Minimization (BWM) problem of sparse matrices. The proposed CM technique is demonstrated to provide very convincing results while attempting to solve the said problems using real public domain data. Pakistan is the fourth largest supplier of cotton in the world. An apparent anomaly has been observed during 1989-97 between cotton yield and pesticide consumption in Pakistan showing unexpected periods of negative correlation. By applying the indigenous CM technique for one-way clustering to real Agro-Met data (2001-2002), a possible explanation of the anomaly has been presented in this thesis

Data mining using the crossing minimization paradigm

Our ability and capacity to generate, record and store multi-dimensional, apparently unstructured data is increasing rapidly, while the cost of data storage is going down. The data recorded is not perfect, as noise gets introduced in it from different sources. Some of the basic forms of noise are incorrect recording of values and missing values. The formal study of discovering useful hidden information in the data is called Data Mining. Because of the size, and complexity of the problem, practical data mining problems are best attempted using automatic means. Data Mining can be categorized into two types i.e. supervised learning or classification and unsupervised learning or clustering. Clustering only the records in a database (or data matrix) gives a global view of the data and is called one-way clustering. For a detailed analysis or a local view, biclustering or co-clustering or two-way clustering is required involving the simultaneous clustering of the records and the attributes. In this dissertation, a novel fast and white noise tolerant data mining solution is proposed based on the Crossing Minimization (CM) paradigm; the solution works for one-way as well as two-way clustering for discovering overlapping biclusters. For decades the CM paradigm has traditionally been used for graph drawing and VLSI (Very Large Scale Integration) circuit design for reducing wire length and congestion. The utility of the proposed technique is demonstrated by comparing it with other biclustering techniques using simulated noisy, as well as real data from Agriculture, Biology and other domains. Two other interesting and hard problems also addressed in this dissertation are (i) the Minimum Attribute Subset Selection (MASS) problem and (ii) Bandwidth Minimization (BWM) problem of sparse matrices. The proposed CM technique is demonstrated to provide very convincing results while attempting to solve the said problems using real public domain data. Pakistan is the fourth largest supplier of cotton in the world. An apparent anomaly has been observed during 1989-97 between cotton yield and pesticide consumption in Pakistan showing unexpected periods of negative correlation. By applying the indigenous CM technique for one-way clustering to real Agro-Met data (2001-2002), a possible explanation of the anomaly has been presented in this thesis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

DNA sequencing by hybridization using semi-degenerate bases

One way to enhance the performance of hybridization microarrrays for DNA de novo sequencing is the use of probing patterns with gaps of unsampled positions. Ideally, such gaps could be realized by the inclusion into microarray oligos (probes) of wild-card compounds, referred to as universal bases (which bind nonspecifically to natural bases). The suggested alternative is to deploy in the gap positions degenerate bases, i.e., uniform mixtures of the four natural bases, with ensuing deterioration of the hybridization signal. In this paper, we show that such signal loss is a minor shortcoming, compared with the fact that degenerate bases cannot be treated as universal. Indeed, the substantial spread of hybridization energies at any microarray feature is such that on overwhelming number of mismatches bind more strongly than legal matches. We observed, however, that much narrower energy spreads are exhibited by pairs of bases in the same strength class (A-T and C-G). We call semi-degenerate a gap position realized with bases in the same energy class and show that well-known sequence reconstruction algorithms can be modified to achieve substantial improvements in sequencing effectiveness. For example, with a 4 9-feature microarray and an acceptable weakening of the hybridization signal, one may achieve lengths of about 4,000 bases (compared with < 250 of the standard uniform method). Our approach also incorporates the use of a spectrum expressed in terms of observed feature melting temperatures (analog spectrum), rather than binary decisions made directly at the biochemical level (digital spectrum). While universal bases represent the ultimate goal of sequencing by hybridization, semidegenerate natural bases are the most effective known substitute

Étude des phénomènes reliés à la présence du brin complémentaire au brin cible lors d'hybridation avec sondes de captures fixées sur support solide

Cette thèse de doctorat présente 3 études reliées à l’hybridation d’ADN fixée à un support solide et les phénomènes qui y sont associés. L’hybridation d’ADN en phase aqueuse est bien caractérisée, cependant plusieurs phénomènes et paramètres restent encore à déterminer lorsqu’un des brins est fixé sur un support solide. Un de ces paramètres est l’influence du brin complémentaire en fonction de la longueur de l’extrémité d’ADN cible immobilisée exposée à la phase aqueuse. Pour mieux comprendre ce phénomène, nous avons établi une méthode pour digérer spécifiquement le brin d’ADN complémentaire pour simplifier l’hybridation sur support solide. La combinaison d’une molécule favorisant la digestion du brin complémentaire et d’une molécule bloquant la digestion du brin cible a permis de produire un ADN cible simple brin à l’aide de l’exonucléase Lambda, permettant d’obtenir des signaux d’hybridation supérieurs à ceux obtenus avec des ADN doubles brins. Ensuite, l’observation en temps réel de l’hybridation d’amplicons simples brins et doubles brins a permis de mieux comprendre les phénomènes de compétition entre le brin complémentaire et la cible. Finalement, les conclusions des deux premières études nous ont permis d’inventer une technologie tirant profit de la compétition entre le brin complémentaire et la sonde de capture et de l’activité exonucléase pour réaliser l’amplification PCR et l’hybridation sur biopuce en un seul site réactionnel et dans un seul tampon réactionnel. La combinaison de ces techniques aura pour effet une simplification des dispositifs et une diminution du nombre de réactifs permettant la fabrication de tests diagnostiques automatisés à moindre coûts. Les travaux de cette thèse ont permis d’approfondir nos connaissances sur les phénomènes reliés à l’hybridation sur support solide et de concevoir une technologie faisant l’objet d’une demande de brevet. La suite logique de ces travaux serait de poursuivre le développement et l’optimisation de la technologie pour augmenter ses capacités de multiplexage tout en l’intégrant à un dispositif microfluidique et à un instrument de détection qui combine amplification PCR et hybridation sur biopuce dans une seule réaction réalisée en une seule étape.This thesis presents three studies related to the phenomenon related to DNA hybridization onto capture probes attached to solid support. The work was carried out in the context of infectious diseases detection, but the conclusions are of interest to any test based on the recognition of genetic material by solid support immobilized capture probes. DNA hybridization in aqueous phase is well characterised, however several parameters and phenomena related to hybridization onto solid supports are still to be determined. One of these parameters is the influence of the complementary strand in relation with the length of the solvent-exposed immobilized target strand. To better understand this phenomenon, we established a method to specifically digest the complementary DNA strands to simplify hybridization onto solid support. The presence of a molecule favorising digestion on the complementary strand and a blocking molecule on the target strand enabled selective digestion of the complementary strand using Lambda exonuclease in PCR buffer. Hybridization of single-stranded target DNA generated this way resulted in significantly higher fluorescence signals than those obtained with double stranded hybridization. Afterwards, observation of real-time hybridization of single-stranded amplicons and double-stranded amplicons to capture probes fixed onto solid support allowed for a better understanding of the competition phenomena between the complementary strand and the capture probe. Finally, the knowledge gained with the first two studies was used to invent a technology taking advantage of the competition between the complementary strand and the capture probe and exonuclease activity to perform single vessel and single buffer PCR amplification and microarray hybridization. The combination of these techniques will simplify devices and require fewer reagents storage and handling, facilitating production of automated diagnostic tests at lower costs. The works of this thesis have expended our understanding of the phenomena associated with hybridization onto a solid support and also resulted in the creation of a new technology covered by a patent application. The logical progression of this work is to further develop and optimize this technology by integrating it into a microfluidic device and an instrument compatible with the detection of microarrays to increase the multiplexing capabilities of molecular diagnostics

Conception automatisée d'amorces et de sondes aux fins de diagnostic moléculaire

Des algorithmes de conception automatisée d’amorces PCR et de sondes d’hybridation aux fins de diagnostic moléculaire ont été créés et implémentés. La vitesse de recherche d’amorces PCR à la fois spécifiques, ubiquitaires et sensibles s’est avérée considérablement accrue par le calcul et l’emploi de consensus binaires d’alignements multiples de séquences d’ADN. La recherche de sondes d’hybridation spécifiques et l’analyse du potentiel d’hybridation en général entre deux séquences a été facilitée par la synthèse de données empiriques d’hybridation trouvées dans divers articles publiés. D’autres programmes servant à faciliter ou automatiser l’exécution de diverses tâches utiles ont également été créés. Tous les programmes résultants ont permis de valider les algorithmes créés et de confirmer leur utilité dans l’exécution des tâches désirées par les membres de l’équipe de recherche auxquels ils étaient prioritairement destinés.Algorithms for automatic design of PCR primers and of hybridization probes for molecular diagnostics have been created and implemented. The search speed for PCR primers that were at the same time specific, ubiquitary and sensitive was greatly improved by both computation and use of binary consensus of multiple alignments of DNA sequences. The search for specific hybridization probes and also analysis of hybridization potential in general between two sequences was facilitated by synthesis of empirical data gathered from different published papers. Other software for facilitating or automating the execution of different useful tasks were also created. All resulting software allowed both for the validation of algorithms and for the confirmation of their usefullness in executing desired tasks by the members of the research team for which they were written in the first place