Pathogenic nucleic acid detection, in aiming to diagnose infectious diseases and thus better provide a better treatment for them is a very important goal for human health. Moreover, as more and more links are discovered between hereditary diseases and DNA mutations, their diagnostic based on DNA mutation detection is increasingly important. Throughout this Ph. D. project, we developed a fluorometric method for nucleic acid detection based on a water-soluble cationic polymer able to transduce DNA hybridization. We demonstrated the applicability of this method to detect DNA oligonucleotides of different lengths as well as single stranded RNA extracted from the flu virus. With the use of optimized experimental conditions and a custom-built fluorimeter, we demonstrated the ability to detect as little as 220 target DNA copies in the probed volume. Since DNA is present in its double stranded form in most organisms, we also developed an experimental method allowing double stranded DNA detection with the polymeric transducer. Furthermore, we modified our DNA detection process by using fluorescence resonance energy transfer. This process, named fluorescence chain reaction and discovered by Dr. Hoang-Anh Ho, is based on a fluorescence signal amplification mechanism and allows the detection of as few as 20 copies of target DNA. With this method, it was possible to detect a single base mutation responsible for type 1 hereditary tyrosinemia in unpurified genomic DNA extracted from patient blood. Subsequently, we concentrated our efforts on the characterization of this fluorescence signal amplification mechanism. Thus, we established that the fluorescence chain reaction happens in rod-like micellar aggregates of 400 nm in length containing between 4000 and 5000 polymer-tagged DNA probe complexes. This aggregated structure maintains the fluorescent donors near the acceptors, thus greatly helping the fluorescence resonance energy transfer mechanism. Moreover, the conjugated structure of the polymeric transducer allows efficient energy transportation along its chain, accelerating the energy transfer mechanism. / Pathogenic nucleic acid detection, in aiming to diagnose infectious diseases and thus better provide a better treatment for them is a very important goal for human health. Moreover, as more and more links are discovered between hereditary diseases and DNA mutations, their diagnostic based on DNA mutation detection is increasingly important. Throughout this Ph. D. project, we developed a fluorometric method for nucleic acid detection based on a water-soluble cationic polymer able to transduce DNA hybridization. We demonstrated the applicability of this method to detect DNA oligonucleotides of different lengths as well as single stranded RNA extracted from the flu virus. With the use of optimized experimental conditions and a custom-built fluorimeter, we demonstrated the ability to detect as little as 220 target DNA copies in the probed volume. Since DNA is present in its double stranded form in most organisms, we also developed an experimental method allowing double stranded DNA detection with the polymeric transducer. Furthermore, we modified our DNA detection process by using fluorescence resonance energy transfer. This process, named fluorescence chain reaction and discovered by Dr. Hoang-Anh Ho, is based on a fluorescence signal amplification mechanism and allows the detection of as few as 20 copies of target DNA. With this method, it was possible to detect a single base mutation responsible for type 1 hereditary tyrosinemia in unpurified genomic DNA extracted from patient blood. Subsequently, we concentrated our efforts on the characterization of this fluorescence signal amplification mechanism. Thus, we established that the fluorescence chain reaction happens in rod-like micellar aggregates of 400 nm in length containing between 4000 and 5000 polymer-tagged DNA probe complexes. This aggregated structure maintains the fluorescent donors near the acceptors, thus greatly helping the fluorescence resonance energy transfer mechanism. Moreover, the conjugated structure of the polymeric transducer allows efficient energy transportation along its chain, accelerating the energy transfer mechanism. / La détection d'acides nucléiques pathogènes afin de faire le diagnostic de maladies infectieuses et ainsi mieux diriger leur traitement est un enjeu très important pour la santé humaine. Aussi, comme les mutations responsables des maladies génétiques sont de mieux en mieux connues, leur détection est maintenant possible. Au cours de ce projet de doctorat, nous avons élaboré une méthode de détection d'acides nucléiques basée sur un polymère cationique soluble dans l'eau capable de traduire la réaction d'hybridation de l'ADN en un signal optique. Nous avons démontré l'applicabilité de cette méthode pour faire la détection par fluorescence d'oligonucléotides d'ADN de différentes longueurs ainsi que d'ARN viral. Avec l'utilisation de conditions expérimentales optimisées et d'un fluorimètre conçu spécialement pour cette application, nous avons pu détecter aussi peu que 220 copies d'ADN. Par la suite, comme l'ADN est présent chez la majorité des organismes vivants sous la forme de double hélice, nous avons élaboré une méthode expérimentale originale permettant la détection directe de cette forme d'ADN. Par la suite, nous avons modifié notre méthode pour augmenter sa sensibilité en utilisant le transfert énergétique résonnant de fluorescence. Un mécanisme d'amplification du signal de fluorescence a ainsi été découvert par le Dr. Hoang-Anh Ho. Cette méthode que nous avons nommée superallumage, nous a permis de faire la détection d'aussi peu que 5 copies d'ADN cible. Avec cette méthode il a été possible de faire la détection d'une seule mutation génétique responsable de la tyrosinémie héréditaire de type 1 dans des échantillons d'ADN génomique non purifiés. Finalement, nous avons concentré nos efforts sur l'étude du phénomène d'amplification de la fluorescence menant au superallumage. Ainsi, nous avons établi que le superallumage se produit dans des agrégats micellaires ayant la forme d'un bâtonnet mesurant 400 nm de long et contenant entre 4000 et 5000 complexes polymère-sonde d'ADN marquée. Cette structure agrégée maintient les fluorophores donneurs très près des accepteurs de fluorescence, ce qui favorise grandement le processus de transfert énergétique résonnant. De plus, la structure conjuguée du polymère transducteur permet un transport efficace de l'énergie le long de sa chaîne, ce qui accélère le transfert énergétique résonnant.
| Identifer | oai:union.ndltd.org:LAVAL/oai:corpus.ulaval.ca:20.500.11794/19031 |
| Date | 12 April 2018 |
| Creators | Doré, Kim |
| Contributors | Boudreau, Denis |
| Source Sets | Université Laval |
| Language | French |
| Detected Language | English |
| Type | thèse de doctorat, COAR1_1::Texte::Thèse::Thèse de doctorat |
| Format | xviii, 172 f., application/pdf |
| Rights | http://purl.org/coar/access_right/c_abf2 |
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