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.
|Titolo:||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|
|Data di pubblicazione:||9-dic-2010|
|Parole Chiave:||whole genome amplification ; buccal cell ; FTA cards|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||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 ; tutore: Cristina Battaglia ; correlatore: Maria Carla Proverbio ; direttore della scuola : Maria Luisa Villa. - Milano : Università degli studi di Milano. Universita' degli Studi di Milano, 2010 Dec 09. ((23. ciclo, Anno Accademico 2010.|
|Digital Object Identifier (DOI):||10.13130/gabrieli-arianna_phd2010-12-09|
|Appare nelle tipologie:||Tesi di dottorato|