This thesis relates to the development of an isothermal method for the detection of DNA hypermethylation. The function of DNA methylation is to silence specific areas of the human genome and its maintenance is necessary to regulate key functions of the cell and of the organism. Aberrant hypermethylation of the promoter regions of several tumor suppressor genes can lead to formation of tumors and its detection, even at very early stages, can be an useful and powerful instrument for the diagnosis of the cancer, the determination of the prognosis and also to gauge the therapy. In this work we developed a molecular assay (MS-LAMP), based on the Loop-mediated Isothermal Amplification, for the rapid and specific amplification and detection of the methylated DNA in three human gene promoters after a sodium bisulfite treatment. The assay, showed PCR-level performance in terms of specificity, sensitivity and selectivity both on synthetic genes and fully methylated and unmethylated genomic DNA. The method was also validated on DNA extracted from clinical samples in comparison with a reference technique. Finally the notable specificity of LAMP was also exploited for the development of a triplex reaction, tested successfully on control DNA and clinical samples, with two possible signal detection approaches.

(2009). Development of an isothermal method for the detection of DNA hypermethylation. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2009).

Development of an isothermal method for the detection of DNA hypermethylation

ZERILLI, FRANCESCO
2009-12-15

Abstract

This thesis relates to the development of an isothermal method for the detection of DNA hypermethylation. The function of DNA methylation is to silence specific areas of the human genome and its maintenance is necessary to regulate key functions of the cell and of the organism. Aberrant hypermethylation of the promoter regions of several tumor suppressor genes can lead to formation of tumors and its detection, even at very early stages, can be an useful and powerful instrument for the diagnosis of the cancer, the determination of the prognosis and also to gauge the therapy. In this work we developed a molecular assay (MS-LAMP), based on the Loop-mediated Isothermal Amplification, for the rapid and specific amplification and detection of the methylated DNA in three human gene promoters after a sodium bisulfite treatment. The assay, showed PCR-level performance in terms of specificity, sensitivity and selectivity both on synthetic genes and fully methylated and unmethylated genomic DNA. The method was also validated on DNA extracted from clinical samples in comparison with a reference technique. Finally the notable specificity of LAMP was also exploited for the development of a triplex reaction, tested successfully on control DNA and clinical samples, with two possible signal detection approaches.
DOGLIA, SILVIA MARIA
ADLERSTEIN, DANIEL
LAMP, methylation, DNA, cancer, diagnosis
FIS/01 - FISICA SPERIMENTALE
English
Scuola di dottorato di Scienze
BIOTECNOLOGIE INDUSTRIALI - 15R
22
2008/2009
(2009). Development of an isothermal method for the detection of DNA hypermethylation. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2009).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/7481
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