Određivanje prisutnosti mutacije V600E gena BRAF u sporadičnim tumorima debelog crijeva metodom detekcije heterodupleksa DNA (HRM)

master's thesis

• Title (english): High resolution melting (HRM) analysis of BRAF V600E mutation in sporadic colon cancer
• Author: Sandra Bulat Lokas
• Mentor: Tamara Čačev (mentor)
• Committee member: Maja Matulić (predsjednik povjerenstva)
• Committee member: Tamara Čačev (član povjerenstva)
• Committee member: Mirjana Kalafatić (član povjerenstva)
• Committee member: Domagoj Đikić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2010-11-19, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Karcinom debelog crijeva nastaje kao rezultat kontinuirane promjene epitelnih stanica debelog crijeva zbog nakupljanja mutacija u nizu onkogena i tumor supresorskih gena. Danas je poznato da je u približno 50 % tumora debelog crijeva prisutan poremećaj signalnog puta MAPK. Iako su mutacije gena koji sudjeluju u ovom signalnom putu poznate već dugi niz godina, važnost njihove detekcije ponovno se aktualizirala razvojem personalizirane medicine i modernih ciljanih antitumorskih lijekova. Konvencionalne metode detekcije mutacija podložne su generiranju lažno pozitivnih ili negativnih rezultata. Stoga se u novije vrijeme kao metoda izbora za detekciju mutacija koristi metoda fluorescentne detekcije heterodupleksa DNA (HRM). U ovom radu metoda HRM uvedena je za detekciju najčešće mutacije gena BRAF, mutacije V600E, u uzorcima sporadičnog karcinoma debelog crijeva. Od ukupno 120 analiziranih uzoraka sporadičnog karcinoma debelog crijeva, 6 uzoraka (5 %) je u analizi metodom HRM pokazalo aberantni profil krivulje mekšanja. Metodom sekvencioniranja je u svih 6 analiziranih uzoraka dokazana prisutnost mutacije V600E. Metoda HRM se pokazala kao jednostavnija i brža alternativa konvencionalnim metodama analize mutacija te se može koristiti i za molekularno profiliranje drugih tipova tumora u kojima mutacija gena BRAF igra značajnu ulogu.

Abstract (english)

Colorectal cancer arises as a consequence of continuous mutation accumulation in a number of oncogenes and tumor suppressor genes. Approximately 50 % of colorectal tumors have an aberrant MAPK signalling. Although mutations of genes involved in MAPK signalling pathway are known for some time, their importance has recently been brought to scientific attention as a consequence of break-troughs in personalized medicine and modern antitumor therapy. Conventional methods of mutation detection are prone to generating false positive or negative results, therefore, in recent years a method of choice for mutation screening is the high resolution melting fluorescent detection of heteroduplexes (HRM). In this study HRM method was introduced in order to detect the most common BRAF gene mutation, V600E in sporadic colon cancer tumors. A total of 120 tumor samples were analyzed and in 6 (5 %) BRAF V600E mutation was detected and confirmed by DNA sequencing. Finally, HRM method has proved itself to be the simplest and faster alternative to conventional methods of mutation detection and can be used for molecular profiling of other tumors where BRAF mutations have an important role.

• Keywords: BRAF; HRM; signalni put MAPK; krivulja mekšanja
• Keywords (english): BRAF; HRM; MAPK signalling pathway; melting curve
• Language: croatian
• URN:NBN: urn:nbn:hr:217:401445
• Study programme: Title: Experimental Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra eksperimentalne biologije (magistar/magistra eksperimentalne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-05-03 12:13:21