Optimizacija izolacije DNK iz tkivnih vzorcev ter analiza somatskih mutacij pri bolnicah z rakom dojk

diplomsko delo univerzitetnega študijskega programa I. stopnje

Janina Gea Cvikl (Author), Uroš Potočnik (Mentor), Katja Repnik (Co-mentor), Iztok Takač (Co-mentor)

Abstract

Rak dojk je biološko in molekularno heterogena bolezen, zato je identifikacija podtipa ključna za pravilno izbiro zdravljenja. Pri tem nam pomagajo tudi somatske mutacije, to so mutacije DNK v somatskih celicah, ki lahko vodijo v razvoj tumorja. Analiza somatskih mutacij v tkivnih vzorcih predstavlja velik izziv. Zato je bil cilj diplomske naloge optimizirati postopek izolacije iz različnih tipov tkivnih vzorcev ter analizirati izbrane somatske mutacije v genih, najpogosteje povezanih z nastankom raka dojk. V prvem delu diplomske naloge smo se osredotočili na izolacijo DNK iz treh različnih tipov vzorcev in sicer iz krvnih limfocitov s fenol-kloroformno ekstrakcijo, iz sveže zamrznjenih tkiv, shranjenih v raztopini RNAlater s predhodno homogenizacijo tkiva ter nadaljno fenol-kloroformno ekstrakcijo, ter iz tkivnih vzorcev vpetih v parafin z uporabo komercialno dostopnega kompleta reagentov. Pri evalvaciji metod smo upoštevali parametre, kot so koncentracija, izkoristek, čistost in integriteta DNK, metode izolacije pa smo ovrednotili tudi stroškovno in časovno, ter primerjali intenziteto dela. Za določanje koncentracije, izkoristka in čistosti izolirane DNK smo uporabili spektrofotometrične meritve, za določevanje integritete DNK pa metodo gelske elektroforeze. Nadalje smo želeli optimizirati genotipizacijo vzorcev za izbrane somatske mutacije na genih BRCA1 (rs55770810), TP53 (rs587778720) in BRCA2 (rs8035971). Pri genotipizaciji smo uporabljali metodi PCR-RFLP in qPCR-HRM. V okviru diplomske naloge smo uspešno izolirali DNK iz vseh treh tipov vzorcev, pri čemer smo najvišji izkupiček in integriteto DNK pričakovano dosegli pri DNK, izolirani iz krvnih limfocitov, vendar je bila čistost izolirane DNK najnižja. Stroškovna in časovna analiza je pokazala, da je postopek izolacije DNK iz tkiva vpetega v parafin najdražji, vendar najhitrejši in najmanj zahteven postopek, predvsem v primerjavi z izolacijo DNK iz svežih tkiv. Pri analizi izbranih somatskih mutacij smo za SNP rs587778720 potrdili večjo frekvenco patogenega alela v somatskih celicah kot v DNK, izolirani iz krvnih vzorcev. Genotipizacija SNP-jev rs55770810 in rs8035971 je bila neuspešna. Oba SNP-ja se nahajata na delu DNK, kjer zaradi velike variabilnosti regije prihaja do številnih drugih variacij, ki interferirajo z genotipizacijo izbranega SNP-ja.

Keywords

rak dojk;SNP;genotipizacija;izolacija DNK;somatske mutacije;diplomske naloge;

Data

Language:	Slovenian
Year of publishing:	2019
Typology:	2.11 - Undergraduate Thesis
Organization:	UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher:	[J. G. Cvikl]
UDC:	601.4:618.19-006(043.2)
COBISS:	22609430
Views:	1164
Downloads:	96
Average score:	0 (0 votes)
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Other data

Secondary language:	English
Secondary title:	Optimization of DNA isolation and analysis of somatic mutations in breast cancer patients
Secondary abstract:	Breast cancer consists of a group of biologically and molecularly heterogeneous diseases, which consequently differ in the clinical implications and treatment strategies of each individual disease. Somatic mutations are DNA mutations, which occur in somatic cells and lead to tumour development. They can sometimes be key to identifying the cancer subtype. Analysis of somatic mutations in tissue samples can be challenging. The aim of this work was to optimize isolation protocols for DNA isolation from different types of tissue and to analyse the selected somatic mutations in genes, most commonly associated with breast cancer. In the first part, we isolated DNA from three different tissue sources; blood lymphocytes with the phenol-chloroform extraction; fresh tissue samples stored in RNAlater solution with the phenol-chloroform extraction on previously homogenized tissue samples; and formalin-fixed paraffin embedded tissues using a commercially available DNA extraction kit. We evaluated all three methods in terms of the concentration, yield, purity and integrity of extracted DNA. In addition, we evaluated cost, time and intensity of labour for each of the methods. Spectrophotometric measurements were used to determine the concentration, yield and purity of extracted DNA, while the integrity of extracted DNA was assessed by agarose gel electrophoresis. In the second part, we aimed to optimize genotyping of the selected somatic mutations on genes BRCA1 (rs55770810), TP53 (rs587778720) in BRCA2 (rs8035971). We used SNP genotyping methods PCR-RFLP and qPCR-HRM. Results show, that all three DNA extraction methods were successful, with DNA isolated from blood lymphocytes showing the highest yield and integrity, but the lowest purity of extracted DNA. Cost, time and labour intensity analysis show, that DNA extraction from FFPE is the quickest and least intensive, but the most expensive type of isolation. Frequency of pathogen allele of SNP rs587778720 was higher in somatic cells of tumor tissue compared to DNA, extracted from blood lymphocytes. Genotyping of SNPs rs55770810 and rs8035971 was unsuccessful. Both SNPs are located in DNA regions, where high variability causes many different variations, which can interfere with successful genotyping.
Secondary keywords:	breast cancer;SNP;genotyping;DNA isolation;somatic mutations;
URN:	URN:SI:UM:
Type (COBISS):	Bachelor thesis/paper
Thesis comment:	Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages:	X, 41 str.
ID:	11185658