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Učinkovitost izolacije DNA iz različnih človeških tkiv ter bakterijske DNA iz brisa ustne sluznice z avtomatizirano metodo na osnovi magnetnih kroglic
ID Buneto, Špela (Author), ID Ambrožič Avguštin, Jerneja (Mentor) More about this mentor... This link opens in a new window, ID Pajič, Tadej (Co-mentor)

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Abstract
Izolacija ustrezno čiste in kakovostne deoksiribonukleinske kisline (DNA) je predpogoj za nadaljnje analize, ki so pomembne za diagnosticiranje bolezni, napovedovanje zdravljenja, spremljanje učinka zdravljenja ter izboljšanje kakovosti življenja posameznika. Na Kliničnem inštitutu za genomsko medicino (KIGM) v Univerzitetnem kliničnem centru v Ljubljani, zagotavljajo možnosti diagnostike in genetskega svetovanja bolnikom in družinam z genetsko pogojenimi boleznimi. S strokovnim in raziskovalnim delom omogočajo prenos najsodobnejših spoznanj v vsakodnevno obravnavo genetsko pogojenih bolezni na področjih, kot so genetska epidemiologija bolezni, ki je pomembna za javno zdravstvo, moške in ženske neplodnosti in nevrogenetike, vključujoč tudi raziskovanje povezanosti mikrobioma z nevrodegenerativnimi boleznimi. Na KIGM vrsto let izvajajo osamitev DNA iz različnih bioloških vzorcev s komercialno dostopnimi reagenčnimi kompleti. Zadnja leta pa želijo povečali pretočnost izolacije DNA in razširili nabor vzorcev, iz katerih bi lahko pridobili zadostne količine kakovostne molekule DNA. Zaradi tega smo se odločili, da v sklopu magistrske naloge preverimo učinkovitost aparata ChemagicTM360, ki omogoča avtomatiziran način izolacije zelo čistih in visokokakovostnih molekul DNA iz različnih človeških vzorcev, kot tudi bakterijske DNA iz brisa ustne sluznice. Z raziskavo smo želeli preveriti, ali je izolacija z avtomatizirano metodo primerljiva z že vpeljanimi metodami izolacije. Z analizami merjenja količin, čistosti in preverjanja učinkovitosti izolacije DNA smo ugotovili, da je avtomatizirana metoda primerljiva z izbranimi, v laboratoriju že vpeljanimi, metodami izolacije in je ustrezna za uporabo v rutinski genetski diagnostiki.

Language:Slovenian
Keywords:molekularna genetika, izolacija nukleinskih kislin, kri, tkivo FFPE, plodovo tkivo, cfDNA, bakterijska DNA, količina DNA, čistost DNA, učinkovitost izolacije
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Place of publishing:Ljubljana
Publisher:[Š. Buneto]
Year:2022
PID:20.500.12556/RUL-137298 This link opens in a new window
UDC:577.21:575.113:616-056.7
COBISS.SI-ID:110764547 This link opens in a new window
Publication date in RUL:10.06.2022
Views:1092
Downloads:106
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Secondary language

Language:English
Title:Efficiency of DNA isolation from various human tissues and of bacterial DNA from oral mucosa swabs by an automated method based on magnetic beads
Abstract:
Isolation of deoxyribonucleic acid (DNA) with a high level of purity and quality is a prerequisite for further analyses, which are of great importance for diagnosing human diseases, predicting treatments, monitoring the impacts of treatments, and improving the quality of life of individuals. At the Clinical Institute of Medical Genetics, a department of the University Medical Centre, they work with molecular genetic tests for the management of genetic diseases and oncological disorders, and among other things, they research the relationship between the microbiome and neurodegenerative diseases. They have established the isolation of nucleic acid from various biological samples using commercially available reagent tests for several years. In recent years they have been endeavoring to increase the flow rate of the samples, broadening the specter of samples from which they can collect DNA and improve the quality of the isolated nucleic acids. Therefore, we decided that validation of the ChemagicTM360 instrument should be a part of my master's thesis. This instrument has an automated system for DNA purification from various samples of human tissue as well as bacterial DNA from oral cavity swabs. As part of the ongoing research, we wanted to verify that the automated method was equivalent to well-established laboratory methods for isolation of nucleic acid. By analysing the quantity, purity, and verifying the efficiency of nucleic acid isolation, we concluded that the validated method can be substituted for reference isolation methods and is suitable for use in routine diagnostics.

Keywords:molecular genetics, nucleic acid isolation, blood, FFPE tissue, fetal tissue, cfDNA, bacterial DNA, amount of DNA, quantity of DNA, efficiency of isolation

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