Original title: Testování mikrometody izolace DNA z listů, plodů a výrobků z ovoce
Translated title: Evaluation of a micromethod for isolation of DNA from plant leaf, fruit and fruit products
Authors: Balažovičová, Nikola ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
Document type: Master’s theses
Year: 2019
Language: slo
Publisher: Vysoké učení technické v Brně. Fakulta chemická
Abstract: [slo] [eng]
Diplomová práca bola zameraná na testovanie mikrometódy izolácie DNA z listov, plodov a výrobkov z ovocia. Pre analýzu rastlinnej DNA v technologicky spracovaných potravinách boli vybraté džemy. Listy, plody a džemy boli homogenizované s použitím plastového kopistu v lyzačnom roztoku s 2% cetyltrimetylammonium bromidom (CTAB) s prídavkom 2,5M chloridu sodného (NaCl). Homogenáty boli použité pre izoláciu rastlinnej DNA metódou založenej na magnetických časticiach. Boli použité magnetické mikročastice poly(hydroxyethylmethakrylát-co-glycidylmethakrylát) – P(HEMA-co-GMA). Izolovaná DNA bola podrobená spektrofotometrickej analýze, kde bola zisťovaná jej koncentrácia a čistota. Následne bola testovaná amplifikovateľnosť rastlinnej DNA v polymerázovej reťazovej reakcii (PCR). Použité boli špecifické priméry pre rastlinnú ribozomálnu DNA - 18S_for a 5,8S_rev (produkt PCR - 700bp), 26S_for a 26S_rev (produkt PCR - 220 bp), 18S_for a 18-S_rev (produkt PCR - 263 bp). Boli optimalizované podmienky PCR a bol sledovaný vplyv dĺžky amplikónu na jeho detekciu. Produkty PCR boli detegované pomocou agarózovej gélovej elektroforézy. Bolo zistené, že z väčšiny listov je možné pomocou zvolených magnetických nosičov izolovať DNA v kvalite vhodnej pre PCR , na rozdiel od plodov. Z väčšiny testovaných džemov bola izolovaná DNA, ktorá sa amplifikovala v PCR s priméry dávajúcimi krátke produkty PCR. Metódu je treba ďalej optimalizovať. The thesis has been focused on testing of micromethod of DNA isolation from leaves, fruits and fruit products. Jams were selected for the analysis of plant DNA in technologically processed foods. Plant leaves, fruits, and jams were homogenized using plastic copist in a lysis buffer containing 2% cetyltrimethylammonium bromide (CTAB) with 2.5M sodium chloride (NaCl). Microisolation of plant DNA was performed using poly(hydroxyethylmethacrylate-co-glycidylmethacrylate) – P(HEMA-co-GMA)microparticles. Isolated the DNA concentration and purity were assessed by UV light aborbance using a spectrophotometer. After that, amplification of the DNA was tested in PCR. Primers specific for plant ribosomal DNA: 18S_for a 5,8S_rev (PCR product - 700bp), 26S_for a 26S_rev (PCR product - 220 bp), 18S_for a 18¬S_rev (PCR product - 263 bp) were used. The PCR conditions were optimized and the effect of the amplicon length on its detection was followed. PCR products were detected by agarose gel electrophoresis. It was shown that DNA isolated from almost all of leaves using magnetic particles was in PCR-ready quality in contrary to the fruits. DNA amplified in PCR with primers giving short PCR products was isolated from almost all tested jams. The method must be optimalised, yet.
Keywords: fruit; highly processed food products; isolation of DNA; jams; leaves; magnetic microparticles; PCR

Institution: Brno University of Technology (web)
Document availability information: Fulltext is available in the Brno University of Technology Digital Library.
Original record: http://hdl.handle.net/11012/177449

Permalink: http://www.nusl.cz/ntk/nusl-401887


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Universities and colleges > Public universities > Brno University of Technology
Academic theses (ETDs) > Master’s theses