Sinteza fragmenta DNA za inaktivaciju gena RAD27 lančanom reakcijom polimerazom

undergraduate thesis

• Title (english): Synthesis of a DNA fragment by polymerase chain reaction used for inactivation of the gene RAD27
• Author: Davor Nestić
• Mentor: Ivan-Krešimir Svetec (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2013-09-13, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Cilj ovoga rada je umnažanje linearnog fragmenta DNA rad27::kanMX4 lančanom reakcijom polimerazom iz kvasca Saccharomyces cerevisiae. Fragment rad27::kanMX4 se zahvaljujući krajevima homolognima dijelu gena RAD27 i selektivnom biljegu kanMX4 moţe koristiti za inaktivaciju gena RAD27 u drugim sojevima kvasca mehanizmom rekombinacije „end-out“ (zamjena gena). Nakon izolacije genomske DNA kvasca, provedena je gel-elektroforeza radi provjere uspješnosti izolacije te je izolirana DNA poslužila kao kalup za sintezu fragmenta lančanom reakcijom polimerazom. Gel-elektroforezom provjerena je uspješnost sinteze fragmenta, a struktura umnoženog fragmenta provjerena je restrikcijskom analizom pomoću tri restrikcijska enzima. Veličina dobivenih fragmenata nakon cijepanja restrikcijskim enzimima bila je u skladu s restrikcijskom mapom fragmenta rad27::kanMX4.

Abstract (english)

The objective of this project was the amplification of a linear DNA fragment rad27::kanMX4 by polymerase chain reaction (PCR) from the yeast Saccharomyces cerevisiae genome. The rad27::kanMX4 fragment carries a kanMX4 selection marker and it’s ends are homologous to yeast RAD27 gene. Hence, it can be used for transformation and inactivation of RAD27 gene in yeast via the „ends-out” recombination (gene replacement). After isolating the yeast genomic DNA, a gel electrophoresis was used to verify the success of the isolation. The isolated DNA served as a template for the PCR reaction, and gel electrophoresis was carried out to verify the success of the amplification. Three restriction enzymes were used for the restriction analysis, which determined that the structure of the amplified rad27::kanMX4 fragment is in accordance with the restriction map.

• Keywords: Saccharomyces cerevisiae; lančana reakcija polimerazom; homologna rekombinacija; zamjena gena.
• Keywords (english): Saccharomyces cerevisiae; polymerase chain reaction; homologous recombination; gene replacement
• Language: croatian
• URN:NBN: urn:nbn:hr:159:985031
• Study programme: Title: Biotechnology; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-05-04 08:35:14