Sinteza i određivanje osjetljivosti digoksigeninom obilježene probe za hibridizaciju DNA metodom po Southern-u

undergraduate thesis

• Title (english): Synthesis and sensitivity determination of digoxigenin-labelled probe for Southern blotting
• Author: Rea Bertoša
• 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: 2014-09-11, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Cilj ovog rada bio je sintetizirati te odrediti osjetljivost digoksigeninom (DIG) obilježene probe, komplementarne genu URA3 kvasca Dekkera bruxellensis, koja se koristi pri hibridizaciji DNA po Southern-u. DIG-obilježena proba sintetizirana je lančanom reakcijom polimerazom (PCR), koristeći kao kalup regiju DNA koja sadrži gen URA3, a uspješnost reakcije provjerena je gelelektroforezom. Kako bi se odredila osjetljivost obilježene probe provedena je gel-elektroforeza s različitim masama plazmida koji sadrži gen URA3 kvasca Dekkera bruxellensis. Uzorci razdvojeni elektroforezom preneseni su na nitroceluloznu membranu koristeći vakuum prijenos u lužnatom mediju. Nakon predhibridizacije i hibridizacije denaturiranom DIG-obilježenom probom, detekcija je provedena kolorimetrijskom metodom pomoću BCIP (toluidinska sol 5-brom-4-klor-3’-indolilfosfata) i NBT (nitro-plavi tetrazolijski klorid) koji su korišteni kao supstrat i indikator za alkalnu fosfatazu. Budući da se hibridizacijski signal na membrani pojavio i u uzorku koji je sadržavao svega 1 ng plazmidne DNA može se zaključiti da je sintetizirana proba dovoljno osjetljiva te se može koristiti za analizu genoma kvasca D. bruxellensis.

Abstract (english)

The aim of this work was to synthesize and to determine the sensitivity of digoxigeninlabelled (DIG-labelled) probe, complementary to the URA3 gene from the yeast Dekkera bruxellensis, used for Southern blotting. DIG-labelled probe was synthetized by polymerase chain reaction (PCR), using as a template DNA region that contained URA3 gene, while the success of a PCR reaction was confirmed by gel electrophoresis. To determine the sensitivity of the probe, gel electrophoresis with various masses of the plasmid DNA, containing URA3 gene from the yeast D. bruxellensis was performed. Samples separated by gel electrophoresis were transferred to a nitrocellulose membrane using alkaline vacuum transfer. DNA was prehybridized and hybridized with DIG-labelled probe while the detection was done by colorimetric method using BCIP (toluidine-salt of 5-bromo-4-chloro- 3-indolyl-phosphate) and NBT (nitro blue tetrazolium chloride), as a substrate and indicator for the alkaline phosphatase, respectively. Since hybridization signal was obtained even with the sample containing only 1 ng of the plasmid DNA, we can conclude that newly synthesised probe is sensitive enough and can be used for yeast D. bruxellensis genome analysis.

• Keywords: hibridizacija po Southern-u; gel-elektroforeza; digoksigeninom obilježena proba
• Keywords (english): Southern blotting; gel-electrophoresis; digoxigenin labelled probe.
• Language: croatian
• URN:NBN: urn:nbn:hr:159:008162
• 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: 2017-04-26 11:46:12