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Identifier 000426964
Title Development of enzymatic molecular tools for RNA analysis
Alternative Title Ανάπτυξη ενζυμικών εργαλείων για ανάλυση RNA μορίων
Author Χεκήμογλου, Μαρία
Thesis advisor Μπουριώτης, Βασίλης
Abstract Synthesis of RNA by in vitro transcription is essential in many analytical techniques. Transcript generation is a simple and efficient process, requiring only an RNA polymerase to transcribe the gene of interest, DNA template downstream of a T7 promoter, ribonucleotides and a ribonuclease inhibitor to protect the RNA product. The current Master Thesis focuses on the development of two enzymatic molecular tools essential for RNA analysis, T7 RNA Polymerase and Human Placental Ribonuclease Inhibitor.T7RNAP is a single subunit RNA polymerase, extremely promoter-specific and able to complete RNA synthesis without additional protein factors. These properties make T7RNAP an ideal enzyme for in vitro transcription. Both the isolation and quality controls have been successfully performed and the enzyme is commercially available by Minotech Biotechnology. Ribonuclease inhibitor binds to pancreatic-type ribonucleases with high affinity and renders them inactive. For this work, an hRI variant resistant to oxidation was selected. Due to the enzymes hydrophobicity and instability that are strongly connected with the existence of 31 cysteine residues, the development of the product was incomplete. Extensive troubleshooting was applied including expression and purification conditions in order to isolate an active enzyme.
Language English
Subject Biotechnology
Enzyme assay
In vitro transcription
In vitro μεταγραφή
Ενζυμική αντίδραση
Issue date 2019-11-29
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Biology--Post-graduate theses
  Type of Work
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