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The mechanisms that underlay the repairs of DNA damages - Thesis Example

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Chapter 2 Tutor College Date Objective The objective of this project is to understand the mechanisms that underlay the repairs of DNA damages induced by nucleoside analogues. This will be observed through assessing the role of DNA repair mechanisms in survival to nucleoside analogue treatment in S…
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The mechanisms that underlay the repairs of DNA damages
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Download file to see previous pages Cytarabine is used in the treatment of acute cases of lymphomas and leukemia. The key mutants to be acted upon are deoxyribonucleoside kinase and deoxycytidine kinase. Using S. pombe provides a greater advantage as it can be easily manipulated both physically and genetically. It also helps to understand the DNA repair mechanism in higher eukaryotes. In its natural form, yeasts do not contain a particular transporter and the particular kinase used to phosphorylate nucleoside analogues. Therefore, in this project, the S. pombe in use is adapted in that it contains deoxynucleotide kinase (dmdHK) which is found in Drosophila melanogaster (fruit fly) and is important for their phosphorylation (the addition of a phosphate molecule on to a protein molecule or any other organic molecule). It also contains the human membrane transporter (hENTI) which allows the nucleoside analogue to directly interfere with the processes taking place within the cell. 2.1.0 Material used in the practical 2.1.1 S. pombe strains The S.pombe strains used in this practical are listed below: WT: h+ ura4-D18::adhdmdNK-NAT-adhhENT1 URA4aim Swi10: (h+ ura4-D18::adhdmdNK-NAT-adhhENT1 swi10::KanMX URA4aim) Removal of damage in nucleotide excision repair. exo1: (h+ ura4-D18::adhdmdNK-NAT-adhhENT1 exo1::URA) Removal of damage in mismatch repair. rhp14: (h+ ura4-D18::adhdmdNK-NAT-adhhENT1 rhp14:: KanMX URA4aim). Recognition of damage in the process of nucleotide excision repair. rad50: (h+ ura4-D18::adhdmdNK-NAT-adhhENT1 rad50:: KanMX URA4aim) of the MRN(Mre11,Rad50,Nbs1) complex, for repair of double strand breaks. rad32D65N: (h+ ura4-D18::adhdmdNK-NAT-adhhENT1 rad32D65N URA4aim). Mre11 nuclease dead, lacks the activity of Mre11 in double strand breaks repair. 2.1.2 Medias Liquid EMM+Glut {(EMM broth without nitrogen 109.2 g + Glutamic acid 15g) liquid + Granulated agar 10g} solid . 2.1.3: Preparing Agarose gel: first PCR Procedure: 1. With the use tape, seal all the borders of a dry and clean glass plate. This is important for forming mold. 2. Prepare 50ml 10X TBE (Tris-boric acid-EDTA) buffer for the preparation of the gel. 3. Add 0.8% of powdered Agarose to the TBE (Tris-boric acid-EDTA) buffer in an Erlenmeyer flask. The buffer should occupy less than 50% of the total flask volume. 4. Heat the solution in a water bath or a microwave oven to facilitate the dissolving of all the Agarose grains. During the heating process, part of the buffer will evaporate. As such it is important to add more buffer solution in order to return the solution to the original volume. 5. After heating, cool the solution to 60°C and immerse the solution in an ethidium bromide solution once the gel solidifies. Ethidium bromide helps to intercalate DNA thus making it visible under ultra violet light. 6. Place the comb 1.0 mm above the place consequently allowing for the formation of a complete well once the Agarose solidifies. It is important to avoid air bubbles that may form between or under the teeth of the comb. Seal the plate with small quantities of the Agarose solution through a Pasteur pipette. Once the plate is sealed, transfer the gel in to the glass plate. It is important to swirl the gel in the flask to ensure uniformity in the temperature before pouring it out into the plate to prevent the gel for hardening unevenly. 7. The gel is expected to have ...Download file to see next pagesRead More
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