A reverse transcription reaction allows the production of complementary DNA (cDNA) using an RNA template and relies on polymerases displaying reverse transcriptase (RT) activity. This process, with major applications in both research and in medical diagnostics, is often limited by the nature of the RTs available. RNA secondary structure can prove problematic where mesophilic retroviral RTs are used while the alternative approach, using thermophilic DNA polymerases with RT activity, often results in error-prone cDNA production. <br /> This project recognised the need to study other possible sources of thermophilic RTs and outlines the study of four previously uncharacterised Group II Intronencoded proteins (IEP), with RT domains, from thermophilic bacteria. While cloning of the IEP genes and their expression on a small scale proved successful, difficulties were encountered when attempting purification. Despite a lack of overall purity, samples containing IEPs from Thermosinus carboxydivorans and Petrotoga mobilis were shown to have RT activity but characterisation of these IEPs was not carried out. However, an IEP from Bacillus caldovelox proved to be an excellent candidate for characterisation as successful purification was achieved. Enzyme engineering was also performed, fusing a Sac7d domain onto the C-terminus of this protein. These enzymes were shown to have optimum RT activity at 54ºC with activity still being displayed at 76ºC. Other studies on these enzymes showed that, unlike the retroviral RTs, the IEPs displayed no DNA-dependent DNA polymerase activity. The Sac7d fusion protein was also studied in terms of possible enhancements to the RT activity of an IEP. However, preliminary studies showed that, although this domain did not prove to be detrimental to the enzyme, it had little effect on improving the processivity of the RTs. <br /> Although this class of RT looks promising in terms of use as an alternative thermophilic RT, the IEPs studied in this report did incur major limitations during cDNA synthesis, which included lower than expected optimum reaction temperatures, very low fidelity and an inability to synthesise cDNA using complex RNA templates.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:538289 |
| Date | January 2011 |
| Creators | Voina, Natasha J. |
| Contributors | Danson, Michael ; Hough, David |
| Publisher | University of Bath |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.0016 seconds