Mutation, Cloning And Expression Analysis Of Thermophilic And Mesophilic Dna Polymerases: Towards Thier Use In DNA Amplification Assays

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dc.contributor.authorSuppan, Malarveli
dc.date.accessioned2016-06-21T07:04:04Z
dc.date.available2016-06-21T07:04:04Z
dc.date.issued2015-10
dc.description.abstractRoutine molecular genetics detection method is based on in vitro DNA amplification via isothermal kits which employ commercially available Bst Large Fragment DNA polymerase. However, these assays lacked accuracy as the DNA polymerase lacks proofreading activity. In an effort to enhance the fidelity of the diagnostic test, a thermophilic enzyme with 3'→5' exonuclease activity was synthetically produced. At the same time, in order to perform on site DNA amplification testing novel mesophilic DNA polymerase was identified. In this study, the native and mutant Bst DNA polymerase genes were constructed via assembly and sequential overlap extension PCR gene synthesis method and cloned into pCR2.1-TOPO vector. The mutant gene was constructed with specific mutations at position 319 (V319D), 325 (E325L), 376 (A376D), 425 (D425F), 450 (K450D) and insertion at 446 (446InY). At the same time, a gene that encodes for mesophilic DNA polymerase from local Proteus mirabilis isolate was amplified and cloned into pCR2.1-TOPO vector for sequence analysis and family prediction. Then, these genes were cloned in pET- 28a(+) vector, expressed and their proteins were purified with two step purification which includes IMAC purification and IEC purification. Then, purified proteins were confirmed with MALDI-TOF/TOF analysis. Their enzymatic characterization showed a significant polymerization activity. Moreover, the Pmi DNA polymerase was able to perform isothermal amplifications at room temperature. While the mutant Bst DNA polymerase showed reduction in isothermal amplification efficiency compared to native enzyme but enhanced amplification fidelity.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/2172
dc.subjectThermophilic DNA Polymerasesen_US
dc.subjectMesophilic DNA Polymerasesen_US
dc.titleMutation, Cloning And Expression Analysis Of Thermophilic And Mesophilic Dna Polymerases: Towards Thier Use In DNA Amplification Assaysen_US
dc.typeThesisen_US
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