Mutation, Cloning And Expression Analysis Of Thermophilic And Mesophilic Dna Polymerases: Towards Thier Use In DNA Amplification Assays
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Date
2015-10
Authors
Suppan, Malarveli
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Abstract
Routine 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.
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Keywords
Thermophilic DNA Polymerases , Mesophilic DNA Polymerases