The stabilization effect of DNA polymerase on DNA duplexes
This study is based upon previous observations of DNA polymerase stabilizing DNA duplexes. With the presence of DNA duplexes and DNA polymerases in many experimental methods in modern biotechnology, it is important to gain knowledge about the interactions between them. In order to acquire this knowl...
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Format: | Dissertation |
Sprache: | eng |
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Zusammenfassung: | This study is based upon previous observations of DNA polymerase stabilizing DNA duplexes. With the presence of DNA duplexes and DNA polymerases in many experimental methods in modern biotechnology, it is important to gain knowledge about the interactions between them. In order to acquire this knowledge, several DNA duplexes have been investigated. These DNA duplexes were designed to have different structures, in order to understand what factors might influence the stabilization provided by DNA polymerase. These alterations were made in order to gain information that can further test the hypothesis that DNA polymerase can stabilize DNA duplexes. The duplexes used in these experiments had three different sequences, as well as three alterations made to them. Including mismatches positioned in different locations on the sequence, a length difference between the strands, and replacing the 3’-hydroxyl on the recessed primer strand with a 3’-phosphate group. The DNA duplexes were put through a melting curve analysis in a qPCR machine, using EvaGreen® as an intercalating agent, to find the melting temperature (Tm) of the different probes, with and without active DNA polymerase. The theoretical Tm was also calculated utilizing an online prediction tool for comparison.
These experiments were performed with two different DNA polymerases, which provided insights into how their stabilization effect may change under different circumstances. In all the experiments, duplexes incubated with FIREpol® showed a slightly higher Tm than those with HOT TERMIpol®. If the primer strand was shorter than the template strand, a Tm shift was observed. The duplexes with a mismatch also had a change in the stability of the duplexes. When comparing the observed and predicted Tms of the DNA duplex, it was detected that the further away the mismatch was from the 3’ end of the recessed nucleotide, the greater the difference between the theoretical Tm and the observed Tm. DNA polymerase did not stabilize duplexes where the 3’-hydroxyl group was replaced by a 3’-phosphate group on the recessed primer strand.
The results achieved in this study demonstrates that DNA polymerase can have a stabilizing effect on DNA duplexes. |
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