4.3. DNA nicking assay
There are not many studies using UV-photolysis of hydrogen
peroxide to generate hydroxyl radical in DNA nicking assay. However,
it worked well in visualizing the DNA strands breakage when coupled
with hydrogen peroxide[17]. When exposed to UV, hydrogen peroxide
will undergo photodecomposition and produce two hydroxyl radicals.
Hydroxyl radicals generated by this reaction can cause oxidative
damage to the DNA and induce breakage in DNA strands to its damaged
forms. Different DNA forms have their own migration pattern. The
fastest moving prominent band corresponded to the native SCP, and
the slowest moving band represented the NCP. Thus, the extent of DNA
breakage could be identified through the presence of specific bands
on gel image. It was important to verify UV-photolysis method in the
presence of H2O2 before carrying on further with the DNA nicking
assay. In conclusion, H2O2 only induced DNA damage when exposed to
UV. This verification showed that UV-induced hydroxyl radical DNA
damage method was plausible and properly working in inducing the
damage to plasmid DNA. There were several advantages of using this
method over Fenton’s reaction. First, it had less uncertainty than latter
where ascorbic acid used as reducing agent in Fenton’s reaction might
inhibit hydroxyl radical[4]. Besides, this method had faster reaction
time (15-minute incubation time) compared to Fenton’s reaction,
which required 20 to 30 min incubation at 37 °C[4].
4.3. DNA nicking assayThere are not many studies using UV-photolysis of hydrogenperoxide to generate hydroxyl radical in DNA nicking assay. However,it worked well in visualizing the DNA strands breakage when coupledwith hydrogen peroxide[17]. When exposed to UV, hydrogen peroxidewill undergo photodecomposition and produce two hydroxyl radicals.Hydroxyl radicals generated by this reaction can cause oxidativedamage to the DNA and induce breakage in DNA strands to its damagedforms. Different DNA forms have their own migration pattern. Thefastest moving prominent band corresponded to the native SCP, andthe slowest moving band represented the NCP. Thus, the extent of DNAbreakage could be identified through the presence of specific bandson gel image. It was important to verify UV-photolysis method in thepresence of H2O2 before carrying on further with the DNA nickingassay. In conclusion, H2O2 only induced DNA damage when exposed toUV. This verification showed that UV-induced hydroxyl radical DNAdamage method was plausible and properly working in inducing thedamage to plasmid DNA. There were several advantages of using thismethod over Fenton’s reaction. First, it had less uncertainty than latterwhere ascorbic acid used as reducing agent in Fenton’s reaction mightinhibit hydroxyl radical[4]. Besides, this method had faster reactiontime (15-minute incubation time) compared to Fenton’s reaction,which required 20 to 30 min incubation at 37 °C[4].
การแปล กรุณารอสักครู่..