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UV-induced DNA damage detection in long oligonucleotide sequences using EvaGreen®
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- Author / Creator
- Ikegwuonu, Ebuka Penticost
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The exposure of DNA to UV radiation can lead to deletions, strand breaks or base modifications such as the formation of cyclobutane pyrimidine dimers (CPDs), [6-4] pyrimidine-pyrimidinones, and photohydrates, which may then lead to skin cancer. Different methods have been developed for the detection of UV-induced DNA damage, including molecular beacons, smart probes, mass spectrometry, high-performance liquid chromatography, comet assays, and electrophoresis. However, these methods have disadvantages, such as rigorous or cumbersome sample preparation procedures which may further damage the DNA, are sequence dependent, and/or are expensive. Recent studies have shown that EvaGreen® (EG®), a DNA intercalating dye, can be used to detect UV-induced DNA damage in short oligonucleotide sequences. In this study, we show a simple mix-and-read method of detecting UV-induced DNA damage using calf thymus DNA (ct-DNA), salmon sperm DNA (ss-DNA) and E. coli DNA samples. The ss-DNA and ct-DNA were approximately 2000 base pairs long. Samples were irradiated anoxically individually and later simultaneously with UVC lamps emitting at 254 nm with a power density of 75 W m-2. Irradiated DNA samples were then hybridized with EG® after various irradiation times and the fluorescence measured using a plate reader at room temperature. The results obtained show that the fluorescence intensities decrease with increasing irradiation time, consistent with EG® being released in its lower fluorescent-intensity form from the damaged DNA. Therefore, EG® is a potential tool for the detection of UV-induced DNA damage in long oligonucleotide sequences, and further shows good prospects of potentially up to and including genomic DNA. Under these conditions for the individual irradiation experiments of ct-DNA and ss-DNA, ct-DNA with an Adenine-Thymine base pair percentage composition (AT %) of 58.1 % is damaged at a faster rate than the ss-DNA of AT % composition of 56.9 %, with average damage time constants of 82 ± 13 min and 143 ± 43 min, respectively.
In the simultaneous irradiation experiments of ss-DNA and ct-DNA under the same conditions, ss-DNA although with a lower AT% composition showed similar average damage time constant of 112 ± 25 min with ct-DNA of an average damage time constant of 101 ± 24 min within experimental errors. The simultaneous UVC irradiation of extracted cellular E. coli DNA, ss-DNA and ct-DNA showed that E. coli DNA with an AT% composition of 49.2 which is lower than that of both ss-DNA and ct-DNA showed a higher average time constant. Results from the simultaneous irradiation of E. coli DNA and ss-DNA showed that E coli DNA had a damage time constant of 69 ± 11 min while 63 ± 6 min was obtained for ss-DNA which are similar within statistical analysis, although E. coli DNA showed an observed higher average damage time constant of 105 ± 29 min when irradiated simultaneously with ct-DNA of average damage time constant 66 ± 12 min, which is consistent with results obtained from previous studies. Finally, the systematic and human error analysis associated with this method was evaluated in this study.
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- Subjects / Keywords
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- Graduation date
- Spring 2021
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- Type of Item
- Thesis
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- Degree
- Master of Science
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.