Multiplex Detection of DNA Damage

• Author / Creator

  Nair,Sindhu G

• A large number of exogenous and endogenous agents can result in DNA damage leading to mutagenesis and cell death. A thorough knowledge and understanding about the effects of these agents on different sequences of DNA is important for elucidating the fundamental chemical mechanisms of DNA damage. Therefore, developing a rapid and inexpensive technique for the detection of damage in multiple samples is of great importance. Numerous DNA detection techniques, involving polymerase chain reaction and gel electrophoresis, have been developed. However, most of these techniques are cumbersome and/or expensive. In this thesis, we develop a simple, sensitive, inexpensive, mix-and-read assay to detect DNA damage with higher throughput and better ease-of-use. The goal of this work is to design simple hybridization assays for DNA damage detection of multiple sequences in single and double stranded DNA on a 96-well microplate platform. We first replaced the conventional cuvette method with the well plate method in order to study UVC-induced ssDNA damage on four sequences simultaneously. In this study, we measured the change in fluorescence intensity of a series of sequence-specific smart probes (SPs) for quantifying the extent of damage. The results show that this method has similar reproducibility as the cuvette method, but designing SPs complementary to each sequence makes the method tedious and expensive. In the second approach we developed a microplate assay coupled with EvaGreen (EG), an intercalating dye to quantify both ssDNA and dsDNA damage in an inexpensive way. This gives maximum sensitivity for detecting damage since the dye gives zero/minimum fluorescence with ssDNA and a maximum fluorescence with dsDNA. This dye gave good sensitivity and selectivity for quantification of dsDNA damaged by both UVC and a Ru cis-platin analog. The calibration curve for the EG probe shows good linearity (R2 = 0.99) with a limit of detection of 2.3 nM for dsDNA. Confirmation that EG can detect damage was done by melting curve and matrix-assisted laser desorption/ ionization time-of-fight mass spectroscopy (MALDI-TOF-MS). We also used our method to study the effect of the number and position of mismatches on the stability of dsDNA with another intercalating dye, Hoechst 33258 (H258). Results show that the sensitivity of the dsDNA detection is determined more by the position of mismatches than by the number of mismatches. We also compared the UVC-induced DNA damage kinetics of ssDNA and dsDNA. Our results are in agreement with in vitro and in vivo studies, which show ssDNA to be more vulnerable to damage than dsDNA. The H258 dye was much cheaper than the EG dye, but its use is limited because of its high selectivity for A-T rich regions of dsDNA. Finally, we explored the ability of our method to detect DNA damage in multiple samples of K-Ras and N-Ras proto-oncogenes. Results show that the K-Ras sequences are more mutagenic than the N-Ras sequences. This result is in excellent agreement with past biological studies performed on K-Ras and N-Ras genes. Thus, our method proves as a simple, inexpensive, mix-and-read assay for the reproducible quantification of DNA damaged induced by different etiological agents.

• Subjects / Keywords

  • Hybridization method
  • UVC and Ru-induced DNA damage
  • DNA damage
  • Multiplex assay
  • Fluorescence detection

• Graduation date

  Spring 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3S17T524

• 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.