Evolving the Methodology for Detection of Primary DNA Damage : Development, adaptation and assessment of the single cell gel electrophoresis (comet) assay

Abstract: Deoxyribonucleic acid (DNA) is one of the most important molecules in nature. It is the fundamental carrier of evolutionary information and constitutes the genetic blueprint of all living organisms. Being the sole source of information, it is vital for the cell to transmit the correct genetic information from generation to generation. DNA damage is a critical precursor to cancer development, highlighting the need for tests to predict genotoxicity and mutagenicity of various agents, including pharmaceuticals and environmental factors. This thesis focuses on enhancing the single cell gel electrophoresis (comet assay) for assessing primary DNA damage.The work was concentrated around several fundamental aspects of the methodology where a novel statistical approach, Uppsala Comet Data Analysis Strategy (UCDAS), was developed for data evaluation. A proportional odds model tailored to continuous outcomes was used, accommodating the experimental design's hierarchy, large zero values, and avoiding data transformation. A revisit of the formulation of the electrophoresis medium led to the introduction of a low conductive lithium hydroxide-based solution, enabling higher field strengths, significantly reducing runtimes and increasing sensitivity compared to the conventional comet assay. A lot of work was done on the investigation of the pH's impact on DNA integrity, revealing elevated background DNA damage at higher pH levels. Extended unwinding at pH >13, typical of the most commonly used versions of alkaline comet assays, jeopardizes the integrity of DNA, resulting in greater background DNA damage than at lower pH values. The study underscores pH's significance for DNA stability, highlighting risks associated with extremely alkaline conditions.A new method was developed, the Polymerase Assisted DNA Damage Assay (PADDA), to label and quantify single- and double-strand DNA breaks selectively in comet heads and tails after exposure to established DNA-damaging agents. This approach also allowed detection of DNA damage inside comet heads, an ability lacking in traditional comet assays.In conclusion, this research enhances DNA damage assessment methodologies, introducing new statistical innovations, novel electrophoresis mediums, and a novel technique for the selective detection and quantification of single- and double-strand breaks. These advancements deepen our understanding of DNA damage's complexities and underscore the crucial role of pH in influencing DNA stability and its implications for genotoxicity assessment.