Abstract
Human genome is continuously susceptible to changes that may lead to undesirable mutations causing various diseases and cancer. Vast majority of techniques has investigated the discrimination between base-pair mismatched nucleic acid, but many of these techniques are time-consuming, complex, expensive, and limited to the detection of specific type of dsDNA mismatches. In this study, we introduce a simple mix-and-read assay for the sensitive and cost-effective analysis of DNA base mismatches and UV-induced DNA damage using Hoechst genosensor dye (H258). This dye is a minor groove binder that undergoes a drastic conformational change upon binding with mismatch DNA. The difference in binding affinity between perfectly matched and mismatched DNA was studied for sequences at different base mismatch locations and finally, extended for the detection of dsDNA damage by UVC radiation in calf thymus DNA. In addition, a comparative DNA damage kinetic study was performed using H258 (minor groove binder) and EvaGreen (intercalating) dye to get insight on assay selectivity and sensitivity with dye binding mechanism. The result shows good reproducibility making H258 genosensor a cheaper alternative for DNA mismatch and damage studies with possibility of extension for in-vitro detection of hot spots of DNA mutations.
Published Version
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