title: Single-step intercalating dye strategies for DNA damage studies.
abstract: Many analytical protocols exist for the quantification of varied types of DNA damage, which span a range of complexity and sensitivity. As an alternative or companion to existing procedures, this article demonstrates the application of quantitative PCR (qPCR) and high-resolution DNA melting analysis (HRMA) to the detection and quantification of intramolecular DNA damage and/or strand breaks. These proven molecular biology methods are essentially single-step processes. When implemented with a third-generation saturating DNA dye, high sensitivity can be obtained. The experiments presented here demonstrate how DNA damage can inhibit amplification of the affected molecules. This corresponding decrease in the initial concentration of amplifiable DNA can be measured with qPCR. In addition, damage in the form of intramolecular dimerization and strand breaks alters the stored energy in the hydrogen bonds between the two strands in the dsDNA molecule. This significantly affects the thermal stability, which can be measured with extreme precision using HRMA. Simplified damage models were used in these experiments: UV-C irradiation to produce photoproducts, and restriction enzyme digestion to simulate double-strand breaks. The findings of this work, however, can be intuitively applied to the broad scope of DNA damage mechanisms.
authors.affiliation: Institute for Micromanufacturing, Louisiana Tech University, 911 Hergot Ave., Ruston, LA 71272, United States. Electronic address: mpa014@latech.edu.
authors.forename: Manasa
authors.initials: M
authors.lastname: Paidipalli
authors.affiliation: Institute for Micromanufacturing, Louisiana Tech University, 911 Hergot Ave., Ruston, LA 71272, United States.
authors.forename: Ilija
authors.initials: I
authors.lastname: Pjescic
authors.affiliation: School of Biological Sciences, Louisiana Tech University, Ruston, LA 71272, United States.
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Hindmarsh
authors.affiliation: Institute for Micromanufacturing, Louisiana Tech University, 911 Hergot Ave., Ruston, LA 71272, United States.
authors.forename: Niel D
authors.initials: ND
authors.lastname: Crews
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification / Q000528:radiation effects; D053960:DNA Breaks / Q000528:radiation effects; D004249:DNA Damage / Q000528:radiation effects; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D007364:Intercalating Agents / Q000737:chemistry*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D044366:Transition Temperature; D014466:Ultraviolet Rays
chemical_list: D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D007364:Intercalating Agents
keywords: CPDs; DNA damage; HRMA; Photoproducts; Strand breaks; qPCR