Subject(s)
DNA Damage , DNA/chemistry , Environmental Monitoring/methods , Environmental Pollution/analysis , Bacteriophage phi X 174/genetics , Cobalt Radioisotopes , DNA/analysis , DNA/radiation effects , DNA, Neoplasm/analysis , DNA, Neoplasm/chemistry , DNA, Superhelical/chemistry , DNA, Superhelical/isolation & purification , DNA, Viral/analysis , DNA, Viral/chemistry , Electrophoresis, Capillary/instrumentation , Electrophoresis, Capillary/methods , Gamma Rays , Humans , Indicators and Reagents , Lasers , Micrococcal Nuclease , Plasmids/chemistry , Plasmids/isolation & purification , Restriction Mapping , Spectrophotometry, Ultraviolet/instrumentation , Spectrophotometry, Ultraviolet/methods , Tumor Cells, CulturedABSTRACT
Bacterial plasmids and the chromosomal DNA of many organisms adopt naturally the negatively supercoiled conformation. Therefore, the irradiation of such plasmids could be used to model conformational changes of chromosomal DNA associated with externally-induced damage. We have applied dynamic size-sieving capillary electrophoresis (CE) to monitor the damage of three DNA plasmids, over an unprecedented base pair (bp) size range (2870-27 500 bp), upon exposure to gamma-radiation (20-400 Gy). Predominantly, CE with UV absorbance detection in the absence of DNA intercalating dyes was employed to preclude undesirable, induced plasmid conformational changes. Plasmid samples and their enzymatic digestion products were analyzed using both CE and slab gel electrophoresis (SGE) in order to verify the conformation of sample components. Relative to SGE, CE analyses revealed more fine structural features of plasmid degradation.
Subject(s)
DNA Damage , DNA/radiation effects , Electrophoresis, Capillary/methods , Gamma Rays , Molecular Weight , Particle Size , Spectrophotometry, UltravioletABSTRACT
Various natural and induced processes cause DNA fragmentation. Examples of these processes include apoptosis, enzymatic digestion, free radical production from ionizing radiation, photoscission by laser radiation and thermal degradation. Slab gel electrophoresis has been used most often to monitor such DNA damage. We have investigated with capillary electrophoresis the use of a new size-sieving polymer solution, TreviSol-CE (TS-CE), to monitor the DNA fragments produced from a variety of degradation processes. This polymer solution provides high run-to-run migration time and peak width reproducibilities and high separation efficiency of double-stranded DNA fragments in the 500 to 7000 base pair size range. Analysis of apoptotic DNA fragments suggested the presence of multiple nucleosomes within each cell type investigated. For irradiated DNA standards, peak-width-at-half-height and peak area were used to monitor the progress of DNA fragmentation. For both apoptotic DNA and irradiated DNA standards, fine structural features of fragmentation were revealed.
