Description
Ionizing radiation can cause single-strand breaks (SSB) and double-strand breaks (DSB) in DNA. The first part of this study investigated the sensitivity of DNA to damage from ionizing radiation for four dilution buffers; TE X10, deionized water, 20.0 mM and 5.0 mM of potassium phosphate (i.e., PP X20 and PP X5, respectively). The second part of the study investigated DNA sensitivity to damage as a function of DNA concentration. We hypothesized that with a decrease in free radical scavenging capacity from the dilution buffer and an increase in DNA concentration, there would be an increase in DNA SSB and DSB damage and detection. The level of strand break damage induced corresponds to the dilution buffer and the final DNA concentration obtained. We used a supercoiled plasmid DNA with a length of 6,000 base pairs obtained from an E. coli glycerol stock with a final concentration of 2.02 mg/ml. The DNA samples were diluted to 0.15 mg/ml concentration and individual 1.0 μL samples were exposed to a radiation dose of 50.0 Gy for the four dilution buffers and DNA concentrations (0.15, 0.3, 0.6, and 0.9 mg/ml) using the buffer that yielded the greatest number of strand breaks. DNA SSBs and DSBs were measured using gel electrophoresis; an analytical tool to evaluate fragmented DNA by its extent of migration through an agarose gel upon the application of an electric potential. An electrophoresis gel imager was used to image the migration of DNA through the gel to correlate the migration signal with the extent of DNA SSB and DSB damage. DNA damage was quantified using the gel quant analysis software (Life Technologies) to obtain average band intensity. The uncertainty of the average band intensity was determined by calculating the standard deviation of the average intensity from triplicate and quadruplicate sample runs. More DNA strand breaks were observed with buffers other than TE X10, which is a free radical scavenger, however there was no statistically significant difference between using DI water, PP X20, and PP X5. Furthermore, DNA sensitivity to damage was optimally observed at a final DNA concentration of 0.15 mg/ml.
