Evaluation of number average length analysis in quantifying double strand breaks in genomic DNAs.
Double strand breaks in DNA can be quantified down to very low frequencies (a few per Gigabase pair) in nanogram quantities of nonradioactive, genomic DNA by dispersing the DNAs on electrophoretic gels, digitizing them by quantitative electronic imaging, and calculating the DNA lengths by number average length analysis. No specific distribution of damages is required for number average length analysis. To test the validity of this approach, we used DNA populations of known absolute lengths and break frequencies as experimental DNAs and calculated the number average lengths and double strand break levels. Experimental DNAs and length standards were dispersed using pulsed field electrophoretic modes (unidirectional pulsed field, contour clamped homogeneous field, or transverse alternating field) appropriate for their size range, stained with ethidium, destained, and a quantitative electronic image obtained. A dispersion curve was constructed from the migration-mobility relationships of the length standard DNAs, and the number average lengths of the experimental DNAs were calculated. The calculated DNA lengths agreed well with the actual lengths. Furthermore, the double strand break frequencies calculated through number average length analysis of DNAs dispersed by these pulsed field gel modes and digitized by quantitative electronic imaging were in excellent agreement with the actual values for populations of DNA over the size range of approximately 4 kbp to approximately 3 Mbp. The use of this approach in quantifying DNA damages is illustrated for double strand breaks and damage clusters (e.g., OxyPurine clusters recognized by Escherichia coli Fpg protein) induced in T7 DNA by ionizing radiation.
Sutherland
Betsy M
BM
Department of Biology, Brookhaven National Laboratory, Building 463, 50 Bell Avenue, Upton, New York 11973-5000, USA. bms@bnl.gov
Bennett
Paula V
PV
Georgakilas
Alexandros G
AG
Sutherland
John C
JC
D017123:Bacteriophage T7 / Q000235:genetics; D004249:DNA Damage; D004279:DNA, Viral / Q000737:chemistry* / Q000528:radiation effects; D016521:Electrophoresis, Gel, Pulsed-Field
D004279:DNA, Viral