Methods for the quantification of DNA double-strand breaks determined from the distribution of DNA fragment sizes measured by pulsed-field gel electrophoresis

B. Cedervall, R. Wong, N. Albright, Joseph Dynlacht, P. Lambin, W. C. Dewey

Research output: Contribution to journalArticle

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Abstract

Different methods were used for evaluating data for DNA double-strand breaks (DSBs), as obtained by pulsed-field gel electrophoresis (PFGE) after X irradiation of Chinese hamster ovary cells. A total of 60 data points in the dose range of 0 to 116 Gy, along with repair data for 30 and 60 Gy, were analyzed by four methods: (1) percentage of DNA released from the plug, (2) specific size markers (percentage of DNA less than specific sizes), (3) fragment size distributions and (4) shape of the molecular weight (M) distributions. With the last method, both the slope and the intercept of the logarithm of the amount of radioactive DNA/ΔM/M plotted as a function of M were used for calculating DSBs/100 Mbp. The slope and the intercept analyses differ in that the former is relatively independent of DNA trapped in the agarose plugs, i.e. cannot be released by doses of 100-150 Gy, whereas the intercept is dependent on the percentage of DNA trapped. Also, calculations of DSBs/100 Mbp for methods 1, 2 and 3 depend on the amount of DNA trapped in the plug. However, the slope method is unreliable for doses below about 20 Gy, and the scatter of data points is much greater than that obtained by the intercept method and by methods 1, 2 and 3. Therefore, the fragment size distribution and the specific size marker methods give the most consistent results, with 0.49 ± 0.03 (95% CI) (DSBs/100 Mbp)/Gy. With the specific size marker method, however, care must be taken in selection of size markers in relation to the levels of DSBs of interest. Assuming randomly distributed DSBs, all four methods gave essentially the same results; i.e., the dose response was linear with a calculated level of 0.5-0.6 (DSBs/100 Mbp)/Gy, which is the same as 0.47-0.62 determined previously by calibrating with 125IdU.

Original languageEnglish (US)
Pages (from-to)8-16
Number of pages9
JournalRadiation Research
Volume143
Issue number1
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

Double-Stranded DNA Breaks
Pulsed Field Gel Electrophoresis
pulsed-field gel electrophoresis
electrophoresis
strands
deoxyribonucleic acid
fragments
gels
DNA
markers
plugs
dosage
slopes
methodology
ovaries
hamsters
calibrating
logarithms
molecular weight
Chinese hamsters

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Radiology Nuclear Medicine and imaging
  • Biophysics
  • Radiation

Cite this

Methods for the quantification of DNA double-strand breaks determined from the distribution of DNA fragment sizes measured by pulsed-field gel electrophoresis. / Cedervall, B.; Wong, R.; Albright, N.; Dynlacht, Joseph; Lambin, P.; Dewey, W. C.

In: Radiation Research, Vol. 143, No. 1, 1995, p. 8-16.

Research output: Contribution to journalArticle

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