MEDICAL AND ENVIRONMENTAL EFFECTS OF UV RADIATION - Environmental UV Radiation: Impact on Ecosystems and Human Health and Predictive Models - page 41

F = L/b = N t /b t - N u /b u (2)
Since each term is divided by the quantity of DNA in that sample, the result does not
depend on the mass of DNA in each sample, i.e., the procedure is "self-normalizing.''
Thus the problem of measuring damage levels becomes one of counting the number of
molecules in each DNA sample before and after treatment with the lesion-specific
agent. One method for "counting'' DNA molecules is to separate them according to
molecular size by agarose gel electrophoresis, and then to measure the quantity of DNA
molecules of each size, computed from a DNA dispersion curve established by
molecular length standard DNAs electrophoresed on the same agarose gel [14]. An
electronic image is obtained of an electrophoretic gel containing sample DNAs stained
with ethidium bromide. A DNA dispersion function is derived from the molecular
length standards in the gel. The size distribution of molecules (or number average
molecular length, which is the ordinary average of the size of each molecule multiplied
by the number of molecules in each size class) in each sample is calculated from the
relation

=

()
()
()

f xdx

L , (3)

n

fx dx
Lx

where L(x) is the length of the DNA molecules that migrated to position x , and f(x)dx is
the intensity of ethidium fluorescence from DNA molecules at that position.
From the L n s of the treated and untreated populations, the frequency of lesions,
I D , is computed from

I D = 1/L n , (+ treatment) - 1/L n (- treatment), (4)
where 1/L n (+ treatment), and 1/L n (- treatment) , are the reciprocals of the L n s of
samples that were treated or not treated, respectively.
Since this method measures the lesion frequency per molecule, the sensitivity
of lesion frequency measurement can be increased substantially by increasing the size
of the molecules examined. In practical terms this means that improved methods for
isolating larger and larger DNA molecules are required. For damages affecting both
strands, this means that the level of double strand breaks induced in DNA isolation
must be minimized. However, for lesions affecting only one DNA strand, this requires
that the level of single strand breaks induced during isolation must be minimized, a
much more rigorous requirement. Isolation of DNA by methods that avoid chemical
extraction, including enzymatic digestion while the DNA is stabilized mechanically
within an agarose "plug'' or "button,'' provides a successful approach to meeting such
requirements [15-17].
With high sensitivity methods of detection (quantitative detection by charge
coupled device imaging [18-20] of fluorescence from a DNA-bound molecule such as
ethidium bromide, whose fluorescence is directly proportional to the mass of DNA over