Methods - Free-Radical-Induced DNA Damage and Its Repair - page 477

13. 2 .11
Immuno Assays

Immuno assays have been developed for a variety of DNA lesion such as UV-
damage (McConlogue et al. 1982; Eggset et al. 1987), Tg (Rajagopalan et al. 1984;
Le et al. 1998) cA (Fuciarelli et al. 1985, 1987), Ade- N 1 -oxide (Signorini et al.
1998), carcinogen-modified DNA (Müller et al. 1982), hydroxylation of guanine
(Kasai and Nishimura 1986), 8-oxo-G (Yin et al. 1995), HMdUra (Lewis et al.
1978), DNA base damage (Lewis and Ward 1978) and X-ray-induced damage
(Waller et al. 1981).
A f fluorescent 90-mer oligonucleotide in combination with laser-induced f flu-
orescence and capillary electrophoresis has been used as a versatile probe for a
detection of DNA damage (Carnelley et al. 2001).

13. 2 .12
Atomic Force Microscopy

Atomic force microscopy has been developed to a stage that DSBs and the length
of the resulting fragments can be detected by this technique (Pang et al. 1996).
In this context it is of interest that neutron irradiation leads to the formation of
very small fragments (Pang et al. 1997).

13. 2 .13
Comet Assay

The comet assay (Ostling and Johanson 1984) makes use of the fact that the
smaller DNA fractions that are formed when the molecular weight of the cellular
DNA is reduced by free-radical-induced DSBs move faster in an applied electric
field (after cell lysis) than the undamaged DNA. Staining the DNA yields a com-
et-li ke pattern. Purif ied repair enzy mes, applied to DNA during t he course of t he
comet assay procedure, can greatly enhance the sensitivity and specificity of the
assay (Collins et al. 1997; Sauvaigo et al. 2002). This assay is now widely applied
( cf. Pouget et al. 1999), and a combination with immunof luorescence detection
techniques allows even the detection specific damage and its repair (Sauvaigo et
al. 1998). For the application of the comet assay in assessing environmental haz-
ards, see Ündeger et al. (1999), for a comparison of

γ -irradiated and BLM-treated

cells, see Östling and Johanson (1987).