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tion (6), where
V
is the steady-state velocity of the fragment,
k
a factor related
the net polyanion charge
Q
,
S
the size of the fragment (also related to
Q
),
E
the
electrical field,
E
a
the activation energy for the viscous f flow,
R
the universal gas
constant and
T
the absolute temperature.
(6)
As an example for the study of DNA damage after irradiation using this tech-
nique may serve reference (Valenzuela et al. 2000).
In pulsed field gel electrophoresis (PFGE), intact DNA is treated with restric-
tion enzymes to generate pieces small enough to resolve by electrophoresis in an
agarose or acrylamide gel. With each reorientation of the electric field relative to
the gel, small-sized DNA will begin moving in the new direction more quickly
than the larger DNA. Thus, the larger DNA lags behind providing a separation
from the smaller DNA (for a review see Anand and Southern 1990). Among oth-
ers, PFGE seems to be the most sensitive technique for the determination of DSBs
in cells (Rothkamm and Löbrich 2003).
13. 2 .7
32
P-Postlabeling
The
32
P-postlabeling technique allows to improve the sensitivity of the detection
of DNA damage (Cadet et al. 1998). The damaged DNA is enzymatically degraded
into nucleotide-3-phosphates [reaction (7)]. The resulting mixture of unchanged
nucleoside-3-phosphates (dNp) and damaged ones (dXP) are separated by HPLC
[reaction (8)]. They are then labeled at the 5
-position with
32
P [reaction (9)] and
′
subsequently dephosphorylated at the 3
-position [reaction (10)]. This allows to
proceed with a second purification and their identification by, for example, two-
dimensional TLC [reactions (11) and (12)].
′
Factors that affect the determination of 8-oxo-G by this technique have been dis-
cussed in some detail (Möller et al. 1998). The determination of Tg by this tech-
nique (Hegi et al. 1989) is one of its most sensitive assays (Weinfeld and Soderlind
1991), many orders of magnitude higher than the earlier determination by HPLC
(Frenkel et al. 1981). A
32
P-postlabeling assay for the cA lesion which blocks gene
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