Biology Reference
In-Depth Information
Several techniques for genomic analysis, initially used for species characterization, can
also be used for nonsequenced species. Such techniques include the random amplifica-
tion of DNA or randomly amplified polymorphic DNA (RAPD; Williams et al. 1990),
arbitrary primed polymerase chain reaction (Welsh and McClelland 1990), DNA amplifi-
cation fingerprinting (Caetano-Anollés et al. 1991), and amplified fragment length poly-
morphism (AFLP; Vos et al. 1995). All these techniques are based on DNA amplification
after random primer annealing. Each primer generates amplimers of different size that
can be separated by electrophoresis. Patterns from control and treated samples are then
compared. The appearance and disappearance of bands, and changes in band intensity
could be due to alterations at DNA level such as mutations, DNA breakages, and DNA
adducts that could interfere with the
Taq
(
Thermus aquaticus
) polymerase. An example
of control and treated RAPD profiles is presented in Figure 13.2. Patterns have been
generated from
M. galloprovincialis
gill DNA exposed to benzo(
a
)pyrene diol epoxyde
or solvent. Figure 13.2 reveals, for instance, the disappearance of bands 6-1 and 8-2, the
appearance of amplimers 8-3 and 8-4, as well as the overexpression of band 5-1 after
amplification with primers OPB8, OPB6, and OPB5 in treated samples (Atienzar et al.
2002). In ecotoxicology, RAPD technology has been used to study the effects of genotox-
ins in environmental species (for review, please refer to Atienzar and Jha 2006). Atienzar
and collaborators originally optimized and validated the technique to finally use it to
detect DNA alterations. They studied the effects of UV on macroalgae (Atienzar et al.
2000) as well as the consequences of benzo(
a
)pyrene exposure on
Daphnia
and on follow-
ing generations (Atienzar et al. 1999; Atienzar and Jha 2004).
Daphnia
species are ideal for
RAPD studies because all individuals are identical at DNA level (reproduction by parthe-
nogenesis). This significantly reduces the variability in control RAPD profiles.
Daphnia
is also an excellent model to study the transgenerational effects of genotoxins. After
exposure to B(
a
)P, changes in RAPD profiles appeared after 3 and 6 days. Nevertheless,
OPB1
OPB5
OPB6
OPB7
OPB8
e e e e
12121212 121212121212M
e
f
f
f
f
f
M
_
_
3
_
2
_
1
_
0.5
_
0.3
FIGURE 13.2
RAPD profiles of genomic DNA from gill cells of
Mytilus galloprovincialis
nonexposed (e) or exposed (f) to
benzo[
a
]pyrene diol epoxyde (677 adducts/10
8
nucleotides). This figure shows disappearance of bands 8-2 and
6-1; appearance of bands 8-3 and 8-4, and an increased intensity of band 5-1. (Well 1 and 2: 20 and 5 ng DNA;
(-): control without DNA;
M
= 1 kb, the molecular size (in kilobases) of selected bands is indicated on the left of
the gel; OPB 1 to 8 10-mer primers (5′→3′) of different sizes). (From Atienzar, F.A. et al.,
Mutat. Res.
,
521, 151-163,
2002. With permission.)
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