placed on the gel and the DNA molecules migrate upward into the membrane via capillary
action. However, a more recent protocol describes a downward Southern blot in which the
gel is placed on the membrane and the molecules move quickly into the membrane as a
result of capillary action and gravity (Fig. 11.5) (Chomczynski 1993). The blot is hybri-
dized with a probe complementary to the GOI or SM, and the probe binds to these hom-
ologous sequences through hydrogen bonds. Probes are usually derived by restriction
digestion of plasmid DNA or PCR amplification of the GOI (Reece 2004; Sambrook and
Russell 2001) and gel purification of the fragment. After hybridization, nonspecifically
bound probe is washed away at high stringency (i.e., high temperature and/or low salt)
and the blot is exposed to X-ray film in autoradiography (Reece 2004; Sambrook and
Russell 2001) or a phosphor screen (Johnston et al. 1990) and scanned to produce a
digital image.
Two different kinds of genomic digests are commonly used to show transgene inte-
gration in the genome:
- Copy number can be determined relative to a border of the introduced DNA. For
example, this type of analysis has been used inAgrobacterium-mediated transformation
experiments ofArabidopsisin which few T-DNA insertions are transferred (Katavic
et al. 1994). It is important to know how many insertion sites are present. Single copies
Figure 11.4.Digestion of plant genomic DNA that is electrophoretically separated for Southern blot
analysis (a) and a phosphorimage of the blot after hybridization with radiolabeled probe
(b). The samples are as follows: Lane 1, plasmid control; lane 2, putative transgenic plant; lane 3,
non-transgenic plant control. Genomic DNA was extracted and digested to completion and run on
a 0.8% agarose gel (a). The DNA was blotted to a membrane, hybridized with a radiolabeled
probe, and exposed to a phosphor screen (b). There is a plasmid band (.10 kb) in lane 1 and one
band in lane 2 at 1.8 kb that have sequences complementary to the probe.
11.4. DEFINITIVE MOLECULAR CHARACTERIZATION 281