Biology Reference
In-Depth Information
nitrocellulose membrane is baked at 80°C in a vacuum to bind the DNA perma-
nently onto the surface of the nitrocellulose filter. Electroblotting or vacuum
blotting are alternative methods for transferring the DNA to a membrane and
require specialized equipment.
To determine whether the DNA of interest is present on the blot requires
probing the DNA on the nitrocellulose membrane (
Figure 5.6
). The
probes
can
be labeled by
32
P or by nonradioactive methods. Probes can consist of RNA, ss
DNA, or a synthetic oligonucleotide that is
complementary
in sequence to the
DNA of interest. The labeled probe must bind specifically to the DNA of interest,
but not bind to nontarget DNA or the nitrocellulose.
Pretreating the bound DNA on the nitrocellulose by placing it in a solution
containing 0.2% each of Ficoll (an artificial polymer of sucrose), polyvinylpyrrol-
idone, and bovine serum albumin (also known as Denhardt's solution) will pre-
vent nonspecific binding. The mixture often includes an irrelevant nucleic acid
such as salmon sperm DNA, which may act by occupying all the available non-
specific binding sites on the membrane.
The temperature at which Southern blotting is conducted is adjusted to maxi-
mize the rate of hybridization of the probe with the immobilized DNA on the
nitrocellulose and to minimize the amount of nonspecific binding. This aspect
of planning the Southern blot is called
stringency
, and a highly stringent
Southern blot will be more specific. After the hybridization step, in which the
labeled probe binds to the immobilized DNA on the membrane, the membrane
is washed to remove any unbound probe. The temperature at which the wash-
ing takes place also determines the stringency of the Southern blot.
The regions on the membrane where hybridization of the labeled probe and
target DNA took place are detected by placing the membrane in contact with
X-ray film if the probe was radiolabeled. The length of time the X-ray film is
exposed to the radioactivity is determined by the amount of DNA in the blot
and the degree of homology between the DNA and the probe. If there is only a
small amount of DNA present, as would be expected for a single-copy gene in a
blot of genomic DNA, the film may be exposed for 2-3 weeks to the blot.
Southern blots can detect single-copy gene sequences in genomic DNA
digests. Modifications of the Southern blot method use nylon membranes as
substrates because they are more robust and can be reused. Thus, one probe can
be removed by high-temperature washing, and the DNA can be reprobed with
a different probe. Another advantage to nylon membranes is that the DNA can
be permanently fixed to the membrane by a brief exposure to UV light, which
cross-links the DNA and fixes it to the membrane.
