Biomedical Engineering Reference
In-Depth Information
Long DNA
fragments
-
Gene X
Fig. 2.6
Mapping restriction sites
around a hypothetical gene sequence
in total genomic DNA by the Southern
blot method.
Genomic DNA is cleaved with a
restriction endonuclease into hundreds
of thousands of fragments of various
sizes. The fragments are separated
according to size by gel electrophoresis
and blot-transferred on to nitrocellulose
paper. Highly radioactive RNA or
denatured DNA complementary in
sequence to gene
X
is applied to the
nitrocellulose paper bearing the blotted
DNA. The radiolabelled RNA or DNA
will hybridize with gene
X
sequences
and can be detected subsequently by
autoradiography, so enabling the sizes
of restriction fragments containing
gene
X
sequences to be estimated from
their electrophoretic mobility. By
using several restriction endonucleases
singly and in combination, a map of
restriction sites in and around gene
X
can be built up.
Restriction
endo-
nuclease
Gel
electro-
phoresis
DNA
fragments
Short DNA
fragments
+
Genomic DNA
Genomic DNA
Agarose gel
(1) Denature in alkali
(2) Blot-transfer, bake
(1) Hybridize nitrocellulose
with radioactive probe
Nitrocellulose
Autoradio-
graphy
(2) Wash
Photographic
film
Images correspond only to
fragments containing gene X
sequences - estimate
fragment sizes from mobility
Radioactive RNA or
denatured DNA containing
sequences complementary
to gene X (radioactive probe)
Single stranded
DNA fragments
The Southern blotting methodology can be extre-
mely sensitive. It can be applied to mapping restric-
tion sites around a single-copy gene sequence in a
complex genome such as that of humans (Fig. 2.6),
and when a 'mini-satellite' probe is used it can be
applied forensically to minute amounts of DNA (see
Chapter 14).
ization with radiolabelled DNA probes. As before,
hybridizing bands are located by autoradiography.
Alwine
et al
.'s method thus extends that of Southern
and for this reason it has acquired the jargon term
northern
blotting.
Subsequently it was found that RNA bands can
indeed be blotted on to nitrocellulose membranes
under appropriate conditions (Thomas 1980) and
suitable nylon membranes have been developed.
Because of the convenience of these more recent
methods, which do not require freshly activated paper,
the use of DBM paper has been superseded.
Northern blotting
Southern's technique has been of enormous value,
but it was thought that it could not be applied
directly to the blot-transfer of RNAs separated by gel
electrophoresis, since RNA was found not to bind to
nitrocellulose. Alwine
et al
. (1979) therefore devised
a procedure in which RNA bands are blot-transferred
from the gel on to chemically reactive paper, where
they are bound covalently. The reactive paper is
prepared by diazotization of aminobenzyloxymethyl
paper (creating diazobenzyloxymethyl (DBM) paper),
which itself can be prepared from Whatman 540
paper by a series of uncomplicated reactions. Once
covalently bound, the RNA is available for hybrid-
Western blotting
The term 'western' blotting (Burnette 1981) refers
to a procedure which does not directly involve nucleic
acids, but which is of importance in gene manipula-
tion. It involves the transfer of electrophoresed
protein bands from a polyacrylamide gel on to a
membrane of nitrocellulose or nylon, to which they
bind strongly (Gershoni & Palade 1982, Renart &
Sandoval 1984). The bound proteins are then avail-
