DETECTION AND ANALYSIS OF NUCLEIC ACIDS - A Guide to Methods in the Biomedical Sciences - page 31

frequency and are very abundant, occurring at a rate of about 1 per
1000 base pairs. SNP genotypes are used in mapping disease suscep-
tibility loci as well as in comparative genetics, evolutionary genetics, and
forensic analysis.
Southern blotting
After resolving complex mixtures of DNA (such as restriction digested
genomic DNA) on a gel, how can you determine which restriction frag-
ment in a smear of DNA contains a particular gene? One method that
was initially used was to manually cut slices out of the gel, isolate the
DNA from the slices, and then hybridize the isolated DNA with a specific
probe. This was not only highly laborious but also extremely inefficient.
A second method, which revolutionized the ability to analyze DNA, was
introduced by E.M. Southern in 1975 (14), and was rapidly adapted to
the analysis of other macromolecules. Southern realized that because
gels are porous, macromolecules within the gel could be transferred to
another medium by a method called “blotting through”. The mediums
used are membranes, usually constructed of nitrocellulose or nylon.
The DNA is denatured (with NaOH, since only ssDNA can transfer), the
membrane is placed onto the gel, and then capillary action or a pump-
based suction method is used to transfer the DNA from the gel onto the
membrane using high salt solutions. While the capillary action method
of transfer is “low tech”, it is highly efficient and still used by many in-
vestigators since it takes longer and allows them the time to go home
and sleep! The DNA is then immobilized on the membrane using heat
(nitrocellulose) or ultraviolet light (nylon), and the entire membrane can
now be probed.
Blotting methods work well for agarose gels but not for polyacrylamide
gels, particularly the higher percentage gels used for proteins. For these,
the transfer is accomplished by electroblotting (see Chapter 1).
Dot blotting and slot blotting
These are methods to immobilize bulk unfractionated DNA onto a
membrane using a suction manifold. The manifolds have circular wells
(dot blots) or slit wells (slot blots). They are frequently used to deter-
mine if a nucleic acid is present in a sample and to determine the rel-
ative abundance of the nucleic acid. They are most frequently used for
RNA analysis. Since fractionation is not used, they do not rely on the in-
tegrity of the RNA. These blotting methods are now rarely used and have
been supplanted almost entirely by polymerase chain reaction methods,
which are more rapid, accurate, and quantitative.