DETECTION AND ANALYSIS OF NUCLEIC ACIDS - A Guide to Methods in the Biomedical Sciences - page 28

Polyacrylamide gel electrophoresis (PAGE)
PAGE gels, as previously mentioned, are used for high resolution
separation of nucleic acids, such as for resolving DNA ladders in DNA
sequencing reactions or for RNase protection assays. For nucleic acid
separation, PAGE gels served as the first matrix used to demonstrate
that restriction fragments of nucleic acids could be resolved in gels,
and their sizes could be calculated from their migration patterns (11).
In general, relatively low percentage gels are used for resolving nucleic
acids.
Agarose gel electrophoresis
Agarose gel electrophoresis is one of the standard methods of molec-
ular biology and is a method used to resolve and separate nucleic acid
fragments of different sizes. The use of agarose as a matrix to separate
DNA was quickly adapted once it was established that DNA fragments
could be separated by gel electrophoresis.
Agarose is a sugar polymer isolated from seaweed; when boiled and
cooled, it forms a “gel”, much like jello. The agarose gel serves to im-
pede the migration of the DNA, so that larger fragments of DNA migrate
slower than smaller fragments. Because DNA migrates in an electric
field as a consequence of the phosphates on the DNA backbone, the
charge to mass ratio is identical for all DNA fragments, irrespective of
their length. As a result, while the separation is due to the charge of the
DNA, the migration of DNA fragments is inversely proportional to the log
(base 10) of the molecular weight of the fragment. Thus, if fragments
of unknown size are compared with a control “ladder” containing frag-
ments of known sizes (these are commercially available), the sizes of
unknown fragments can be readily calculated. Depending on the con-
centration of agarose in the gels, DNA fragments from under 100 base
pairs (bp) to 30,000 base pairs (30 kilobases, or kb) can be resolved
by standard agarose gel electrophoresis. Following separation of the
DNA, the fragments can be visualized in the gel using fluorescent dyes
that intercalate (i.e., insert) between the bases of the DNA helix. The
most common dye used is ethidium bromide, which appears red upon
exposure to ultraviolet light, but other dyes, such as SYBER green, are
becoming increasingly popular.
Pulsed field electrophoresis
This is a method that allows for the separation of much larger frag-
ments of DNA than standard agarose gel electrophoresis. In this method,