DETECTION AND ANALYSIS OF NUCLEIC ACIDS - A Guide to Methods in the Biomedical Sciences - page 29

the direction of the electrical current is altered periodically, allowing for
the separation of fragments from 30 kb to several million base pairs.
Alkaline agarose gels
Agarose gel electrophoresis works well for separating double
stranded DNA fragments and for the separation of RNA, but does not
resolve single stranded DNA species. The incorporation of a base such
as sodium hydroxide into the buffers used in the gels permits good res-
olution of single stranded DNAs.
Restriction enzymes and restriction fragments
Restriction endonucleases are enzymes that cleave DNA at specific
sites. They are one of the most important tools in the analysis of DNA
and for recombinant DNA technology. Restriction enzymes come from
bacteria. They are a component of the “restriction-modification systems”
of bacteria. These modification systems are thought to have evolved
to help protect bacteria against foreign DNA, whether introduced by
viral infection (by bacteriophages) or by DNA from other species of
bacteria taken up by the host bacterium (see below). The fundamen-
tal work on restriction modification systems by Werner Arber, includ-
ing the discovery of restriction enzymes, helped lead to the first in-
sights into their sequence specificity by Kelly and Smith in 1970 (12)
and to their use to characterize restriction fragments of viral DNA by
Danna and Nathans in 1971 (11). These studies on restriction enzymes
earned the Nobel Prize for Arber, Hamilton Smith and Daniel Nathans
in 1978.
Hundreds of restriction enzymes (over 600 at this point) have been iso-
lated. Restriction enzymes recognize short specific sequences in dou-
ble stranded DNA. The sequences are generally palandromic, meaning
they read the same (have the same nucleotides) in opposite orientations
on the complementary DNA strands. Palandromic sequences can oc-
cur because of the base-pairing that is required in double stranded DNA
(dsDNA). The 4 nucleotide bases adenine (A), cytosine (C), guanine (G)
and thymine (T) exist in dsDNA as A-T and G-C complementary pairs.
When the appropriate sequence is recognized, the restriction enzymes
cut the DNA at specific sites within this recognition site, giving rise to
a restriction fragment, which represents the piece(s) of DNA produced
after cleaving the DNA with one or more restriction enzymes.
The actual cuts produced by a restriction enzyme can be “blunt” (that
is, they cut at the same place on both strands of DNA) whereas others
make symmetrical cuts with “overhangs”. For example, the restriction