Gene Cloning

128 Gene Cloning
Box 5.3 Types of DNA Labeling
In Chapter 3 we saw that DNA can be visualized by staining with ethidium
bromide (Section 3.11). However, many techniques require either the
specificity or sensitivity that can be obtained by attaching a label to DNA
molecules. Once a DNA molecule is labeled it can be detected in a mixture
of unlabeled DNA molecules, for instance when it is being used as a probe
(Section 5.5). Additionally, as most methods of detecting the labels involve
amplification of the signal it is possible to detect very small amounts of
labeled DNA for example in DNA sequencing (Section 7.3). Commonly
used DNA labels include radioactive isotopes, haptens, which can be
detected immunologically, and fluorescent dyes.
Radioactive labels
Radioactive isotopes such as phosphate can be incorporated into nucleosides
( 32 P or 33 P) (a) or can replace elements in the nucleoside ( 35 S) (b).
When DNA is being labeled for analytical purposes the end result of the
experiment is often an electrophoresis gel where the labeled DNA is
detected by autoradiography. The gel is placed in direct contact with a
sheet of photographic film for several hours, the energy released by the
radioactive isotopes causes the formation of dark silver grains and when
the film is developed these appear as bands or spots.
Labels which are detected immunologically
For reasons of safety and convenience non-radioactive labels are increasingly
replacing radioactive ones. Haptens such as biotin and digoxigenin
are small molecules which can be coupled to nucleotides and can then be
incorporated into nucleic acid molecules by DNA polymerases. These
molecules are immunogenic and can be detected by specific antibodies
conjugated with a moiety which can be visualized by fluorescence, chemiluminescence
or a color reaction (c).