Biology Reference
In-Depth Information
5.15 Labeling DNA or RNA Probes
In molecular biology, many DNA manipulation techniques depend on hybridiz-
ing a nucleic-acid probe to a target DNA or RNA sequence. Probes are required
in Southern and Northern blots, dot blots, colony/plaque blots, and
in situ
hybridization. Dot blots can be used to identify unfractionated DNA or RNA
molecules that have been immobilized on a nitrocellulose membrane. Plaque/
colony blots detect DNA released from lysed bacteria or phage after immobi-
lizing the DNA on a nitrocellulose membrane.
In situ
hybridization is used to
detect DNA or RNA molecules in cytological preparations.
Nucleic-acid probes can be labeled by several methods. One method to uni-
formly label double-stranded DNA probes is described here.
Nick translation
describes the incorporation of a nick (or break in one strand) of a ds DNA mol-
ecule (
Figure 5.7
). Nicks are introduced at widely separated, random sites along
Figure 5.7
32
P-labeling of double-stranded DNA can be carried out by nick translation. The aster-
isks indicate the location of radiolabeled phosphate groups that are inserted into the strand.
