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| Figure 9-1 DNA hybridization. |
In DNA hybridization assays, DNA from a virus or cell is denatured
with alkali to separate the strands. The single strands of DNA are then attached
to a solid support such as a nitrocellulose or nylon membrane so
that the strands do not reanneal (Figure 9-1). The DNA is attached to
the membrane by its sugar-phosphate backbone with the nitrogenous
bases projecting outward. To characterize or identify the target DNA , a single-stranded DNA or RNAmolecule of known origin, called a probe,
is added to the membrane in a buffered solution. This allows the formation
of hydrogen bonds between complementary bases. The probe, so
called because it is used to seek or probe for DNA sequences, is labeled
with a reporter group, which may be a radioactive atom or an enzyme
whose presence can be easily detected.
The probe is allowed to react with the target DNA ; then any unreacted
probe is removed by washing in buffered solutions. After the washes,
all that remains on the nitrocellulose is the target DNA and any probe
molecules that have attached to complementary sequences in the target
DNA , forming stable hybrids.
Hybridization of target and probe DNA s is detected by assaying for the probe's reporter group. If the reporter group is detected, hybridization
has taken place. If no reporter group is detected, it can be assumed that
the target molecule does not have sequences that are complementary to
those of the probe, and hence, the gene or DNA segment sought is not
present in the sample.
Three common formats are used in solid-phase hybridization assays;
dot blot, Southern blotting, and in situ hybridization. In the dot blot assay,
a specified volume of sample or specimen is spotted onto a small area
of nitrocellulose membrane, which is then carried through the procedure
described above. Southern hybridization assays (Figure 9-2) involve
restriction enzyme digestion and agarose gel electrophoresis of the target
DNA prior to the hybridization assay. The different bands on the agarose
gel are transferred by capillary action onto a nitrocellulose or nylon membrane
in a blotting apparatus. During the transfer, each of the DNA bands
is transferred onto the membrane in the same relative position that it had
in the gel. After the transfer, the target DNA is probed and detected, as in
the dot blot assay. In situ hybridization assays involve the probing of intact
cells or tissue sections affixed to a microscope slide. This type of solid-
phase assay has the advantage that one cannot only detect the presence
of target DNA in intact cells but also determine the location of such target
DNA within a tissue. An important application of in situ hybridization
is for the detection of viruses and certain types of bacteria within infected
cells.
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| Figure 9-2 Southern hybridization analysis. |
Notes
Four components of DNA hybridization:
1. Target DNA
2. Probe
3. Detection system
4. Format