Biology Reference
In-Depth Information
synthetic oligonucleotides, isolate or analyze DNA
<
1 kb, and resolve small RNA
molecules by two-dimensional or pulsed-field gel electrophoresis. CAUTION:
Polyacrylamide gel ingredients are highly toxic and should not be inhaled or
touched without gloves.
5.13 Detecting, Viewing, and Photographing Nucleic Acids in Gels
Ethidium bromide is a useful dye to detect both single- and double-stranded
nucleic acids in both agarose and polyacrylamide gels (
Figure 5.5
). Agarose gels
are less sensitive in detecting small amounts of DNA than are polyacrylamide
gels. The sensitivity for DNA is 5- to 10-fold less.
Ethidium bromide (EtBr) can be incorporated into the gel and running buffer
during electrophoresis. Alternatively, gels can be stained after electrophoresis by
placing them in buffer containing EtBr for 30 minutes. CAUTION: EtBr is muta-
genic so the experimenter must be extremely cautious when handling it. Gloves
must be worn and care must be taken to avoid contaminating laboratory surfaces.
As little as 0.05
μ
g of DNA can be visualized in one band when the gel is
exposed to ultraviolet (UV) light (
Figure 5.5
). The EtBr-nucleic acid complex
absorbs UV irradiation at
≈
260 nanometers (nm) or 300nm. The fluorescence
of EtBr stacking in duplex DNA is 10 times greater than that of free EtBr, and
the emission is at 590nm (red orange). UV light sources (transilluminators) are
used to view DNA stained with EtBr at 254 or 366 nm. Although the short-wave
model can detect smaller amounts of sample DNA, it damages DNA by nicking
or dimerization, and the DNA is unsuitable for most cloning work. CAUTION:
Experimenters should wear safety glasses around UV light sources to protect
their eyes. Agarose and polyacrylamide gels can be photographed to document
the results of the experiment.
5.14 Identifying Specific DNA by Southern Blot Analysis
It is often necessary to identify specific DNA sequences. One method to do so was
invented by
Southern (1975)
, and the “
Southern blot
” has been a fundamental
and versatile tool for genetic engineers ever since. The original paper published
by Southern was the most-cited paper ever published in the
Journal of Molecular
Biology
(
Southern 2000
). Variations on Southern blots have been developed to
identify RNA or proteins in gels. These modifications are called
Northern
(RNA)
and
Western
(protein)
blotting
, respectively. There is no “Eastern” blot.
In Southern blotting, DNA fragments that have been separated by electro-
phoresis in an agarose gel are denatured into a single-stranded form by alkali
treatment (
Figure 5.6
). The gel is then laid on top of buffer-saturated filter
