Biology Reference
In-Depth Information
Method 2, electroelution, allows recovery of a wide size range of DNA, but it
is inconvenient. DNA is separated by electrophoresis in agarose gel containing
ethidium bromide (EtBr). The band of interest is located with an ultraviolet lamp,
and the band is cut from the gel with a razor blade. The gel fragment contain-
ing the DNA of interest is then placed in a piece of dialysis tubing, sealed, and
placed into an electrophoresis tank. Electric current is passed through the bag to
elute the DNA out of the gel and onto the inner wall of the bag. The polarity of
the current is reversed to release the DNA from the wall of the bag, the bag is
opened and the buffer containing the DNA is transferred to a clean tube.
Method 3 uses low-melting-temperature agarose gels. DNA of interest is elec-
trophoresed, the band of interest is detected by staining with ethidium bromide,
cut out, and placed into a clean tube. A buffer is added, and then the mixture
is heated to 65°C to melt the agarose and centrifuged to leave the DNA in the
aqueous phase and the agarose in the interface. The DNA in the aqueous phase
can be purified with phenol. The DNA in the aqueous phase is then precipitated
by ammonium acetate and cold (temperature) ethanol. At this point, the DNA is
sufficiently pure to be digested by restriction enzymes or modified by ligases.
None of these methods is fully satisfactory in producing large amounts of
pure DNA. Problems include the presence of inhibitors of enzymatic inhibitors in
the agarose, which can affect subsequent DNA manipulations. Large fragments
(
>
5kb) of DNA are often inefficiently recovered from agarose gels because
these longer fragments bind very tenaciously to purification matrices such as the
DEAE-cellulose membrane. Small (
<
500 nanograms [ng]) amounts of DNA are
recovered inefficiently from gels. The methods are labor-intensive so that recov-
ery of DNA from gels is not readily performed on large numbers of samples.
Kits, such as spin-columns, can be purchased from commercial sources to purify
DNA from gels and remove impurities more efficiently than the three methods
described here.
5.17 Restriction-Site Mapping
So far in this experiment, DNA has been cloned into a plasmid, amplified in
E. coli
, and specific sequence(s) have been identified by Southern blot analysis
using a probe. Specific bands of DNA have been isolated and the DNA purified
from the agarose gel.
Information about the cloned DNA can now be obtained by restriction-site
mapping, DNA sequencing, and translation of the DNA into proteins using an
expression vector. DNA sequencing is described in Chapter 7, and translation
and expression of DNA are discussed in Chapter 6.
