Biology Reference
In-Depth Information
the DNA molecule by treating the DNA with small amounts of DNase. A nick
exposes a free 3 -OH group and DNA polymerase I of E. coli will then remove
nucleotides from the 5 side of the nick. The simultaneous removal of nucleo-
tides from the 5 side and the addition of labeled nucleotides to the 3 side
by DNA polymerase I results. If deoxynucleoside-5 -triphosphates (dNTPs) are
radiolabeled with 32 P and the nicks are random, the duplex DNA molecule will
become labeled uniformly along its length as it incorporates radiolabeled dNTPs.
The reaction may be carried out to label all four dNTPs or only one dNTP. Nick
translation is particularly useful for producing large amounts of probe for mul-
tiple hybridization reactions and where a high probe concentration is required.
Nick translation kits are available from a number of commercial sources and
provide instructions, a stock mixture of DNA polymerase I and DNase I, and a
series of buffers lacking one or more unlabeled dNTPs. The radioactive dNTPs
must be obtained fresh (within a few days) before the labeling reaction is set up
because 32 P-labeled dNTPs decay rapidly.
The use of radioactive probes requires that the experimenter obtain training
in their safe use. Safety protocols are required to prevent contamination of the
laboratory and to ensure safe disposal of the radioactive dNTPs. Most organiza-
tions require the training and licensing of scientists using radioactivity and regu-
lar inspections and reports to confirm its safe use and disposal ( Davies 1994 ).
Kits are available to label DNA without using radioactivity, thereby reduc-
ing potential risks to the experimenter and eliminating the need for special
disposal methods and the short half-life associated with radioactivity ( Allefs
et al. 1990 ). Although nonradioactive labels may not be as sensitive in detecting
very small amounts of target DNA, they can be more rapid and less hazardous.
Chemiluminescence and fluorescence provide a signal when an enzyme reacts
with a chemiluminescent or chromogenic substrate. Alternatively, a signal can
occur through excitation and emission of a fluorophore-labeled probe.
5.16 Removing DNA from Agarose Gels after Electrophoresis
Several methods have been developed to recover DNA from agarose gels:
1) electrophoresis onto a diethylaminoethyl(DEAE)-cellulose membrane, 2) elec-
troelution into dialysis bags, and 3) low-melting temperature agarose gels. With
method 1, fragments of DNA are separated by electrophoresis, a slit is cut in the
gel immediately ahead of the DNA fragment of interest, and a sliver of DEAE-
cellulose membrane is inserted. Electrophoresis is continued until the DNA in the
band has been transferred to the membrane. The membrane is removed from
the slit and washed and the DNA is eluted from the membrane.
Search WWH ::




Custom Search