Gene Cloning

130 Gene Cloning
Box 5.4 Methods for Incorporating Labels into DNA
Molecules
Random prime labeling
This method involves incorporating a labeled nucleotide during in vitro
DNA synthesis and is used to make DNA probes. Remember from Chapter
3 that DNA polymerase requires a free 3′ hydroxyl; usually provided by a
primer. One of the most straightforward ways to provide this is to use random
hexanucleotides in a process called random prime labeling.
(a)
(b)
(c)
The target DNA is denatured by heating. a) Random hexanucleotide
primers are added and the mixture is cooled to allow the primers to
anneal. b) DNA synthesis, in the presence of four dNTPs, is initiated by the
primers and labeled nucleotides are incorporated. c) The DNA is heated to
separate the strands before being used as a probe. Because the hexanucleotides
will anneal to random positions on the target DNA a whole series
of labeled overlapping fragments will be produced. The advantage of this
technique is that you do not have to make specific primers for each fragment
to be labeled; the random hexanucleotide mixtures are commercially
available.
Nick translation
Another approach to labeling DNA fragments is nick translation. In this
case DNA synthesis is initiated at a single-strand break in the DNA duplex;
the remains of the other strand is displaced as synthesis proceeds.
DNase I introduces single-stranded nicks into the DNA, these provide a
free 3′ hydroxyl for DNA synthesis. In this case DNA polymerase I is used
in preference to the Klenow fragment as the 5′ and the 3′ exonuclease
activity will extend the regions which can be labeled.
One of the advantages of techniques where a labeled nucleotide is incorporated
during in vitro DNA synthesis is that many labeled nucleotides are
incorporated into each probe molecule giving a stronger signal than with
an end-labeled probe.