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4. Introduction
Feinbereg and Vogelstein (1,2) introduced the use of random
sequence hexancleotides to prime DNA synthesis on denatured
template DNA at numerous sites along its length. The primer-
template complex is a substrate for the ‘Klenow’ fragment of DNA
polymerase 1. By substituting a radiolabelled nucleotide for a non-
radioactive equivalent in the reaction mixture newly synthesized
DNA is made radioactive (see Figure 1). The absence of the 5’–3’
exonuclease activity associated with DNA polymerase 1 ensures
that labelled nucleotides incorporated by the polymerase are not
subsequently removed as monophosphates. Very small amount of
input DNA can be labelled, enabling very high specific activity DNA
probes to be produced with relatively small quantities of added
nucleotides. These radioactive labelled fragments can then be used
as sensitive hybridization probes for a wide range of filter based
applications (3-6).
Previous protocols for the random primer labelling of DNA have
required reaction times of at least 30 minutes. GE Healthcare’s
Magaprime DNA labelling system allows the labelling of template
DNA to the same high specific activity but at a greatly accelerated
rate. Probes of specific activity 1.9x10
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dpm/µg can be produced with
the majority of DNA substrates, using the standard protocol, after
10 minutes incubation at 37°C. This rapid labelling is achieved by
the use of nonamer primers rather than the conventional hexamers
(Figure 1). Nonamers allow for more efficient priming from the
template DNA at 37°C, resulting in fast and efficient labelling of the
DNA. A new alternative protocol has further reduced the variability
in labelling which can occur with DNA template from a variety
of sources. Both the standard Megaprime protocol and the new
protocol are given as options in this booklet. The labelling of DNA in
low melting point agarose takes only 15–30 minutes in contrast to
conventional systems where overnight incubation are necessary.
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