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A suitable nucleotide mixture containing dITP is included in the kit for use with
templates prone to gel compression artifacts. To use dITP simply substitute the
dITP Nucleotide Mix for the dGTP Nucleotide Mix. All other aspects of the
sequencing protocol remain unchanged except that when using dITP, reduce
the termination temperature from 72°C to 60°C and increase the time to
approximately 5 minutes or longer (see figure 3). The use of dITP will result in
less uniform band intensities, but will completely resolve even the strongest
compressions. A 40% formamide gel will also eliminate almost all compressions
(see ‘Denaturing gel electrophoresis’ section).
Figure 2. Compression artifacts can be eliminated using dITP in place of dGTP without
interference by stops or other artifact bands. Shown are two severely ‘compressed’
regions of secondary structure (see arrows). The sequences run using dITP in place of
dGTP are accurate and unambiguous.
dGTP dITP dGTP dITP
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