DNA
hybridisation
This is based on the property
that complementary nucleic acid strands will 'bind' to each other
(hydrogen bond) and that these bonds can be broken by high temperature
(melting) and stabilised in solutions containing salt (ionic strength).
Therefore a small nucleic acid sequence (either a DNA or RNA molecule)
can be used as a probe to detect complementary
sequences within a mixture of different nucleic acid sequences (see
Southern
Blotting and DNA
library screening methods). For instance, we can chemically synthesise
an oligonucleotide of the same sequence as (or complementary to) the gene
of interest (target sequence). Oligonucleotide probes
can be as small as 20 nucleotides. Detection is achieved by labelling the
probe - often by incorporating radioactivity
into it (visualised by exposure to film).
Specificity is important
It is important to remember that
the probe must be specific for the
gene of interest and should not cross-react with other DNA (or RNA) sequences
- that is anneal to other DNA sequences that are present. The probability
of a specific 20 nucleotide sequence occurring is very low, i.e. 1 in 420
(1 in ~1012) nucleotides.
Labelling the probe
The probe
can be labelled with a radioactive phosphate (32P) from radiolabelled
ATP. This is done by removing the 5' phosphate of the oligonucleotide using
alkaline
phophatase, and replacing it with a radiolabelled phosphate
using polynucleotide
kinase.
Fig. 7-20, Lodish et al. (4th
ed.)
An alternative is to incorporate
one or more radiolabelled nucleotides to the 3' end of the probe
using
terminal transferase.
Incubation & removal
of unbound probe
The membrane is soaked with a
solution of the radiolabelled probe,
to allow it to hybridise (anneal) to the correct DNA (or RNA) sequence.
The membrane is washed extensively to remove non-specifically bound probe,
and the positions where the probe remains
bound are visualised by autoradiography.
Probe can be non-radioactively
labelled
Recent technologies have allowed
development of techniques for DNA hybridisation
that do not rely on radioactive labelling. One such method uses a probe
which contains modified nucleotide bases that have a steroid-like molecule
attached. After blotting to the membrane, this can be targeted by an antibody
which is fused to a reporter enzyme
e.g. Alkaline phosphatase. The reporter
enzyme catalyses a colour-forming reaction at the positions on the filter/membrane
to which the antibody binds - which tells us, for instance, where the DNA
fragments recognised by the probe are
located.
Hybridisation and washing conditions
The hybridisation and washing
conditions for the membrane are critical. If the probe
and the target sequence to which it hybridises are 100% identical - then
a high stringency hybridisation can
be perfomed. The stringency of the
blot is determined by the temperature at which the membrane is incubated
in the probe-containing hybridisation
buffer, and by the ionic strength (salt concentration) of the buffer.
High temperature and low salt make for high stringency
- since only perfect hybrids between the probe and the target sequence
will remain formed under these conditions.
For probes that are not identical
to the target sequence, the stringency
must be reduced to a level that allows hybridisation between non-perfectly
matched DNA. However, if the stringency
is too low, then there may be too many non-specific interactions observed,
i.e. numerous bands that do not reflect meaningful DNA-DNA interactions.
The hybridisation and washing
steps should normally be carried out at 12°C below the theoretical
melting temperature of the probe (and
target) DNA. The melting
temperature (Tm) is the temperature required to separate
the double helix of a DNA fragment into two separate strands.
A formula for determining the
Tm of the probe fragment
is:
Tm = 69.3°C
+ 0.41[%(G+C)] - 650/l
Where l = the length (in nucleotides)
of the probe
So, for a probe
of 30 nucleotides with 50% (G+C) content, the theoretical Tm
is:
(69.3 + 20.5 - 21.7) =
68.1 ° C
Including formamide
in the incubation buffer effectively reduces the temperature required for
hybridisation by up to ~25°C