Biomedical Engineering Reference
In-Depth Information
Box 2.1
continued
oligonucleotides. It is standard practice to use
oligonucleotides to analyse putative mutants
following a site-directed mutagenesis experiment
where the difference between parental and mutant
progeny is often only a single base-pair change
(see p. 132
et seq
.).
The availability of the exact sequence of
oligonucleotides allows conditions for hybridization
and stringency washing to be tightly controlled so
that the probe will only remain hybridized when
it is 100% homologous to the target. Stringency is
commonly controlled by adjusting the temperature
of the wash buffer. The 'Wallace rule' (Lay Thein &
Wallace 1986) is used to determine the appropriate
stringency wash temperature:
T
m
=
4
×
(number of GC base pairs)
+
2
×
(number
of AT base pairs)
In filter hybridizations with oligonucleotide probes,
the hybridization step is usually performed at 5°C
below
T
m
for perfectly matched sequences. For every
mismatched base pair, a further 5°C reduction is
necessary to maintain hybrid stability.
The design of oligonucleotides for hybridization
experiments is critical to maximize hybridization
specificity. Consideration should be given to:
• probe length - the longer the oligonucleotide, the
less chance there is of it binding to sequences other
than the desired target sequence under conditions
of high stringency;
• oligonucleotide composition - the GC content
will influence the stability of the resultant hybrid
and hence the determination of the appropriate
stringency washing conditions. Also the presence
of any non-complementary bases will have an effect
on the hybridization conditions.
Stringency control
Stringency can be regarded as the specificity with
which a particular target sequence is detected by
hybridization to a probe. Thus, at high stringency,
only completely complementary sequences will be
bound, whereas low-stringency conditions will allow
hybridization to partially matched sequences.
Stringency is most commonly controlled by the
temperature and salt concentration in the post-
hybridization washes, although these parameters
can also be utilized in the hybridization step.
In practice, the stringency washes are performed
under successively more stringent conditions
(lower salt or higher temperature) until the desired
result is obtained.
The melting temperature (
T
m
) of a probe-target
hybrid can be calculated to provide a starting-point
for the determination of correct stringency. The
T
m
is the temperature at which the probe and
target are 50% dissociated. For probes longer than
100 base pairs:
T
m
=
81.5°C
+
16.6 log
M
+
0.41 (% G
+
C)
where
M
= ionic strength of buffer in moles/litre.
With long probes, the hybridization is usually carried
out at
T
m
−
25°C. When the probe is used to
detect partially matched sequences, the
hybridization temperature is reduced by 1°C
for every 1% sequence divergence between
probe and target.
Oligonucleotides can give a more rapid
hybridization rate than long probes as they can
be used at a higher molarity. Also, in situations
where target is in excess to the probe, for example
dot blots, the hybridization rate is diffusion-limited
and longer probes diffuse more slowly than
are detected autoradiographically by placing the
membrane in contact with X-ray film (see Box 2.2).
A common approach is to carry out the hybridiza-
tion under conditions of relatively low stringency
which permit a high rate of hybridization, followed
by a series of post-hybridization washes of increasing
stringency (i.e. higher temperature or, more com-
monly, lower ionic strength). Autoradiography
following each washing stage will reveal any DNA
bands that are related to, but not perfectly comple-
mentary with, the probe and will also permit an
estimate of the degree of mismatching to be made.
