Biomedical Engineering Reference
In-Depth Information
After transfer, the nucleic acid needs to be fixed to
the membrane and a number of methods are avail-
able. Oven baking at 80°C is the recommended
method for nitrocellulose membranes and this can
also be used with nylon membranes. Due to the
flammable nature of nitrocellulose, it is important
that it is baked in a vacuum oven. An alternative
fixation method utilizes ultraviolet cross-linking. It
is based on the formation of cross-links between a
small fraction of the thymine residues in the DNA
and positively charged amino groups on the surface
of nylon membranes. A calibration experiment must
be performed to determine the optimal fixation period.
Following the fixation step, the membrane is placed
in a solution of labelled (radioactive or non-radioactive)
RNA, single-stranded DNA or oligodeoxynucleotide
which is complementary in sequence to the blot-
transferred DNA band or bands to be detected.
Conditions are chosen so that the labelled nucleic
acid hybridizes with the DNA on the membrane.
Since this labelled nucleic acid is used to detect and
locate the complementary sequence, it is called the
probe
. Conditions are chosen which maximize the
rate of hybridization, compatible with a low back-
ground of non-specific binding on the membrane
(see Box 2.1). After the hybridization reaction has
been carried out, the membrane is washed to remove
unbound radioactivity and regions of hybridization
Weight < 0.75 kg
Glass plate
Paper tissues
3 sheets filter paper
Membrane
Gel
Plastic tray
Fig. 2.5
A typical capillary blotting apparatus.
uniform transfer of a wide range of DNA fragment
sizes, the electrophoresed DNA is exposed to a short
depurination treatment (0.25 mol/l HCl) followed by
alkali. This shortens the DNA fragments by alkaline
hydrolysis at depurinated sites. It also denatures the
fragments prior to transfer, ensuring that they are in
the single-stranded state and accessible for probing.
Finally, the gel is equilibrated in neutralizing solution
prior to blotting. An alternative method uses posit-
ively charged nylon membranes, which remove the
need for extended gel pretreatment. With them the
DNA is transferred in native (non-denatured) form
and then alkali-denatured
in situ
on the membrane.
Box 2.1 Hybridization of nucleic acids on membranes
The hybridization of nucleic acids on membranes is a
widely used technique in gene manipulation and
analysis. Unlike solution hybridizations, membrane
hybridizations tend not to proceed to completion.
One reason for this is that some of the bound nucleic
acid is embedded in the membrane and is inaccessible
to the probe. Prolonged incubations may not generate
any significant increase in detection sensitivity.
The composition of the hybridization buffer can
greatly affect the speed of the reaction and the
sensitivity of detection. The key components of these
buffers are shown below:
Rate enhancers
Dextran sulphate and other polymers act as volume excluders to increase both the rate and the
extent of hybridization
Detergents and blocking agents
Dried milk, heparin and detergents such as sodium dodecyl sulphate (SDS) have been used
to depress non-specific binding of the probe to the membrane. Denhardt's solution
(Denhardt 1966) uses Ficoll, polyvinylpyrrolidone and bovine serum albumin
Denaturants
Urea or formamide can be used to depress the melting temperature of the hybrid so that reduced
temperatures of hybridization can be used
Heterologous DNA
This can reduce non-specific binding of probes to non-homologous DNA on the blot
continued
