DNA sequencing

In many of the techniques outlined above, no specific
information is gained about the exact nature of the alteration in
the DNA. In some cases, the change detected may turn out to
be a polymorphism that has no direct bearing on the condition
under investigation. The exception to this is the protein
truncation test (PTT), which detects mutations that shorten the
protein product and are therefore more likely to be pathogenic.
In chemical cleavage of mismatch analysis, particular types of
base mismatch are cleaved specifically by the different chemicals
employed; this yields limited information about the type of
change observed.

making a copy of the DNA

The technique relies on making a copy of the DNA in
the presence of modified versions of the four bases (A, C, G,
and T) which are fluorescently labelled with their own specific
tag. The sequencing products are then separated with the use
of long polyacrylamide gels with a laser being used to
automatically detect the fluorescent molecules as they migrate.
A computer program is then used to generate the DNA
sequence. Recent improvements in DNA sequencing have seen
polyacrylamide gels being replaced by capillary columns
allowing the method to be further automated.

Hybridisation methods and “gene-chip” technology

In most of the methods described above, the specific site of a
mutation within a gene is not known until after DNA
sequencing has been completed. If the mutation is very
common, however, methods may be used that specifically
interrogate the site of the mutation. One of the simplest ways
of doing this is by using a restriction enzyme (see above);
however, this is not applicable in all situations.

use of DNA probe technology

Another possibility is the use of DNA probe technology.
This utilises the tendency of two complementary singlestranded
DNA molecules to anneal together to produce a
double-stranded duplex. This method involves the DNA under
investigation being immobilised onto a solid support such as
nylon. A labelled single-stranded DNA probe may then be used
to determine whether a specific sequence is present. This
technique is often referred to as forward dot-blotting.

labelled target DNA

Alternatively, the probes may be immobilised to the
membrane and hybridised with the labelled target DNA, that is
free in solution (the reverse dot-blot approach). It is this basic
principle that has been developed into the so-called “gene
chip” technology. In this technique, literally thousands of short
DNA probe molecules are first attached to silica-based support
materials. The DNA under investigation is then fluorescently
labelled and hybridised to the probe matrix. The large number
of probes used enables the pattern of hybridisation to be
translated into sequence information. At present, however, the
high cost of this approach means that it is of limited value for
the analysis of rare disease genes in a diagnostic setting.

Non-PCR based analysis

Not every gene can be studied using PCR. In some conditions,
the mutation itself is large, and may have even deleted the
entire gene. In other cases, the gene may be very rich in G and
C bases, which makes conventional PCR difficult. In these
situations, the older methods of analysis are invaluable,
although generally more time-consuming than PCR-based
methods.

Southern blotting

Although largely replaced by PCR-based methods, Southern
blotting is still necessary to detect relatively large changes in
the DNA that exceed the limits of PCR. Genomic DNA is first
cut using restriction enzymes and the digested fragments
fractionated using gel electrophoresis. The DNA is then
transferred by capillary blotting onto nylon membrane before
radiolabelled probes are used to investigate the region of
interest.