Gene Cloning

Forensic and Medical Applications 425
develop. In diagnosis of Huntington’s disease it is therefore essential to be
able to determine the number of repeats as this correlates with age of
onset and severity of the condition.
With single gene disorders the mutation is confined to a specific region of
the human genome, a gene or its regulatory sequences. However, there
may be many different mutations possible within this region, some of
which are common and others much less so. For example in the case of
cystic fibrosis the ΔF508 mutation is by far the most common mutation
being found on 75% of CF chromosomes in the UK. The next most common
mutation G551D is found in only 3% of CF chromosomes. There are
some 20 mutations found routinely in the UK and hundreds more which
are only rarely found. The incidence of the ΔF508 mutation also varies,
occurring in only 20% of CF chromosomes in Turkey but in 90% in
Denmark. Another common inherited disorder, β thalassemia, results
from point mutations and small deletions in the β globin gene, causing
anemia of varying degrees of severity. It is prevalent in people who originate
in the Mediterranean, the Middle East, India and South-East Asia.
Again there is a variety of mutations and their prevalence varies between
ethnic groups.
Usually, primers specific for both normal and mutant alleles are used in
separate PCR reactions so that both alleles can be detected. This makes it
possible to identify heterozygous carriers as well as affected individuals.
This technique can be used to detect a range of different types of mutations
which give rise to genetic diseases including deletions and insertions as
well as single nucleotide polymorphisms as they will all create mismatches
between primer and template.
The sensitivity and efficiency of this technique can be improved by
labeling the allele specific primer with a fluorophore (Box 5.3). As with DNA
profiling the amplification products can then be analyzed by capillary electrophoresis,
this improves detection of weakly amplified products. By
labeling each allele with a different fluorophore it is possible to detect both
alleles in a single reaction.
Fluorescent PCR for detection of expanded trinucleotide repeats
This is in fact exactly the same technique as that used in DNA profiling
(Section 13.3). The expanded trinucleotide repeats responsible for diseases
like Huntington’s disease (Box 13.2) are in effect STRs and testing for them
involves PCR amplification of the repeat region in a PCR reaction with one
fluorescently labeled primer. The sensitivity gained by using fluorescent
PCR and capillary electrophoresis is particularly important in the context
of Huntington’s disease as it is important to be able to determine the