Gene Cloning

Forensic and Medical Applications 443
A13.5. The overall length and the GC content are the two key factors here
(Box 3.6). The longer the region of complementarity and the greater the proportion
of GC residues, the more stable the hybrid will be.
Q13.6. What types of mutation are likely to introduce stop codons into
a gene?
A13.6. Any single base pair substitution which changes an amino acid
encoding codon into a stop codon, for example TTA or TTG (which encode
leucine) need only a single substitution of a T for an A in the middle position
to give the stop codons TAA and TAG, respectively. Also, any mutation which
results in the loss or gain of a number of nucleotides that is not divisible by
three, will almost certainly give rise to a stop codon. Deletion of three
nucleotides (or multiples of three) results in the loss of a single amino acid;
unless this particular amino acid has a critical role in the protein the protein
may still be able to function normally. However, deletion of other numbers
of nucleotides results in a shift in the reading frame. This means that from
this point onwards a different sequence of codons is being read which will
encode a nonsense protein. However, because stop codons are relatively common
in non coding reading frames (see Figure 8.2) the most likely effect is in
fact that a stop codon will be encountered shortly after the point of the mutation
and a shortened or truncated protein will be produced.
Q13.7. Why is multiplex PCR an important tool in genetic diagnosis?
A13.7. The ability to amplify DNA by PCR is the key to genetic testing, particularly
where the sample has been taken from a fetus or early embryo and
may consist of very few cells. Multiplex PCR makes it possible to test for a
range of different mutations in the same PCR reaction, permitting testing
either for several different diseases or for a range of mutations which cause
the same disease.
Q13.8. What advantages do DNA-based diagnostic tests for bacterial and
viral infections offer over conventional tests?
A13.8. In both cases DNA-based tests overcome some of the problems associated
with the requirement of conventional tests to culture the organisms. In
the case of bacteria problems may arise because some bacteria are hard to
culture, because bacteria are present in mixtures or simply because of the
time and expertise required to grow bacteria in the laboratory. Many viruses
cannot easily be cultured in the laboratory and diagnostic tests often rely on
detecting antibodies in patient serum and these may not be detectable in the
early stages of infection. In DNA-based tests PCR amplification of DNA
replaces laboratory culture. Techniques such as the 5′ exonuclease
(TaqMan®) and molecular beacon assays allow for quantification in addition
to identification. This can be very useful to detect the presence of small
numbers of one pathogen in the presence of a more abundant one and also