of these techniques is that they allow animal models of human diseases to be created. This
is useful since the physiological and biochemical consequences of a disease are often
complex and difficult to study impeding the development of diagnostic and therapeutic
strategies.6.8.5 Modulating gene expression by RNAi
There are a number of ways of experimentally changing the expression of genes.
Traditionally methods have focussed on altering the levels of mRNA by manipulation
of promoter sequences or levels of accessory proteins involved in control of expres-
sion. In addition post-mRNA production methods have also been employed such as
antisense RNA, where a nucleic acid sequence complementary to an expressed mRNA
is delivered into the cell. This antisense sequence binds to the mRNA and prevents its
translation. A development of this theme and a process that is found in a variety of
normal cellular processes is termedRNA interference(RNAi) and uses microRNA.
Here a number of techniques have been developed that allow the modulation of gene
expression in certain cells. This type of cellular-based gene expression modulation
will no doubt extend to many organisms in the next few years.6.8.6 Analysing genetic mutations
There are several types of mutations that can occur in nucleic acids, either transiently or
those that are stably incorporated into the genome. During evolution, mutations may be
inherited in one or both copies of a chromosome, resulting in polymorphisms within the
population (Section 5.3). Mutations may potentially occur at any site within the genome;
however, there are several instances whereby mutations occur in limited regions. This is
particularly obvious in prokaryotes, where elements of the genome (termedhypervari-
able regions) undergo extensive mutations to generate large numbers of variants, by
virtue of the high rate of replication of the organisms. Similar hypervariable sequences
are generated in the normal antibody immune response in eukaryotes. Mutations may
have several effects upon the structure and function of the genome. Some mutations may
lead to undetectable effects upon normal cellular functions, termed conservative muta-
tions. An example of these are mutations that occur in intron sequences and therefore
play no part in the final structure and function of the protein or its regulation. Alterna-
tively, mutations may result in profoundeffects upon normal cell function such as altered
transcription rates or on the sequence of mRNAs necessary for normal cellular processes.
Mutations occurring within exons may alter the amino acid composition of the
encoded protein by causing amino acid substitution or by changing the reading frame
used during translation. These point mutations were traditionally detected by South-
ern blotting or, if a convenient restriction site was available, byrestriction fragment
length polymorphism(RFLP) (Section 5.9). However, the PCR has been used to great
effect in mutation detection since it is possible to useallele-specific oligonucleotide
PCR(ASO–PCR) where two competing primers and one general primer are used in the
reaction (Fig. 6.45). One of the primers is directly complementary to the known point
mutation whereas the other is a wild-type primer; that is, the primers are identical249 6.8 Analysing genes and gene expression