Gene Cloning

142 Gene Cloning
diseases, where the absence of a functional gene, or the presence of a defective
gene, can lead to disability or death. In such cases, the identification of
the gene concerned can help in early diagnosis, including early screening in
pregnancy and screening of embryos created by in vitro fertilization (IVF),
and has great potential in devising treatments for the condition.
We can illustrate this point with one of the best known examples: the
disease cystic fibrosis (CF). This disease has long been known to be caused
by mutations in a single gene. These mutations are recessive, but if an individual
carries two copies of the gene both of which are defective, then they
will develop CF. The defective gene is inherited in a classical Mendelian
fashion for a single gene, so if both parents are carriers (i.e. heterozygous
for the defective gene) then one in four of their children, on average, will
develop the disease, and half of their children, on average, will be carriers.
The symptoms of the disease were well known for many years, but the
underlying cause of the disease – the nature of the gene, its role in its normal
non-defective state, and how mutation of the gene causes the disease
– were mysteries until the gene was successfully cloned. We will return to
this classic story in Section 6.10.
Thus, methods have had to be developed for cloning genes when nothing
is known in advance about the sequence of the gene or the nature of the
protein which it encodes. These methods can be divided into two groups,
which are very different in their approach and applicability. The first of
these, gene tagging, involves generating mutations in such a way that the
gene is marked so that it can be recovered in subsequent cloning experiments.
The second, map-based or positional cloning, requires the development
of powerful maps of the genome which can be used to focus the
search for a particular gene on smaller and smaller regions. We will consider
each of these methods in turn in the next two sections.
6.2 Gene Tagging: A Method That Both Mutates and Marks
Genes
Mutations in genes often produce changes in phenotype. To identify a gene
which has been mutated and where the phenotype has consequently
changed, it would be useful to be able to identify the gene purely on the basis
that it carries a mutation. This is the idea behind gene tagging, which involves
generating mutations using something which not only randomly inactivates
genes, but at the same time marks them so that they can be easily detected,
for example by screening a gene library. It involves essentially four steps.
In the first step, a large number of mutations are generated, using as a
mutagen a piece of mobile DNA that can insert at random in the genome,
disrupting and blocking the function of any gene into which it inserts. This
results in a population of organisms where the only difference between
them is the position of the mutation. The mobile DNA used for the mutagenesis
is usually a transposon; that is, a piece of DNA which is able to