Biomedical Engineering Reference
In-Depth Information
CHAPTER 14
Applications of recombinant
DNA technology
Introduction
Theme 1: Nucleic acid sequences
as diagnostic tools
Biotechnology is not new. The making of beer, wine,
bread, yoghurt and cheese was practised by ancient
civilizations, such as the Babylonians, the Romans
and the Chinese. Much, much later came vaccines,
the production of basic chemicals (e.g. glycerol,
citric acid, lactic acid) and the development of anti-
biotics. In each of these examples,
existing
properties
of microorganisms were exploited. For example,
Penicillium
species naturally make penicillin. What
the scientists have done is to increase the yield of
penicillin by repeated rounds of mutation and
selection, coupled with optimization of the growth
medium. Similarly, sexual crosses between related
plant species have created high-yielding and disease-
resistant varieties of cereals. These improved cereals
represent new combinations of genes and alleles
already existing in wild strains.
With the development of gene manipulation tech-
niques in the 1970s, there was a major paradigm
shift. For the first time microorganisms could be
made to synthesize compounds that they had never
synthesized before, e.g. insulin production in
E. coli
( Johnson 1983). Soon all sorts of commercially or
therapeutically useful proteins were being made in
bacteria, principally
E. coli
, and thus the modern
biotechnology industry was born. As the techniques
developed for manipulating genes in bacteria were
extended to plants and animals there was a con-
comitant expansion of the biotechnology industry
to exploit the new opportunities being provided.
Today there are many different facets to the com-
mercial exploitation of gene manipulation tech-
niques as shown in Fig. 14.1. Rather than discuss
all these topics in detail, for that would take a topic
in itself, we have chosen to focus on six inter-
disciplinary themes that reflect both the successes
achieved to date and the likely successes in the next
decade.
Introduction to theme 1
Nucleic acid sequences can be used diagnostically in
two different ways. The first is to determine whether
a particular, relatively long sequence is present in or
absent from a test sample. A good example of such
an application is the diagnosis of infectious disease.
By choosing appropriate probes, one can ascertain
in a single step which, if any, microorganisms are
present in a sample. Alternatively, a search could be
made for the presence of known antibiotic-resistance
determinants so that an appropriate therapeutic
regime can be instituted. In the second way in which
sequences are used diagnostically, the objective is to
determine the similarity of sequences from different
individuals. Good examples of this approach are
prenatal diagnosis of genetic disease and forensic
profiling ('DNA fingerprinting').
Detection of sequences at the gross level
Imagine that a seriously ill individual has a disorder
of the gastrointestinal tract. A likely cause is a
microbial infection and there are a number of candid-
ate organisms (Table 14.1). The question is, which
organism is present and to which antibiotics is it
susceptible? The sooner one has an answer to these
questions, the sooner
effective
therapy can begin.
Traditionally, in such a case, a stool specimen would
be cultured on a variety of different media and would
be examined microscopically and tested with vari-
ous immunological reagents. A simpler approach
is to test the sample with a battery of probes and
determine which, if any, hybridize in a simple dot-
blot assay. With such a simple format it is possible to
vary the stringency of the hybridization reaction to
accommodate any sequence differences that might
