Biomedical Engineering Reference
In-Depth Information
CHAPTER 1
Gene manipulation:
an all-embracing technique
Introduction
Sequence analysis
Occasionally technical developments in science
occur that enable leaps forward in our knowledge
and increase the potential for innovation. Molecular
biology and biomedical research experienced such
a revolutionary change in the mid-70s with the
development of gene manipulation. Although the
initial experiments generated much excitement, it is
unlikely that any of the early workers in the field
could have predicted the breadth of applications to
which the technique has been put. Nor could they
have envisaged that the methods they developed
would spawn an entire industry comprising several
hundred companies, of varying sizes, in the USA
alone.
The term gene manipulation can be applied to
a variety of sophisticated
in vivo
genetics as well as
to
in vitro
techniques. In fact, in most Western
countries there is a precise
legal
definition of gene
manipulation as a result of government legisla-
tion to control it. In the UK, gene manipulation is
defined as
the formation of new combinations of heritable
material by the insertion of nucleic acid molecules,
produced by whatever means outside the cell,
into any virus, bacterial plasmid or other vector
system so as to allow their incorporation into a
host organism in which they do not naturally
occur but in which they are capable of continued
propagation.
The definitions adopted by other countries are sim-
ilar and all adequately describe the subject-matter
of this topic. Simply put, gene manipulation per-
mits stretches of DNA to be isolated from their host
organism and propagated in the same or a different
host, a technique known as
cloning.
The ability to
clone DNA has far-reaching consequences, as will
be shown below.
Cloning permits the isolation of discrete pieces of a
genome and their amplification. This in turn enables
the DNA to be sequenced. Analysis of the sequences
of some genetically well-characterized genes led to
the identification of the sequences and structures
which characterize the principal control elements
of gene expression, e.g. promoters, ribosome bind-
ing sites, etc. As this information built up it became
possible to scan new DNA sequences and identify
potential new genes, or
open reading frames
, because
they were bounded by characteristic motifs. Initially
this sequence analysis was done manually but to
the eye long runs of nucleotides have little meaning
and patterns evade recognition. Fortunately such
analyses have been facilitated by rapid increases
in the power of computers and improvements in soft-
ware which have taken place contemporaneously
with advances in gene cloning. Now sequences can
be scanned quickly for a whole series of structural
features, e.g. restriction enzyme recognition sites,
start and stop signals for transcription, inverted
palindromes, sequence repeats, Z-DNA, etc., using
programs available on the Internet.
From the nucleotide sequence of a gene it is easy
to deduce the protein sequence which it encodes.
Unfortunately, we are unable to formulate a set of
general rules that allows us to predict a protein's
three-dimensional structure from the amino acid
sequence of its polypeptide chain. However, based
on crystallographic data from over 300 proteins,
certain structural motifs can be predicted. Nor does
an amino acid sequence on its own give any clue to
function. The solution is to compare the amino acid
sequence with that of other better-characterized pro-
teins: a high degree of homology suggests similarity
in function. Again, computers are of great value
since algorithms exist for comparing two sequences
or for comparing one sequence with a group of other
