Biomedical Engineering Reference
In-Depth Information
CHAPTER 2
Basic techniques
and in the absence of replication will be diluted out of
their host cells. It should be noted that, even if a DNA
molecule contains an origin of replication, this may
not function in a foreign host cell.
There is an additional, subsequent problem. If the
early experiments were to proceed, a method was
required for assessing the fate of the donor DNA. In
particular, in circumstances where the foreign DNA
was maintained because it had become integrated in
the host DNA, a method was required for mapping the
foreign DNA and the surrounding host sequences.
Introduction
The initial impetus for gene manipulation
in vitro
came about in the early 1970s with the simultan-
eous development of techniques for:
• genetic transformation of
Escherichia coli
;
• cutting and joining DNA molecules;
• monitoring the cutting and joining reactions.
In order to explain the significance of these devel-
opments we must first consider the essential require-
ments of a successful gene-manipulation procedure.
The basic problems
The solutions: basic techniques
Before the advent of modern gene-manipulation
methods there had been many early attempts at
transforming pro- and eukaryotic cells with foreign
DNA. But, in general, little progress could be made.
The reasons for this are as follows. Let us assume
that the exogenous DNA is taken up by the recipient
cells. There are then two basic difficulties. First,
where detection of uptake is dependent on gene
expression, failure could be due to lack of accurate
transcription or translation. Secondly, and more
importantly, the exogenous DNA may not be main-
tained in the transformed cells. If the exogenous
DNA is integrated into the host genome, there is no
problem. The exact mechanism whereby this integ-
ration occurs is not clear and it is usually a rare
event. However this occurs, the result is that the
foreign DNA sequence becomes incorporated into
the host cell's genetic material and will subsequently
be propagated as part of that genome. If, however,
the exogenous DNA fails to be integrated, it will
probably be lost during subsequent multiplication of
the host cells. The reason for this is simple. In order
to be replicated, DNA molecules must contain an
origin of replication
, and in bacteria and viruses there
is usually only one per genome. Such molecules are
called
replicons.
Fragments of DNA are not replicons
If fragments of DNA are not replicated, the obvious
solution is to attach them to a suitable replicon.
Such replicons are known as
vectors
or
cloning
vehicles.
Small plasmids and bacteriophages are the
most suitable vectors for they are replicons in their
own right, their maintenance does not necessarily
require integration into the host genome and their
DNA can be readily isolated in an intact form. The
different plasmids and phages which are used as
vectors are described in detail in Chapters 4 and 5.
Suffice it to say at this point that initially plasmids
and phages suitable as vectors were only found in
E
.
coli
. An important consequence follows from the use
of a vector to carry the foreign DNA: simple methods
become available for purifying the vector molecule,
complete with its foreign DNA insert, from trans-
formed host cells. Thus not only does the vector
provide the replicon function, but it also permits the
easy bulk preparation of the foreign DNA sequence,
free from host-cell DNA.
Composite molecules in which foreign DNA has
been inserted into a vector molecule are sometimes
called DNA
chimeras
because of their analogy with
the Chimaera of mythology - a creature with the
head of a lion, body of a goat and tail of a serpent.
The construction of such composite or
artificial
