Biology Reference
In-Depth Information
particularly with the bacterium
Escherichia coli
. This bacterium has become
a molecular biology workhorse because it can be induced to produce large
amounts of recombinant DNA molecules by inserting plasmids, the bacterio-
phage
λ
or genetically engineered variants of these agents into it. Genetic engi-
neers use enzymes from different organisms to modify, ligate, or splice DNA. Basic
tools include purifying plasmids from
E. coli
, visualizing DNA by electrophoresis
through agarose or polyacrylamide gels, Southern blot analyses, and producing
labeled probes. This chapter describes the steps involved in inserting a foreign
gene into a plasmid, inserting the plasmid into
E. coli
, and isolating and analyz-
ing the amplified DNA by Southern blot analysis and restriction-site mapping.
“Northern” and “Western” blot analyses allow the researcher to evaluate RNA
and proteins, respectively. Many of these procedures have been simplified by the
availability of commercial kits, but understanding the concepts behind them will
allow you to “trouble shoot.” Cloning of DNA has five essential components:
1) a method for generating exogenous DNA fragments, 2) reactions to join exog-
enous DNA fragments to a vector, 3) a method to introduce the vector into a
host cell where the vector ensures the exogenous DNA is replicated, 4) methods
for selecting or identifying the vectors that contain the introduced DNA (recom-
binant molecules), and 5) methods for analyzing the cloned DNA.
5.2 Introduction to a Basic Molecular Biology Experiment
Molecular genetic techniques, some arising from research on basic topics, have
become crucial tools that allow scientists to manipulate DNA from living organ-
isms. As a result, it is difficult to predict to what use some basic research can
be applied. The molecular genetic revolution began in 1970. Before 1970, there
was no way to cut a DNA molecule into discrete and predictable fragments,
nor could specific DNA fragments be joined together. The discovery of enzymes
called
restriction endonucleases
and
ligases
changed this. Much of genetic engi-
neering technology is dependent upon our ability to cut DNA molecules at spe-
cific sites and combine them into new molecules by base pairing and ligation.
Another significant development was the harnessing of
plasmids
and
bacte-
riophages
as vehicles (
vectors
) to replicate foreign DNA within the bacterium
Escherichia coli
, which allowed nearly unlimited amounts of specific DNA to be
produced for study and manipulation. Techniques for monitoring the results of
such manipulations were developed so that researchers could identify as small as
a single-base modification in DNA.
A simple cloning project, as would have been conducted in 1985 when many
of the current kits were unavailable, is outlined in
Figure 5.1
. The methods
