Essential Cell Biology 5th edition

338 CHAPTER 10 Analyzing the Structure and Function of Genes
Figure 10–6 A DNA fragment is inserted
into a bacterial plasmid using the enzyme
DNA ligase. The plasmid is first cut open
at a single site with a restriction enzyme
(in this case, one that produces staggered
ends). It is then mixed with the DNA
fragment to be cloned, which has been
cut with the same restriction enzyme. The
staggered ends base-pair, and when DNA
ligase and ATP are added, the nicks in the
DNA backbone are sealed to produce a
complete recombinant DNA molecule. In
the accompanying micrographs, we have
colored the DNA fragment red to make
it easier to see. (Micrographs courtesy of
Huntington Potter and David Dressler.)
circular,
double-stranded
plasmid DNA
(cloning vector)
CLEAVAGE WITH
RESTRICTION
ENZYME
DNA fragment
to be cloned
COVALENT
LINKAGE
BY DNA LIGASE
recombinant DNA
200 nm
200 nm
The vectors used for cloning are streamlined versions of plasmids that
occur naturally in many bacteria. Bacterial plasmids were first recognized
by physicians and scientists because they often carry genes that render
their microbial host resistant to one or more antibiotics. Indeed, historically
potent antibiotics—penicillin, for example—are no longer effective
against many of today’s bacterial infections because plasmids that confer
resistance to the antibiotic ECB5 have E10.08/10.06 spread among bacterial species by horizontal
gene transfer (see Figure 9−15).
To insert a piece of DNA into a plasmid vector, the purified plasmid DNA
is opened up by a restriction enzyme that cleaves it at a single site, and
the DNA fragment to be cloned is then spliced into that site using DNA
ligase (Figure 10–6). This recombinant DNA molecule is now ready to be
introduced into a bacterium, where it will be copied and amplified.
To accomplish this feat, investigators take advantage of the fact that
some bacteria naturally take up DNA molecules present in their surroundings.
The mechanism that controls this uptake is called transformation,
because early observations suggested it could “transform” one bacterial
strain into another. Indeed, the first proof that genes are made of DNA
came from an experiment in which DNA purified from a pathogenic strain
of pneumococcus was used to transform a harmless bacterium into a
deadly one (see How We Know, pp. 192−194).
In a natural bacterial population, a source of DNA for transformation is
provided by bacteria that have died and released their contents, including
DNA, into the environment. In a test tube, however, bacteria such as
E. coli can be coaxed to take up recombinant DNA that has been created
in the laboratory. These bacteria are then suspended in a nutrient-rich
broth and allowed to proliferate.
Each time the bacterial population doubles—every 30 minutes or so—the
number of copies of the recombinant DNA molecule also doubles. Thus,
in 24 hours, the engineered cells will produce hundreds of millions of
copies of the plasmid, along with the DNA fragment it contains. The bacteria
can then be split open (lysed) and the plasmid DNA purified from
the rest of the cell contents, including the large bacterial chromosome
(Figure 10–7).
The DNA fragment can be readily recovered by cutting it out of the plasmid
DNA with the same restriction enzyme that was used to insert it,
and then separating it from the plasmid DNA by gel electrophoresis (see
Figure 10–3). Together, these steps allow the amplification and purification
of any segment of DNA from the genome of any organism.