Essential Cell Biology 5th edition

Isolating and Cloning DNA Molecules
339
bacterial
cell
DOUBLE-STRANDED
RECOMBINANT
PLASMID DNA
INTRODUCED INTO
BACTERIAL CELL
cell culture produces
hundreds of millions of
new bacteria
many copies of purified
plasmid isolated from
lysed bacteria
Figure 10–7 A DNA fragment can be
replicated inside a bacterial cell. To clone
a particular fragment of DNA, it is first
inserted into a plasmid vector, as shown
in Figure 10–6. The resulting recombinant
plasmid DNA is then introduced into a
bacterium, where it is replicated many
millions of times as the bacterium multiplies.
For simplicity, the genome of the bacterial
cell is not shown.
An Entire Genome Can Be Represented in a DNA Library
When a whole genome ECB5 is E10.09/10.07
cut by a restriction enzyme, a large number
of different DNA fragments is generated. This collection of DNA fragments
can be ligated into plasmid vectors, under conditions that favor
the insertion of a single DNA fragment into each plasmid molecule. These
recombinant plasmids are then introduced into E. coli at a concentration
that ensures that no more than one plasmid molecule is taken up
by each bacterium. The resulting collection of cloned DNA fragments,
present in the bacterial culture, is known as a DNA library. Because the
DNA fragments were derived by digesting chromosomal DNA directly
from an organism, the resulting collection—called a genomic library—
should represent the entire genome of that organism (Figure 10–8). Such
genomic libraries often provide the starting material for determining the
complete nucleotide sequence of an organism’s genome.
For other applications, however, it can be advantageous to work with a
different type of library—one that includes only the coding sequences of
genes; that is, a library that lacks intronic and other noncoding sequences
that make up most eukaryotic DNA. For some genes, the complete
genomic clone—including introns and exons—is too large and unwieldy
to handle conveniently in the laboratory (see, for example, Figure 7−19B).
What’s more, the bacterial cells typically used to amplify cloned DNA are
unable to remove introns from mammalian RNA transcripts. So if the
goal is to use a cloned mammalian gene to produce a large amount of
the protein it encodes, for example, it is essential to use only the coding
sequence of the gene.
In this case, investigators generate a cDNA library. A cDNA library is
similar to a genomic library in that it also contains numerous clones containing
many different DNA sequences. But it differs in one important
respect. The DNA that goes into a cDNA library is not genomic DNA;
it is DNA copied from the mRNAs present in a particular type of cell.
To prepare a cDNA library, all of the mRNAs are extracted, and doublestranded
DNA copies of these mRNAs are produced by the enzymes
reverse transcriptase and DNA polymerase (Figure 10–9). The resulting
complementary DNA—or cDNA—molecules are then introduced into
bacteria and amplified, as described for genomic DNA fragments (see
Figure 10–8).
CLEAVE WITH
RESTRICTION
ENZYME
INTRODUCTION
OF PLASMIDS
INTO BACTERIA
human
DNA
millions of
genomic
DNA
fragments
DNA FRAGMENTS
INSERTED INTO PLASMIDS
USING DNA LIGASE
recombinant
DNA molecules
Figure 10–8 Human genomic libraries containing DNA fragments
representing the whole human genome can be constructed using
restriction enzymes and DNA ligase. Such a genomic library consists
of a set of bacteria, each carrying a different small fragment of human
DNA. For simplicity, only the colored DNA fragments are shown in
the library; in reality, all of the different gray fragments will also be
represented.
genomic library