This animation demonstrates the details of
the molecular cloning of DNA sequences. The image shown is a circular plasmid
DNA molecule known as a cloning vector. The vector contains two important
regions – the cloning site and the drug resistance marker. The cloning site
is the DNA region where the gene to be studied is inserted,
and the resistance marker contains a gene which allows cells that contain
the plasmid to survive in the presence of antibiotics. Often one of the first
steps involved is to insert the gene of interest into a cloning vector. To
accomplish this, the plasmid vector must be cut with a restriction enzyme at
the cloning site. The enzyme, shown here as a pair of scissors, recognizes a
specific DNA sequence within the cloning site and often makes an uneven, or
staggered, cut at this location. This leaves some of the bases at the ends of
the DNA unpaired or “overhanging” with respect to the rest of the molecule –
these are referred to as “sticky ends” since they can base pair or “stick” to
a DNA fragment with a complementary sequence. Next, the DNA sequence to be
cloned, sometimes containing a functional gene, is added to the cut plasmid
vector. Since it too has been cut with a restriction enzyme, it may have
“sticky ends” which are complementary to the overhanging bases of the cloning
vector. Therefore, with the help of an enzyme called ligase, the DNA fragment
can be inserted, or ligated, into the vector. Then, the cloning vector that
now contains a copy of the gene of interest is used to transform a bacterial
cell such as E. coli, shown in green. The term “transform” refers to
the uptake of DNA by a living cell. The circular DNA vector including the
inserted gene is replicated by the bacterium until several copies are
present, and when the cell divides, many more copies, or clones, of the
vector and genes are produced. These can then be selected via the drug resistance
marker and isolated from bacterial cells, such that many copies of the gene
of interest are available for study.
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