Biotechnological Tools and Techniques N 8(1)

BIOTECHNOLOGICAL TOOLS AND TECHNIQUES
Molecular biologists can cut, join, and replicate DNA. Because of this, DNA sequences
are treated as modules and can be moved from one DNA molecule to another, forming
recombinant DNA. Recombinant DNA consists of fragments of DNA composed of
sequences originating from at least two different sources. Scientists use restriction
enzymes (also called restriction endonucleases), which occur naturally in prokaryotic
cells.
Restriction Enzymes
Restriction enzymes, are molecular

scissors that can cut double-stranded
DNA at a specific base-pair sequence.
Each type of restriction enzyme
recognizes a specific sequence of
nucleotides that is known as its
recognition site. Recognition sites are
usually four to eight base pairs long and
are characterized by a complementary
palindromic sequence. It is
palindromic because both strands have
the same base sequence when read in the
5’ to 3’ direction. The restriction enzyme EcoRI scans a DNA molecule and only stops
when it is able to bind to its recognition site. Once bound, it disrupts via a hydrolysis
reaction (catabolic), the phosphodiester bond between the guanine and adenine
nucleotides on each strand. The hydrogen bonds of complementary base pairs are also
disrupted. EcoRI produces sticky ends; both fragments have DNA nucleotides that lack
complementary bases. Restriction endonucleases that produce sticky ends are a more
useful tool because sticky-end fragments can be joined more easily to other sticky-
end fragments through complementary base pairing. Another restriction
endonuclease, SmaI, produces blunt ends, which means that the ends of the DNA
molecule fragments are fully base paired.
Restriction enzymes are examples of biological tools

because they are isolated and purified from bacteria.
Restriction enzymes protect the immune system of
bacteria. When a virus attacks a bacterial cell, it injects
its DNA into the bacterium and leaves its protein coat
outside the cell wall. The bacteria’s restriction
endonucleases start to scan the foreign viral DNA,
looking for recognition sites. When recognition sites are
encountered EcoRI cleaves the viral DNA into fragments
thus rendering it inactive.
Restriction enzymes are named according to the bacteria from which they originate. For
example, the restriction enzyme BamHI is named as follows
B represents the genus Bacillus
am represents the species amyloliquefaciens
H represents the strain
I means that it was the first endonuclease isolated from this strain
Methylases
Restriction endonucleases must be able to distinguish between foreign
DNA and the genetic material of their own cells; otherwise a bacterium’s
DNA would be in jeopardy of being cleaved by its own immune system.
Methylases are enzymes that modify the recognition site of a restricition
endonuclease by placing a methyl group on one of the bases in bacteria,
preventing the restriction endonuclease from cutting the DNA into
fragments. When foreign DNA is introduced into the bacterium, it is not
methylated, rendering it defenseless against the bacterium’s restriction
enzymes.
DNA Ligase
If two fragments of nucleic acids have been generated using the same restriction enzyme,
they will naturally be attracted to each other at their complementary sticky ends via
hydrogen bonding between complementary base pairs. DNA ligase is an enzyme used to
reform the phosphodiester linkage between the backbones of the double strands using a
condensation reaction (anabolic).
Plasmids
Recombinant DNA technology allows scientists to equip an organism with DNA
that is not normally found in that
organism. This new information, which
has been introduced into the host, can
then be used to cause the cell to produce
a specific protein. One of the first
successful transfers involved the human
gene for insulin. Insulin is a protein
produced by the pancreas that controls
blood glucose.
A segment of human DNA containing

the insulin gene is isolated in the lab.
A plasmid (small ring of genetic
material considered to be extra DNA) is
removed from an E. coli bacterial cell.
Both strands of DNA are cut using the
same restriction enzyme. Recognition
sites are present only once in the plasmid
and, therefore, the restriction enzyme can
only make one cut in the DNA resulting
in the circular plasmid becoming linear.
Since the foreign DNA has been open
using the same restriction enzyme that
was used on the plasmid both segments
of DNA will possess complementary
sticky ends that when placed together
will anneal. The sticky ends of the two
different DNA molecules are recombined
to form a single intact plasmid. DNA
ligase is then added to re-form
phosphodiester bonds between the
fragments. The recombinant plasmid is
then inserted into another E. coli host cell. A cell that is able to take up foreign
DNA, such as an E. coli cell, is called a competent cell. A plasmid that has been
designed to be a vehicle for transferring foreign genetic materials into a cell is
called a vector.
When the E. coli divides, the recombinant plasmid is replicated and is thus capable
of producing insulin. The bacterial cell becomes a “mini-factory” capable of
producing insulin for diabetics. The gene has been cloned because, as the plasmid
replicates, many copies of the recombinant DNA are produced, each of which
includes a copy of the insulin gene.
This cloning method is still used today as a means of amplifying larger DNA
sequences. For shorter fragments PCR is used.

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BIOTECHNOLOGICAL TOOLS AND TECHNIQUES

Restriction enzymes, are molecular

Restriction enzymes are examples of biological tools

A segment of human DNA containing

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