Recombinant DNA Technology Has Revolutionized All Aspects of Biology

recombinant DNA technology, which has permitted biology to move from an exclusively analytical science to a
synthetic one. New combinations of unrelated genes can be constructed in the laboratory by applying recombinant DNA
techniques. These novel combinations can be cloned amplified manyfold by introducing them into suitable cells,
where they are replicated by the DNA-synthesizing machinery of the host. The inserted genes are often transcribed and
translated in their new setting. What is most striking is that the genetic endowment of the host can be permanently
altered in a designed way.
Restriction Enzymes and DNA Ligase Are Key Tools in Forming Recombinant
DNA Molecules
Let us begin by seeing how novel DNA molecules can be constructed in the laboratory. A DNA fragment of interest is
covalently joined to a DNA vector. The essential feature of a vector is that it can replicate autonomously in an
appropriate host. Plasmids (naturally occurring circles of DNA that act as accessory chromosomes in bacteria) and
bacteriophage l , a virus, are choice vectors for cloning in E. coli. The vector can be prepared for accepting a new DNA
fragment by cleaving it at a single specific site with a restriction enzyme. For example, the plasmid pSC101, a 9.9-kb
double-helical circular DNA molecule, is split at a unique site by the EcoRI restriction enzyme. The staggered cuts made
by this enzyme produce complementary single-stranded ends, which have specific affinity for each other and hence are
known as cohesive or sticky ends. Any DNA fragment can be inserted into this plasmid if it has the same cohesive ends.
Such a fragment can be prepared from a larger piece of DNA by using the same restriction enzyme as was used to open
the plasmid DNA
The single-stranded ends of the fragment are then complementary to those of the cut plasmid. The DNA fragment and
the cut plasmid can be annealed and then joined by DNA ligase, which catalyzes the formation of a phosphodiester bond
at a break in a DNA chain. DNA ligase requires a free 3 -hydroxyl group and a 5 -phosphoryl group. Furthermore, the
chains joined by ligase must be in a double helix. An energy source such as ATP or NAD+ is required for the joining
reaction,
This cohesive-end method for joining DNA molecules can be made general by using a short, chemically synthesized
DNA linker that can be cleaved by restriction enzymes. First, the linker is covalently joined to the ends of a DNA
fragment or vector. For example, the 5 ends of a decameric linker and a DNA molecule are phosphorylated by
polynucleotide kinase and then joined by the ligase from T4 phage . This ligase can form a covalent bond
between blunt-ended (flush-ended) double-helical DNA molecules. Cohesive ends are produced when these terminal
extensions are cut by an appropriate restriction enzyme. Thus, cohesive ends corresponding to a particular restriction
enzyme can be added to virtually any DNA molecule. We see here the fruits of combining enzymatic and synthetic chemical approaches in crafting new DNA molecules.