Chapter 17: Recombinant DNA Technology
17.1: Key Discoveries Led to the Development of Recombinant DNA Technology
Recombinant DNA is DNA with a new nucleotide sequence. Such DNA is formed by joining
fragments from two or more different sources.
These enzymes, known as restriction enzymes or restriction endonucleases, recognize and
cleave specific sequences about four to eight base pairs long (figure 17.2). Restriction
enzymes identify specific DNA sequences called recognition sites. Each restriction enzyme
has its own recognition site. Hundreds of different restriction enzymes have been purified and
are commercially available.
When EcoRI cleaves between the G and A residues, unpaired 5′-AATTC-3′ remains at the end of
They isolated the enzyme reverse transcriptase (RT) from retroviruses. These viruses have an
RNA genome that is copied into DNA prior to replication. The mechanism by which reverse
transcriptase accomplishes this is outlined in figure 17.5. Processed mRNA can be used as a
template for complementary DNA (cDNA) synthesis in vitro. The resulting cDNA can then be
cloned, without the need for RNA processing
Agarose and polyacrylamide gel electrophoresis is now routinely used to separate and visualize
An oligonucleotide (Greek oligo, few or scant) probe, is a fragment of ssDNA complementary to
the DNA of interest.
• Streptavidin-Biotin Binding and Biotechnology -
Egg white contains many proteins and glycoproteins with unique properties. One of the most
interesting, which binds tenaciously to biotin, was isolated in 1963. This glycoprotein, called
avidin due to its “avid” binding of biotin, was suggested to play an important role: making
egg white antimicrobial by “tying up” the biotin needed by many microorganisms. Avidin,
which functions best under alkaline conditions, has the highest known binding affinity
between a protein and a ligand. Several years later, scientists at Merck & Co., Inc.,
discovered a similar protein produced by the actinomycete Streptomyces avidini, which binds
biotin at a neutral pH and does not contain carbohydrates. These characteristics make this
protein, called streptavidin, an ideal binding agent for biotin, and it has been used in an
almost unlimited range of biotechnology applications. The streptavidin protein is joined to a
probe. When a sample is incubated with the biotinylated binder, the binder attaches to any
available target molecules. The presence and location of target molecules can be determined
by treating the sample with a streptavidin probe because the streptavidin binds to the biotin
on the biotinylated binder, and the probe is then visualized. This detection system is
employed in a wide variety of biotechnological applications, including use as a nonradioactive probe in hybridization studies and as a critical component in biosensors for a
wide range of environmental monitoring and clinical applications.
17.2: Polymerase Chain Reaction Amplifies Targeted DNA
polymerase chain reaction (PCR): An in vitro technique used to synthesize large quantities of
specific nucleotide sequences from small amounts of DNA. It employs oligonucleotide primers
complementary to specific sequences in the target gene and special heat-stable DNA
polymerases (e.g., Taq polymerase).
Quite simply, it enables the rapid synthesis of billions of copies of a specific DNA fragment
from a complex mixture of DNA molecules.
Suppose that one wishes to make large quantities of a particular gene or other DNA sequence, a
process known as gene or DNA amplification.
17.3: Cloning Vectors Are Needed to Create Recombinant DNA
Plasmids make excellent cloning vectors because they replicate autonomously (i.e.,
independently of the chromosome) and are easy to purify.
These plasmids are called shuttle vectors because they can be transferred, or “shuttle,” from one
host to another.
Alternatively, two different, unique sites may be cleaved and the DNA sequence between the two
sites replaced with cloned DNA. Plasmids used for cloning have been designed with many
restriction sites clustered in a single region called the mu