CHAPTER
8
Recombinant DNA Technology
Chapter Outline
The Role of Recombinant DNA Technology in Biotechnology (p. 238)
The Tools of Recombinant DNA Technology (pp. 238–242)
Mutagens
The Use of Reverse Transcriptase to Synthesize cDNA
Techniques of Recombinant DNA Technology (pp. 242–247)
Multiplying DNA In Vitro: The Polymerase Chain Reaction
Selecting a Clone of Recombinant Cells
Separating DNA Molecules: Gel Electrophoresis and the Southern Blot
DNA Microarrays
Inserting DNA into Cells
Applications of Recombinant DNA Technology (pp. 247–254)
Interactive Microbiology: Human Microbiome
Genetic Mapping
Microbial Community Studies
The Ethics and Safety of Recombinant DNA Technology (pp. 254–255)
Chapter Summary
The Role of Recombinant DNA Technology in Biotechnology
(p. 238)
Biotechnology is the use of microorganisms to make practical products, such as bread, wine,
paper, and antibiotics. Since the 1990s, scientists have become increasingly adept at
intentionally modifying the genomes of organisms, by natural and artificial processes, for a
Instructor’s Manual for Microbiology with Diseases by Body System, 5e
variety of practical purposes. This recombinant DNA technology has expanded the
possibilities of biotechnology. Scientists who manipulate genomes have three main goals:
The Tools of Recombinant DNA Technology (pp. 238–242)
Scientists use a variety of physical agents, naturally occurring enzymes, and synthetic molecules
to manipulate genes and genomes and create gene libraries.
Mutagens
Mutagens are chemical and physical agents used to create changes in a microbe’s genome to
The Use of Reverse Transcriptase to Synthesize cDNA
Transcription is the transmission of genetic information from DNA to RNA. Reverse
transcriptase is an enzyme that transcribes DNA nucleotides from an RNA template. Genetic
Synthetic Nucleic Acids
The enzymes of nucleic acid synthesis can be used to produce DNA and RNA in cell-free
solutions. Computer technology has allowed for the development of machines that synthesize
molecules of DNA and RNA with any nucleotide sequence entered onto the machine’s
Restriction Enzymes
Restriction enzymes (restriction endonucleases) are enzymes that cut DNA at locations with
specific and usually palindromic nucleotide sequences called restriction sites. They are used by
bacterial cells to protect against phages by cutting phage DNA into nonfunctional pieces.
Chapter 8 Recombinant DNA Technology
Vectors
In recombinant DNA technology, a vector is a small DNA molecule (such as a viral genome,
transposon, or plasmid) that carries a particular gene into a cell so that the cell will develop a
new phenotype. Vectors must be small enough to manipulate in the laboratory, be able to
Gene Libraries
A gene library is a collection of bacterial or phage clones, each of which carries a fragment
Techniques of Recombinant DNA Technology (pp. 242–247)
Multiplying DNA In Vitro: PCR
PCR is a technique by which scientists produce a large number of identical molecules of DNA
in vitro. PCR is a repetitive process that alternately separates and replicates the two strands of
DNA. Each cycle consists of three steps: denaturation (separation of the two strands of DNA),
priming (addition of DNA primer mixture followed by cooling), and extension (heating to
increase the rate of replication by polymerase). With each repetition of the cycle, the number of
Instructor’s Manual for Microbiology with Diseases by Body System, 5e
Selecting a Clone of Recombinant Cells
Researchers use probes to find clones containing the DNA of interest. They then isolate and
culture cells that have the desired gene as indicated by binding of the labeled probe.
Separating DNA Molecules: Gel Electrophoresis and the Southern Blot
Electrophoresis separates molecules (including fragments of nucleic acids) by size, shape, and
electrical charge. Gel electrophoresis is a technique for separating DNA molecules which have
an overall negative charge by drawing them through a semisolid gel by an electric current
DNA Microarrays
DNA microarrays consist of molecules of single-stranded DNA that have been immobilized on
glass slides, silicon chips, or nylon membranes. A single array may contain thousands of
different sequences, either all from one organism or from numerous species. Single strands of
Inserting DNA into Cells
In addition to using vectors and the natural methods of transformation of competent cells,
transduction, and conjugation, scientists have developed several artificial methods to introduce
DNA into cells, including the following:
Electroporation uses an electrical current to puncture microscopic holes through a cell’s
cytoplasmic membrane so that DNA can enter. Thick cell walls are first removed
enzymatically to form protoplasts.
Chapter 8 Recombinant DNA Technology
Applications of Recombinant DNA Technology (pp. 247–254)
Interactive Microbiology: Human Microbiome
Recombinant DNA technology has a wide range of applications.
Genetic Mapping
Genetic mapping involves locating genes on a nucleic acid molecule, frequently by using DNA
probes to identify restriction fragments (restriction fragmentation) or clones containing the gene
of interest. Scientists have elucidated complete gene maps of numerous viruses, bacteria, and
eukaryotic organisms. When a gene unique to an organism is identified, the technique known as
fluorescent in situ hybridization (FISH) can be used to locate the organism in samples
(diagnostic or environmental) using a single-strand sequence tagged with fluorescent molecules.
(NGS) methods like four-color reversible termination sequencing are now used. In this method
each of the four types of nucleotides is modified by the addition of a different color fluorescent
dye as well as a terminating group. Single-strand DNA fragments (produced by PCR) are
attached to glass slides and DNA synthesis begins. When a modified nucleotide is incorporated
in a growing DNA strand, synthesis is halted. Light detectors record the colors emitted when a
Microbial Community Studies
Investigations estimate that 99% of microbes have never been cultured in the laboratory. NGS
can be used to detect unique DNA fingerprints or even genetic sequences of such organisms.
These discoveries may lead to a better understanding of the organisms’ impact on health and
Instructor’s Manual for Microbiology with Diseases by Body System, 5e
Pharmaceutical and Therapeutic Applications
Pharmaceutical and therapeutic applications of recombinant DNA technology include the
following:
Protein Synthesis. Scientists have inserted genes for insulin, interferon, and other proteins
Vaccines. Vaccines contain the antigens of a pathogen that stimulate an immune response.
Scientists synthesize subunit vaccines—which use a portion of a pathogen rather than the
pathogen itself—by introducing genes for a pathogen’s polypeptides into vectors. When the
vectors, or the proteins they produce, are injected into a human, the body’s immune system is
Gene Therapy. In gene therapy, missing or defective genes are replaced with normal genes,
with a goal of curing the genetic disease. Gene therapy can be difficult because therapeutic
levels of a functioning gene can be hard to achieve, nonetheless patients have been
successfully treated for a form of severe combined immunodeficiency disease (SCID) and a
type of blindness.
Medical Diagnosis. Clinical microbiologists now use PCR, fluorescent genetic probes, and
Agricultural Applications
Recombinant DNA technology has been applied to the realm of agriculture to produce
transgenic organisms (genetically modified organisms, GMOs), recombinant plants and
The Ethics and Safety of Recombinant DNA Technology
(pp. 254–255)
Among the ethical and safety issues surrounding recombinant DNA technology are concerns
over the accidental release of altered organisms into the environment, the ethics of altering
animals for human use, and the potential for creating genetically modified biological weapons.