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CHAPTER
8
Recombinant DNA Technology
Chapter Summary
The Role of Recombinant DNA Technology in
Biotechnology (p. 237)
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 variety of practical purposes. This recombinant DNA technology has expanded the possibilities of biotechnology. Scientists who manipulate genomes have three main goals:
1. To eliminate undesirable phenotypic traits in humans, animals, plants, and microbes
2. To combine beneficial traits of two or more organisms to create valuable new organisms
3. To create organisms that synthesize products that humans need
The Tools of Recombinant DNA Technology (pp. 237–242)
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 effect desired changes
in the microbe’s phenotype. For example, researchers exposed the fungus Penicillium to mutagenic agents and then
selected strains that produce greater amounts of penicillin.
The Use of Reverse Transcriptase to Synthesize cDNA
Reverse transcriptase is an enzyme that transcribes DNA nucleotides from an RNA
template. Genetic researchers use it to make complementary DNA (cDNA), so called
because it is complementary to an RNA template. Since eukaryotic cDNA lacks noncoding sequences, scientists can
insert it into prokaryotic cells, making it possible for the prokaryotes to produce eukaryotic proteins such as human
growth factor, insulin, and blood-clotting factors.
Synthetic Nucleic Acids
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 keyboard. Scientists used synthetic nucleic acids to elucidate the genetic code, and now use them to create genes for specific proteins. They also use them to synthesize DNA
and RNA probes, which are nucleic acid molecules with a specific nucleotide sequence that have been labeled with
radioactive or fluorescent chemicals so that their locations can be detected. Probes help locate specific nucleic acid
sequences such as genes for particular polypeptides. Finally, synthetic nucleic acids can be used to make antisense
nucleic acid molecules that bind to and interfere with genes and mRNA molecules.
Restriction Enzymes
Restriction enzymes are enzymes used by bacterial cells to protect against phages by cutting phage DNA into nonfunctional pieces. Scientists use restriction enzymes to cut DNA at locations with specific and usually palindromic
nucleotide sequences called restriction sites. They then combine these bits of DNA with ligase to form recombinant
DNA molecules.
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 and a recognizable gene marker into a cell so that the cell will develop a new phenotype—for example, the ability to synthesize growth hormone. Vectors must be small enough to manipulate in the
laboratory, be able to survive inside cells, contain a recognizable genetic marker, and may provide the required
genetic elements to ensure expression.
Gene Libraries
A gene library is a collection of bacterial or phage clones, each of which carries a fragment (typically a single gene)
of an organism’s genome. Alternatively, a cDNA library may contain the full set of cDNA complementary to an organism’s mRNA. Genetic researchers create each of the clones in a gene library by using restriction enzymes to generate fragments of the DNA of interest and then ligase to synthesize recombinant vectors. They insert the vectors
into bacterial cells, which are then grown on culture media. Many gene libraries are now commercially available.
Techniques of Recombinant DNA Technology (pp. 242–246)
Mutagens, restriction enzymes, vectors, and other tools of recombinant DNA technology are used in a variety of
techniques to multiply, identify, manipulate, isolate, map, and sequence the nucleotides of genes.
Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)
The polymerase chain reaction (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 DNA molecules increases exponentially.
After 30 cycles in a few hours, PCR produces over a billion copies of the original DNA molecule.
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
Gel electrophoresis is a technique for separating molecules (including fragments of nucleic acids) by size, shape,
and electrical charge. The technique involves drawing DNA molecules, which have an overall negative charge,
through a semisolid gel by an electric current toward the positive electrode within an electrophoresis chamber.
Smaller DNA fragments move faster and farther than larger ones. In genetic engineering, scientists use the technique
to isolate fragments of DNA molecules that can then be inserted into vectors, multiplied by PCR, or preserved in a
gene library.
The Southern blot technique begins with the procedures of gel electrophoresis just
described, but allows researchers to stabilize specific DNA sequences and then localize them using DNA dyes or
probes. The Southern blot is used for genetic fingerprinting, diagnosis of infectious disease, and other purposes.
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. Single strands of fluorescently labeled DNA in a sample washed over an array adhere only to locations on the array where there are complementary DNA sequences. DNA microarrays can be used in a number of ways including monitoring gene expression,
diagnosing infection, and identifying organisms in an environmental sample.
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.
 Protoplast fusion is a process in which the cytoplasmic membranes of protoplasts encountering each other fuse to
form a recombinant cell.
 Injection is a process in which scientists use a gene gun to inject a cell with a tungsten or gold bead coated with
DNA. In microinjection, a glass micropipette is used.
Applications of Recombinant DNA Technology (pp. 246–253)
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. The nucleotide sequence
of the gene is then determined. The technique has been speeded up by an automated machine that distinguishes
among fluorescent dyes attached to each type of nucleotide base. When a sequence unique to an organism is known,
the technique known as FISH (fluorescent in site hybridization) can be used to locate the organism in samples (diagnostic or environmental) using a single strand sequence tagged with fluorescent molecules. Genomics is the sequencing, analysis, and comparison of genomes. Scientists have elucidated complete gene maps of numerous viral,
bacterial, and eukaryotic organisms. Another use for genomics is to relate DNA sequence data to protein function.
Environmental Studies
Recombinant techniques can be used to address many environmental questions. Southern blotting, FISH (fluorescent
in situ hybridization), and microarrays can identify the unique DNA sequences (signatures or DNA fingerprints). Investigations have revealed that huge numbers of microbes that have never been cultured in the laboratory are present
in environments as diverse as soil and the human mouth. These discoveries may lead to a better understanding of the
organisms’ impact on disease or the environment and the means to reduce undesirable impacts.
Pharmaceutical and Therapeutic Applications
Pharmaceutical and therapeutic applications of recombinant DNA technology include the
following:
 Protein synthesis. Scientists have inserted genes for insulin and other proteins into
bacteria and yeast cells so that the cells synthesize these proteins in vast quantities.
“Genetically engineered” proteins are safer and less expensive than proteins isolated from donated blood or from
animals.
 Vaccines. 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 polypeptides they produce, are injected into a human, the body’s immune system is exposed to and reacts against relatively harmless antigens instead of the potentially harmful pathogen.
 Genetic screening. Genetic mutations cause some diseases such as inherited forms of breast cancer and Huntington’s disease. In genetic screening, laboratory technicians use DNA microarrays to screen a patient’s blood or
other tissues for these genetic mutations before the patient shows any sign of the disease.
 DNA fingerprinting. Medical laboratory technicians and forensic investigators use gel electrophoresis and
Southern blotting for so-called genetic fingerprinting or DNA fingerprinting, to identify individuals or organisms
by their unique DNA sequences. The technique is used in paternity investigations, crime scene forensics, and diagnostic microbiology.
 Gene therapy. In gene therapy, missing or defective genes are replaced with normal genes, with a goal of curing
the genetic disease. For example, patients have been successfully treated for a form of severe combined immunodeficiency disease (SCID). Unfortunately, gene therapy has also caused patient deaths because some patients’
immune systems have reacted uncontrollably to the presence of the vectors.
 Medical diagnosis. Clinical microbiologists now use PCR, fluorescent genetic probes, and DNA microarrays in
diagnostic applications such as examining patient specimens for sequences unique to certain pathogens.
 Xenotransplants. Xenotransplants introduce animal cells, tissues, or organs into the
human body. In xenotransplants involving recombinant DNA technology, human genes are inserted into animals
to produce cells, tissues, or organs that are then introduced into the human body, reducing the likelihood of rejection.
Agricultural Applications
Recombinant DNA technology has been applied to the realm of agriculture to produce
transgenic organisms, recombinant plants and animals that have been altered for specific purposes by the addition
of genes from other organisms. Agricultural uses of recombinant DNA technology include advances in herbicide resistance, salt tolerance, freeze resistance, and pest and disease resistance, as well as improvements in nutritional value and yield.
The Ethics and Safety of Recombinant DNA Technology
(pp. 253)
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. Some opponents of recombinant DNA techniques argue that transgenic organisms could trigger allergies
or cause harmless organisms to become pathogenic, whereas others caution that the long-term effects of transgenic
manipulations are unknown, and that unforeseen problems arise from every new technology. Various agencies and
research organizations have conducted numerous research projects that found few indications of increased risk from
transgenic organisms produced for agricultural use. The same tools could be used to deliberately produce biological
weapons. In addition, emergent recombinant DNA technologies raise numerous other ethical issues, debating the
rights of governments, employers, or insurers to routinely screen people for certain diseases, the rights of individuals
to refuse either screening or treatment for genetic disease, and the rights of both institutions and individuals to privacy and confidentiality of genetic data. Our society will have to confront these and other ethical issues as the genomic
revolution continues.