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16
Cleaving and Rejoining DNA
• Recombinant DNA technology is the
manipulation and combination of DNA molecules
from different sources.
• Recombinant DNA technology uses the
techniques of sequencing, rejoining, amplifying,
and locating DNA fragments, making use of
complementary base pairing.
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Cleaving and Rejoining DNA
• Bacteria defend themselves against invasion by
viruses by producing restriction enzymes which
catalyze the cleavage of DNA into small fragments.
• The enzymes cut the bonds between the 3
hydroxyl of one nucleotide, and the 5 phosphate of
the next.
• There are many such enzymes, each of which
recognizes and cuts a specific sequence of bases,
called a recognition sequence or restriction site
(4 to 6 base pairs long).
Figure 16.1 Bacteria Fight Invading Viruses with Restriction Enzymes
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Cleaving and Rejoining DNA
• Host DNA is not damaged due to methylation of
certain bases at the restriction sites; this is
performed by enzymes called methylases.
• The enzyme EcoRI cuts DNA with the following
paired sequence:
 5 ... GAATTC ... 3
 3... CTTAAG ... 5
• Notice that the sequence is palindromic: It reads
the same in the 5-to-3 direction on both strands.
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Cleaving and Rejoining DNA
• Using EcoRI on a long stretch of DNA would
create fragments with an average length of 4,098
bases.
• Using EcoRI to cut up small viral genomes may
result in only a few fragments.
• For a eukaryotic genome with tens of millions of
base pairs, the number of fragments will be very
large.
• Hundreds of restriction enzymes have been
purified from various organisms, and these
enzymes serve as “knives” for genetic surgery.
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Cleaving and Rejoining DNA
• The fragments of DNA can be separated using gel
electrophoresis. Because of its phosphate groups,
DNA is negatively charged at neutral pH.
• When DNA is placed in a semisolid gel and an
electric field is applied, the DNA molecules
migrate toward the positive pole.
• Smaller molecules can migrate more quickly
through the porous gel than larger ones.
• After a fixed time, the separated molecules can
then be stained with a fluorescent dye and
examined under ultraviolet light.
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 3)
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Cleaving and Rejoining DNA
• Electrophoresis gives two types of information:
 Size of the DNA fragments can be determined
by comparison to DNA fragments of known
size added to the gel as a reference.
 A specific DNA sequence can be determined
by using a complementary labeled singlestranded DNA probe.
• The specific fragment can be cut out as a lump of
gel and removed by diffusion into a small volume
of water.
Figure 16.3 Analyzing DNA Fragments
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Cleaving and Rejoining DNA
• Some restriction enzymes cut DNA strands and
leave staggered ends of single-stranded DNA, or
“sticky” ends, that attract complementary
sequences.
• If two different DNAs are cut so each has sticky
ends, fragments with complementary sticky ends
can be recombined and sealed with the enzyme
DNA ligase.
• These simple techniques, which give scientists the
power to manipulate genetic material, have
revolutionized biological science in the past 30
years.
Figure 16.4 Cutting and Splicing DNA
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Getting New Genes into Cells
• The goal of recombinant DNA work is to produce
many copies (clones) of a particular gene.
• To make protein, the genes must be introduced,
or transfected, into a host cell.
• The host cells or organisms, referred to as
transgenic, are transfected with DNA under
special conditions.
• The cells that get the DNA are distinguished from
those that do not by means of genetic markers,
called reporter genes.
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Getting New Genes into Cells
• Bacteria have been useful as hosts for
recombinant DNA.
 Bacteria are easy to manipulate, and they
grow and divide quickly.
 They have genetic markers that make it easy
to select or screen for insertion.
 They have been intensely studied and much of
their molecular biology is known.
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Getting New Genes into Cells
• Bacteria have some disadvantages as well.
 Bacteria lack splicing machinery to excise
introns.
 Protein modifications, such as glycosylation
and phosphorylation, fail to occur as they
would in a eukaryotic cell.
 In some applications, the expression of the
new gene in a eukaryote (the creation of a
transgenic organism) is the desired outcome.
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Getting New Genes into Cells
• Saccharomyces, baker’s and brewer’s yeast, are
commonly used eukaryotic hosts for recombinant
DNA studies.
• In comparison to many other eukaryotic cells,
yeasts divide quickly, they are easy to grow, and
have relatively small genomes (about 20 million
base pairs).
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Getting New Genes into Cells
• Plants are also used as hosts if the goal is to
make a transgenic plant.
• It is relatively easy to regenerate an entire plant
from differentiated plant cells because of plant cell
totipotency.
• The transgenic plant can then reproduce naturally
in the field and will carry and express the gene on
the recombinant DNA.
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Getting New Genes into Cells
• New DNA can be introduced into the cell’s
genome by integration into a chromosome of the
host cell.
• If the new DNA is to be replicated, it must become
part of a segment of DNA that contains an origin
of replication called a replicon, or replication
unit.
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Getting New Genes into Cells
• New DNA can be incorporated into the host cell by a
vector, which should have four characteristics:
 The ability to replicate independently in the host
cell
 A recognition sequence for a restriction enzyme,
permitting it to form recombinant DNA
 A reporter gene that will announce its presence in
the host cell
 A small size in comparison to host chromosomes
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Getting New Genes into Cells
• Plasmids are ideal vectors for the introduction of
recombinant DNA into bacteria.
• A plasmid is small and can divide separately from
the host’s chromosome.
• They often have just one restriction site, if any, for a
given restriction enzyme.
• Cutting the plasmid at one site makes it a linear
molecule with sticky ends.
• If another DNA is cut with the same enzyme, it is
possible to insert the DNA into the plasmid.
• Plasmids often contain antibiotic resistance genes,
which serve as genetic markers.
Figure 16.5 (a) Vectors for Carrying DNA into Cells
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Getting New Genes into Cells
• Only about 10,000 base pairs can be inserted into
plasmid DNA, so for most eukaryotic genes a vector
that accommodates larger DNA inserts is needed.
• For inserting larger DNA sequences, viruses are
often used as vectors.
• If the genes that cause death and lysis in E. coli are
eliminated, the bacteriophage  can still infect the
host and inject its DNA.
• The deleted 20,000 base pairs can be replaced by
DNA from another organism, creating recombinant
viral DNA.
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Getting New Genes into Cells
• Bacterial plasmids are not good vectors for yeast
hosts because prokaryotic and eukaryotic DNA
sequences use different origins of replication.
• A yeast artificial chromosome, or YAC, has
been made that has a yeast origin of replication, a
centromere sequence, and telomeres, making it a
true eukaryotic chromosome.
• YACs have been engineered to include
specialized single restriction sites and selectable
markers.
• YACs can accommodate up to 1.5 million base
pairs of inserted DNA.
Figure 16.5 (b) Vectors for Carrying DNA into Cells
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Getting New Genes into Cells
• Plasmid vectors for plants include a plasmid found
in the Agrobacterium tumefaciens bacterium,
which causes the tumor-producing disease, crown
gall, in plants.
• Part of the tumor-inducing (Ti) plasmid of A.
tumefaciens is T DNA, a transposon, which
inserts copies of itself into the host chromosomes.
• If T DNA is replaced with the new DNA, the
plasmid no longer produces tumors, but the
transposon still can be inserted into the host cell’s
chromosomes.
• The plant cells containing the new DNA can be
used to generate transgenic plants.
Figure 16.5 (c) Vectors for Carrying DNA into Cells
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Getting New Genes into Cells
• When a population of host cells is treated to
introduce DNA, just a fraction actually incorporate
and express it.
• In addition, only a few vectors that move into cells
actually contain the new DNA sequence.
• Therefore, a method for selecting for transfected
cells and screening for inserts is needed.
• A commonly used approach to this problem is
illustrated using E. coli as hosts, and a plasmid
vector with genes for resistance to two antibiotics.
Figure 16.6 Marking Recombinant DNA by Inactivating a Gene
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Getting New Genes into Cells
• Other methods have since been developed for
screening.
• The gene for luciferase, the enzyme that makes
fireflies glow in the dark, has been used as a
reporter gene.
• Green fluorescent protein, which is the product of a
jellyfish gene, glows without any required substrate.
• Cells with this gene in the plasmid grow on
ampicillin and glow when exposed to ultraviolet
light.
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Sources of Genes for Cloning
• Gene libraries contain fragments of DNA from an
organism’s genome.
• Restriction enzymes are used to break
chromosomes into fragments, which are inserted
into vectors and taken up by host cells.
Figure 16.7 Construction of a Gene Library
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Sources of Genes for Cloning
• Using plasmids for insertion of DNA, about one
million separate fragments are required for the
human genome library.
• Phage , which carries four times as much DNA
as a plasmid, is used to hold these random
fragments.
• It takes about 250,000 different phage to ensure a
copy of every sequence.
• This number seems large, but just one growth
plate can hold as many as 80,000 phage colonies.
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Sources of Genes for Cloning
• A smaller DNA library can be made from
complementary DNA (cDNA).
• Oligo dT primer is added to mRNA tissue where it
hybridizes with the poly A tail of the mRNA
molecule.
• Reverse transcriptase, an enzyme that uses an
RNA template to synthesize a DNA–RNA hybrid, is
then added.
• The resulting DNA is complementary to the RNA
and is called cDNA. DNA polymerase can be used
to synthesize a DNA strand that is complementary
to the cDNA.
Figure 16.8 Synthesizing Complementary DNA
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Sources of Genes for Cloning
• If the amino acid sequence of a protein is known,
it is possible to synthesize a DNA that can code
for the protein.
• Using the knowledge of the genetic code and
known amino acid sequences, the most likely
base sequence for the gene may be found.
• Often sequences are added to this sequence to
promote expression of the protein.
• Human insulin has been manufactured using this
approach.
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Sources of Genes for Cloning
• With synthetic DNA, mutations can be created
and studied.
• Additions, deletions, and base-pair substitutions
can be manipulated and tracked.
• The functional importance of certain amino acid
sequences can be studied.
• The signals that mark proteins for passage
through the ER membrane were discovered by
site-directed mutagenesis.
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Some Additional Tools for DNA Manipulation
• Homologous recombination is used to study the
role of a gene at the level of the organism.
• In a knockout experiment, a gene inside a cell is
replaced with an inactivated gene to determine the
inactivated gene’s effect.
• This technique is important in determining the roles
of genes during development.
Figure 16.9 Making a Knockout Mouse (Part 1)
Figure 16.9 Making a Knockout Mouse (Part 2)
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Some Additional Tools for DNA Manipulation
• The emerging science of genomics has to
contend with two difficulties:
 The large number of genes in eukaryotic
genomes
 The distinctive pattern of gene expression in
different tissues at different times
• To find these patterns, DNA sequences have to be
arranged in an array on some solid support.
• DNA chip technology provides these large arrays
of sequences for hybridization.
Figure 16.10 DNA on a Chip
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Some Additional Tools for DNA Manipulation
• Analysis of cellular mRNA using DNA chips:
 In a process called RT-PCR, cellular mRNA is
isolated and incubated with reverse
transcriptase (RT) to make complementary
DNA (cDNA). The cDNA is amplified by PCR
prior to hybridization.
 The amplified cDNA is coupled to a fluorescent
dye and then hybridized to the chip.
 A scanner detects glowing spots on the array.
The combinations of these spots differ with
different types of cells or different physiological
states.
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Some Additional Tools for DNA Manipulation
• DNA chip technology can be used to detect
genetic variants and to diagnose human genetic
diseases.
• Instead of sequencing the entire gene, it is
possible to make a chip with 20-nucleotide
fragments including every possible mutant
sequence.
• Hybridizing that sequence with a person’s DNA
may reveal whether any of the DNA hybridized to
a mutant sequence on the chip.
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Some Additional Tools for DNA Manipulation
• Base-pairing rules can also be used to stop
mRNA translation.
• Antisense RNA is complementary to a sequence
of mRNA.
• The antisense RNA forms a double-stranded
hybrid with an mRNA, which inhibits translation.
• These hybrids are broken down rapidly in the
cytoplasm, so translation does not occur.
• In the laboratory, antisense RNA can be made
and added to cells to block translation.
Figure 16.11 Using Antisense RNA and RNAi to Block Translation of mRNA
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Some Additional Tools for DNA Manipulation
• A related technique uses interference RNA (RNAi)
which inhibits mRNA translation in the inactive X
chromosome of mammals.
• Scientists can synthesize a small interfering RNA
(siRNA) to inhibit translation of any known gene.
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Some Additional Tools for DNA Manipulation
• The two-hybrid system allows scientists to test
for protein interactions within a living cell.
• A two-hybrid system uses a transcription factor
that activates the transcription of an easily
detectable reporter gene.
• This transcription factor has two domains: one
that binds to DNA at the promoter, and another
that binds to the transcription complex to activate
transcription.
• An example is the yeast two-hybrid system.
Figure 16.12 The Two-Hybrid System
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Biotechnology: Applications of DNA Manipulation
• Biotechnology is the use of microbial, plant, and
animal cells to produce materials—such as foods,
medicines, and chemicals—that are useful to
people.
• The use of yeast to create beer and wine and
bacterial cultures to make yogurt and cheese are
examples of centuries-old biotechnology.
• Gene cloning techniques of modern molecular
biology have vastly increased the number of these
products beyond those that are naturally made by
microbes.
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Biotechnology: Applications of DNA Manipulation
• Expression vectors are typical vectors, but they
also have extra sequences needed for the foreign
gene to be expressed in the host cell.
• An expression vector might have an inducible
promoter, which can be stimulated into expression
by responding to a specific signal such as a
hormone.
• A tissue-specific promoter is expressed only in a
certain tissue at a certain time.
• Targeting sequences are sometimes added to
direct the protein product to an appropriate
destination.
Figure 16.13 An Expression Vector Allows a Foreign Gene to Be Expressed in a Host Cell
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Biotechnology: Applications of DNA Manipulation
• Many medical products have been made using
recombinant DNA technology.
• For example, tissue plasminogen activator (TPA),
is currently being produced in E. coli by
recombinant DNA techniques.
• TPA is an enzyme that converts blood
plasminogen into plasmin, a protein that dissolves
clots.
• Recombinant DNA technology has made it
possible to produce the naturally occurring protein
in quantities large enough to be medically useful.
Figure 16.14 Tissue Plasminogen Activator: From Protein to Gene to Drug
Table 16.1 Some Medically Useful Products of Biotechnology
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Biotechnology: Applications of DNA Manipulation
• Selective breeding has been used for centuries to
improve plant and animal species to meet human
needs.
• Molecular biology is accelerating progress in these
applications.
• There are three major advantages over traditional
techniques:
 Specific genes can be affected.
 Genes can be introduced from other organisms.
 Plants can be regenerated much more quickly
by cloning than by traditional breeding.
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Biotechnology: Applications of DNA Manipulation
• Insecticides tend to be nonspecific, killing both
pest and beneficial insects. They can also be
blown or washed away to contaminate and pollute
non-target sites.
• Bacillus thuringiensis bacteria produce a protein
toxin that kills insect larvae pests and is 80,000
times more toxic than the typical chemical
insecticide.
• Transgenic tomato, corn, potato, and cotton plants
have been made that produce a toxin from B.
thuringiensis.
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Biotechnology: Applications of DNA Manipulation
• The process of producing pharmaceuticals using
agriculture is nicknamed “pharming.”
• Transgenic sheep are being used to produce
human a-1-antitrypsin (a-1-AT) in their milk; this
protein inhibits the enzyme elastase, which
breaks down connective tissue in the lungs.
Treatment with a-1-AT alleviates symptoms in
people suffering from emphysema.
• Other products of “pharming” include blood
clotting factors and antibodies for treating colon
cancer.
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Biotechnology: Applications of DNA Manipulation
• Crops that are resistant to herbicides:
 Glyphosate (“Roundup”) is a broad-spectrum
herbicide that inhibits an enzyme system in
chloroplasts that is involved in the synthesis of
amino acids.
 A bacterial gene, which confers resistance to
glyphosate, is inserted into useful food crops
(corn, cotton, soybeans) to protect them from
the herbicide, which otherwise would kill them
along with the weeds.
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Biotechnology: Applications of DNA Manipulation
• Grains with improved nutritional characteristics:
 Genes from bacteria and daffodil plants are
transferred to rice using the vector
Agrobacterium tumefaciens.
 Now a genetically modified strain of rice
produces -carotene, a molecule that is
converted to vitamin A in animals.
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Biotechnology: Applications of DNA Manipulation
• Crops that adapt to the environment:
 A gene was recently discovered in the thale
cress (Arabidopsis thaliana) that allows it to
thrive in salty soils.
 When this gene is added to tomato plants,
they can grow in soils four times as salty as
the normal lethal level.
 This finding raises the prospect of growing
useful crops on previously unproductive soils
with high salt concentration.
 Biotechnology may allow us to adapt plants to
different environments.
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Biotechnology: Applications of DNA Manipulation
• There is public concern about biotechnology:
 Genetically modified E. coli might share their
genes with the E. coli bacteria that live
normally in the human intestines.
 Researchers now take precautions against
this. For example, the strains of E. coli used in
the lab have a number of mutations that make
their survival in the human intestine
impossible.
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Biotechnology: Applications of DNA Manipulation
• There are concerns that genetic manipulation
interferes with nature, that genetically altered
foods are unsafe, and that genetically altered
plants might allow transgenes to escape to other
species and thus threaten the environment.
• Regarding safety for human consumption,
advocates of genetic engineering note that
typically only single genes specific for plant
function are added.
• As plant biotechnology moves from adding genes
to improve plant growth to adding genes that
affect human nutrition, such concerns will become
more pressing.
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Biotechnology: Applications of DNA Manipulation
• The risks to the environment are more difficult to
assess.
• Transgenic plants undergo extensive field testing
before they are approved for use, but the
complexity of the biological world makes it
impossible to predict all potential environmental
effects of transgenic organisms.
• Because of the potential benefits of agricultural
biotechnology, most scientists believe we should
proceed, but with caution.
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Biotechnology: Applications of DNA Manipulation
• With the exception of identical twins, each human
being is genetically distinct from all other human
beings.
• Characterization of an individual by DNA base
sequences is called DNA fingerprinting.
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Biotechnology: Applications of DNA Manipulation
• Scientists look for DNA sequences that are highly
polymorphic.
• Sequences called VNTRs (variable number of
tandem repeats) are easily detectable if they are
between two restriction enzyme recognition sites.
• Different individuals have different numbers of
repeats. Each gets two sequences of repeats, one
from the mother and one from the father.
• Using PCR and gel electrophoresis, patterns for
each individual can be determined.
Figure 16.17 DNA Fingerprinting
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Biotechnology: Applications of DNA Manipulation
• The many applications of DNA fingerprinting
include forensics and cases of contested
paternity.
• DNA from a single cell is sufficient to determine
the DNA fingerprint because PCR can amplify a
tiny amount of DNA in a few hours.
• PCR is used in diagnosing infections in which the
infectious agent is present in small amounts.
• Genetic diseases such as sickle-cell anemia are
now diagnosable before they manifest
themselves.