Applications of Biotechnology: Industry

Applications of Biotechnology: Test tube Meat

Applications of Biotechnology: Forensics
Applications of Biotechnology: Medicine
Applications of Biotechnology: Personalized
Medicine
Applications of Biotechnology: Biology
 DNA Technology

Genetic Engineering (Recombinant DNA Technology)

DNA Sequencing, PCR, and DNA finger printing

Human Genome Project and Gene Therapy

Dolly and Golden Rice

Tools used in rDNA Technology
1. Enzymes
- cut and join DNA fragments from
different organisms

2. Vectors
- carry foreign DNA fragments in to
host cells

3. Hosts
- cells allow to propagate rDNA
Bacteria Fight Invading Viruses
with Restriction Enzymes
Restriction Endonucleases
Source : Bacteria
Types
Recognition site Restriction site

Type I
Recognition & Restriction site

Type II

http://rebase.neb.com/rebase/rebase.html enzyme
recognition seq., genome information, cut patterns, etc
Restriction Endonucleases
Arthrobacter
luteus Blunt end

Haemophilus Dyad /
aegyptius twofold
rotational
Bacillus symmetry
amyloliquefacies (palindromic)

Haemophilus
influenzae
Sticky /
Staggered /
Escherichia Cohesive
coli End
 Plasmid

Bacteriophage
Vectors for Carrying DNA into Cells
Vectors for Carrying DNA into Cells
Construction of a Gene Library
 DIABETES

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Pharmaceutical Applications: Humulin

Herbert Boyer and Stanley Cohen

Pharmaceutical Applications: Humulin

Herbert Boyer Robert Swanson

 Recombinant DNA Technology
Clone of cells
containing the
Some uses gene of interest Some uses
of genes of proteins

The gene
Genes may and protein Harvested
be inserted of interest proteins may
Genes for into other are isolated be used Protein for
cleaning up organisms. from the directly “stone-washing”
toxic waste bacteria. jeans

Protein for
Gene for pest dissolving
resistance clots
Recombinant DNA (rDNA) -
Genetically engineered
DNA made by
recombining fragments
of DNA from different
organisms

DNA that has been altered by joining

genetic material from two
different sources.
It usually involves putting a gene
from one organism into the
genome of a different organism
Enzymes
Restriction Endonucleases
enzymes cleave DNA
at specific sites by
breaking
phosphodiester
bonds that link
adjacent nucleotides

DNA ligase
enzymes catalyze the formation of a
phosphodiester bond between adjacent
phosphate & hydroxyl groups of DNA
nucleotides
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).
 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.
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

 Visualizing DNA fragments
• 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.
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.
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.
 Genetic Engineering
• Consider a typical genetic engineering
challenge:
• A genetic engineer at a pharmaceutical
company identifies a gene of interest that
codes for a valuable protein, such as a
potential new drug.
• The biologist wants to manufacture the
protein on a large scale.

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 Pharmaceutical Applications
• Humulin is human insulin produced by genetically
modified bacteria.
• In humans, insulin is a protein normally made by the
pancreas.
• Because human insulin is not readily available,
diabetes was historically treated using insulin from
cows and pigs.
• In 1978, scientists working at a biotechnology
company chemically synthesized DNA fragments
and linked them to form the two genes that code for
the two polypeptides that make up human insulin.

© 2016 Pearson Education, Inc.

 Pharmaceutical Applications
• DNA technology is used to produce medically
valuable molecules, including
• human growth hormone (HGH),
• the hormone erythropoietin (EPO), which stimulates
production of red blood cells, and
• vaccines, a harmless variant or derivative of a
disease-causing microbe—such as a bacterium or
virus—that is used to prevent an infectious disease.
• Genetically modified whole animals are also used to
produce drugs.

© 2016 Pearson Education, Inc.

 DNA Technology

Genetic Engineering (Recombinant DNA Technology)

DNA Sequencing, PCR, and DNA finger printing

Human Genome Project and Gene Therapy

Dolly and Golden Rice

DNA Sequencing
 DNA Sequencing
• A specially engineered DNA replication reaction.
– The DNA of interest is the template.
– Uses DNA polymerase
– Uses a primer (gives DNA polymerase a place to start)
– Uses regular nucleotides (A,T,G,C)
– Uses special fluorescently-labeled dideoxynucleotides
• When these are added to the growing chain, replication will stop.
• The fragment will be fluorescently labeled as well.
– Fragments are separated by electrophoresis, and the sequence can
be read.
 Crime scene Suspect 1 Suspect 2
1 DNA isolated

2 DNA amplified

3 DNA compared
The Polymerase Chain Reaction (PCR)

Initial
DNA
segment

1 2 4 8
Number of DNA molecules
PCR
 Variable Number Tandem
Repeats (VNTR)
• Variable number tandem
repeats are regions of DNA
that are repetitive sequences.
• Each person has a slightly
different number of repeats.
• Therefore, if these regions
are cut with restriction
enzymes, each person will
have a different set of
fragments.
DNA Fingerprinting
in a Paternity Case
 DNA Fingerprinting
• Uniquely identifies individuals on the basis of DNA fragment
lengths.
– Fragments are generated by restriction enzymes that cut
DNA at specific sites.
– Each individual’s DNA is different enough that these
enzymes will generate different lengths of fragments in two
different individuals.
 DNA Fingerprinting Technique

• DNA is obtained from two sources:

– Blood at a crime scene
– Hair from the crime suspect
• Polymerase chain reaction is used to make many copies of
a VNTR region.
• Restriction enzymes are used to cut the VNTRs into
fragments.
• The fragments are separated by electrophoresis.
• The patterns of the two DNA samples are compared.
 DNA Fingerprinting
Applications
• DNA fingerprinting can identify if two
samples of DNA came from the same
person.
• Used in the prosecution of crime suspects
• Used in paternity cases
 DNA Profiling Techniques
The Polymerase Chain Reaction (PCR)
• The polymerase chain reaction (PCR)
• is a technique by which a specific segment of DNA can be amplified:
targeted and copied quickly and precisely, and permits a scientist to
obtain enough DNA from even minute amounts of blood or other
tissue to allow a DNA profile to be constructed.

• In principle, PCR is simple.

• A DNA sample is mixed with nucleotides, the DNA replication
enzyme DNA polymerase, and a few other ingredients.
• The solution is then exposed to cycles of heating (to separate the
DNA strands) and cooling (to allow double-stranded DNA to re-
form).
• During these cycles, specific regions of each molecule of DNA are
replicated, doubling the amount of that DNA.
• The result of this chain reaction is an exponentially growing
population of identical DNA molecules.
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 The Polymerase Chain Reaction (PCR)

• A DNA molecule within a starting sample is likely to be very long. But,

most often, only a very small target region of that large DNA molecule
needs to be amplified.

• The key to amplifying one particular segment of DNA and no others is

the use of primers, short (usually 15–20 nucleotides long), chemically
synthesized single-stranded DNA molecules.

• The primers bind to sequences that flank the target sequence, marking
the start and end points for the segment of DNA to be amplified.

• In addition to forensic applications, PCR can be used in the treatment

and diagnosis of disease. PCR can be used to
• amplify, and thus detect, HIV in blood or tissue samples and
• diagnose hundreds of human genetic disorders by being used with
primers that target the genes associated with these disorders.

•
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 DNA Technology

Genetic Engineering (Recombinant DNA Technology)

DNA Sequencing, PCR, and DNA finger printing

Human Genome Project and Gene Therapy

Dolly and Golden Rice

The Human Genome Project
Chromosome

Chop up with
restriction enzyme

DNA fragments

Sequence fragments

Align fragments

Reassemble
full sequence
Genes Known to be on Human Chromosome
21
The Human Genome
 The Human Genome
• Begun in 1990, the Human Genome Project was a massive scientific
endeavor to
• determine the nucleotide sequence of all the DNA in the human
genome and
• identify the location and sequence of every gene.

• At the completion of the project,

• more than 99% of the genome had been determined to 99.999%
accuracy, about 3 billion nucleotide pairs were identified, about
21,000 genes were found, and about 98% of the human DNA was
identified as noncoding.

• As of today, the complete genomes of many individuals have been

completed.

• Whereas sequencing the first human genome took 13 years and cost
$100 million, we are rapidly approaching the day when an individual’s
genome can be sequenced in a matter of hours for less than $1,000.
 The Human Genome Project

• A massive collaborative effort to determine the

sequence of DNA in all human chromosomes.
• Started with physical maps of chromosomes
• Determines the location of specific markers on the
chromosomes

• DNA sequencing determined the exact nucleotide

sequence between each marker.
 Human Genome Project Applications

• Disease diagnosis

• Discovery of new family of proteins

• Better understanding of basic biology

• The human genome project found that there are far

fewer genes in the human genome than previously
predicted.
• Each gene can be alternatively spliced to code for several
proteins.
Other Genomes
• The genomes of other organisms have also been
sequenced.
 Genome-Mapping Techniques

• The DNA sequences determined by many research groups

in the United States are deposited in GenBank, a database
that is available to anyone via the Internet.

• You can browse it yourself at the website for the National

Center for Biotechnology Information:
www.ncbi.nlm.nih.gov.

• The database is constantly updated, and the amount of data

it contains doubles every 18 months.

• Bioinformatics has revolutionized evolutionary biology by

opening a vast new reservoir of data that can test
evolutionary hypotheses.

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Human Gene Therapy
 Human Gene Therapy

Normal
human gene

1 An RNA version is
inserted into a virus

RNA genome of virus

Inserted human RNA
Healthy
person
 Human Gene Therapy
2 Bone marrow
cells are infected
3 Viral DNA inserts into
the cell’s chromosome
Human chromosome
Bone marrow
cell from the patient

Bone of person
with disease
4 The engineered Bone marrow
cells are injected
 Human Gene Therapy

• Human gene therapy is intended to treat disease

by introducing genes into an afflicted person.
• In cases where a single defective gene causes a
disorder, the mutant version of a gene may be
replaced or supplemented with the normal allele.
• This could potentially correct a genetic disorder,
perhaps permanently.
• In other cases, genes are inserted and expressed
only long enough to treat a medical problem.

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 Human Gene Therapy
• The promise of gene therapy thus far exceeds actual results, but there have
been some successes.

• In 2009, an international research team conducted a trial that focused on a form

of progressive blindness linked to a defect in a gene responsible for producing
light-detecting pigments in the eye.
• The researchers found that a single injection of a virus carrying the normal
gene into one eye of affected children improved vision in that eye,
sometimes enough to allow normal functioning, without significant side
effects. The other eye was left untreated as a control.

• From 2000 to 2011, gene therapy also cured 22 children with severe combined
immunodeficiency (SCID), a fatal inherited disease caused by a defective gene
that prevents development of the immune system, requiring patients to remain
isolated within protective ―bubbles.‖

• The treatment cured the patients of SCID, but there have been some serious
side effects:
• Four of the treated patients developed leukemia, and one died after the
inserted gene activated an oncogene, creating cancerous blood cells.

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Prevalence of Vitamin A Deficiency

https://commons.wikimedia.org/wiki/File:Vitamin_A_deficiency.PNG
 Applications of rDNA technology

Dr.Ingo Potrykus
Golden rice (yellow) with
standard rice (white)
Worldwide, 7% of children suffer vitamin A deficiency,
many of them living in regions in which rice is a
staple of the diet.
 Genetically Engineered Golden Rice
Beans Aspergillus fungus Wild rice Daffodil

Ferritin gene Phytase gene Metallothionin b-carotene enzyme

from from Gene from Synthesis genes from
beans. fungus. Wild rice. daffodils.

Rice A1 A2 A3 A4
Fe Pt S
chromosome

Ferritin protein Phytate, which Metallothionin b-carotene, a

increases iron inhibits iron protein supplies precursor to
content of rice. extra sulfur to vitamin A, is
reabsorption, increase iron synthesized.
is destroyed by the uptake.
phytase enzyme.
Insect Resistant GM Corn
AquAdvantage: Atlantic Salmon

http://aquabounty.com/our-salmon/in-the-kitchen/
 Cloning of Plants

Single
cell

Cells removed Cells in growth Cell division Young plant Adult plant
from orchid plant medium in culture
 Reproductive Cloning of Animals

Reproductive cloning

Donor
Nucleus from
cell
donor cell
Implant embryo in Clone of
surrogate mother donor is born

Therapeutic cloning

Remove Add somatic Grow in culture

nucleus cell from to produce a
from adult donor ball of cells Remove embryonic Induce stem
egg cell stem cells from cells to form
embryo and specialized cells
grow in culture for therapeutic
use
 Cloning - Dolly

In 1997 Dolly the sheep

became the first
vertebrate cloned from
the cell of an adult
animal.

Prof. Ian Wilmut and his colleagues

at the Roslin Institute in Edinburgh, Scotland

Failed 276 times and could succeed in

their 277th attempt
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The first clone Cloning for medical use

Clones of endangered animals

Mouflon lamb with Banteng Gaur

mother
 Therapeutic Cloning and Stem Cells

Adult stem Blood cells

cells in
bone marrow

Embryonic Cultured Nerve cells

stem cells embryonic
in early stem cells
embryo

Heart muscle cells

Different culture Different types of
conditions differentiated cells
Genetically Modified Organisms in Agriculture

• Since ancient times, people have selectively bred agricultural crops to

make them more useful.

• Today, DNA technology is quickly replacing traditional breeding

programs as scientists work to improve the productivity of agriculturally
important plants and animals.

• In the United States today, more than 80% of the corn crop, more than
90% of the soybean crop, and about 75% of the cotton crop are
genetically modified.

• Growing insect-resistant plants reduces the need for chemical

insecticides.

• ―Golden rice 2‖ is a transgenic variety of rice that carries genes from

daffodils and corn. This rice could help prevent vitamin A deficiency and
resulting blindness, especially in developing nations that depend on rice
as a staple crop.
 Genetically Modified Organisms

• Genetic engineers are targeting agricultural animals as well as plant

crops.
• Although no transgenic animals are currently being sold as food, the
Food and Drug Administration (FDA) has issued regulatory
guidelines for their eventual introduction.

• AquAdvantage is a trade name for Atlantic salmon that have been

genetically modified to reach market size in half the normal time (18
months vs. 3 years).
• The FDA is currently reviewing AquAdvantage salmon.
• It may become the first GM animal approved for consumption in the
United States.

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 Plant Cloning

• All body cells contain a complete complement of genes, even if they are
not expressing all of them. A single differentiated plant cell can undergo
cell division and give rise to a complete adult plant.

• Plant cloning is now used extensively in agriculture.

• For some plants, such as orchids, cloning is the only commercially
practical means of reproducing plants.
• In other cases, cloning has been used to reproduce a plant with
specific desirable traits, such as high fruit yield or resistance to
disease.
• Seedless plants (such as seedless grapes, watermelons, and
oranges) cannot reproduce sexually, leaving cloning as the sole
means of mass-producing these common foods.

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 Animal Cloning

• Regeneration is the regrowth of lost body parts.

• When a salamander loses a leg, certain cells in the leg stump reverse
their differentiated state, divide, and then differentiate again to give rise
to a new leg.

• Nuclear transplantation involves replacing the nucleus of an egg cell

or a zygote with a nucleus removed from an adult body cell.
• If the animal to be cloned is a mammal, further development
requires implanting the early embryo into the uterus of a surrogate
mother.
• This type of cloning is called reproductive cloning because it
results in the birth of a new animal.

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 Reproductive Cloning of Animals

• In 1996, researchers used reproductive cloning to produce the first

mammal cloned from an adult cell, a sheep named Dolly.
• The researchers fused specially treated sheep cells with eggs from
which they had removed the nuclei.
• After several days of growth, the resulting embryos were implanted
in the uteruses of surrogate mothers.
• One of the embryos developed into Dolly—and as expected, Dolly
resembled the nucleus donor, not the egg donor or the surrogate
mother.
• Since the first success in 1996, researchers have cloned many
species of mammals, including mice, horses, dogs, mules, cows,
pigs, rabbits, ferrets, camels, goats, and cats.

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 Practical Applications of Reproductive Cloning
• Why is reproductive cloning used?
• In agriculture, farm animals with specific sets of desirable traits
might be cloned to produce identical herds.
• In research, genetically identical animals can provide perfect
―control animals‖ for experiments.

• Reproductive cloning is used to restock populations of endangered

animals. Cloning may also create new problems.
• Conservationists argue that cloning
• may detract from efforts to preserve natural habitats.
• does not increase genetic diversity, and
• is therefore not as beneficial to endangered species as natural
reproduction.
• An increasing body of evidence suggests that cloned animals are
less healthy than animals produced via fertilization.

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 Therapeutic Cloning and Stem Cells
• The purpose of therapeutic cloning is not to produce a living organism
but rather to produce embryonic stem cells.

• In mammals, embryonic stem cells (ES cells) are obtained by

• removing cells from an early embryo and growing them in laboratory
culture. Embryonic stem cells can divide indefinitely and, under the
right conditions, can (hypothetically) develop into a wide variety of
different specialized cells.
• If scientists can discover the right conditions, they may be able to
grow cells for the repair of injured or diseased organs.

• Adult stem cells

• are further along the road to differentiation than ES cells,
• can therefore give rise to only a few related types of specialized
cells, and can also generate replacements for some of the body’s
cells. Because no embryonic tissue is involved in their harvest, adult
stem cells are less ethically problematic than ES cells.
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 Human Cloning

• The cloning of various mammals has heightened

speculation that humans could be cloned.
• Critics point out the many practical and ethical
objections to human cloning.
• Practically, cloning of mammals is extremely
difficult and inefficient.
• Only a small percentage of cloned embryos develop
normally and
• they appear less healthy than naturally born kin.

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 Biotechnology Ethics

• The recent advances in biotechnology

have raised a number of ethical
questions.
• Will the technology be used safely?
• Who will benefit?
• Who will suffer?
• Should the technology be used to make a
profit?
• Just because we can, should we?
 What are the consequences?
• One approach to ethics is to weigh the ―pros‖ and
―cons‖ of the particular technology or application.
• Genetic testing allows us to predict disease before
it happens.
• Pro: This allows one to seek treatment early.
• Con: If insurance companies obtain this
information, they may deny coverage.

• Cloning technology allows us to generate

nutritionally advanced foods.
• Pro: This provides underdeveloped nations
with better food sources.
• Con: The GMO organisms may impact the
ecosystem negatively.
 Is it inherently wrong?
• Another approach to ethics is to ask if the technology
and its application violates principles that are valued
by society.
• Does it threaten someone’s human rights?
• Does it threaten someone’s Bill of Rights?
• Does it violate someone’s religious beliefs?
• Does it diminish someone’s quality of life?

• Does it violate animal rights? Is it inherently wrong

to…
• Produce genetically modified organisms?
• Manipulate genes?
• Manipulate or destroy embryos to obtain stem
cells?