Applications of Biotechnology: Industry

DNA Technology

Genetic Engineering (Recombinant DNA Technology)

DNA Sequencing, PCR, and DNA finger printing

Human Genome Project

Dolly and Golden Rice

Tools used in Recombinant DNA 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
Recombinant DNA (r-DNA)
 Bacteria Fight Invading Viruses with
Restriction Enzymes

• 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).
Restriction Endonucleases
Source : Bacteria
Types
Recognition sequence

Type I

Restriction site

Type II

Recognition sequence
Restriction Endonucleases
Arthrobacter
luteus
Blunt end
Haemophilus
aegyptius Palindromic
sequence
Bacillus
amyloliquefacies
Sticky /
Haemophilus
influenzae
Staggered /
Cohesive
Escherichia End
coli
GAATTC GAATTC
CTTAAG CTTAAG
 Vectors

Plasmids are ideal vectors for the introduction of r-DNA into bacteria.

• A plasmid is small and can divide separately from the host’s

chromosome.
• Each plasmid contains an origin of replication called a replicon, or
replication unit.
• They often have a restriction site, 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.
Vectors for Carrying DNA into Cells
Pharmaceutical Applications: Humulin

Herbert Boyer and Stanley Cohen

 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.
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 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.
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 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
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.
Restriction Endonucleases Cleave DNA
at specific DNA sequences
 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.
DNA Sequencing
 The first round of PCR
5’ 3’ 5’ 3’ 5’ 3’

**
5’ 3’

3’ 5’
94°C 37-65°C 70-75°C

3’ 5’ 3’ 5’ 3’ 5’

Denaturation Annealing Extension

or Elongation

**
Taq polymerase is thermostable as is obtained from a thermophilic bacterium
Thermus aquaticus, mostly found in hot springs. It’s optimum temperature is 75–
80 °C, where the enzyme is maximally active, and can polymerize 150 nucleotides per
second per enzyme molecule. It has a half life of 2 hours at 92.5 °C and 40 minutes at
95 °C.
PCR increases the yield of DNA
exponentially
 The Polymerase Chain Reaction (PCR)

Initial
DNA
segment

Cycle No: 0 1 2 3
Number of double stranded
DNA molecules 1 2 4 8
 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.

© 2016 Pearson Education, Inc.

 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.

© 2016 Pearson Education, Inc.

 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 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 Fingerprinting using
Variable Number Tandem Repeats (VNTR)
• Variable number tandem repeats
are regions of short repetitive RES1 RES2
nucleotide sequences, found on
many chromosomes. RES1 RES2

• Each individual has variable no. of RES1 RES2

repeats in their chromosomes.

RES1 RES2

• Therefore, if these regions are cut

with restriction enzymes, (or RES1 RES2
amplified by PCR), each person will
have a different set of fragments. RES1 RES2

Restriction Enzyme Recognition Site,

• Thus VNTRs are useful in parental (RES) (or Restriction site, RS)1 and 2
identifications, forensics and DNA are intervened by different no. of
fingerprinting. tandem repeats in 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 OR
• 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.
 Crime scene Suspect 1 Suspect 2
1 DNA isolated

2 DNA amplified

3 DNA compared
Variable Number of Tandem Repeat (VNTR) analysis is
commonly used in forensics

For a homologous pair of

chromosome, tandem repeat in one
chromosome is contributed by one of
the parents, tandem in the other
chromosome is contributed by the
other parent.
DNA Fingerprinting
in a Paternity Case
Using VNTR to compare forensic and suspect samples

Each individual has unique

set of tandem repeats
considering all of them in
various chromosomes.
The Human Genome Project
Chromosome

Chop up with
restriction enzyme

DNA fragments

Sequence fragments

Align fragments

Reassemble
full sequence
 The Human Genome Project
• 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.

• 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.
 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.
Reproductive Cloning of Animals
The first clone Cloning for medical use

Clones of endangered animals

Mouflon lamb with Banteng Gaur

mother
 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.

© 2016 Pearson Education, Inc.

 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.
© 2016 Pearson Education, Inc.
 Reproductive and Therapeutic Cloning
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
egg cell
stem cells from Induce stem cells to
embryo and form specialized
grow in culture cells for therapeutic
use
 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
 Therapeutic Cloning and 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.

© 2016 Pearson Education, Inc.

© 2016 Pearson Education, Inc.
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

PowerPoint® Lectures created by Edward J. Zalisko for

Campbell Essential Biology, Sixth Edition, and
Campbell Essential Biology with Physiology, Fifth Edition
– Eric J. Simon, Jean L. Dickey, Kelly A. Hogan, and Jane B. Reece
 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?