Download 7. Biotechnology- Using Molecular Biology and Genetic Engineering

Biotechnology
Introduction
Biotechnology is essentially
the use of living organisms and their
products for health, social or economic
purposes.
Biotechnology is widely considered to be
the growth technology of the 21st
century which will lead to huge growth in
the Biotechnology industry and exciting
opportunities for graduates
Application of Biotechnology
Its use and application ranges from fields
like agriculture to industry (food,
pharmaceutical, chemical, bioproducts,
textiles etc.), medicine, nutrition,
environmental conservation, animal
sciences etc. making it one of the fastest
growing fields.
The work is generally carried out in the
laboratories, as it is a scientific research
oriented field.
Application of Biotechnology
Applications of biotechnology are widespread,
including the following:
 diagnosis and treatment of human diseases.
 improved production of therapeutic agents.
 development of improved crop plant species.
 Development of improved farm animals
 development of improved pest/pathogen
control processes
Application of Biotechnology
development of biosensors for
environmental pollutants.
development of improved waste treatment
processes and methods for remediation
contaminated sites.
production of transgenic organisms for
production of new drugs, improved
transplantation success and improved
animal and plant.
Selective Breeding
 Humans use selective
breeding, which takes
advantage of naturally
occurring genetic
variation in plants,
animals, and other
organisms, to pass
desired traits on to the
next generation of
organisms
Selective Breeding
 Breed only those
plants or animals with
desirable traits
 People have been
using selective
breeding for 1000’s of
years with farm crops
and domesticated
animals.
Selective Breeding
Nearly all domestic
animals -- including
horses, cats, and
farm animals – and
most crop plants
have been produced
by selective breeding
No freaking way!
Hybridization
Louis Burbank was the
greatest selective
breeder of all time. He
developed the
disease-resistant
potato and more than
800 varieties of plants.
Louis Burbank used the technique
of hybridization and bred
dissimilar individuals to combine
the best traits of both parents.
The hybrids produced by these
crosses were hardier than their
parents
Inbreeding
 To maintain the desired
characteristics of a line of
organisms, breeders often use
the technique of inbreeding.
 Inbreeding is the continued
breeding of individuals with
similar characteristics
Increasing Variation
In order for selective breeding to be
successful, there must be a lot of genetic
variation in the population
Breeders increase the genetic variation
in a population by inducing mutations,
which are the ultimate source of genetic
variability
Increasing Variation
Breeders increase the mutation rate by
using radiation and chemicals
Molecular Biology
Molecular Biology
 Molecular biology refers to the field of study
regarding the investigation and manipulation of
biological structures, processes, and
phenomena at the molecular level.
 Involves several classical basic techniques such
as restriction enzymes, gel electrophoresis, and
PCR, as well as more complex methods such as
DNA fingerprinting, DNA sequencing, and
genetic engineering
Outline
 Restriction-enzyme analysis
 Gel electrophoresis
 The polymerase chain reaction (PCR)
 DNA Fingerprinting
 DNA sequencing
 Blotting techniques
 Recombinant DNA
Restriction Enzyme Analysis
aka restriction endonucleases
Recognizes specific base sequences and
cleave the nucleic acid
PALINDROMES
Two-fold rotational symmetry
Generates fragments of DNA
Restriction Enzyme and Gel Electrophoresis
 DNA fragments
produced by
restriction enzymes
can be separated by
gel electrophoresis
Agarose (>20 kb)
PAGE (1 kb)
 Visualization
Autoradiography
Ethidium bromide
Gel Electrophoresis
A technique for separating DNA (or
protein) molecules on the basis of size.
DNA plus
restriction enzyme
Power source
Longer
fragments
Mixture of
DNA
fragments
Gel
Shorter
fragments
Gel Electrophoresis
Utilizes agarose or polyacrylamide gels for
the separation of DNA
DNA plus
restriction enzyme
Power source
Longer
fragments
Mixture of
DNA
fragments
Gel
Shorter
fragments
Electrophoresis
Electrophoresis
• Electrophoresis is used to map the structure of a DNA
fragment
Electrophoresis
 Stained gel
result
 Visualization
may be
achieved
through UV
dyes or
radioactive
agents
Polymerase Chain Reaction
 A rapid and versatile in vitro method to amplify
defined target DNA within a heterogeneous
collection of DNA sequences (genomic DNA or
cDNA)
PCR Requirements
 Template (genomic DNA or cDNA population)
 Oligonucleotide primers
 DNA polymerase (Taq polymerase)
 dNTP
 Thermal cycler
Cycles (25 – 30)
 Denaturation
95o C
Separate strands
 DNA Synthesis
70 – 75 oC (ideal temp for Taq polymerase)
Thermus aquaticus (Taq)
 Annealing
50 – 70 oC (~5o C lower than Tm)
Utility of PCR in Medical Diagnostics
 Detection of bacteria and viruses by specific
primers
HIV virus in people who have not mounted an
immune response
Mycobacterium tuberculosis bacilli
 Detection of certain cancer cells
ras genes and leukemias caused by chromosomal
rearrangement
Monitoring cancer chemotherapy
Utility of PCR in Forensics
 DNA fingerprinting
 Restriction fragment length polymorphisms
 PCR-Based analysis
 Can be used to determine biological parentage
 Can be used to settle assault and rape cases
DNA Fingerprinting: A tool for
forensics and paternity cases
 DNA analysis can be used for catching criminals, establishing
parentage, finding how closely organisms are related and many
other applications.
 The pattern of bands in a gel electrophoresis is known as a DNA
fingerprint or a ‘genetic fingerprint’ or ‘genetic profile’
 If a DNA fingerprint found in a sample of blood or other tissue at
the scene of a crime matches the genetic fingerprint of a suspect,
this can be used as evidence
 A DNA sample can be obtained from the suspect using blood, cheek
epithelial cells taken from the mouth lining or even the cells clinging
to the root of a hair
 Steps:
1. Get DNA sample
2. Amplify with PCR
3. Cut with
restriction enzyme
4. Run resulting
fragments on gel
electrophoresis
5. Analyze result
 A sample with the
shorter DNA
fragments travels
through the gel faster
than a sample with
the larger fragments
V
S
S1
S2
S3
DNA
profiles
V Victim
S Sample from crime scene
S1 Suspect 1
S2 Suspect 2
S3 Suspect 3
More than 20 fragments
from Suspect 1 match those
taken from the crime scene
Genetic fingerprint of …
1 mother
2 child
3 possible father A
4 possible father B
There is a match between one of
the child’s restriction fragments
and one of the mother’s.
There is also a match between
the child’s other fragment and
one from possible father A.
1
2
3
4
Starting position of sample
Neither of the child’s restriction
fragments match those of possible
father B
Famous cases
 In 2002 Elizabeth
Hurley used DNA
profiling to prove that
Steve Bing was the
father
of her child Damien
Famous Cases
 Colin Pitchfork was
the first criminal
caught based on DNA
fingerprinting
evidence.
 He was arrested in
1986 for the rape and
murder of two girls
and was sentenced in
1988.
Famous Cases
 O.J. Simpson was
cleared of a double
murder charge in
1994 which relied
heavily on DNA
evidence.
 This case highlighted
lab difficulties.
Sequencing by Sanger Dideoxy Method
 Controlled termination
of replication
Uses 2’,3’ dideoxy
analog of nucleotide
Sequencing by Sanger Dideoxy Method
Electrophoresis
Fluorescence Detection
Automated DNA Sequencing
Southern blotting
 Identification of restriction fragment
Southern blotting
 Identification of restriction fragment
Research
Molecular biology
Genetic Engineering
Outline
Cell Transformation
Transforming Bacteria
Transforming Plant Cells
Transforming Animal Cells
Applications of Genetic Engineering
Transgenic Organisms
Cloning
Introduction
Through genetic engineering scientists
can combine DNA from different sources
and this process is called “Recombinant
DNA technology”
The secrets of DNA structure and
functions have led to gene cloning and
genetic engineering, manipulating the
DNA of an organism
Genetic Engineering
A set of techniques used to manipulate
DNA in order to elicit a desired
characteristic in the target organism
Recombinant DNA technology, or the
creation of recombinant DNA, is a
necessary component of genetic
engineering
Cutting DNA & Making Recombinant DNA
 How Restriction enzymes work:
The Enzymes recognize specific sequences on
Human and Bacterial Plasmids
The Enzymes cut the strands.
The cut produces DNA fragments with short strands
on each end that are complementary to each other
“Sticky Ends”
Both the human DNA and the Plasmid “Open Up”
with the same sticky ends remaining
They Bind Together
Restriction enzyme cleaving
Recognition
sequences
DNA sequence
Recognition sequences
DNA sequence
Restriction enzyme EcoRI cuts
the DNA into fragments.
Sticky end
Vectors = carriers of DNA fragments
 Plasmids
Naturally occurring circular, double-stranded
DNA that act as accessory chromosomes in
bacteria; DNA fragments (15, 000 bp)
 λ phage
Bacteriophage (virus) DNA; for larger
DNA fragments (23, 000 bp)
 Yeast and bacterial artificial chromosome
Laboratory-designed carriers for larger DNA
fragments
Confirmation of a Cloned Gene
 Southern Blot can be used to identify a
specific gene:
1. Cut DNA from bacteria with restriction
enzymes.
2. DNA fragments are separated by a gel
soaked in a chemical solution.
Confirmation of a Cloned Gene
3. The DNA separated is then transferred to a
membrane (blotted) and a probe solution is
added.
 Probes: radioactive RNA or single-stranded
DNA pieces that are complementary to the gene
of interest
4. Only DNA fragments complementary to the
probe will form and bind bands
Producing Recombinant Bacteria
1.
Remove bacterial DNA
(plasmid).
2.
Cut the Bacterial DNA with
“restriction enzymes”.
3.
Cut the DNA from another
organism with “restriction
enzymes”.
4.
Combine the cut pieces of DNA
together with another enzyme
and insert them into bacteria.
5.
Reproduce the recombinant
bacteria.
6.
The foreign genes will be
expressed in the bacteria.
When a bacteria or other cell takes
in a foreign piece of DNA such as a
plasmid, the process is called
transformation
If transformation is successful, the
recombinant DNA is integrated into
one of the chromosomes of the cell.
Creating HGH
Recombinant
DNA
Gene for human
growth hormone
Gene for human
growth hormone
Human Cell
Sticky
ends
Bacterial Cell
Bacterial
chromosome
Plasmid
DNA
recombination
DNA
insertion
Bacterial cell for containing gene
for human growth hormone
What Can You Do with a Cloned Gene?
Applications of Recombinant DNA Technology
Some Benefits of Recombinant
Bacteria
1. Bacteria can make human insulin or
human growth hormone.
1. Bacteria can be engineered to “eat” oil
spills.
Genetically Engineered Drugs and Vaccines
Today, many pharmaceutical companies
around the world produce important
proteins using genetic engineering.
Vaccine: a solution containing all or part of a
harmless version of a pathogen; used to
prevent viral diseases (don’t respond to
drugs)
Many vaccines are made using genetic
engineering
The DNA of plants and animals
can also be altered.
PLANTS
1. disease-resistant and
insect-resistant crops
2. Hardier fruit
3. 70-75% of food in
supermarket is
genetically modified.
Improving Crops
Genetic engineers can add favorable
characteristics to a plant
Plants become resistant to insects
(no longer need pesticides); resistant
to weed killer (so crops won’t die, but
weeds will); improved nutrition – rice +
corn
Pest Resistance: Bt Corn
Plant Transformation
Gene to be
transferred
Inside plant cell,
Agrobacterium
inserts part of its
DNA into host cell
chromosome
Agrobacterium
tumefaciens
Cellular
DNA
Recombinant
plasmid
Plant cell
colonies
Complete plant is
generated from
transformed cell
Transformed bacteria introduce
plasmids into plant cells
How to Create a Genetically
Modified Plant
1.Create recombinant
bacteria with
desired gene.
2. Allow the bacteria
to “infect" the plant
cells.
3. Desired gene is
inserted into plant
chromosomes.
Genetically modified animals are
called transgenic animals.
TRANSGENIC ANIMALS
1.
Mice – used to study human
immune system
2.
Chickens – more resistant to
infections
3.
Cows – increase milk supply
and leaner meat
4. Goats, sheep and pigs –
produce human proteins in
their milk
Transgenic Goat
Human DNA in
a Goat Cell
.
This goat contains a human
gene that codes for a blood
clotting agent. The blood
clotting agent can be harvested
in the goat’s milk.
Transgenic Cows
 Growth hormones are given to cows to
produce more milk
 Human genes are added to farm animals in
order to have human proteins in their milk
The human proteins are extracted from
milk and sold to pharmacy companies.
Useful for complex proteins that can’t be
made in bacteria
Transgenic animals
How it works: an intact nucleus from an
embryonic cell (whose DNA has recombined
with a human gene) is placed into an egg
whose nucleus has been removed.
The “new” egg is then placed into the uterus
of an animal.
How to Create a
Transgenic Animal
Animal Cloning
A clone is a member of a population of
genetically identical cells produced from a
single cell
The goal of cloning is to mass produce a
certain individual with desired characteristics
No need for breeding, and the desired
characteristics can be preserved.
How it works: an intact nucleus from a cell is
removed
Cloning Animals
The nucleus is fused with a egg cell (whose
nucleus has been removed) taken from
another adult
The fused cell begins to divide and the
embryo is placed in the uterus of a foster
mother.
The “new” egg is then develops normally.
Cloning
A donor cell is taken from a
sheep’s udder.
Donor Nucleus
Egg Cell
These two cells are fused using an
electric shock.
Fused Cell
The nucleus of the egg cell is removed.
The fused cell begins dividing
normally.
Cloned Lamb
An egg cell is taken from an
adult female sheep.
The embryo is placed in the uterus of
a foster mother.
Embryo
The embryo develops normally into a lamb—
Dolly
Foster Mother
PERPETUATE: A pet cloning company
A company founded in 1998 by Dr. Heather
Bessoff and Ron Gillespie
“PERPETUATE's packaged service
provides the pet owner with the following
three biotechnologies performed on their
pet's cell biopsy”
Cell culturing,
DNA preservation,
Genotyping.