Introduction to genetic engineering
(rDNA technology)
Abera J. ([Link], MSc in Clinical Pharmacy)
Chapter Outline
– Introduction
– Tools of genetic engineering
• Enzymes, cloning vectors, cloning hosts
– Basic techniques
• Gene cloning, protein expression
– Application of rDNA technology
• PCR and other techniques of biotechnology
• The four bases in DNA are adenine (A), cytosine (C), guanine (G), and
thymine (T).
• These bases form specific pairs (A with T, and G with C).
Introduction
• Genetic engineering……..………….also known as rDNA technology
– Means altering the genes in a living organism to produce a
Genetically Modified Organism (GMO) with a new genotype.
• Various kinds of genetic modification are possible;
– Inserting a foreign gene from one species into another,
– Forming a transgenic organism;
– Altering an existing gene so that its product is changed; or
– Changing gene expression so that it is translated more often or not
at all.
Introduction…
• Genetic engineering………..involves the manipulation of genes
within a cell or organism to bring about a change in the
genetic makeup of an organism
• There are several methods of gene manipulation currently
used;
– Most of which include the removal and insertion of genetic material
into organisms
Tools of genetic engineering
Manipulating Genes
• Sexual reproduction is often preferred over asexual reproduction.
– For example, in human beings, both adults will contribute half
their genes (23 chromosomes) to their child.
– Through this method of reproduction, no human is ever exactly
the same.
– The one exception to this is when a couple happens to conceive
twins.
• Identical twins are exactly the same genetically, and can also
be referred to as "nature's clones".
Manipulating Genes…
• By human intervention, this same phenomena can also be
reproduced.
– Through cloning, the nucleus of a selected egg is removed, and
the nucleus of another cell (usually a body cell) is inserted
inside.
– The cell usually needs an outside stimulus (chemicals or
electric) to start up cell division.
– The genetic makeup of the child will be that of the person who
contributed the cell.
Manipulating Genes…
Types of cloning
• Recombinant DNA Cloning………..……describes the extraction of
a DNA desired and placing it in a organism which has the
ability to replicate it's genetic material.
– This is basically the cloning of a gene.
• Reproductive Cloning……………....it uses the nuclear DNA of one
organism to create another life form that is exactly the same.
Manipulating Genes…
• Therapeutic Cloning……………human embryos are produced and
used for research.
– Uses the clones as a method of studying the effects of stem cells and
how they can be used to treat disease.
– Stem Cells have the ability to become any type of cell in the body.
– These cells will eventually be manipulated and be used to replace any
damaged or disfunctioning cells in the body.
– However, the extraction of stem cells are only available in the
embryo stage, and this causes ethical problems as the embryo is
destroyed once they do this.
Manipulating Genes
A. Restriction Enzymes……..bacterial enzymes that cut long strands of
nucleotides into smaller segments.
1. Recognize specific sequences of nucleotides (bases) and cut the DNA at
a specific site within the sequence.
2. In one chain, the sequence runs left to right and on the complementary
chain the sequence runs right to left.
Manipulating Genes…
3. Single chain “tails” of DNA called sticky ends are created on each
DNA segment by the action of the restriction enzymes.
4. Sticky ends readily bind to complementary chains of DNA.
5. Pieces of DNA that have been cut with the same restriction enzyme
can bind together to form a new sequence of nucleotides.
6. Restriction enzymes can be used to isolate (cut out) a specific gene.
Manipulating Genes…
B. Cloning Vectors………………A DNA carrier used to clone a gene and
transfer it from one organism to another.
1. Once a gene has been isolated, that small piece of DNA can be placed
into a cloning vector and can be introduced into an organism.
2. Many bacteria contain a cloning vector called a plasmid.
Ideal cloning vector Should;
Be capable of replicating in host cell
have convenient RE sites for inserting DNA of interest
have a selectable marker to indicate which host cells
received recombinant DNA molecule
be small and easy to isolate
Non toxic to the host
I. Manipulating Genes…
Plasmid…………a ring of DNA found in bacteria in addition to it’s
main chromosome.
• It can be used to transport genes from one organism to
another.
Manipulating Genes…
Process of using a cloning vector;
a) A plasmid is removed from a bacterial cell
b) Using a restriction enzyme, the plasmid is cut and a donor gene
is spliced in.
• Donor gene……………………….a specific gene that is isolated
from another organism and introduced into a cloning vector.
Manipulating Genes…
c) the plasmid is returned to the bacterial cell,
where it replicates as the bacterial cell
divides.
• This clones the donor gene.
d) the bacteria containing clones of the donor
gene can then be used to infect other organisms
and transfer the gene to them.
Transplanting Genes
A. In some cases, plasmids are used to clone a specific gene so
that the bacteria will produce a specific protein.
For example: insulin is a protein that controls sugar
metabolism.
Transplanting Genes…
a) People who are diabetic do not make the hormone
insulin.
• Insulin allows sugars to travel from the blood into
the cells where they are transformed into energy.
b) A large volume of insulin can be produced by cutting
out the human gene and cloning it using a plasmid.
Transplanting Genes…
B. Process
1. Isolating a gene
a. DNA is removed from a human cell to isolate the gene
that produces insulin.
b. A restriction enzyme is used to cut the human DNA into
separate genes, this includes the gene for insulin.
Transplanting Genes…
2. Producing Recombinant DNA
a) Recombinant DNA…………….combination of DNA from
two or more sources.
b) Inserting a donor gene (human gene for insulin) into a
cloning vector (bacterial plasmid) results in a recombinant
DNA molecule.
Transplanting Genes…
3. Cloning DNA
a) The plasmid containing recombinant DNA is inserted into
a host bacterium.
b) The transgenic bacteria is placed into a nutrient medium
where it can grow and reproduce.
c) Within each bacterium, the plasmid is copied many times,
and the donor gene for insulin is cloned.
Transplanting Genes…
d) Thousands of bacteria are produced very quickly
through mitosis.
e) The transgenic bacteria can be used to produce large
amounts of insulin.
Basic techniques
Expression of Cloned Genes
1. Once a donor gene is transferred to a host cell, it is
transcribed and translated as though it were in its own
cell.
a) Not all of the genes in a cell’s genome are expressed.
b) Genes are often turned off until the proteins they code
for are needed
DNA Fingerprints
Uses
1. The banding patterns of DNA fragments from two different
individuals may be compared to see if they are related
(Example……Paternity tests)
2. DNA fingerprints of members of two different species can be
compared to determine how closely species are related.
3. Using DNA fingerprint to compare samples of blood, hair, or tissue
found at a crime scene with a suspect’s blood sample may help in
solving crimes.
Making a DNA Fingerprint
1. RFLP (Restriction Fragments Length
Polymorphism) Analysis: method for
preparing a DNA fingerprint.
a) Involves extracting DNA from
blood, hair, or tissue and cutting it
into fragments using restriction
enzymes.
b) Fragments of DNA are then
separated using a technique called
gel electrophoresis.
Making a DNA Fingerprint…
2. Gel electrophoresis
• Separates nucleic acids or proteins
(amino acids) according to their size
and charge.
a) Samples of DNA being compared are
placed in wells made on the gel
b) An electric current is then run
through the gel
Making a DNA Fingerprint…
c) DNA fragments (“-” charge) migrate towards the “+“ end of
the gel
d) The smaller DNA fragments migrate faster and further
down the gel than the longer fragments.
e) The DNA fragments that have been separated on the gel are
stained in order to be seen.
Making a DNA Fingerprint…
3. Accuracy of a DNA fingerprint
• The complete nucleotide sequence of each individual is
unique for each person (except identical twins).
• Therefore, everyone’s DNA fingerprint will be different.
• DNA fingerprints are 99.9999% accurate.
Application of rDNA technology
Polymerase Chain Reaction (PCR)
• Can be used to make copies of selected segments of DNA for a
DNA fingerprint.
Requires
a) A primer……….artificially made single-stranded sequence of DNA.
b) When these ingredients (DNA and Primer) are combined and
incubated, the selected regions of DNA quickly double.
Polymerase Chain Reaction (PCR)…
c) Every five minutes the sample of DNA doubles again, resulting in
many copies of a sample in a short amount of time.
d) The new copies of the DNA sample can then be used to make a DNA
fingerprint.
e) Used in
– Paternity tests
– Diagnosis of genetic disorders
– Study of ancient fragments of DNA
Human Genome Project
A. Goals
1. To Determine the nucleotide sequence of the Human genome
and to map the location of every gene on each chromosome.
2. To compare the human genome with genomes of other
organisms in an effort to provide insight into fundamental
questions about how genomes are:
a. Organized
b. How cellular differentiation and growth are under genetic
control
c. How gene expression is controlled, and
d. How evolution occurs.
Human Genome Project…
B. It is hoped that the knowledge gained from the Human
Genome Project will improve diagnosis, treatments, and
even provide cures for genetic diseases.
1. Identifying these genes and defective proteins for which
they code may make it possible to design therapies aimed at
correcting the gene defects responsible for the disorder.
Gene Therapy and Cloning
• Treating a genetic disorder by introducing a normal gene into a
cell or by correcting a gene defect in a cell’s genome.
A. Is well suited for treating genetic disorders that result from a
deficiency of a single enzyme or protein.
Example: Cystic fibrosis, lung CA, AIDS, ovarian CA, brain CA
Gene Therapy and Cloning…
E.g Role of Gene therapy in CA
• Gene transfer is a new treatment modality that introduces
new genes into a cancerous cell or the surrounding tissue to
cause cell death or slow the growth of the CA.
• This treatment technique is very flexible, and a wide range of
genes and vectors are being used in clinical trials with
successful outcomes.
Gene Therapy and Cloning…
A. Many medicine are proteins that can be mass-produced in a less
expensive way through DNA technology.
Examples:
1. Insulin: controls sugar metabolism; used to treat diabetes.
2. Colony-Stimulating Factors: used to treat immune deficiency by
stimulating the production of white blood cells
3. Erythropoietin: used to treat anemia by stimulating the production
of red blood cells.
Gene Therapy and Cloning…
4. Human Growth Hormone: used as a treatment for
dwarfism; causes bones to elongate.
5. Interferon: used to treat viral infections and CA by
preventing the replication of viruses
6. Interleukins: used to treat HIV and CA by activating and
stimulating different kinds of white blood cells.
Gene Therapy and Cloning…
• Genetically Engineered Vaccines
• Many diseases are combated by prevention, using
vaccines made by genetic engineering.
Gene Therapy and Cloning…
• The examples given above are also classified as
therapeutic cloning
• Therapeutic cloning uses genetic engineering
to help treat and cure diseases.
Increasing Agricultural Yields
[Link] technology has been used to develop new strains of
plants, which in turn can be used to improve food and
crop yields.
A. Examples: Tomatoes and hornworms:
-by transferring genes for enzymes that are harmful to
hornworms into tomato plants, scientists can make
tomato plants toxic to hornworms and effectively
protect the plants from these pests.
Increasing Agricultural Yields…
B. Wheat, cotton, and soybeans:
• have been created to be resistant to weed controlling
chemicals
1) herbicide: weed controlling chemicals.
2) such herbicide resistant crops can be protected from weeds
more easily and less expensively than crops that are
susceptible to the herbicides
Safety and Environmental Issues
• Many people are concerned about the safety of genetically
engineered foods.
• The concern is that the food produced by genetic
engineering could contain toxic proteins or substances that
can cause allergies in people who consume them.
Safety and Environmental Issues…
• Foods produced by transgenic crops can be sold
without special permits or labels if the product is
identical to products produced by non-transgenic
crops.
Example: corn, tomatoes
Safety and Environmental Issues…
Concerns
a) genetically engineered crops could spread into the wild and
wipe out native plant species.
b) transgenic crops could transmit their new genes to other
species in neighboring areas.
Example: superweeds produced by rice and lawn grasses
exchanging pollen with native species
Safety and Environmental Issues…
Reproductive Cloning
• Involves creating an animal that is genetically identical to a donor
animal through somatic cell nuclear transfer.
• Involves the making of a whole new organism from a still living
organism
• There is no sexual reproduction or fertilization involved
• Sheep, donkeys, and a number of plants have been cloned
• Human cloning is still far off in the future
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