SHOT-GUN APPROACH

OBJECTIVES Students should understand the general principles of

biotechnology.

⮚ Outline the principles of restriction enzyme use to

‘cut’ sections of DNA and ligase enzyme to ‘paste’
them together.
⮚ Explain the use of advanced genome editing tools like
CRISPR-Cas9.
⮚ Explain the basic steps in recombinant DNA technology.
⮚ Discuss the medical, agricultural, ethical and social
implications of the use of gene therapy and
genetically modified organisms on humans and the
environment.
Other names:
✔GENETIC MODIFICATION RECOMBINANT DNA TECHNOLOGY DNA
 INTRODUCTIO
N

What is Genetic Engineering? - YouTube

https://www.youtube.com/watch?v=BK12dQ
q4sJw
 RESTRICTION ENDONUCLEASES - ‘MOLECULAR SCISSORS’

◼For the gene cloning procedure to occur, a piece of DNA

containing the gene of interest must be cut out of a
chromosome and “pasted” into a bacterial plasmid.
◼The cutting tools are bacterial enzymes called restriction
endonucleases or restriction enzymes.
◼In nature, these enzymes protect bacterial cells against
intruding DNA from other organisms or viruses.
◼They work by chopping up the foreign DNA, a process
that restricts foreign DNA from surviving in the cell.
 RESTRICTION ENDONUCLEASES
(ENZYMES)
◼Each restriction enzyme is very specific,
recognizing a particular short DNA sequence
(usually four to eight nucleotides long). These
are called restriction sites.
◼Once the DNA sequence is recognized, the
restriction enzyme cuts both DNA strands at
specific points within the sequence.
◼Some leave blunt ends and other leave sticky or
ECORI- Escherichia coli restriction enzyme I
 RESTRICTION ENZYMES
STICKY ENDS e.g. BLUNT ENDS e.g.
ECORI HAE III
RESTRICTION ENZYMES
Restriction enzymes commonly recognize DNA sequences that are palindromes.
A palindrome is a nucleotide sequence in the double-stranded DNA that reads the same backwards and forwards.
ECOR1
RESTRICTION ENZYMES AND DNA LIGASE ‘molecular glue’

◼Creating recombinant DNA using a restriction

enzyme and DNA ligase:
◼Restriction enzymes cut up the DNA producing
pieces called restriction fragments.
◼The foreign and original DNA fragments are spliced
or joined together permanently by the “pasting”
enzyme DNA ligase.
◼A recombinant DNA – DNA from two different
sources – is formed.
 SHOTGUN APPROACH OF GENE CLONING
◼ Steps to a way to make many copies of the gene using the
techniques of recombinant DNA technology:

1. The biologist isolates two kinds of DNA: the bacterial plasmid that will serve
as the vector (gene carrier) and the DNA containing the desired gene.
2. The researcher treats both the plasmid and the desired DNA with the same
restriction endonuclease enzyme. An enzyme is chosen that cleaves the
plasmid in only one place. Restriction fragments will be produced.
3. The desired DNA is mixed with the cut plasmid. The sticky ends of the
plasmid base pairs with the complementary sticky ends of the desired DNA
fragment.
 SHOTGUN APPRAOCH OF CLONING GENES IN
RECOMBINANT PLASMIDS
4.The enzyme DNA ligase joins the two DNA molecules
by covalent bonds, and the result is a recombinant
plasmid containing the desired gene.
5. The recombinant plasmid is added to a bacterium.
Under the right conditions, the bacterium takes up the
plasmid DNA by transformation.
6. The bacterium is allowed to reproduce, forming a
clone of cells that all carry the recombinant plasmid.
7. The transformed bacteria are selected and cultured.
SHOTGUN APPROACH OF CLONING GENES IN RECOMBINANT PLASMIDS
SUMMARY

1. Gene isolation
2. Gene cutting with restriction endonucleases
3. Gene and plasmid mixing
4. Gene splicing or Plasmid transformation
5. Bacterial transformation
6. Gene (bacterial) cloning
7. Bacterial selection and culturing
Summar
y of the
process
 How are vectors used in cloning?

🠶 Two naturally occurring vectors that are

used to insert foreign DNA into bacterial
cells are bacteriophages and plasmids.
🠶 Bacteriophages (or phages, for short) are
viruses that infect bacterial cells by injecting
their genetic material into the bacterial cell.
🠶 Plasmids are small circles of DNA that are
sometimes present in bacteria in addition to the
larger circle of DNA that constitutes the main
bacterial genome. Many bacteria readily absorb
plasmids from the environment under
appropriate conditions.
 pBR322
engineered plasmid used in GE
• pBR322 contains the genes for
resistance to ampicillin and
tetracycline.
• Disruption of any of the site
renders the gene inactive.

• For example, if a segment of DNA

was added to the Bam HI site, then
the tetracycline gene would be
disrupted and stop working.
 pUC 18

This plasmid includes 2

genes.
• One gene confers
resistance to the
antibiotic ampicillin.
Bacteria that contain this
gene are able to grow in the
presence of ampicillin, while
bacteria that lack this gene
are not.
• The second gene is called β–galactosidase converts lactose to glucose
and galactose
the lacZ gene. The lacZ
Selecting the
transformed
cells
with recombinant DNA

https://www.youtube.com/watch?v=nfC689ElUVk
Steps involved

• The linearized plasmids and the foreign DNA are

mixed together.
• Complementary sticky ends allow the pieces of DNA
An enzyme called DNA ligase is used to join the
to anneal in three
annealed possible
pieces together. combinations.
• Plasmid-plasmid Reclosed plasmid
Recombinant plasmid

• An enzyme called DNA ligase is used to join the annealed

pieces.
Selecting the transformed bacteria
SELECTING THE
TRANSFORMED
BACTERIA
Colorimetric
identification If the plasmid closed on
itself, then the lacZ gene
will not be disrupted and
β–galactosidase will
convert X-Gal into the blue
compound

If the foreign DNA is

inserted at any site in
the LacZ gene, then the
X-Gal X-Gal gene is disrupted and
β–galactosidase will not
be made hence X-Gal
will not be converted to
the blue compound and
the colony will remain
white.
X-Gal is 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.
Colorimetric identification

No
recombinant
plasmid.

Recombinant
plasmid.
APPLICATIONS OF GENETIC ENGINEERING

◼Forensic science
◼Medicine
◼Gene therapy
◼Genetically modified organisms
FORENSIC SCIENCE

Forensic science is the scientific analysis of evidence for crime scene and other
legal investigations, and DNA technology now plays an important role.
FORENSIC SCIENCE

Is defendant D guilty or
innocent?
Give a reason for your answer.
DNA FINGERPRINTS FROM A MURDER CASE
 1. Insulin/Humulin
▪ E. Coli can be used to
make human insulin to
MEDICINAL treat diabetics
APPLICATIONS

2. Diagnosis of diseases
▪ Amniocentesis
▪ Chorionic villi sampling
MEDICINAL USES
3) Tissue plasminogen activator (TPA)
◼Converts plasminogen to plasmin- a protein that dissolves
blood clots.
◼Treat patients at risk of stroke and heart attacks
4) Erythropoietin
◼Produced by the kidneys
◼Needed by the stem cells in bone marrow that become red
blood cells.
◼Kidney dialysis patients lose erythropoietin during process
◼Risk of anaemia
GENE THERAPY
• Techniques to manipulate DNA for treating diseases -
alteration of an afflicted individual’s genes.

• Two types: somatic and germline

• Somatic: The new allele could be inserted into somatic
body cells of the tissue affected by the disorder
• Germline: The normal allele would have to be transferred
to germ cells that is passed from parents to offspring.
GENE THERAPY PROCEDURE

• One possible procedure for gene therapy in an individual whose bone

marrow cells do not produce a vital protein product because of a defective
gene:
1. The normal gene is cloned and then inserted into the nucleic acid of a
retrovirus vector that has been rendered harmless.
2. Bone marrow cells are taken from the patient and infected with the virus.
3. The virus inserts its nucleic acid, including the human gene, in the cells’
DNA.
4. The engineered cells are then injected back into the patient.
*If the procedure succeeds, the cells will multiply throughout the patient’s
life and produce the missing protein. The patient will be cured!
GENE THERAPY – TECHNICAL AND ETHICAL ISSUES

• Technical issues
• How can researchers build in gene control mechanisms to
ensure that cells with the transferred gene make
appropriate amounts of the gene product at the right
time and in the right parts of the body?
• And how can they be sure that the gene’s insertion does
not harm some other necessary cell function?
GENE THERAPY – TECHNICAL AND ETHICAL ISSUES
• Ethical issues:
• Who will have access to it?
• Should gene therapy be reserved for treating serious diseases?
• And, what about its potential use for enhancing athletic ability,
physical appearance, and even intelligence?
• Should we try to eliminate genetic defects in children and their
descendants?
• Genetic variation is a necessary ingredient for the survival of a
species as environmental conditions change with time.
• Genes that are damaging under some conditions may be
advantageous under others (one example is the sickle-cell allele)
 https://www.youtube.com/watch?v=vp9QkB4nLBY

What are Genetically Modified Organism? | Biology | Extraclass.com – YouTube

 GM
PLANTS
Genetically Modified Organisms

• Social concerns:
• Creation of new pathogens.
• One safety measure is a set of strict laboratory procedures
designed to protect researchers from infection by engineered
microbes and to prevent the microbes from accidentally
leaving the laboratory.
• Today, most public concern about possible hazards centres not on
recombinant microbes but on genetically modified (GM) crops.
• Hazardous to human health or the environment???
• One specific concern is that genetic engineering could
transfer allergens to plants people eat.
Ecological concerns of GMO
🠶 GMOs may become invasive species, out-compete local species and damage
ecosystems if they are released into environment.
🠶 The transfer of genes through cross-pollination and fertilisation may occur between GMOs
and closely related species. This is a form of genetic pollution or contamination.
🠶 GMOs engineered for virus resistance may generate new diseases. Virus resistance is
achieved by introducing into plants a gene from certain viruses which cause disease.
Virus resistance makes plants less susceptible to diseases caused by these viruses and
so plants give higher crop yields. These virus genes may accidentally escape into other
organisms they were not intended for and cause a disease.
🠶 Foods containing ingredients derived from GMOs may cause allergies in susceptible
persons.
🠶 Farmers could be impacted negatively if they rely on imported genetically modified
seeds (e.g. the companies that produce the herbicide Roundup™ also hold patents for
seeds that contain herbicide-resistant genes). Companies can initiate court action
against farmers who develop their own seeds using seeds that contain the genetically
Advantages of GE

1. It allows for a faster growth rate.

2. It can create an extended life.
3. Specific traits can be developed.
4. New products can be created.
5. Greater yields can be produced.
6. Risks to the local water supply are
reduced.
7. It is a scientific practice that has
been in place for millennia.
Disadvantages

1.The nutritional value of foods can be

less.
2. Pathogens adapt to the new genetic
profiles.
3. There can be negative side effects that
are unexpected.
4. The amount of diversity developed
can be less favorable.
5.Copyrighted genetic engineering can
have costly consequences.
6. This knowledge and technology can
be easily abused.
 CRISPR-Cas9
The new gene editing tool

https://www.youtube.com/watch?v=2pp17E4E-
O8
CRISPR (/ˈkrɪspər/) (clustered regularly interspaced short
palindromic repeats) is a family of DNA sequences found within
the genomes of prokaryotic organisms that identify and destroy
viral DNA. These sequences play a key role in the antiviral defence
system of prokaryotes.

Cas9 (or "CRISPR-associated 9") is an enzyme that uses CRISPR

sequences as a guide to recognize and cleave specific strands of
DNA that are complementary to the CRISPR sequence.

Cas9 enzymes together with CRISPR sequences form the basis of a

technology known as CRISPR/Cas9 that can be used to edit genes
within organisms.
 https://wordwall.net/resource/12578794/geneti
c-engineering
Evaluation
Non-interactive version