This is a presentation that covers on Module 2 Aspects of Genetic Engineering from the syllabus. For Cape biology Unit 1, students I hope this presentation is helpful and clear for you to get an idea of how to cut and paste DNA, The process of Recombinant Data Technology etc.

1. a s p e c t s o f
G e n e t i c
e n g i n e e r i n g
GROUP 2

2. introduction
Genetic engineering is a field of biotechnology that involves the manipulation of an
organism's genetic material, often to introduce or enhance desirable traits. This powerful
and rapidly advancing technology has numerous applications across various sectors,
including medicine, agriculture, and industry. Here are key aspects of genetic engineering:
DNA Manipulation
1.
Recombinant DNA Technology
2.
Gene Cloning
3.
Genetic Modification in Agriculture:
4.
Medical Applications
5.
Gene Therapy
6.
Ethical and Social Implications
7.
CRISPR-Cas9
8.
Understanding the various aspects of genetic engineering is essential for navigating the
ethical, scientific, and societal challenges associated with this powerful technology. As
genetic engineering continues to evolve, ongoing discussions and regulations are crucial to
ensuring responsible and beneficial use of these techniques.

3. VECTORS
A vector, as related to molecular biology, is a DNA molecule (often plasmid or
virus) that is used as a vehicle to carry a particular DNA segment into a host
cell as part of a cloning or recombinant DNA technique
DEFINITION
RESTRICTION ENZYMES
PLASMIDS
Restriction enzymes are molecular scissors used in molecular biology for
cutting DNA sequences at a specific site. It plays an important role in
gene manipulation.
Plasmid is an extra-chromosomal DNA molecule in bacteria that is capable of
replicating, independent of chromosomal DNA. They serve as a vehicle to carry a
foreign DNA sequence into a given host cell.

4. RESTRICTION
ENZYMES
01

5. KEY POINTS: Restriction enzymes are
DNA-cutting enzymes. Each
enzyme recognizes one or a few
target sequences and cuts DNA
at or near those sequences.
Many restriction enzymes make
staggered cuts, producing ends
with single-stranded DNA
overhangs. However, some
produce blunt ends.
DNA ligase is a DNA-joining
enzyme. If two pieces of DNA
have matching ends, ligase can
link them to form a single,
unbroken molecule of DNA.
In DNA cloning, restriction
enzymes and DNA ligase are used
to insert genes and other pieces
of DNA into plasmids.

6. In DNA cloning, researchers make many copies of
a piece of DNA, such as a gene. In many cases,
cloning involves inserting the gene into a piece
of circular DNA called a plasmid, which can be
copied in bacteria.
HOW DO YOU CUT
AND PASTE DNA?
How can pieces of DNA from different sources
(such as a human gene and a bacterial plasmid)
be joined together to make a single DNA
molecule? One common method is based on
restriction enzymes and DNA ligase.

7. A restriction enzyme is a DNA-cutting enzyme
that recognizes specific sites in DNA. Many
restriction enzymes make staggered cuts at or
near their recognition sites, producing ends with
a single-stranded overhang.
If two DNA molecules have matching ends, they
can be joined by the enzyme DNA ligase. DNA
ligase seals the gap between the molecules,
forming a single piece of DNA.
Restriction enzymes and DNA ligase are often
used to insert genes and other pieces of DNA into
plasmids during DNA cloning.
01
02

8. Restriction enzymes are found in bacteria (and other prokaryotes). They
recognize and bind to specific sequences of DNA, called restriction sites.
Each restriction enzyme recognizes just one or a few restriction sites.
When it finds its target sequence, a restriction enzyme will make a
double-stranded cut in the DNA molecule. Typically, the cut is at or near
the restriction site and occurs in a tidy, predictable pattern.
RESTRICTION ENZYMES

9. As an example of how a restriction enzyme recognizes and cuts at a DNA sequence, let's consider
EcoRI, a common restriction enzyme used in labs. EcoRI cuts at the following site:
EXAMPLE
When EcoRI recognizes and cuts this site, it always does so in a very specific pattern that produces
ends with single-stranded DNA “overhangs”:

10. If another piece of DNA has matching overhangs (for instance, because it has also been cut by
EcoRI), the overhangs can stick together by complementary base pairing. For this reason, enzymes
that leave single-stranded overhangs are said to produce sticky ends. Sticky ends are helpful in
cloning because they hold two pieces of DNA together so they can be linked by DNA ligase.

11. Not all restriction enzymes produce sticky ends. Some are
“blunt cutters,” which cut straight down the middle of a
target sequence and leave no overhang. The restriction
enzyme SmaI is an example of a blunt cutter:
Blunt-ended fragments can be joined to each other by DNA ligase. However,
blunt-ended fragments are harder to ligate together (the ligation reaction is less
efficient and more likely to fail) because there are no single-stranded overhangs
to hold the DNA molecules in position.

12. DNA LIGASE
If you’ve learned about DNA replication, you may already have met DNA
ligase. In DNA replication, ligase’s job is to join together fragments of
newly synthesized DNA to form a seamless strand. The ligases used in
DNA cloning do basically the same thing. If two pieces of DNA have
matching ends, DNA ligase can join them together to make an unbroken
molecule.
How does DNA ligase do this? Using ATP as an energy source, ligase
catalyzes a reaction in which the phosphate group sticking off the 5’ end of
one DNA strand is linked to the hydroxyl group sticking off the 3’ end of
the other. This reaction produces an intact sugar-phosphate backbone.

13. RECOMBINANT DNA
TECHNOLOGY
02

14. The first and the initial step in Recombinant DNA technology is
to isolate the desired DNA in its pure form i.e. free from other
macromolecules.
ISOLATION OF
GENETIC MATERIAL.
CUTTING THE GENE
AT THE RECOGNITION
SITES.
The restriction enzymes play a major role in determining the
location at which the desired gene is inserted into the vector
genome. These reactions are called ‘restriction enzyme
digestions’.
01
02
PROCESS OF
RECOMBINANT DNA
TECHNOLOGY

15. It is a process to amplify a single copy of DNA
into thousands to millions of copies once the
proper gene of interest has been cut using
restriction enzymes.
AMPLIFYING THE GENE
COPIES THROUGH
POLYMERASE CHAIN
REACTION (PCR).
LIGATION OF DNA
MOLECULES.
In this step of Ligation, the joining of the two
pieces – a cut fragment of DNA and the vector
together with the help of the enzyme DNA ligase.
03
04

16. In this step, the recombinant DNA is introduced
into a recipient host cell. This process is termed as
Transformation. Once the recombinant DNA is inserted into the
host cell, it gets multiplied and is expressed in the form of the
manufactured protein under optimal conditions.
NSERTION OF
RECOMBINANT DNA
INTO HOST.
As mentioned in Tools of recombinant DNA
technology, there are various ways in which this
can be achieved. The effectively transformed
cells/organisms carry forward the recombinant
gene to the offspring.
05

17. Insulin is synthesized as a single polypeptide known as preproinsulin in
pancreatic beta cells and plays a key role in regulating carbohydrate
amd fat metabolism in the body.
PRODUCTION OF INSULIN

18. DNA CLONING
03

19. KEY TERMS DNA cloning is a molecular
biology technique that makes
many identical copies of a piece
of DNA, such as a gene.
In a typical cloning experiment,
a target gene is inserted into a
circular piece of DNA called a
plasmid.
The plasmid is introduced into
bacteria via a process called
transformation, and bacteria
carrying the plasmid are
selected using antibiotics.
Bacteria with the correct
plasmid are used to make more
plasmid DNA or, in some cases,
induced to express the gene and
make protein.

20. DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a
typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a
medically important human protein) is first inserted into a circular piece of DNA called a plasmid.
The insertion is done using enzymes that “cut and paste” DNA, and it produces a molecule of
recombinant DNA, or DNA assembled out of fragments from multiple sources.
Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the plasmid are
selected and grown up. As they reproduce, they replicate the plasmid and pass it on to their
offspring, making copies of the DNA it contains.
OVERVIEW OF DNA CLONING

21. Our goal in cloning is to insert a target gene (e.g., for human insulin) into
a plasmid. Using a carefully chosen restriction enzyme, we digest:
The plasmid, which has a single cut site
The target gene fragment, which has a cut site near each end
Then, we combine the fragments with DNA ligase, which links them to
make a recombinant plasmid containing the gene.
STEPS IN DNA CLONING
01
DNA cloning is used for many purposes. As an example,
let's see how DNA cloning can be used to synthesize a
protein (such as human insulin) in bacteria. The basic
steps are:
CUTTING AND PASTING
DNA

23. Our goal in cloning is to insert a target gene (e.g., for human insulin) into
a plasmid. Using a carefully chosen restriction enzyme, we digest:
The plasmid, which has a single cut site
The target gene fragment, which has a cut site near each end
Then, we combine the fragments with DNA ligase, which links them to
make a recombinant plasmid containing the gene.
STEPS IN DNA CLONING
01
DNA cloning is used for many purposes. As an example,
let's see how DNA cloning can be used to synthesize a
protein (such as human insulin) in bacteria. The basic
steps are:
CUTTING AND PASTING
DNA

24. Plasmids and other DNA can be introduced into bacteria, such as
the harmless E. coli used in labs, in a process called
transformation. During transformation, specially prepared
bacterial cells are given a shock (such as high temperature) that
encourages them to take up foreign DNA.
02 BACTERIAL TRANSFORMATION
AND SELECTION

25. A plasmid typically contains an antibiotic resistance gene, which allows
bacteria to survive in the presence of a specific antibiotic. Thus,
bacteria that took up the plasmid can be selected on nutrient plates
containing the antibiotic. Bacteria without a plasmid will die, while
bacteria carrying a plasmid can live and reproduce. Each surviving
bacterium will give rise to a small, dot-like group, or colony, of
identical bacteria that all carry the same plasmid.
02 BACTERIAL TRANSFORMATION
AND SELECTION

26. Not all colonies will necessarily contain the right plasmid. That’s
because, during a ligation, DNA fragments don’t always get “pasted”
in exactly the way we intend. Instead, we must collect DNA from
several colonies and see whether each one contain the right plasmid.
Methods like restriction enzyme digestion and PCR are commonly
used to check the plasmids.
BACTERIAL TRANSFORMATION
AND SELECTION
02

27. Once we have found a bacterial colony with the right plasmid, we can grow a
large culture of plasmid-bearing bacteria. Then, we give the bacteria a
chemical signal that instructs them to make the target protein.
The bacteria serve as miniature “factories," churning out large amounts of
protein. For instance, if our plasmid contained the human insulin gene, the
bacteria would start transcribing the gene and translating the mRNA to
produce many molecules of human insulin protein.
03
PROTEIN PRODUCTION

28. Once the protein has been produced, the bacterial cells can be split
open to release it. There are many other proteins and macromolecules
floating around in bacteria besides the target protein (e.g., insulin).
Because of this, the target protein must be purified, or separated from
the other contents of the cells by biochemical techniques. The purified
protein can be used for experiments or, in the case of insulin,
administered to patients.
03
PROTEIN PRODUCTION

29. USES OF DNA CLONING
BIOPHARMACEUTICALS.
DNA cloning can be used to make human proteins with biomedical applications,
such as the insulin mentioned above. Other examples of recombinant proteins
include human growth hormone, which is given to patients who are unable to
synthesize the hormone, and tissue plasminogen activator (tPA), which is used to
treat strokes and prevent blood clots. Recombinant proteins like these are often
made in bacteria.
LDNA molecules built through cloning techniques are used for many purposes in
molecular biology. A short list of examples includes:

30. GENE ANALYSIS
In basic research labs, biologists often use DNA cloning to build artificial,
recombinant versions of genes that help them understand how normal
genes in an organism function.
USES OF DNA CLONING
In some genetic disorders, patients lack the functional form of a particular gene.
Gene therapy attempts to provide a normal copy of the gene to the cells of a
patient’s body. For example, DNA cloning was used to build plasmids containing a
normal version of the gene that's nonfunctional in cystic fibrosis. When the
plasmids were delivered to the lungs of cystic fibrosis patients, lung function
deteriorated less quickly
GENE THERAPY

31. GENOME EDITING WITH CRISPR-CAS9

32. REFERENCE
KHAN ACADEMY.RESTRICTION ENZYMES & DNA LIGASE
KHAN ACADEMY. OVERVIEW: DNA CLONING
BYJUS. RECOMBINANT DNA TECHNOLOGY
https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-
tutorial/a/restriction-enzymes-dna-ligase
https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-
cloning-tutorial/a/overview-dna-cloning
https://byjus.com/biology/recombinant-dna-technology/
MCGOVERN INSTITUTE (5 NOV 2014). GENOME EDITING WITH CRISPR-CAS9
https://youtu.be/2pp17E4E-O8?si=-iSk8xlI0khZDiHw

33. THANK YOU!
THIS PRESENTATION WAS DONE BY:
DANESHA NEWMAN
ANNA-LISA DIXON
JASMINE DANVERS