1

Genetic engineering is the process of altering the genetic makeup of an

organism. Traditionally, humans have manipulated genomes indirectly by
controlling breeding and selecting offspring with desired traits. Genetic
engineering involves the direct manipulation of one or more genes.

2
• Recombinant DNA: a gene from another
species is added to an organism's genome to
give it a desired phenotype. The main aim is
to produce new genetic combinations that
are of value to science, medicine, agriculture
and industry. It’s carried out on bacteria and
on plant cells.
• Hybridoma: production of antibodies.
• Transgenic animals: A transgenic animal is
one whose genome has been changed to
carry genes from another species.
• Gene therapy: a normal gene is introduced
into cells to compensate for an abnormal one
in order to treat/prevent a disease. It’s
carried out on mammalian cells.
• Gene editing: the introduction of a desired
change to the sequence of genomic DNA of a
living organism.

3
4
 Recombinant DNA Technology
rDNA technology is a major arm of genetic engineering which has been applied
to the joining together of DNA molecules from different organisms and species
and inserting it into a host organism to produce new genetic combinations that
are of value to science, medicine, agriculture and industry.

5
Bacterial cell

6
Bacterial cell

7
A plasmid is a small circular DNA
molecule found in bacteria. Plasmids
are physically separate from
chromosomal DNA and replicate
independently. They typically have a
small number of genes — notably,
some associated with antibiotic
resistance — and can be passed from
one cell to another.

8
 Recombinant DNA Technology
Recombinant DNA technology comprises altering genetic material outside an
organism to obtain enhanced and desired characteristics in living organisms or as
their products. This technology involves the insertion in procaryotic cells of DNA
fragments from a variety of sources, having a desirable gene
sequence via manipulation of a plasmid.

9
 DNA cloning
DNA cloning allows to create and amplify new DNA molecules by isolating
and recombining DNA fragments of different origin.

Example: isolation of a gene sequence from an eukaryotic genome and

insertion into a DNA plasmid that allows its replication and / or autonomous
expression within prokaryotic cells.

Its implementation requires the

ability to:
• Fragment DNA.
• Separate DNA fragments.
• Isolate and amplify fragment.
• Recombine them together to
create new molecules.

10
In 1972 Boyer at the University of California in collaboration with Cohen of Stanford
University for the first time inserted a rDNA into bacteria in such a way that the
foreign DNA would replicate naturally.

11
12
 Main steps
Central to the technology are the following manipulations:

1. Cleavage of DNA at specific sites for the isolation of the gene of interest.
2. Amplification of the gene.
3. Insertion of the gene into a DNA cloning vector.
4. DNA ligation, which makes it possible to seamlessly join together DNA molecules
from widely different sources.
5. rDNA tranfer in host cell.

13
 Restriction enzymes
How could a gene be separated from all the others?
The solution to this problem began to emerge with the discovery of restriction nucleases.
These enzymes, which are purified from bacteria, cut the DNA double helix at specific sites
defined by the local nucleotide sequence, thereby cleaving a long, double-stranded DNA
molecule into fragments of strictly defined sizes. The bacterium’s own DNA is protected from
cleavage by methylation of these same sequences, thereby protecting a bacterium’s own
genome from being overrun by foreign DNA.

14
 Restriction enzymes
Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA
• Endonuclease: cleave nt in the middle of DNA molecules.
• Exonuclease: cleave nt from the end of DNA molecule

15
 Restriction nucleases
Restriction nucleases cleave DNA at specific nucleotide sequences.
Restriction nucleases are usually obtained from bacteria, and their names reflect their
origins: for example, the enzyme EcoRI comes from Escherichia coli. There are
currently hundreds of different restriction enzymes available; they can be ordered
from companies that commercially produce them.

16
 Restriction enzymes
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.

EcoRI is a common restriction enzyme used in labs. EcoRI cuts at the following site:

When EcoRI recognizes and cuts this site, it always does so in a very specific pattern that
produces ends with single-stranded DNA “overhangs”:

Enzymes that leave single-stranded overhangs are

said to produce sticky ends.
17
 Restriction enzymes
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:

18
Gel Electrophoresis Separates DNA Molecules of
Different Sizes

Larger DNA fragments will

migrate more slowly because
their progress is impeded to a
greater extent by the gel matrix.
Over several hours, the DNA
fragments become spread out
across the gel according to size,
forming a ladder of discrete
bands, each composed of a
collection of DNA molecules of
identical length

19
20
 Main steps
Central to the technology are the following manipulations:

1. Cleavage of DNA at specific sites for the isolation of the gene of interest.
2. Amplification of the gene.
3. Insertion of the gene into a DNA cloning vector.
4. DNA ligation, which makes it possible to seamlessly join together DNA molecules
from widely different sources.
5. rDNA tranfer in host cell.

21
Polymerase chain reaction

22
23
24
 Main steps
Central to the technology are the following manipulations:

1. Cleavage of DNA at specific sites for the isolation of the gene of interest.
2. Amplification of the gene.
3. Insertion of the gene into a DNA cloning vector.
4. DNA ligation, which makes it possible to seamlessly join together DNA molecules
from widely different sources.
5. rDNA tranfer in host cell.

25
 Vector
The selected genes is inserted into a plasmid , creating a so-called recombinant plasmid. This
plasmid can be introduced into a bacterium by way of the process called transformation. Then,
because bacteria divide rapidly, they can be used as factories to copy DNA fragments in large
quantities.

26
27
 Recombinant plasmid

To insert a DNA fragment into a plasmid, both the fragment and the circular plasmid are
cut using a restriction enzyme that produces compatible ends.

28
 Main steps
Central to the technology are the following manipulations:

1. Cleavage of DNA at specific sites for the isolation of the gene of interest.
2. Amplification of the gene.
3. Insertion of the gene into a DNA cloning vector.
4. DNA ligation, which makes it possible to seamlessly join together DNA molecules
from widely different sources.
5. rDNA tranfer in host cell.

29
DNA ligase

30
 DNA ligase
DNA ligase can join together any two DNA fragments in vitro to produce
recombinant DNA molecules.

31
32
 Main steps
Central to the technology are the following manipulations:

1. Cleavage of DNA at specific sites for the isolation of the gene of interest.
2. Amplification of the gene.
3. Insertion of the gene into a DNA cloning vector.
4. DNA ligation, which makes it possible to seamlessly join together DNA molecules
from widely different sources.
5. rDNA tranfer in host cell.

33
A DNA fragment can be replicated inside a bacterial cell.

34
Overview of chemical approaches for gene delivery to cells.

35
Overview of Physical approaches for gene delivery to cells.

36
 Human genomic libraries containing
DNA fragments that represent the
whole human genome can be
constructed using restriction nucleases
and DNA ligase.

37
• DNA is extracted from the donor
organism, it is enzymatically split
(restriction enzymes) to isolate the
gene of interest.
• the isolated gene is amplified (PCR)
and conjugated to another DNA entity
(plasmid) forming a new recombined
DNA molecule.
• the construct is transferred and kept
inside a host cell (bacterial cell).

38
 Recombinant insulin
Porcine and bovine pancreatic tissue was the source of the hormone for many years,
followed by semisynthetic human insulin obtained by modification of animal insulin. With
the development of recombinant DNA technology, recombinant human insulin became
available in large amounts by biosynthesis in microorganisms (bacteria) providing reliable
supplies of the hormone worldwide at affordable prices.

39
Biosysnthetic proteins

40
Recombinant DNA techniques make it possible to move experimentally from gene
to protein and from protein to gene.
Recombinant DNA in Agriculture

42
Recombinant DNA in Agriculture

43
 Recombinant DNA in Agriculture
The crops are often grown extensively and in monoculture. Increased risk of epidemic diseases.

The toxins produced by Bacillus thuringiensis (spore-forming bacterium) are harmless to humans but
are capable of damaging the larvae of Diptera and Lepidoptera. Bacterial spores or toxin isolates are
already available as organic insecticides.

Some crops (cotton, corn, potato, etc.) have been

transformed with different genes that produce
active toxins against some insects.

44
Golden rice is a genetically modified, biofortified crop. Biofortification increases the nutritional value
in crops. Golden rice is genetically modified in order to produce beta carotene, which is not normally
produced in rice. Beta carotene is convereted into Vitamin A when metabolized by the human
body. Vitamin A deficiency (VAD) is prevalent in developing countries whose diets are dependent on
rice or other micronutrient-poor carbohydrate foods, which do not contain vitamin A. The World
Health Organization estimates that about 250 million preschool children are affected by VAD and
about 2.7 million children die because of the deficiency.

Genes responsible for the synthesis of beta-carotene precursors have been isolated from narcissus
45
and introduced into rice.
 Edible vaccines
Edible vaccines are obtained by incorporating a particular gene of interest into the
plant, which produces the desirable encoded protein.

Edible vaccines differ from

traditional vaccines in the way
they stimulate the production of
antibodies in the recipient which,
in the case of edible vaccines,
occurs during digestion.
47
48