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Contents
• Introduction
• Tools of a genetic engineer
• Enzymes
• Vectors
• Genetic Markers
• Gene transfer techniques
• Cloning
• Bioinformatics
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Genetic Engineering
• The aim of genetic engineering is to remove a gene (or genes) from one
organism and transfer it into another so that the gene is expressed.
• The DNA that has been altered by this process and which now contains lengths
of nucleotides from two different organisms is called recombinant DNA
(rDNA).
• The organism which now expresses the new gene or genes is known as a
transgenic organism or a genetically modified organism (GMO); the DNA may
be from another species or from an organism of the same species.
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Genetic Engineering
• Genetic engineering provides a way of overcoming barriers to gene transfer
between species.
• Genes are often taken from an organism in a different domain or kingdom,
such as a bacterial gene inserted into a plant or a human gene inserted into a
bacterium.
• Unlike selective breeding, where whole sets of genes are involved, genetic
engineering often results in the transfer of a single gene.
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Toolkit of a genetic engineer
1) Enzymes
2) Vectors
3) Genetic markers
4) DNA transfer techniques
1) Enzymes – Restriction Endonucleases
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• A class of enzymes from bacteria which

recognise and break down the DNA of
invading viruses known as bacteriophages.
• Bacteria make enzymes that cut phage DNA
into smaller pieces.
• REs cut the sugar-phosphate backbone of
DNA at specific places within the molecule
hence they are known as endonucleases.
• Their role in bacteria is to restrict a viral
infection, hence the name restriction
endonuclease or restriction enzyme.
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1) Enzymes - RE contd.
• Each restriction enzyme binds to a specific target site on DNA and cuts at that
site.
• Bacterial DNA is protected from such an attack either by chemical markers or
by not having the target sites.
• Many, but not all, restriction sites are palindromic.
• Restriction enzymes either cut straight across the sugar-phosphate backbone to
give blunt ends or they cut in a staggered fashion to give sticky ends.
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1) Enzymes - RE contd.
• These target sites, or restriction sites, are specific sequences of bases.
• Ex: - The restriction enzyme BamHI always cuts DNA where there is a
GGATCC sequence on one strand and its complementary sequence,
CCTAGG, on the other. As the sequence reads the same in both directions:
it is a palindrome.
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1) Enzymes - RE contd.
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1) Enzymes - RE contd.
• Sticky ends are short lengths of unpaired
bases.
• They are known as sticky ends because
they can easily form hydrogen bonds with
complementary sequences of bases on
other pieces of DNA cut with the same
restriction enzyme.
• Sticky ends are quite useful for cloning
techniques.
1) Enzymes – Ligase
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• 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.
1) Enzymes – Ligase contd.
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• Using ATP as an energy source, ligase catalyses 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.
1) Enzymes – Reverse Transcriptase
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• Reverse transcription is the synthesis of DNA from

an RNA template.
• RNA‐dependent DNA polymerase (reverse
transcriptase) is involved in this process.
• Like other DNA polymerases, reverse transcriptase
are primer and template‐dependent.
• Reverse transcriptase degrade the RNA template
after it uses for synthesis of the first DNA strand.
• The enzyme then can copy the first strand of DNA
to make a double‐stranded molecule.
1) Enzymes – Reverse Transcriptase
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• However, reverse transcription is error prone relative to DNA replication

because reverse transcriptase don't have an editing (3′‐5′) exonucleolytic
activity.
• In gene cloning, RT is used to generate cDNA of genes from isolated
mRNA strands from specific tissues
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2) Vectors
• Vectors are DNA molecules that accept DNA fragments and replicate
inserted DNA fragments when vectors are placed into host cells.
• Many different vectors are available for cloning.
• Vectors differ in terms of the host cells they are able to enter and in the size
of inserts they can carry, but most DNA vectors have several key properties.
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Ideal characteristics of a Vector
1) A vector contains several restriction sites that allow insertion of the DNA
fragments to be cloned.

2) Vectors must be introduced into host cells to allow for independent

replication of the vector DNA and any DNA fragment it carries.

3) To distinguish host cells that have taken up vectors from host cells that have
not, the vector should carry a selectable marker gene (usually an antibiotic
resistance gene) or the gene for an enzyme absent from the host cell).
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Ideal characteristics of a Vector
4) The vector and its inserted DNA fragment should be easy to isolate from
the host cell to recover cloned DNA for different applications.
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a) Plasmids
• Small circular pieces of double-stranded DNA.

• Plasmids occur naturally in bacteria and often contain genes for antibiotic
resistance.

• They can be exchanged between bacteria - even between different species

of bacteria.
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a) Plasmids contd.
• If a genetic engineer inserts a piece of DNA
into a plasmid, then the plasmid can be used
to take the DNA into a bacterial cell.
• To get the plasmids, the bacteria containing
them are treated with enzymes to break
down their cell walls.
• The ‘naked’ bacteria are then spun at high
speed in a centrifuge, so that the relatively
large bacterial chromosomes are separated
from the much smaller plasmids.
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2)Bacteriophages
• Phage vector systems were among the earliest
vectors used in addition to plasmids.
• These included genetically modified strains of λ
phage.
• The genome of λ phage, a virus that infects E. coli,
has been completely mapped and sequenced, and
the λ phage genome has been modified to
incorporate many of the important features of
cloning vectors.
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2)Bacteriophages contd.
• Phage vectors can carry inserts up to 45 kb,
more than twice as long as DNA inserts in
most plasmid vectors.
• DNA fragments are ligated into the phage
vector to produce recombinant λ vectors that
are subsequently packaged into phage
protein heads in vitro and introduced into
bacterial host cells growing on petri plates.
• Inside the bacteria, the vectors replicate and
form many copies of infective phage, each of
which carries a DNA insert.
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2)Bacteriophages contd.
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3) Genetic markers
• The processes required for successful cloning of DNA happen at a relatively
low frequency, and identifying these events in an efficient manner requires a
method for selecting the desired events from undesired events.
• Ideally the selection process will either prevent the growth of host cells that
do not contain the desired vectors with inserted DNA or allow easy
visualization of the cells that do contain the desired recombinant DNA.
• An essential component of cloning vectors is a gene that facilitates this
process either by providing the cell with resistance to an antibiotic or
allowing the cell to produce an essential nutrient missing from the growth
medium.
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3) Genetic Markers - ARG
Antibiotic resistance genes
• Antibiotic resistance genes produce proteins that allow cells containing these
genes to grow on a toxic substance.
• Following transformation, only the cells that have taken up a vector
encoding antibiotic resistance will be able to grow on media supplemented
with that antibiotic.
• Antibiotics such as ampicillin, tetracycline, kanamycin, and streptomycin are
commonly used to select for transformed cells and to demonstrate the
construction of recombinant DNA plasmids.
3) Genetic Markers – ARG contd
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Antibiotic resistance genes

• Vectors that contain two independent antibiotic resistance genes with
different cloning sites inside each of these genes allow for both selection of
transformed host cells and identification of specific clones containing insert
DNA.
• In recombinant DNA molecules, the insert interrupts expression of that gene
making the host sensitive to that antibiotic, called insertional inactivation.
3) Genetic Markers – ARG contd
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Antibiotic resistance genes

• For example, the cloning vector pBR322
contains genes that confer resistance to
both ampicillin and tetracycline.
• Host cells transformed with pBR322 with
insert DNA cloned into a restriction site in
the tetracycline resistance gene are
resistant to ampicillin but are killed by
tetracycline.
• Cells containing the plasmid with no insert
are able to grow in the presence of both
antibiotics.
3) Genetic Markers – ARG contd
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Replica Plating
• Replica plating involves growing identical patterns of bacterial colonies on
agar plates (Petri dishes) with different media.
• It allows in identifying colonies that cannot survive without a particular nutrient.
3) Genetic Markers – ARG contd
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Replica Plating
3) Genetic Markers – ARG contd
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Replica Plating
Grow Bacteria
on Ampicillin
Medium

Colonies are
visible of
bacteria who
are resistance
to Amp

Ampicillin Medium Tetracyclin Medium

Transformed bacteria cannot

grow on Tetracycline medium
Colonies will be selected from the
Ampicillin medium who shows tetracycline
sensitivity
3) Genetic Markers – Colorimetric
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Colorimetric selection (Blue-white colony selection)

• Colorimetric selection is a more efficient and more commonly used
procedure for identifying clones with recombinant DNA.
• Blue-white selection uses plasmid vectors containing a portion of the lacZ
gene to produce an active β-galactosidase enzyme in host cells.
• The activity of this enzyme produces a blue product from the colourless
substrate X-Gal.
• Introduction of insert DNA into the cloning site in the lacZ gene inactivates
the enzyme, producing white colonies
3) Genetic Markers – Colorimetric
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Colorimetric selection (Blue-white colony selection)

3) Genetic Markers – Fluorescence
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Fluorescence
• Because of the risk of creating pathogenic antibiotic resistant bacteria, there
is now much less use of antibiotic resistance gene
• Current method uses enzymes that produce fluorescent substances.
• For example, enzymes obtained from jellyfish make a protein called GFP
(green fluorescent protein) that fluoresces bright green in ultraviolet light.
3) Genetic Markers – Fluorescence
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Fluorescence
• The gene for the enzyme is inserted into the plasmids.
• So all that needs to be done to identify the bacteria that have taken up the
plasmid is to shine ultraviolet light onto them.
• The ones that glow green are the genetically modified ones.
• The same marker gene can be used in a range of organisms.
3) Genetic Markers – Fluorescence
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Fluorescence
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4) Gene delivery systems
a) Transformation
• Transformation is the uptake of genetic material from the environment by
bacterial cells.
• It’s a very simple method and works for yeast as well.
• In nature, this genetic material often comes from adjacent lysed bacteria
and can include plasmid DNA or fragmented DNA released into the
environment.
• Though not all bacteria are naturally competent to take up DNA, they can
be made competent through chemical manipulation in the lab.
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4) Gene delivery systems
a) Transformation
• This is commonly done using calcium chloride which permeabilizes the cell
membrane so the bacteria can easily uptake your plasmid of interest.
• Scientists can also use electroporation, the application of an electrical
charge to cells, to increase cell membrane permeability and thus
transformation efficiency
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4) Gene delivery systems
a) Transformation
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4) Gene delivery systems
a) Transformation
• As it’s not a very efficient process, making selectable markers on the cloning
vectors is very important.
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4) Gene delivery systems
b) Viral vectors - Transduction
• The insertion of a viral vector into a cell
is called transduction and utilise the
ability of a virus to inject its DNA into a
host cell.
• Viruses integrate their DNA directly into
the host genome, which can have both
beneficial and detrimental
consequences.
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4) Gene delivery systems
b) Viral vectors - Transduction
• Harmless viruses can be engineered to carry a desirable gene and then
used to infect an animal’s cells thus introducing the desirable DNA.
• Viral vectors are mostly used in gene therapy to introduce modified genes
into the host organism.
• However, viruses that are used in genetic engineering can cause an
immune response in some people in the same way that pathogenic viruses
stimulate an immune response.
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4) Gene delivery systems
c) Liposomal wrapping
• Gene to be inserted is wrapped
in liposomes (spheres formed
from a lipid bilayer).
• The liposomes fuse with the
target cell membrane and can
pass through it to deliver the
DNA into the cytoplasm.
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4) Gene delivery systems
c) Liposomal wrapping
• Even though liposome wrapping is not as effective at transferring the DNA
as viral vectors (1 in every 1000 genes that enter a cell in a liposome enter
the nucleus to be transcribed), fewer side-effects and less immune responses
makes the technique to be used in gene therapy.
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4) Gene delivery systems
d) Microinjection
• Introducing DNA by injecting it into
a cell through a very fine
micropipette.
• This is manipulated using a
micromanipulator, because even the
steadiest hand would tremble enough
to destroy the cell.
• The method is not very efficient
because many cells have to be
injected before one accepts the DNA
successfully.
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4) Gene delivery systems
e) Gene guns
• Used to shoot DNA carried on very small gold or tungsten pellets (balls) into
the cell at high speed.
• Some cells survive this treatment and accept the DNA as part of the genetic
material.
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Cloning