Genetic Engineering

GENETIC ENGINEERING
RECOMBINANT DNA
TECHNOLOGY
DEFINITION
• Genetic engineering, also known as
recombinant DNA technology, refers
to altering the genes in a living
organism to produce a Genetically
Modified Organism (GMO) with a new
genotype.
Genetic Modification
• GMOs may be obtained in several ways;
1. Inserting a foreign gene from one species
into another. This creates a transgenic
organism.
2. Altering an existing gene and thereby
changing its product.
3. Altering the gene expression so that it may
be translated more often or even not
translated at all.
Why Genetic Modification?
1. Making crops resistant to certain diseases and

pests
2. Improving nutritional properties.
3. Make bacteria to absorb and metabolise toxins
Steps in Genetic Engineering
1. Isolating the gene of interest

2. Make a copy of it.
3. Inserting this gene into a vector.

4. Introducing the vector into a host cell.
5. Cloning the DNA
What do we need before we can begin these steps to GE?
• RESTRICTION ENDONUCLEASES
– These are enzymes that cut DNA at specific
sequences known as restriction sites.
– The restriction sites contain palindromic
sequences made up of 4 – 8 bases. Each
restriction enzyme has its specific palindromic
restriction site.
(Remember enzymes are specific)
Endo – internally
Nuclease – cuts nucleic acids at specific points.
Palindromic Sequence
Restriction enzymes are derived from specific bacterial
strains and are employed as a defense mechanism for
phage infection.
Danna and Nathan discovered that some bacterial strains

were either resistant or non-resistant to phage infection.
They also observed that periodically a resistant strain of

bacterial could become infected.
Kathleen Danna Daniel Nathan

Bacterial Host Defense:
1) Virusesthat infect bacterial cells are

called bacteriophages. Their main
goal is to produce more
bacteriophages by injecting their
genome into a bacterial host cell,
1) Bacteria will destroy invading

phage DNA by digesting it into
tiny pieces.
2) How is the bacteria DNA

protected?
• PALINDROMES(Palindromic sequences)
– This is a DNA base sequence that reads
the same on both strands in the 5’ to 3’
direction.
The restriction
site on the right
is recognised
by the enzyme
EcoR1
http://medimicro.blogspot.com/2008/01/
genetic-engineering.html
• STICKY ENDS – single stranded
overhangs created when DNA is digested
with certain restriction enzymes.
Overhangs may be on the 5’ or the 3’ of
DNA.
• BLUNT ENDS – the result of DNA being

cut at the same point on both strands.
Restriction Enzymes
• 5’A/AGCTT3’ HindIII
• CGAT/CG3 PvuI
• G/AATTC EcoRI
• G/GATCC BamHI
• TTAAT/TAA PacI
Terms used in GE
• VECTOR
A length of DNA that carries the gene of interest
into the host cell. Several different vectors are
employed in GE.
TYPE OF VECTOR MAX LENGTH OF DNA
INSERT
PLASMID 10kbp
BACTERIOPHAGE /ADENOVIRUSES 30kbp
BACTERIAL ARTIFICIAL 500kbp

CHROMOSOME
Terms used in GE
• A vector:
– Is big enough to hold the gene of interest.
– Is a closed loop (circular) making it less likely
to be broken down.
– Contains control sequences such as ORIs
and promoter sequences.
– Contains a marker gene so that cells
containing the vector can be identified.
• PLASMIDS
– These are circular bits of DNA that occur
naturally in bacteria.
– Plasmids usually contain 3 -5 genes, an ORI,
and multiple cloning sites.
– They are separate from the chromosomal
DNA and they replicate independently when
the cell divides.
– Ideal vectors are readily taken into most
bacterial cells.
• BACTERIOPHAGE
viruses that can
infect bacteria by
injecting their DNA
into them. They are
effective in delivering
large genes.
• RECOMBINANT DNA (rDNA)
This is DNA that is formed from a combination
of DNA from two different sources.
STEPS IN GENETIC
ENGINEERING
Isolating the gene of interest
This can be done in either of three ways.
1. cDNA – complementary DNA is made from
mRNA. The enzyme reverse transcriptase is
used to make an artificial gene of cDNA.
2. Shotgun approach – This involves cutting
the DNA with restriction enzymes.
3. Gene synthesis – The primary structure of
the protein is determined and this knowledge
is used to synthesis DNA.
Inserting this gene into a vector
Using a plasmid as the vector.
1. The DNA containing the gene of interest is
cut with a restriction enzyme.
2. The plasmid is cut with the same restriction
enzyme.
3. The two are mixed, the genomic DNA is the
incorporated into the plasmid.
4. DNA ligase is added, it joins the DNA
together forming rDNA.
Introducing the vector into a host cell
(Transformation)
• Vectors containing the genes must be
incorporated into living cells(host cells).
Cells that have successfully incorporated
the vector are said to be transformed.
• Transformation can be done in several
different ways, depending on the type of
host cells.
Transformation
• Heat shock – cells are incubated with the
vector in solution containing calcium ions
at 0 degrees Celsius. The temperature is
then suddenly raised to about 40 degrees.
This heat shock causes some of the cells
to accept the vector. This works well on
bacteria and animal cells.
Transformation
• Electroporation – Cells are subjected to a
high voltage pulse, which temporarily
disrupts the membrane and allows the
vector to enter the cell. This is the most
efficient method for transforming bacterial
cells.
Transformation
• Viruses – Bacteriophages, adenoviruses
and retroviruses are used as vectors.
– Bacteriophages can deliver large genes into
bacteria cells in culture
– Adenoviruses (such as the common cold) are
used to deliver genes to living patients in
gene therapy.
– Retroviruses insert the gene into the host
cells genome.
Selecting Transformed cells
Selection for transformed bacteria and bacteria containing
plasmids with inserted DNA.
Cloning the DNA
• Markers, contained on the plasmid, make
it possible to identify transformed cells.
E.g. resistance to ampicillin or tetracycline.
• The lacZ gene is a marker that is very
useful because when it is functioning it
creates a blue colony but when it isn’t
functioning the colony is white.
• Transformed cells = white.
Cloning the DNA
• Bacterial cells that have been transformed
are grown in large quantities to make
specific proteins from the gene that have
been inserted.
• In essence the host cell, in dividing
naturally, makes clones of the inserted
gene.
WEBSITES
• http://medimicro.blogspot.com/2008/01/
genetic-engineering.html

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GENETIC ENGINEERING

1. Making crops resistant to certain diseases and

1. Isolating the gene of interest

3. Inserting this gene into a vector.

Danna and Nathan discovered that some bacterial strains

They also observed that periodically a resistant strain of

Kathleen Danna Daniel Nathan

1) Virusesthat infect bacterial cells are

1) Bacteria will destroy invading

2) How is the bacteria DNA

• BLUNT ENDS – the result of DNA being

BACTERIAL ARTIFICIAL 500kbp

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