Download Genetic Engineering

Genetic Engineering
An Overview
What is it???
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Applied techniques of genetics and
biotechnology (“Wet lab procedure”). Much
trial and error.
Involves the “isolation, manipulation and
reintroduction of DNA into cells or model
organisms, usually to express a protein’’.
DNA taken from one organism and inserted
(transformed) into another (transgenic)
organism
Heritable, directed alteration of an
organism. Altering DNA or adding new DNA
allows us to change the characteristics of a
cell or cells.
Why do it?
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It aims to introduce new
characteristics or attributes
physiologically or physically, such as
making a crop resistant to a herbicide,
introducing a novel trait, or producing
a new protein or enzyme.
For example…
How does it work?
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Detect gene within organism of interest
Isolate and “Splice out” DNA
“Ligate” into “Vector” of choice
Introduce into bacteria
Isolate colonies containing vector with gene
of interest
Grow up bacteria colony to produce multiple
copies of gene which is expressed
Isolating and splicing DNA of
Interest…
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Conventionally entire genome
fragmented using “Restiction
Enzymes”
– E.g. BamH1
Hindiii
– Target Palindromic sequences:
AGGTACCT
TCCATGGA
– Can result in “Staggered”/”Sticky” ends
AG
TCCATG
GTACCT
GA
– Can result in “Blunt” ends
AGGT
TCCA
ACCT
TGGA
– Enzymes can create sticky ends from blunt ones
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PCR can be used to specifically target
gene of interest
– “Enzymatic amplification of specific DNA
fragment using repeated cycles of DNA
denaturation, primer annealing and
Chain extension”
– Knowledge of full sequence not required
– Can produce large amount of copies from
>minute quantity of target DNA
>Partially damaged DNA
Inserting Gene fragments
into Vector of choice
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Many types of Vectors depending on
needs
– Bacterial Artificial Chromosomes
– Yeast Artificial Chromosomes
– Plasmids
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Use RE to create complimentary sticky
ends in the vector
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Possible outcomes post digestion with
R.E.
– Plasmid reforms
– Fragmented genomic DNA joins up with
itself
– Plasmid and fragmented DNA for a hybrid
Contains gene of interest
 Contains other gene (unfortunately, most
times!!!)
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Insert into Bacteria/Host
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Allows expression of protein product
Allows multiple copies of the gene to
be made
Several methods
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Heat shock
Electric Pulse (electroporation)
Microinjection/microprojectiles
Viruses
Lysosomes
Identifying the right
colony
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3 levels of identification:
– Bacteria which have taken up a plasmid
– Bacteria which have taken up a
recombinant plasmid
– Bacteria containing the wanted gene
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Achieved by using antibiotic resistence
genes and gene probes.
Finally…
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Once the correct colony is identified it
is cultured and gene expression
encouraged.
Protein product can be harvested!
However… it is not always successful…
Much patience is required!!!
Some examples of current
applications of genetic
engineering
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Synthesis of insulin. Not always
effective due to a eukaryotic protein
being expressed in a prokaryotic cell.
Introduction of resistance genes in
crop plants
Flavr Savr Tomato
Gene therapy (e.g. Cystic Fibrosis)