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Gene technology
- what is it?
- what is it used for?
- how does it work?
How would you treat a diabetes sufferer?
Where does the insulin come from?
Overview
• Genetic engineering involves the transfer
of the DNA of one organism into the DNA
of another.
• Several stages involved:
– Isolating required gene e.g. insulin gene
– Inserting the gene in a ‘vector’.
– Transformation – the gene is delivered into
the required cell for protein growth
– Identification of host cells that have taken up
the gene
– Grow cells with new gene on a large scale.
Stage One:
Isolation of gene
Method 1
Enzyme used: Reverse transcriptase.
- RNA is taken from a cell that produces the
required protein
- The enzyme reverse transcriptase is found in
retroviruses like HIV. It catalyses a reaction in
which complementary DNA (cDNA) is made
from mRNA + DNA nucleotides. The result is
a single strand of cDNA.
- DNA polymerase and free nucleotides are
used to produce a double strand of cDNA.
Isolation of gene
Method 2
Enzyme used: Restriction endonuclease
• Gene can be removed from the chromosome
using restriction enzymes (restriction
endonucleases).
• Different restriction enzymes cut the DNA at a
different base sequence. This is called a
recognition sequence.
• Restriction enzymes are made by bacteria.
They are used to destroy the DNA of
bacteriophages (viruses that infect bacteria).
Sticky ends Vs Blunt ends
• Some endonucleases cut across the
double strand of DNA in a straight line.
This produces ‘blunt ends’:
Sticky ends Vs Blunt ends
• Some endonucleases cut across the
double strand of DNA in a zig zag line.
This produces ‘sticky ends’:
• What do you notice about the sequence?
Splicing a gene
into a vector (in vivo)
Stage Two:
• Insertion of the required gene into the cell
that will make the protein (e.g. a bacterial
cell).
• A vector is a ‘gene carrier’.
• Plasmids from bacteria are
often used.
• Plasmids are short circular DNA strands.
Splicing a gene into a vector
• Plasmid must first be cut using the same restriction
enzyme used to remove the required gene.
• Why?
• To produce complementary sticky ends to the required
gene.
• Cut genes & cut
plasmids are mixed
together.
• Under the right
conditions, the sticky
ends of the gene join
with the sticky ends of
the plasmid.
• A ligase enzyme
catalyses this process
(called ligation)
• Plasmid containing a
human gene is called
recombinant DNA
(rDNA).
Review
1. Which enzymes can be used to isolate gene
fragments? (2 marks)
2. Briefly explain how both of them isolate the
fragments (6 marks)
3. What is commonly used as a ‘gene carrier’? (1
mark)
4. Explain the significance of ‘sticky ends’ in gene
fragments (2 marks)
5. Name the enzyme that helps to join the gene
fragment with the gene carrier (1 mark)
Stage
Three:
Transformation:
Transferring rDNA to host cell
•Difficult
process
– uptake
ofare
plasmid
Host
bacterial
cells containing
rDNA
called
organisms.
bytransgenic
bacterial
cells can be as low as
•0.0025%
Many methods have been tried to introduce
rDNA into the host bacterial cell.
• Successful method:
1. Soak bacteria in ice-cold calcium chloride solution
containing recombinant plasmids
Some
plasmids may have also closed
2. Incubate for 2 mins at 42˚C
up
before they took the gene
3. Bacterial cells take up recombinant plasmids
fragment in.
Stage
Four:
Identification:
Finding the GM bacteria with
the plasmids + new gene
• Some bacteria will have taken up plasmids
that DO NOT contain the desired gene –
why?
• These need to be identified & destroyed
so only the bacteria with the desired gene
are cultured and grown.
• Several options using other useful genes
on the plasmids (gene markers):
– Antibiotic resistance genes
– Genes that make particular enzymes
– Genes that produce fluorescent proteins.
Gene markers –
fluorescent proteins
• Genes that code for fluorescent proteins (e.g.
green fluorescent protein – GFP -from jellyfish)
can also be spliced into a plasmid.
• The desired gene is placed in the plasmid in the
middle of the GFP gene. What will the effect of
this be on the GFP?
• How can this be used to identify the plasmids &
bacteria that contain the desired gene?
Gene markers –
enzymes
• Like before, a gene that produces an enzyme
can be introduced into the plasmid e.g. a
gene that produces lactase.
• Lactase will turn its colourless substrate blue.
• How could is be used to identify which
bacteria have successfully taken up the
plasmid + desired gene?
Gene markers –
antibiotic resistance
• Technique called replica plating.
• This time a gene for antibiotic resistance is
cut in order to make space for the desired
gene (e.g. tetracycline resistance)
• The bacteria are grown on agar plates
containing tetracycline.
• What will happen to these bacteria?
• How is this problem overcome?
Replica plating
• Cells that contain the plasmids are grown on
agar plates. Each cell will grow into a separate
colony.
• A small sample from each colony is taken and
grown on another (replica) plate. All colonies are
carefully placed in the same spot as the original
plate.
• Replica plate contains tetracycline – what
happens to the new colonies that contain the
desired gene?
• Colonies that did NOT grow on the replica plate
must therefore contain the desired gene. The
colonies from the original plate can now be used
to grow the bacteria on a large scale.
Stage
Identification
:
Four:
Finding the GM bacteria with the plasmids
.Produce a story board/flow diagram to
explain and summarise how antibiotic
resistance genes can be used to identify
host cells containing the desired plasmids.