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Replica plating
1- After the plasmids have been
placed near the bacteria we
add electric shock and calcium.
Some of the bacteria take up
the plasmid.
2- We grow colonies of these
bacteria in a petridish.
3- We transfer theses colonies
onto an agar gel which has
ampicillin . the bacteria which
take up the plasmid survive the
ampicillin antibiotic
4- These colonies are then
transferred to another plate
which contains tetracillin. We
use Replica plating
5- The human gene is placed in-
between the tetracycline gene.
6- This stops the tetracycline gene
from working.
7- If the bacterial cell has taken up
the human gene it stops
working.
8- This colony will die.
9- We can identify if by looking at the
ampicillin plate.
Polymerase chain reaction PCR
- Complex method
needs a week to make
the copy
- Needs a host eg bacteria
PCR
disadv
Can make only short
chains
-High number of errors
-mRNA can not be
copied so we cant make
proteins this way
-risk of contamination
Adv-
- Automated process
- No need for host
Thousands of copies
made-
Easier faster process-
No need to isolate gene
Microorganism-
Make hormones eginsulin
Make antibiotics from bacteria
Make- enzymes for biological
washing powders , making beer
etc
3 transgenic animals
Disease resistant
animals
Fast growing animals
Animals which
produce proteins in
their milk
Eg transgenic goat
Transgenic chicken
Genetically modified tomato
Gene therapy.
We can replace a
defective gene _Gene
replacement
Or we can insert a
new gene next to the
defective one so it can
produce some healthy
protein Gene
supplementation
There are two different treatments
Germ line therapy genetically
altering sperm , ova or zygote . genes
will be transferred to all cells and to
the next generation. This treatment
is permanent
A healthy gene is added next to a
defective gene in a fertilized zygote.
All cells will produce healthy CFTR
protein channels next to unhealthy
ones.
Somatic gene therapy
Altering only affected cells with
healthy genes. Not all cells will have
this gene.
The treatment is temporary . it has
to be repeated.
The genes are not transferred to the
offspring
Process
Healthy CFTR gene is added next to
unhealthy CFTR gene.
Insert DNA into plasmid with same
restriction endonuclease
And DNA ligase to form
phosphodiester bond
Place plasmid into host cell use
electric shock and calcium
Check to see if transformation has
occurred using a maker
Clone host cells to produce
recombinant plasmid
Extract plasmid
We can insert plasmids into
a) Viruses
b) Liposomes
Viruses :
Use the Adenovirus which infects the
lungs when we get a cold
Make the virus harmless by
inactivating the gene for replication
Place the viruses next to plasmids and
epithelial cells in the laboratory
The viruses take up the plasmid
Use a marker to see if transformation
has occurred
The virus is inhaled or injected into
the lungs using an aersol spray
Liposomes
Insert plasmids in lipid soluble
molecules called liposomes.
The liposomes fuse with the
phospholipid membrane in the
epithelial cells in the lungs.
The plasmid is then inserted into the
DNA of the lungs
Healthy CFTR protein channels are
produced.
Liposomes are tranfered via aerosol
sprays.
Problems
Treating SCID
Restriction Mapping
Genetic screening
Find the order of nucleotides of the
mutated gene using DNA sequencing
Make a gene probe which is
complementary to the mutated
gene.
A radioactive marker is used so it
can be identified by X-Ray
Make many copies - PCR
Sample of DNA is made single
stranded and poured onto the DNA
probes. If the person has a mutated
gene then hybridization will occur.
Complementary base pairing.
We use an X-ray to identify the DNA
probe
We can identify more than one
genetic disease.
Genetic finger printing
Process.
1-Extract DNA
2-place in PCR to multiply
3-use restriction endonuclease which
cut the
DNA very near to the Non-Coding
Introns
or core sequence but not within the
introns.
3- These fragments of introns have
different lengths .
4- They are passed through the
elcertopheresis gel .
5- Shorter chains will move further
than longer ones.
6- Make the strands separate.
7- Place a nylon sheet onto and
absorbant paper and UV light
8- The strands stick to the nylon sheet
9- Apply DNA probes .
10- If they are complentary they will
hybridise
11- Use Autoradiography to detect.
DNA PROBE
A single strand of DNA 20 bases
long with a base sequence
complementary to the target gene
which is labeled with a gene
marker. Used for genetic screening
and finger printing
Primer- single strand of DNA
starts a base sequencekeeps two
strands separate
Promotor- Trascription factor
attaches to it to start or stop
transcription .Part of the DNA is
switched on