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Sequence of events

1-Isolation cut fragments (Reverse


transcriptase,
Restriction endonuclease )
2-INSERTION insert fragment into
plasmid
3-Transformation- place plasmid
into host
4-Identification use gene maker

1-Reverse transcriptase-Make CDNA -


Reverse transcriptase enzyme from HIV
virus Aligns free nucleotides with
mRNA DNA polymerase forms
phosphodiester bond.Alkali is added to
break the Hydrogen bonds . CDNA
(complementary DNA ) is made. Add
more nucleotides to make a double
strand.

What is the difference between


cDNA and DNA ?cCDNA has no
introns
What is the advantage of using mRNA
Instead of cDNA.
-mRNA has no introns.
-mRNA is in the cytoplasm and it
produces specific enzyme such as
insulin.
-We dont have to use DNA which has
many different genes with introns

Using Restriction endonuclease


Isolate the DNA using Restriction
endonuclease cuts DNA at
recognition site or
pallindromicsequence .Sticky ends
are formed-
The same Restriction endonuclease
enzyme is used to cut the plasmid.
The two complementary sticky
ends are joined by ligase .Forms
phosphodiester bond. A
recombinant DNA plasmid is
formed. Plasmid is inserted into
bacteria by electric shock and
Calcium. Transformation occurs
but not always

Using antibiotic reistance markers

Bacteria plasmids have antibiotic


resistant genes in their plasmids .they
have genes resistant to ampicillin and
tetracycline

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

1- Heat DNA to 90 degrees to break H-


bonds to make them single stranded
2- Allow to cool to 55 C so primers can
anneal.
Primers are short sequence of DNA
which start a sequence , and keep
strand separate. They are
complementary to the DNA sample
-the primers anneal to the single
stranded DNA.
3- Free nucleotides attach by
complementary base pairing.
4- The temperature is lowered to 72 C
5- DNA-polymerase enzymes from hot
springs are added (they donot
denature )
6- Two double strands form
7- The cycle is repeat

Invivo- using bacteria

-Can make long chains


of DNA
-very accurate copies
of DNA made. This is
very important
-Can make copies of
MRNA and DNA
(egmakeinsulin )
-Low risk of
contamination
Disad

- 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

Used for copies mad


; fast
Easier process
making protein eg No need to
insulin isolate gene .
genetic engineering eg
genetically modified Used for
crops
Genetic screening
DNA fingerprinting
Making DNA probes
Uses of gene technology (using
invivo )

Microorganism-
Make hormones eginsulin
Make antibiotics from bacteria
Make- enzymes for biological
washing powders , making beer
etc

2 genetically modified crops GM


crops
Genetically modified tomato prevent
softening of tomato
Improve nutrient content by adding
genes to produce vitamins eg
Golden rice
Make crops resistant to herbicides
,pests ,
Make crops resistant to extreme
temperature
Make vaccines

3 transgenic animals
Disease resistant
animals
Fast growing animals
Animals which
produce proteins in
their milk

Eg transgenic goat
Transgenic chicken
Genetically modified tomato

A gene is made which is


complementary to the gene which
softens the tomato.
It is inserted next to that gene
The normal gene undergoes
transcription
The artificial gene also undergoes
transcription
The mRNA of the artificial gene is
complementary to the normal gene.
It attaches to it and stops
translation.
The tomato does not produce a
protein for softening

Transgenic goat to produce milk


with antithrombin drug ( prevents
clotting )

We remove the egg from the female


and fuse with the sperm by IVF
We insert a gene which produces
antithrombin protein drug next to
the gene which produces milk.
We use a micropipete to insert the
gene
The offspring are now grown and
interbred to make a herd
The offspring produce milk and
protein in that milk

Transgenic chicken Human genes


can be placed next to genes which
produce eggs.When eggs are laid the
contain a protein.We have managed
to produce protein for the treatment
of multiple sclerosis.

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.

The process needs to be repeated


every few weeks.

Problems

Adenovirus may cause infection


Liposome may not be fine enough to
be taken up ny epithelial cell
Treatment has to be repeated
regularly

Treating SCID

SCID is is a rare inherited disease


where the individual does not
produce antibodies. It is due to a
mutation of the ADA gene
When the person is infected there
are no antibodies. The condition is
lethal. They have to live in a bubble.
Gene therapy
The normal ADA gene is isolated
with Restriction endonuclease and
put into a plasmid. The plasmid is
inserted into the RETRO Virus

The Retro virus is made harmless


and is grown in the laboratory to
increase its number. The Retrovirus
now injects a copy of the ADA gene
into T-CELLS .
The T-Cells are reintroduced into the
patients blood.
T-Cells produce normal antibodies.
The process needs to be repeated.
This is also Somatic therapy
Gene therapy is experimental.
The genes are not always expressed .
Virus my cause infection
Gene therapy needs to be repeated

Name Two ways of finding the base


sequence is
1- DNA sequencing
2- Restriction mapping

DNA Sequencing (find the base


sequence using sanger method )

1. Add a single strands of DNA into


4 test tubes
2. Add primers to start the
sequence and to keep strand
separate
3. Add free nucleotides to each test
tubes 99%
4. Add terminator nucleotides
which are fluorescently labelled.
The terminator nucleotides are
special as they only form
phosphodiester bonds on one
side so they dont allow another
base next to them
5. In the first test tube add only
Adenine . In the second guanine
and third Cytosine and forth
Thymine.
6. Allow hybridistion to occur.
7. Increase the number of
fragments using PCR
8. Place DNA fragments in
electrophoresis gel and apply
voltage.
9. The negative phosphate is
attracted to the positive charge.
10. Find the sequence.

Restriction Mapping

Why do we use restriction mapping.


Restriction mapping is a process to
identify the number of sites where a
plasmid is cut at the palindromic
sequence. And
The type of restriction sites which is
cut by different restriction
endonucleases.

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

Genetic finger printing works by


examining the sequence of introns
between the genes.
They are called the Core sequence.
Or Non- Coding repeats.
it is based on the fact that no two
people except identical twins have
the same sequence of non coding
repeats introns.

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.

This technique is used to for


1-forensic science to investigate a crime scene
2-find parternity
3-gentic diversity
(a) 1 DNA is cut;
2 Using restriction enzyme
; cut the DNA very near to the Non-Coding Introns
3 Use electrophoresis;
4 Separates according to length/mass;
5 use Southern blotting.Transfer to (nylon) membrane;
6 Make single-stranded with Alkali;
7 Apply probe;
8 Radioactive/fluorescent;
9 Reference to tandem repeats/VNTRs/minisatellites;
10 Autoradiography/eq;
8 and 10 should be consistent

a) isolate wanted gene/DNA from another organism/mRNA from


cell/organism;
using restriction endonuclease/restriction enzyme/reverse transcriptase to
get DNA;
produce sticky ends;
use ligase to join wanted gene to plasmid;
also include marker gene;
example of marker e.g. antibiotic resistance;
add plasmid to bacteria to grow (colonies);
(replica) plate onto medium where the marker gene is expressed;
bacteria/colonies not killed have antibiotic resistance gene and
(probably) the wanted gene;
bacteria/colonies expressing the marker gene have the wanted
gene as well
Codon triplet code of bases on
mRNA for anti codons on tRNA to
bind which code for an Amino Acid.
Degenerate code- one amino acid
is coded by more than one triplet
code
Universal code- the same triplet
code , codes for the same Amino
Acid in all living organisms
Sticky ends-At the end of the DNA one
strand is longer than the other. The
unpaired bases at the ends are
complementary to the plasmid
Invivo- DNA is placed into a host
cell using a vector
nvitro- making a copy of DNA
using a machine eg PCR

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

Transcription factor- binds to a


promoter at a specific region on
the DNA and stimulates or inhibits
transcription. SO gene switches on
or off
Vector- Strand of DNA that carries
a foreign gene into a host
Recombinant DNA- DNA from two
different species
Clone gnetically identical copy
Plasmid- short strand of circular
DNA -separate from the bacteria
gene

Gene therapy- replace or


supplement a defective gene with
an healthy gene
Germ line therapy- genetically
altering a zygote with a new gene.
All cells will have altered genes
Somatic cell therapy- altering only
affected cells with healthy
cells.Does not transfer gene to all
cells and offspring.- needs to be
repeated.

Stem cell or totipotent cell-


Undifferentiated cell which can
differentiate into any type of cell.
They continually divide

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