2.
STEPS INVOLVED IN THE GENETIC ENGINEERING OF
BACTERIA TO SYNTHESIZE HUMAN INSULIN.
IDENTIFYING & OBTAINING THE GENE REQUIRED
◦ The gene for insulin is active in the B-cells of the
pancreas.
◦ If a gene is active it usually produces thousands of
mRNA molecules which are complementary to the
gene.
◦ Therefore the mRNA is isolated from the B cells of the
pancreas.
◦ Reverse transcriptase is used to make cDNA and DNA
polymerase clones the DNA.
◦ Restriction enzymes/restriction endonucleases cut the
DNA at specific base sequences (restriction site).
◦ They leave sticky ends (short lengths of unpaired
bases).
PUTTING THE GENES INTO A VECTOR
◦ The same restriction enzymes that were used to cut the donor
should be used to cut the plasmid DNA leaving sticky ends.
◦ The wanted genes are mixed together with the plasmid DNA and
they join up at the sticky ends.
◦ The initial attraction is due to hydrogen bonding.
◦ DNA ligase ensures that the gene is permanently added to the
plasmid forming recombinant DNA.
3.
INTRODUCING VECTOR DNA INTO THE HOST CELL
◦ The plasmids containing the human insulin gene are mixed
together with the bacterial cells.
◦ Those bacteria that take up the plasmids are said to be
transformed.
CLONING THE DNA
◦ The bacteria that are transformed are cloned to produce
genetically identical offspring.
◦ Every time a bacterium divides, it will replicate the human insulin
gene.
SELLECTING THE TRANSFORMED BACTERIA
◦ Once the plasmid and DNA are mixed, not all the bacteria are
transformed and not all the plasmid will have taken up the foreign
DNA.
◦ This can be avoided by using: A gene resistant to a particular
antibiotic-If the bacteria are grown on a medium containing that
antibiotic, only the transformed ones will multiply and form
colonies. The DNA that survive growing on the antibiotic are then
grown on X-gal and those that do not contain the donor DNA
appear blue but those that contain it will appear colourless and
they can be isolated for further cloning.
4.
ADVANTAGES OF TREATING DIABETICS WITH HUMAN
INSULIN PRODUCED BY GENE TECHNOLOGY.
Back in the day before bacteria were used to produce human
insulin gene, people with insulin dependent diabetes were
injected with insulin from pigs or cattle.
Pig or cattle insulin is similar to human insulin but it is not
identical.
ADVANTAGES
It is chemically identical, so there is little chance of an
immune response.
It is an exact fit in the human insulin receptors in human cell
surface membranes, hence causing rapid response.
Like in human insulin, the response is much shorter than the
cow or pig insulin.
It overcomes problems related to the development of
tolerance to cow or pig insulin.
It avoids any ethical issues, such as religious objections or
from vegetarians .
5.
WHY PROMOTERS NEED TO BE TRANSFERRED
ALONG WITH THE DESIRED GENES.
Not all the genes in a cell are switched
on at any one time
Certain regions of DNA called promoter
regions are next door to the genes that
have to be activated before a gene is
expressed.
If a human insulin gene is transferred in
a prokaryote DNA without adding the
required prokaryote promoter, it will not
be transcribed and hence will not be
expressed.
The promoter initiates transcription of
the gene so that the desired product is
expressed.
6.
WHY FLOURESCENT MARKERS OR (EASILY STAINED
SUBSTANCES) ARE NOW USED INSTEAD OF ANTIBIOTIC
RESISTANT MARKERS.
HOW ARE ANTIBIOTIC MARKERS USED?
Not all the bacteria are transformed and there needs to be a way to
identify those which have taken up the plasmids and those that have not.
The first methods were based on antibiotic resistant markers.
The method used to identify the bacteria with the required DNA
recombinant IS:
◦
◦
◦
The original selected plasmid has antibiotic resistance genes to two
antibiotics A and B and those bacteria that have the plasmid will grow
successfully in the presence of these antibiotics but those lacking will be
killed.
Restriction enzymes are used to cut the middle of one of the antibiotic
resistance gene, B. Those bacteria that had been transformed will no longer
have a working copy of antibiotic B as a result of being interrupted by the
cDNA insulin gene.
Many plasmids have a working copy of the antibiotic resistance gene to
antibiotic B, showing that the plasmids have failed to form recombinant DNA.
However those that have taken up the recombinant DNA do not have a
working copy of antibiotic A and there are ways to identify them. This is:
7. The bacteria are spread on an agar plate containing antibiotic A and
those that have taken up the plasmid with recombinant DNA survive.
A sponge is used to touch briefly on the agar, picking up some bacteria
and these are touched onto a sterile agar plate containing antibiotic B
and those that have the recombinant DNA are killed by this antibiotic so
that their original location on the plate is known with the help of the
original agar plate that had been refrigerated.
One potential problem of using antibiotic markers in this
way is that:
The antibiotics are present on the plasmids meaning
that if these genetically engineered bacteria come into
contact with pathogenic bacteria, the resistance genes
can easily be transferred to them making it very difficult
to control the spread of such bacteria.
Fluorescent markers are quicker and produce a higher
yield proportion of transformations.
8.
THE BENEFITS AND HAZARDS OF GENE
TECHNOLOGY
BENEFITS
It is fast
It is possible to produce genetically engineered
organism for a specific purpose.
It involves unrelated organisms
Specific products are got, avoids dependence on cows
and pigs.
Reduce use of herbicides and pesticides
Can be used to treat CF as well as in cancer treatment.
HAZARDS
Transfer of genes inserted, to other bacterial species
can occur causing resistance
Crops may end up producing wild relatives(Mexican wild
maize)
Food may trigger toxic or allergic reactions when
consumed.
9.
THE SOCIAL AND ETHIAL IMPLICATIONS OF GENE
TECHNOLOGY
SOCIAL
Enhance crop yields(crops can grow outside normal location
and season)
Enhance nutritional content of food.
Leads to effective and cheaper medicines through gene
manipulation.
Produce supper weeds.
Increases costs of seeds
Reduce crop biodiversity by out competing natural crops
Causes antibiotics to become less useful and causes allergic
reactions
ETHICAL
It is good to conduct such as technological improvements are
done.
It may be wrong as some aspects of it may not be
understood.
It is wrong as some organisms may escape and pause risks.
It is wrong as it cannot be reversed and future implications
are unknown.
10.
THE USE OF ELECTROPHORESIS IN GENETIC
FINGERPRINTING AND DNA SEQUENCING.
ELECTROPHORESIS
It is a method of separating and analyzing molecular structure
based on the rate of movement of fragments of DNA in a liquid
medium while under the influence of a magnetic field.
The type we use is the gel one. The gel has spaces between it
and the molecules can move freely under the influence of a
magnetic field. Electrodes are placed on either side of the gel.
Restriction enzymes are used to cut the DNA into fragments of
different lengths.
The direction of movement is due to the DNA molecules being
negatively charged hence move to the anode.
The distance moved in a given time will depend on the size of
the molecule/fragment.
When DNA is cut by restriction enzymes, the fragments are
similar but not exactly the same.
As DNA is transparent and invisible we have to:
Stain the DNA with methyl blue.
Create a gene probe: this is a single stranded DNA molecule
with a base sequence complementary to the DNA you wish to
identify. To identify where the DNA has attached itself, the
probe must be labeled by:
◦ Making the probe radioactive
◦ Staining the probe with fluorescent stain eg. Vital red
11.
GENETIC FINGERPRINTING
Once the DNA fragments have been separated
by gel electrophoresis they can be compared
with other samples of DNA, hence allowing the
determination of the source of DNA.
DNA SEQUENCING
The best example is the genome project.
Electrophoresis is used to separate DNA
fragments to enable the determination of the
order of the bases within the genes and
chromosomes.
12.
THE CAUSES AND SYMPTOMS OF CYSTIC
FIBROSIS
CAUSES
It is caused by the gene coding for the
transmembrane protein that transports chloride
ions through the cell surface membrane.
Its inheritance is autosomal and recessive.
The gene is located on chromosome 7.
EFFECTS OF CF
Reduced chloride transport-which leads to the
production of thick, sticky mucus which affects
the lungs, pancreas and reproductive organs.
The mucus blocks the pancreatic duct
preventing amylase and protease enzymes from
reacting in the ileum, hence affecting digestion
and nutrition. The mucus remains there and
leads to wheezing and repeated infections. The
mucus may block the sperm ducts.
13.
PROGRESS TOWARDS TREATING CF WITH
GENE TECHNOLOGY
Since it is the mucus in the lungs that limits life span, it is
these cells that have been the focus of efforts. It is
thought that if even a small proportion of lung cells could
be given a working copy of the cells that would thin the
mucus sufficiently.
For CF, a vector must be use to deliver the DNA
containing the functional CFTR gene.
They could use vectors such as:
Viral delivery system- using viruses, the intension being
to infect the lung surface cells which releases the genetic
material into the cells where it is expressed.
Non-viral delivery systems
Gene therapy has not been successful yet because:
◦ Current viral vectors have been found to cause infections.
◦ Some have been found inefficient.
◦ The effect of therapy only lasts a few days.
14.
GENETIC SCREENING AND COUNCELLING
The pattern of inheritance varies according to whether the allele is dominant,
recessive or sex-linked.
GENETIC SCREENING
It is the testing of a sample of DNA from a group of people to identify the
presence or absence of particular alleles. Such screening may be:
Carrier screening- potential parents may be screened; all individuals in a
family may be screened as a result of one member of the family developing
a genetic condition.
Prenatal screening-screening the genetic makeup of an unborn child to
detect genetic conditions.
New-born screening-screened for genetic conditions to maintain stability.
Genetic conditions that may be screened for are:
◦ Chromosomal abnormalities- Down’s syndrome, trisomy 13 and 18.
◦ Single gene disorders- haemophilia, sickle ell anaemia and cf.
◦ Neural tube defects- spina bifida and anencephaly.
Once the results of a genetic test are known it is necessary for those
involved to receive genetic counseling.
15.
GENETIC COUNCELLING
It is required to give an explanation of the results, probabilities,
dangers, diagnosis and treatment.
For the individual and for couples who are aspiring to have
children.
ETHIAL CONSIDERATIONS
Who decides who to be screened or tested?
Which specific disorder should be screened?
Who should be providing the screening?
Should we screen for disorders that have no cure?
Should there results be confidential?
If not should they have access to the information?
Or should they be made available to potential employers, insurers
etc.
