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Gene Technology
Definition
• The manipulation of genes for our
advantage.
• Methods of creating new combinations of
genes is called genetic engineering.
• To create new gene combinations genetic
engineers need to:
– Locate a specific gene
– Isolate this gene
– Transfer it into a host cell in a way that it can
be expressed
Isolating the gene
• Reverse Transcription
– mRNA for a certain polypeptide is isolated
– Complimentary DNA (cDNA) can be
synthesised from it by using an enzyme called
reverse transcriptase
– The mRNA acts as the template
– mRNA removed and DNA polymerase adds
DNA nucleotides to make the second strand
of DNA.
– Result is double strand of DNA identical to the
original in the cell
Inserting the gene
• The DNA sequence is inserted into a plasmid
from a bacteria
• A plasmid is a small ring of DNA that replicates
independently of the main bacterial DNA
• It is cut using enzymes with a specific active site.
• The active site corresponds to a sequence of
nucleotide bases and will only cut at that
sequence.
• Restriction Endonucleases
– Cut the DNA leaving sticky ends
• DNA Ligase sticks pieces of DNA back together
Make a model
Insulin Production
• First human protein to be manufactured by
gene technology
• It produces fewer side effects than insulin
prepared from cow or pig extracts.
– Allergic reactions
– Diseases passed on
– Faster reaction
• Insert gene in plasmid
• Insert plasmid into bacteria
• Reproduce bacteria
Antibiotic resistance genes
• Only bacteria that are carrying the
required gene are needed.
• An antibiotic resistant gene is also
attached to the plasmid
• When the particular antibiotic is then
added to the bacteria colony only those
with the resistant gene (and therefore the
insulin gene) survive.
Cloning and harvesting
• Bacteria are grown in industrial fermenters
• Bacteria multiply rapidly in the growth
phase
• A key enzyme is then added to the
fermenter to ‘switch on’ the donor gene.
• The product is then made by the bacteria
and then separated out.
• The fermenters have cooling jackets.
Markers for genetic engineering
• In order to identify bacteria that have taken
up the plasmid, markers are used
• Antibiotic resistant genes could lead to
pathogens gaining resistance
• So they use fluorescent or easily stained
substances.
• Fluorescence comes from jellyfish.
Promoters
• As well as the gene to be inserted
most constructs contain
a promoter and terminator region as well
as a selectable marker gene.
• The promoter region initiates transcription
of the gene and can be used to control the
location and level of gene expression,
while the terminator region ends
transcription.
Project – benefits, hazards,
social and ethical implications
Benefits of gene technology
• there is more variety in gene combinations.
• Natural sexual reproduction limits the variety of
gene combinations.
• Genetic technology can solve many of the
world's problems.
• For example, researchers have tried to develop
– super-antibiotics,
– designer virus or hunters that will only attack certain
diseases like cancer,
– grass that will only grow 2 inches so it would never
need to be mowed,
– food that can be grown in any climate
• In agriculture
• improving plant breeds
• and utilizing nitrogen from the atmosphere to
improve the efficiency of crop plants.
• The production of new organs by
xenotransplantation may help to overcome
shortages in organs for transplant surgery, so
prolonging life.
• There are potentials for making
human therapeutic chemicals in other animals,
e.g. human serum albumin used to treat burns,
could be made in huge quantities in cows milk.
Hazards of gene technology
• Issues include:
• genetic manipulation can be used to create biological
warfare
• organisms made through genetic engineering to clean oil
spills or toxic wastes, if released in the environment will
wreak havoc and will be disastrous to human life.
• ‘super-weeds’, resistant to herbicides and spreading
uncontrollably,
• genes might transfer into other closely related wild
species, forming a different kind of ‘super-weed’,
• they might reduce biodiversity by genetic contamination
Social and ethical implications
• The social impact of gene technology is to do with its potential and
actual impact of human society and individuals.
– enhance crop yields and permit crops to grow outside their usual
location or season so that people have more food
– enhance the nutritional content of crops so that people are better fed
– permit better targeted clean-up of wastes and pollutants
– lead to production of more effective and cheaper medicines and
treatments
– reduce crop biodiversity by out-competing natural crops so that people
are less well fed
Ethical Issues
• do we really have the right to modify life?
• It is wrong to continue such research when the
potential impact of the technology is unknown
and many aspects of it remain to be understood.
• It is wrong to use the results of such research
when this involves release of gene technology
into the environment as once it is released it
cannot be taken back – the genes are selfperpetuating, and the risks that they might cause
in future are unknown
Electrophoresis
• Electrophoresis is
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a method of separating substances
and analyzing molecular structure
based on the rate of movement of each component
in a liquid medium
while under the influence of an electric field.
• In genetic fingerprinting and DNA sequencing,
the components being separated are fragments
of DNA.
• In this case, the type of electrophoresis
used is
– gel electrophoresis
• the gel appears solid but is actually a
colloid in which there are spaces between
the molecules through which other
molecules can move.
• Electrodes are placed at either end of the
gel, as a result of which the DNA
molecules move under the influence of an
electric current.
• DNA is cut into fragments using a
restriction enzyme.
• The sites are randomly distributed so the
fragments are of varied lengths.
• Fragments of DNA are negatively charged
and so move towards the positive
electrode (anode).
• The distance moved in a given time
will depend on the mass of the
fragment. The smaller fragments move
further in a given time.
• The DNA is transparent and invisible, so the fragments
must be treated to make them visible.
• There are two key ways of doing this:
1. staining all of the DNA fragments,
2. creating a gene probe that is complementary:
– either to a commonly repeated bit of DNA that will therefore be
present on many of the fragments,
– or to a base sequence that is specific to a particular gene or
allele of a gene which will therefore be present on no more than
one of the fragments.
The gene probe is a single stranded piece
of DNA with a base sequence
complementary to the DNA that you wish
to identify.
In order to make it possible to locate which
fragment the gene probe has attached itself
to, the gene probe must be labelled.
The most common forms of labelling are:
• With radioactivity, using X-ray photographs
• With fluorescence stain that will fluoresce with
bright visible light when placed in ultraviolet light,
• In humans the base sequence of every
chromosome 1 in every human is similar,
but not identical due to the existence of
mutations and therefore of different alleles
of genes.
• What this means is that when the DNA is
fragmented with a restriction enzyme, the
fragments are similar but not exactly the
same in DNA from different people.
DNA Sequencing
• DNA sequencing determines the order of
the nucleotide bases in a molecule of DNA.
Process
• A mixture is made containing:
– A DNA template
– A primer to start the reaction
– DNA polymerase, an enzyme that drives the
synthesis of DNA
– Four deoxynucleotides (G, A, C, and T)
– One dideoxynucleotide, either ddG, ddA, ddC,
or ddT
• dideoxynucleotides are missing a special group of
molecules, called a 3'-hydroxyl group, needed to
form a connection with the next nucleotide
• DNA polymerase moves along the
template and adds base after base.
• Until a dideoxynucleotide is added,
blocking further elongation.
• Only a small amount of a
dideoxynucleotide is added to each
reaction, allowing different reactions to
proceed for various lengths of time until by
chance, DNA polymerase inserts a
dideoxynucleotide, terminating the
reaction.
• Therefore, the result is a set of new
chains, all of different lengths.
• The molecules generated in the presence
of ddATP are loaded into one lane of the
gel,
• the other three families, generated with
ddCTP, ddGTP, and ddTTP, are loaded
into three adjacent lanes.
• After electrophoresis, the DNA sequence
can be read directly from the positions of
the bands in the gel.
• Variations of this method have been developed
for automated sequencing machines.
• In cycle sequencing, the dideoxynucleotides are
tagged with different coloured fluorescent dyes;
thus, all four reactions occur in the same tube
and are separated in the same lane on the gel.
• As each labelled DNA fragment passes a
detector at the bottom of the gel, the colour is
recorded, and the sequence is reconstructed
from the pattern of colours representing each
nucleotide in the sequence.
Cystic Fibrosis
• Recessive genetic condition
• Thick mucus produced by lungs and other
parts of the body.
• Prone to bacterial infection
• Need daily therapy
• Pancreatic duct may become blocked.
Causes
• Caused by a recessive allele which codes for a
transporter protein called CFTR.
• CFTR allows chloride ions to pass out of cells.
• Cells lining airways pump chloride ions out to allow a
high concentration of chloride ions outside of the cell.
• This lowers water potential so water moves out by
osmosis.
• It mixes with the mucus making it thin enough for the
cilia to move it.
• The recessive allele codes for a faulty version of this
protein.
Treatment
• The traditional treatments of CF depend on the
severity of the disease and the organs involved.
• Lungs - vigorous percussion on the back using
cupped hands to dislodge the thick mucus from
the lungs
• Varying the positions of the patient and where
the therapy is given drains the mucus from
various parts of the lung.
• Antibiotics help with respiratory infections
• Digestive system - supplements replace the
enzymes needed for digestion.
Gene Therapy for CF
• Gene therapy is the insertion
of genes into an individual's cells and
tissues to treat a disease, such as an
hereditary disease in which a deleterious
mutant allele is replaced with a functional
one.
How?
• basic concept is to identify the defective gene
and to correct the defect with a normal gene.
• There are two forms of gene therapy
1. germ line gene therapy.
– This helps the individual and his or her
children. It would change the genetic pool
2. somatic gene therapy.
– Somatic means of, relating to or
affecting the body.
– This therapy involves changing the
defective gene in the individual but the
change won't be inherited by the next
generation.
– Somatic gene therapy would be the
therapy of choice since it doesn't have
the ethical considerations that germ line
therapy creates.
How?
• There are two vectors that could be used to
transport the CFTR gene.
• A harmless virus which will deposit the gene
into cells as viruses naturally do.
– A new gene is injected into a virus vector, which is
used to introduce the modified DNA into a human cell.
• Or a liposome which would be inhaled and
hopefully would pass the gene through the cells.
• If the treatment is successful, the new gene will
make a functional protein.
Liposome
• Subsequent studies have tested other
methods of gene delivery, such as: fat
capsules, synthetic vectors, nose drops, or
drizzling cells down a flexible tube to
CFTR cells lining the airways of lungs.
• Researchers are now testing aerosol
delivery using nebulizers.
Issues with Gene Therapy
• con - germ line therapy changes the gene expression
forever, what will this do to the gene pool that exists
afterwards.
• - researchers aren't sure how long the results of gene
therapy will work or how often it would have to be
repeated to get results.
• - ethics involved in changing a human beings make-up.
Is this the right thing to do, even though it may help a
person afflicted with CF?
• - it isn't permanent as epithelial cells have a short life
and are constantly being renewed, so the treatment
would have to be taken regularly and this would be
expensive.
• - CF affects the entire body; and we can’t
treat everything and whilst the lungs may be temporarily
cured, the pancreas still will not function properly and the
sufferer will still need to take enzyme supplements.
Genetic Screening
• “systematic searches for persons with a specific
genotype” or “tests to identify persons who have
an inherited predisposition to a certain
phenotype or who are at risk of producing
offspring with inherited diseases or disorders”.
• a sample of a patient’s DNA is tested for
mutated sequences by comparing a sequence of
DNA of a mutated patient to that of a normal
version of the gene.
Process
DNA extracted from a body cell (often skin or wbc).
Mixed with restriction enzyme to cut the strands into
particular sequences of bases.
DNA fragments separated by electrophoresis, and the
correct fragment for the gene concerned detected
according to how far it moves on the gel.
Correct fragment extracted and mixed with a DNA
probe (known sequence of bases) which has a
fluorescent molecule added which will activate if
the bases pair up, indicating the presence of the
defective gene.
• There are many uses of genetic screenings, which can
take place to help against
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infertility,
miscarriage,
stillbirths,
neonatal deaths,
multiple malformations,
retardation in growth and development,
mental illness, and mental retardation.
• Prenatal screening is done on a mother’s fetus to see if
there are any risks, or problems with the baby.
• Carrier screening tests are done to determine whether
an individual is a carrier of a certain disease.
• Susceptibility testing is used in many workplaces to see
if their employees are susceptible to different toxins in
the work place and could have devastating effects later
on in life.
Discussion
• Many religious groups feel that genetic screening is a
very bad idea because they feel that you should not alter
the course of your life, everything happens for a reason.
• The importance of the debate about what constitutes a
disease is underscored by the two extensive questions
that underlay the current debate,
– who decides whether testing is done;
– and what happens to that information?
• Genetic discrimination. Some people may feel that
people with genetic flaws, which may not show up as
dysfunctions, may be denied life insurance.
Genetic Counselling
• The genetic counsellor can help a person or family
understand their risk for genetic conditions (such as
cystic fibrosis, cancer, or Down syndrome), educate the
person or family about that disease, and assess the risk
of passing those diseases on to children.
• A genetic counselor will often work with families to
identify members who are at risk.
• If it is appropriate, they will discuss genetic testing,
coordinate any testing, interpret test results, and review
all additional testing, surveillance, surgical, or research
options that are available to members of the family.