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11.4 – Applications of Gene Technology
Gene technology has important applications in many areas including biotechnology, medicinal drug
production, gene therapy, agriculture and horticulture
Genetic engineering can generate many potential ________________ for humans. But there are
hazards too.
The economic advantages may be outnumbered by environmental and ________________
drawbacks or dangers. The issues require balanced and informed judgements
Principles of Genetic Engineering
The first step in genetic engineering usually involves inserting a short piece of donor ____________
into the DNA of a recipient organism.
The recipient organism is unrelated to the donor e.g. from human to bacterium.
The recipient organism then acquires the ability to synthesise the _____________for which the donor
DNA codes.
Transferring a gene…..
T.G. AS Module 2 – Genes and Genetic Engineering – 11.4 – Applications of Gene Technology – Student Notes 29/06/17 Page 1
The outcomes are new varieties of organisms – mostly _____________organisms
Microoganisms are preferred because they are easier to modify
To remove the DNA from a cell the cell membrane needs to be _______________ and the nucleus
broken open. The method used depends on the type of cell.
One common way to disrupt a cell is a ________________ called sodium dodecyl sulphate (SDS)
which breaks down cell membranes (including the nuclear membrane) and cell walls
The protein framework of the chromosomes is removed by incubation with a ______________
Once the DNA has been isolated from the rest of the cell, the part of the DNA molecule which
contains the required gene has to be cut out and the rest of the DNA discarded. (There are on
average 2200 genes on each human chromosome)
It is important to know the base sequence of the gene required – so that either a gene ____________
can be used and/or the appropriate restriction endonuclease selected
Genes are isolated using enzymes which cut across DNA molecules at particular positions –
______________________________ ENDONUCLEASE ENZYMES.
Several __________________ types of restriction endonuclease exist
Restriction endonuclease enzymes are found naturally in ________________ where their function is
to chop up and destroy the DNA of any viruses that infect the cell. They are so named because the
RESTRICT the multiplication of viruses
A given restriction endonuclease cuts a bacterial plasmid open at a ________________ site which is
determined by the base sequence in that region
The same enzyme will cut donor DNA wherever an identical base sequence occurs
It is characteristic of most restriction endonucleases that they cut the 2 strands of DNA at slightly
different points – the result is that each end of the foreign DNA segment has a short row (4 bases
long) of unpaired bases which match the complementary bases at each end of the opened up
plasmid. These are called ___________________ _______________
Example of how a restriction endonuclease works:
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Genetically Engineered Microorganisms
The next stage is to insert the isolated gene into a _________________. A vector is a piece of DNA
that can take the gene into the chosen microbe.
A common vector is a small circular molecule of DNA called a ___________________. These occur
naturally in bacteria in addition to the larger molecule of chromosomal DNA
Plasmids are easily isolated from a bacterium and they can be re-introduced into another bacteria
relatively easily too.
As mentioned above, the ______________ restriction endonulclease is used to cut the plasmid DNA
as was used for to isolate the gene from the donor DNA. This means that the sticky ends produced
will fit together
The donor gene is then inserted into the plasmid loop using the enzyme __________________.
Ligase catalyses the LIGATION reaction which joins 2 sections of DNA together – the hydrogen
bonds form between complementary bases.
Ligase occurs naturally in nuclei where it ‘repairs’ DNA damaged in replication
Inserting the gene into the plasmid:
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The new DNA is called ________________________ DNA
Once in position, the foreign DNA (donor gene) replicates along with the rest of the plasmid every
time the bacterial cell divides
Plasmids containing the donor gene must now be transferred into the ________________
A culture of the intended bacterial recipients is placed in cold calcium chloride solution for 30 mins
This changes the membranes of the bacteria – making them more _______________ to the plasmids
Plasmids with the recombinant DNA are added to the culture medium and warmed up for a short
time.
Some of the bacteria take up plasmids – those that do are said to have undergone
_________________________________
This process is ____________ very efficient – only a small proportion of the bacteria are transformed
Obtaining transformed bacteria
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Genetic Markers
The transformed cells must now be identified and isolated.
One technique for doing this involves inserting a ___________________ gene into the plasmids as
well as the donor gene
This marker gene may make the bacteria ___________________ to a particular antibiotic
If the culture containing the transformed bacteria is grown on a medium containing the antibiotic,
bacteria with the plasmids that have the antibiotic resistance marker gene (and the donor gene) will
__________________ and grow better than those without it
Replica plating can the be done
Replica Plating………..
This ___________________ colonies without the resistance gene (and therefore no donor gene)
The donor gene also has to be made to start working once it has been transferred to the recipient
bacteria
Not all genes in a bacterium are switched on all the time
It is possible to overcome this by using ___________________ GENES that control the expression of
the main gene.
They indicate where transcription should begin and ensure mRNA is translated by the ribosomes
The promoter genes are inserted into the plasmid, with the donor gene and the marker gene
_____________________ transformation
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Large Scale Culturing
Since the gene product is often needed in _________________ amounts the transformed bacteria
are cultured on a large scale in an industrial fermenter
The medium in it contain all the nutrients the bacteria need for rapid growth and reproduction and
supplies the oxygen they need for respiration
In favourable conditions the bacteria will divide as often as every _________ hour
A huge clone of the bacteria can be produced within days
As the bacteria grow, the gene within the plasmids synthesises its _________________
The product is extracted from the fermenter for commercial use
Industrial Fermentation
Useful substances produced by genetic engineering include antibiotics,
___________________(insulin, human growth hormone, factor VIII) and _________________(those
in biological washing powders, produced for use in food industry)
Genetic engineering is also used to make pest _________________ varieties of crops – genes
producing chemicals which confer resistance to attack by fungi or insects can increase yields and
reduce the amount of pesticide which needs to be used
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The Polymerase Chain Reaction (PCR)
PCR is like a nuclear chain reaction in that it proceeds at an ever – increasing rate
PCR can amplify a ____________ amount of DNA into quantities large enough for scientific analysis
(e.g. at the scene of a crime just a few white blood cells or a tiny sample of sperm may be found)
It is the basis of genetic fingerprinting / DNA fingerprinting / genetic profiling (all same thing!)
Other uses of PCR include amplifying DNA from samples of tissues from extinct animals (e.g.
Tasmanian wolf) to establish their closest ______________and amplifying DNA from buried human
bodies where some soft tissue has been preserved. This helps to understand the migration of early
human populations
PCR uses the enzyme DNA ___________________– this is the enzyme which occurs naturally in
cells and catalyses the replication of DNA in the nucleus
The first stage of PCR is to heat the DNA to 95oC – this makes the 2 polynucleotide strands separate
DNA nucleotides are added and the mixture is cooled to 40oC. The DNA polymerase attaches the
new nucleotides to each strand (as in normal replication)
The process can then be repeated _____________ – producing 2,4,16,32 DNA molecules and so on
A single PCR cycle:
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Once a large enough sample has been generated using PCR it can be analysed and compared with
samples from ________________sources
This allows us to ________________ DNA from the same or different species.
Forensic scientists often have to compare DNA found at the crime scene with DNA from suspects
In each case the first stage is to cut the DNA into short lengths with ____________ enzymes
This produces DNA fragments of _________________ lengths – because the sequence of bases
where the enzymes cut occur at irregular intervals along the DNA molecules
The lengths of these fragments will also be different in different individuals – since we all have a
unique sequence of nucleotides in our DNA. The same restriction endonuclease will chop up our
DNA in different places
The DNA fragments are then separated using ____________________
The mixture of DNA fragments is placed at one end of a long piece of agar jelly in a trough containing
a dilute solution of ionic salts
Electrodes are placed in the solution at either end and a ______________________is applied
The phosphate groups in the fragments of DNA give them a ________________ charge – so they
are attracted through the gel towards the positive electrode. The smaller fragments move more
_________________ than the larger ones – so the different sized fragments are separated in a
similar way to molecules in chromatography
The pattern of fragments is like a ‘fingerprint’ - this fingerprint is _____________ to each of us. The
only exception is identical twins – they share the same pattern
To be able to compare fingerprints they must be made visible.
The pattern of DNA fragments in the gel is transferred to a nitrocellulose sheet
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This is done overnight and produces an ‘imprint’ of the pattern of the fragments from the original gel
onto the nitrocellulose sheet
A ________________ labelled with radioactivity is then used to reveal the position of the bands on
the sheet that contain the DNA sequence you are interested in.
To be able to pick out a particular gene or genes using a proble you first need to know its
___________ _____________________
The gene probe can then be made which will seek out the particular gene. The gene probe may be
labelled – often done by using a radioactive isotope of __________________ (32P) as a component
of the phosphate groups
A general probe can be used which make all the bands show up or a specific probe can be used. A
specific probe is a piece of single stranded DNA that is complementary to the base sequence of the
specific stretch of DNA
The nitrocellulose sheet is incubated with the probe in a sealed plastic bag containing a buffer
solution. The probes then bind (base pair) to the appropriate DNA fragments
The sheet is removed and placed next to an unexposed piece of ___________________ film
The radioactivity in the probe causes a band to show up on the film
Analysing DNA samples
Other examples of where the technique has been used:

Paternity cases

Relationships within families

Social behaviour in animals

Immigration cases
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Gene Therapy
This is the field of medicine that aims to treat people with genetic disorders by giving them a copy of
a __________________gene to overcome the problems produced by the mutated gene
There are 2 types of cells in our bodies
1. _________________ cells – give rise to eggs and sperm
2. _________________ cells – ‘normal’ body cells
Genetic changes to somatic cells ___________________be passed onto future generations.
Changes to germ line cells _______________ be passed onto future generations
The genetic engineering of somatic cells (somatic gene therapy) is legal, but tightly regulated.
The genetic engineering of germ-line cells (germ-line therapy) is _________permitted in any country
Gene therapy is being developed to treat the genetic disease c__________ f____________
Common in white North Americans and Northern Europeans
1 in 25 carriers
1 in 2000 sufferers
Most people have a gene that produces a protein called the CYSTIC FIBROSIS TRANSMEMBRANE
REGULATOR (CFTR)
This is a complex molecule made up of 1480 amino acids and it is one of the essential channel
proteins in cell membranes
The function of this particular protein is to transport _____________ ions through the cell membrane
Cause is a recessive mutation of chromosome 7
In CF sufferers this protein does not function because there are _________ copies of the faulty gene
Responsible gene was cloned in 1989
Usually the cause is a _________________mutation – 3 base pairs are missing so codon 508 in the
mRNA is missing
As a result, the amino acid phenylalanine (F) is not inserted at position 508 in the 1480 aa protein
The mutation is ^F508 (^ = d for deletion)
There are at least 400 other mutations of this protein but this is the most common
This mutation affects the __________ shape of the channel protein so that it can no longer transport
chloride ions through a membrane
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Symptoms
Normal epithelial cells form mucus glands which secrete mucus
In CF patients this mucus is unusually _____________ and sticky
In CF the normal outward flow of Cl- ions from the cell is prevented
So, chloride ions build up, a negative charge builds up inside the cell so more sodium ions (+’ve)
move in to balance the –‘ve charge
The high ion concentration in the cell prevents ______________ leaving the cell by osmosis and so
mucus is abnormally thick and sticky
Transport of ions by CFTR:
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The lungs, ________________ and liver are the most affected organs
Sweat produced is unusually salty
The thick mucus _____________ up airways in the lungs, branches of the pancreatic duct and bile
duct from the liver and so there are repeated lung infections and digestive problems
50% of sufferers die by age 20, but average life expectancy has now increased to about 20-30
Treatment
Vigorous __________________ to dislodge the mucus from the lungs
_________ supplements to aid digestion
______________ to fight infections (in respiratory
and digestive systems)
Heart lung transplants for severe cases
Clinical trials are underway for gene therapy, attempting to insert CFTR gene into lung / tracheal
epithelial cells to replace the defective genes
2 techniques are being tested
1. wrapping the CFTR genes in lipids that can be absorbed through the csm
2. inserting CFTR genes into harmless viruses that are then allowed to ‘infect’ the cells
1. wrapping the CFTR genes in lipids that can be absorbed through the csm
This method uses tiny lipid droplets called ____________________
These can fuse with the phospholipid molecules that make up the csm and so carry genes into the
target cells
CFTR genes are isolated from healthy _______________ cells, cloned and inserted into plasmids
using recombinant DNA technology
The genes are inserted into liposomes
___________ sprays are used to get the liposomes into the lungs – like inhalers for asthma sufferers
In the lungs, the liposomes fuse with the csm of _____________ cells and the genes are transported
into the cell
One problem with the technique is developing a __________ enough spray to get the liposome
through the narrow bronchioles in the lungs and into the alveoli
Also, only a ______________ proportion of the genes that are absorbed into cells are actually
expressed
These are problems which must be overcome before this form of gene therapy becomes widely
available as a treatment for CF
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2. inserting CFTR genes into harmless viruses that are then allowed to ‘infect’ the cells
In this method viruses called _____________________ are used
These viruses reproduce themselves by injecting their DNA into host cells
The viral DNA uses the cell’s enzymes and ribosomes to replicate and produce copies – which then
reconstruct the rest of the virus before being released from the cell
Adenoviruses infect the cells of the airways in the ____________, and they are adapted for
replication in the epithelial cells
They normally cause colds and other respiratory diseases
The adenoviruses used for gene therapy have been modified so they infect the cells but
_______________cause the diseases
The modification is that the genes which allow the virus to replicate are __________________
The modified viruses are cultured in epithelial cells grown in the laboratory and exposed to plasmids
that have the CFTR gene
The CFTR gene is incorporated into the viral DNA
The adenoviruses containing the CFTR gene are then extracted from the epithelial cells, purified and
sprayed into the lungs
Here they infect the epithelial cells, taking the CFTR gene into the cells
The CFTR channel protein is synthesised as normal – but as the viruses cannot replicate – they do
not damage the cells
Problems include not completely inactivating the genes controlling viral replication and the patients
becoming ill. Also, patients treated repeatedly may develop _______________ than make them
immune the virus and resistant to the treatment
Perfecting Gene Therapy Techniques
Neither of the above methods provides a permanent cure for CF but both have the potential to
alleviate some of the symptoms
Before using either method on people, research has to be done to ensure gene therapy is safe
The first tests, carried out on laboratory animals like mice aimed to find out the following:
1. The best way of getting the gene into cells
2. Check that the gene was expressed in the target cells
3. Detect any ill effects of the therapy itself
The next step was to start testing the delivery systems on _______________
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This was initially done by giving small doses of liposomes containing the CFTR gene applied to the
lining of the nose to volunteers – (since it is easier to ________ the cells here than deep in the lungs)
The next stage – which is currently ongoing – involve clinical trials with CF patients
Many trials must be done before gene therapy becomes an officially approved treatment for CF
The Future of Gene Therapy
Progress is proceeding at a very _____________rate
Soon it will be possible to
1. Incorporate a healthy gene into one of the chromosomes before putting it into a cell – the
chromosomes would be replicated as the cell divides – providing a longer lasting treatment
2. Treat rapidly dividing cancer cells with genes that kill them
3. Provide protection for viral infections by inserting genes that interfere with virus replication into
cells
More effective ways of getting genes _________ _______________ will make treatment of genetic
diseases more effective
The 2 methods described for CF have to be repeated at regular intervals because copies of the gene
are not passed form cell to cell during mitosis
Epithelial cells divide ________________ so this is a major limitation
If however the healthy gene was inserted into a chromosome then it would be replicated each time
the divided
One problem here though is inserting the gene into the DNA in a chromosome may damage other
genes
Gene therapy could help in the fight against c________________ – There are genes which cause
cells death – so if they could be inserted into cancer cells – tumours cold be destroyed
Reproduction of viruses in cells could also be prevented by inserting genes which produce mRNA
that is complementary to the viral RNA
This would inactivate the virus
The most effective treatment for genetic disorders is to replace the defective genes completely
This could be done using in vitro fertilisation (similar to method for producing transgenic animals)
The dangers of damaging the embryo though means that this technique is not suitable for humans
Such a genetic change would affect the ______________________
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This means the genes of sex cells would be altered and the genetic change would be passed on to
future generations
One other proposed method is to create an __________________ chromosome – containing only
healthy extra genes and the promoter genes needed to express them
Once added to the nucleus this chromosome would be replicated and take part in mitosis in the
normal way
There is less chance it will cause danger to other chromosomes and genes
Once the full human genome is known further developments will be able to be made
At present there are trials using somatic gene therapy for 100’s of diseases including cancers,
Duchenne muscular dystrophy and haemophilia
Some of the possibilities raise difficult moral and ethical questions
Balancing the benefits for the emerging technology against the potential for its abuse will take many
years of consultation and consideration
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Genetically Modified Animals
Animals which have been given genes from another species are called _______________________
organisms
Transgenic mammals are useful because they can express _________________ genes
It is not always possible for bacteria to express human genes since prokaryotic bacterial cells do not
have the ribosomes and other cellular machinery necessary to make complex mammalian proteins
Tracey is the name of a famous transgenic sheep
When she was an ________________, human genes were transferred into her cells
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The human gene to make the human protein alpha – 1- antitrypsin (____________) was isolated
from a culture of human cells
The gene was then combined with a promoter sequence that that allows the gene to be expressed in
sheep mammary glands
Mature egg cells were removed from the ovary of a sheep and fertilised in vitro. The AAT gene and
its promoter sequence were injected into the nucleus of the fertilised egg cells
Once the zygote had divided to form a small embryo – this was placed in the uterus of a sheep
_____________________ mother
When the sheep grew to adulthood she was able to produce the protein human alpha – 1- antitrypsin
(AAT) in her milk. This could then be collected easily and used
AAT is used to treat ___________________and cystic fibrosis
The normal function of the ATT glycoprotein is to inhibit an enzyme called elastase that is produced
by some types of white blood cell
Elastase, if not inhibited, breaks down the elastic tissue in the lungs, causing emphysema
In emphysema, the walls of the alveoli disintegrate, so there is less SA available for the absorption of
oxygen and fluid leaks into the air spaces
The fluid disrupts normal functioning of the lungs and can cause infections
Emphysema can be caused by a number of factors.
Having the Z allele for AAT is rare. More common causes are smoking and working with fine dust
particles
There are many different alleles of the AAT gene
About 3% of the population have the Z allele – this codes for a version of AAT that differs from the
normal form by one amino acid. This is a defective form and patients often develop emphysema
Affected people can be treated with an aerosol spray containing AAT
When inhaled this stops the breakdown of the alveoli and helps breathing
But it is not a permanent cure – patients must have treatment for the whole of their lives
The blood clotting protein Factor VIII is also produced in this way and is used to treat haemophilia
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Evaluation of Genetic Engineering
Rapid developments in genetic engineering are making it possible to alter living organisms in a
variety of ways
It is in theory possible to transfer genes from almost ____________organism to _________ another
There are many ways in which DNA recombinant technology can be used in food production and
medicine
However, many people are suspicious of these developments and are concerned about the possible
ways in which the technology might go wrong

There are concerns about the release of genetically engineered organisms into the environment.
Once released, these organisms cannot be recalled – No one can be sure of what effect they may
have on other organisms in the environment

Genetically modified crop plants have also caused concern e.g. potatoes have been produced
containing a gene which makes them resistant to a herbicide

This herbicide can be sprayed onto the crop and kills weeds but not the potatoes Might this gene
somehow spread to wild plants and affect the balance of the ecosystem in some way – creating
‘superweeds’.

There are concerns GM foods may be harmful to health

There are concerns genetically modifying crops will INCREASE our current use of pesticides

Will a gene, added to a genome behave in an unforeseen manner? Could it trigger a disease?

Should the money used to fund research into GE by developed countries be used to provide basic
things like food and clean water in developing countries?

Is DNA fingerprinting reliable? What about contamination of samples used in evidence? (OJ
Simpson trial)

Are we interfering with nature?

For gene therapy, most people agree that it could be used to relieve the symptoms of a genetic
disease. But the possibility of inserting the ‘correct’ genes into someone’s cells opens up all sorts
of possibilities

In theory, any gene could be inserted into the human egg. What types of gene should be
inserted? Designer babies?
At the moment there is a complete ban on performing any genetic engineering on sex cells. This is
likely to continue for the foreseeable future
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