Genes and Genetic Engineering - Applications of Gene Technology


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T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes



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 benefit
s for humans. But there are hazards too.

The economic advantages may be outnumbered by environmental and ethical 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 DNA

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
protein for which the donor DNA

The outcomes are new varieties of organisms

mostly microorganisms

Microoganisms are preferred because they are easier to modify

To remove the DNA from a cell the cell membrane needs to be disrupted and the nucl
eus broken
open. The method used depends on the type of cell.

One common way to disrupt a cell is a detergent 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 protease

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 22
00 genes on each human chromosome)

It is important to know the base sequence of the gene required

so that either a gene probe can be
used and/or the appropriate restriction endonuclease selected

Genes are isolated using enzymes which cut across DNA mol
ecules at particular positions


Several different types of restriction endonuclease exist

T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes


Restriction endonuclease enzymes are found naturally in bacteria 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 specific 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 STICKY ENDS

Genetically Engineered Microorganisms

The next stage is to insert the isolated gene into a VECTOR. A vector is a piec
e of DNA that can
take the gene into the chosen microbe.

A common vector is a small circular molecule of DNA called a PLASMID. These occur naturally in
bacteria in addition to the larger molecule of chromosomal DNA

Plasmids are easily isolated from a ba
cterium and they can be re
introduced into another bacteria
relatively easily too.

As mentioned above, the same 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 en
ds produced will fit

The donor gene is then inserted into the plasmid loop using the enzyme LIGASE. Ligase catalyses
the LIGATION reaction which joins 2 sections of DNA together

the hydrogen bonds form between
complementary bases.

Ligase occu
rs naturally in nuclei where it ‘repairs’ DNA damaged in replication

The new DNA is called RECOMBINANT DNA

Once in position, the foreign DNA (donor gene) replicates along with the rest of the plasmid every
time the bacterial cell divides

Plasmids contai
ning the donor gene must now be transferred into the microbe

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 permeable to the plasmids

lasmids with the recombinant DNA are added to the culture medium and warmed up for a short

Some of the bacteria take up plasmids

those that do are said to have undergone

This process is not very efficient

only a small proportio
n of the bacteria are transformed

T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes


Genetic Markers

The transformed cells must now be identified and isolated.

One technique for doing this involves inserting a marker gene into the plasmids as well as the donor

This marker gene may make the bacter
ia resistant 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
survive and gro
w better than those without it

Replica plating can the be done

This eliminates 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

Not all genes in a bacterium are switched on all the time

It is possible to overcome this by using PROMOTER GENES that control the expression of the main

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 before

Large Scale Culturing

Since the gene product is often needed in large amounts the transformed bacteria are cultured on a
large scale in an i
ndustrial 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 /2 hour

A huge clone

of the bacteria can be produced within days

As the bacteria grow, the gene within the plasmids synthesises its protein

The product is extracted from the fermenter for commercial use

Useful substances produced by genetic engineering include antibiotics,

hormones (insulin, human
growth hormone, factor VIII) and enzymes (those in biological washing powders, produced for use in
food industry)

T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes


Genetic engineering is also used to make pest resistant 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

The Polymerase Chain Reaction (PCR)

PCR is like a nuclear chain reaction in that it proceeds at an ever

increasing rate

PCR can amp
lify tiny 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 profil
ing (all same thing!)

The uses of PCR include amplifying DNA from samples of tissues from extinct animals (e.g.
Tasmanian wolf) to establish their closest relatives and amplifying DNA from buried human bodies
where some soft tissue has been preserved. Th
is helps to understand the migration of early human

PCR uses the enzyme DNA polymerase

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 (95

this makes the 2 polynucleotide strands

DNA nucleotides are added and the mixture is cooled to 40
C. The DNA polymerase attaches the
new nucleotides to each strand (as in normal replication)

The process can then be repeated endlessly

ucing 2,4,16,32 DNA molecules and so on

Once a large enough sample has been generated using PCR it can be analysed and compared with
samples from known sources

This allows us to compare DNA from the same or different species.

Forensic scientists often h
ave 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 restriction enzymes

This produces DNA fragments of varying lengths

because the sequence of bases where the

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
NA in different places

The DNA fragments are then separated using electrophoresis

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 soluti
on at either end and a voltage is applied

T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes


The phosphate groups in the fragments of DNA give them a negative charge

so they are attracted
through the gel towards the positive electrode. The smaller fragments move more rapidly than the
larger ones

so t
he different sized fragments are separated in a similar way to molecules in

The pattern of fragments is like a ‘fingerprint’

this fingerprint is unique 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

This is done overnight and produces an ‘imprint’ of the pattern of the fragments from the original gel

the nitrocellulose sheet

A probe 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 you first need to know its
base sequence

The gene probe can then be made which will seek out the particular gene. The gene probe may be

often done by using a radioactive isotope of phosphorus (
P) as a component of the
phosphate groups

A general probe can be used whic
h 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 pl
astic 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 photographic film

The radioactivity in the probe causes a band to show up on the film

Other examples of where the technique has been used:

Paternity cases

Relationships within families

Social behaviour in animals

Immigration cases

Gene Therapy

This is the field of medicine that aims to treat people with genetic disorders by giving t
hem a copy of
a healthy gene to overcome the problems produced by the mutated gene

This procedure is called GENE THERAPY

There are 2 types of cells in our bodies

T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes



germ line cells

give rise to eggs and sperm


somatic cells

‘normal’ body cells

c changes to somatic cells cannot be passed onto future generations. Changes to germ line
cells can be passed onto future generations

The genetic engineering of somatic cells (somatic gene therapy) is legal, but tightly regulated.

The genetic engineer
ing of germ
line cells (germ
line therapy) is not permitted in any country

Gene therapy is being developed to treat the genetic disease cystic fibrosis

Common in white North Americans and Northern Europeans

1 in 25 carriers

1 in 2000 sufferers

Most peo
ple have a gene that produces a protein called the CYSTIC FIBROSIS TRANSMEMBRANE

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 pro
tein is to transport chloride ions through the cell membrane

Cause is a recessive mutation of chromosome 7

The gene on this chromosome codes for the CHLORIDE CHANNEL PROTEIN


Fibrosis Transmembrane Regulator)

It is located in the cell su
rface membranes and allows diffusion of chloride ions into and out of
epithelial cells

In CF sufferers this protein does not function because there are 2 copies of the faulty gene

Responsible gene was cloned in 1989

Usually the cause is a deletion muta

3 base pairs are missing so codon 508 in the mRNA is

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 3D shape of the channel protein so that it can no longer transport chloride
ions through a membrane


Normal epithelial cells form mucus glands which secrete mucus

T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes


In CF patient
s this mucus is unusually thick 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 hig
h ion concentration in the cell prevents water leaving the cell and so mucus is abnormally
thick and sticky

The lungs, pancreas and liver are the most affected organs

Sweat produced is unusually salty

The mucus clogs 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


Vigorous physiotherapy to dislodge

the mucus from the lungs

Enzyme supplements to aid digestion

Antibiotics to fight infections (in respiratory and digestive

Heart lung transplants for severe cases

Clinical trials are underway for gene therapy, attempting to insert CFTR gene i
nto lung / tracheal
epithelial cells to replace the defective genes

2 techniques are being tested


wrapping the CFTR genes in lipids that can be absorbed through the csm


inserting CFTR genes into harmless viruses that are then allowed to ‘infect’ the cel

1. wrapping the CFTR genes in lipids that can be absorbed through the csm

This method uses tiny lipid droplets called LIPOSOMES

These can fuse with the phospholipid molecules that make up the csm and so carry genes into the
target cells

CFTR genes a
re isolated from healthy human cells, cloned and inserted into plasmids using
recombinant DNA technology

The genes are inserted into liposomes

Aerosol 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 epithelial cells and the genes are transported into the

T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes


One problem with the technique is developing a fine enough spray to get the liposome through the
narrow bronchioles in the lungs and into the alveoli

Also, o
nly a small 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


inserting CFTR genes into harmless viruses
that are then allowed to ‘infect’ the cells

In this method viruses called ADENOVIRUSES 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 lungs, and they are adapted for replication in the
epithelial cells

They normally cause colds and other respiratory di

The adenoviruses used for gene therapy have been modified so they infect the cells but do not cause
the diseases

The modification is that the genes which allow the virus to replicate are disabled

The modified viruses are cultured in epithelial ce
lls 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


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 controlli
ng viral replication and the patients
becoming ill. Also, patients treated repeatedly may develop antibodies than make them immune the
virus and resistant to the treatment

Perfecting Gene Therapy Techniques

Neither of the above methods provides a perman
ent 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

T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes


The first tests, carried out on laboratory animals like mice aimed to find out the


The best way of getting the gene into cells


Check that the gene was expressed in the target cells


Detect any ill effects of the therapy itself

The next step was to start testing the delivery systems on people

This was initially done by gi
ving small doses of liposomes containing the CFTR gene applied to the
lining of the nose to volunteers

(since it is easier to study the cells here than deep in the lungs)

The next stage

which is currently ongoing

involve clinical trials with CF pati

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 fast rate

Soon it will be possible to


Incorporate a healthy gene into one of the chromosomes
before putting it into a cell

chromosomes would be replicated as the cell divides

providing a longer lasting treatment


Treat rapidly dividing cancer cells with genes that kill them


Provide protection for viral infections by inserting genes that i
nterfere with virus replication into

More effective ways of getting genes into cells 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 p
assed form cell to cell during mitosis

Epithelial cells divide frequently 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

Gene therapy could help in the fight against cancer

There are genes which cause cells death

so if
they could be inserted into cancer cells

tumours cold be destroyed

Reproduction of viruses i
n 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

T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes


This could be do
ne 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 GERMLINE

This means the genes of s
ex cells would be altered and the genetic change would be passed on to
future generations

One other proposed method is to create an artificial chromosome

containing only healthy extra
genes and the promoter genes needed to express them

Once added to t
he 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 presen
t 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 ag
ainst the potential for its abuse will take many
years of consultation and consideration

Genetically Modified Animals

Animals which have been given genes from another species are called TRANSGENIC organisms

Transgenic mammals are useful because they can

express human 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 transg
enic sheep

When she was an embryo, human genes were transferred into her cells

The human gene to make the human protein alpha


antitrypsin (AAT) was isolated from a culture
of human cells

The gene was then combined with a promoter sequence that tha
t 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
surrogate mother

T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes


When the sheep grew to adulthood she was able to produce the protein human alpha


(AAT) in her milk. This could then be collected easily and

AAT is used to treat emphysema 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

There are many different alleles of the AAT gene

About 3% of the population have the Z allele

s 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

Evaluation of Genetic Enginee

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 any organism to any another

There are many ways in which DNA recombinant technolo
gy can be used in food production and

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 organ
isms 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 produce
containing a gene which makes them resistant to a herbicide

T.G. AS Module 2

Genes and Genetic Engineering


Applications of Gene Technology

Teaching Notes


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


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?

uld 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 t
heory, 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 futu