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4.4 Genetic Engineering & Biotechnology
15/04/2011 06:13:00
Topic 4: Genetics
4.4: Genetic Engineering & Biotechnology
4.4.1 Outline the use of polymerase chain reaction (PCR) to copy and amplify
minute quantities of DNA.
4.4.2 State that, in gel electrophoresis, fragments of DNA move in an electric
field and are separated according to their size.
4.4.3 State that gel electrophoresis of DNA is used in DNA profiling.
4.4.4 Describe the application of DNA profiling to determine paternity and also
in forensic investigations.
4.4.5 Analyse DNA profiles to draw conclusions about paternity or forensic
investigations.
4.4.6 Outline three outcomes of the sequencing of the complete human genome.
4.4.7 State that, when genes are transferred between species, the amino acid
sequence of polypeptides translated from them is unchanged because the
genetic code is universal.
4.4.8 Outline a basic technique used for gene transfer involving plasmids, a
host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases)
and DNA ligase.
4.4.9 State two examples of the current uses of genetically modified crops or
animals.
4.4.10 Discuss the potential benefits and possible harmful effects of one
example of genetic modification.
4.4.11 Define clone.
4.4.12 Outline a technique for cloning using differentiated animal cells.
4.4.13 Discuss the ethical issues of therapeutic cloning in humans.
4.4.1 PCR
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4.4.1 Outline the use of polymerase chain reaction (PCR) to copy and amplify
minute quantities of DNA. (Details of method are NOT required).
Orange book  pg. 163
Green book  pg. 64
WS1: Polymerase Chain Reaction
To do:
Remember - as you view the following animations – you do NOT need to know the
process, the idea is to get you to understand that the purpose if to copy and
amplify small quantities of DNA.
http://www.dnalc.org/resources/animations/pcr.html
http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf
http://www.maxanim.com/genetics/PCR/PCR.htm
http://www.sumanasinc.com/webcontent/animations/content/pcr.html
http://learn.genetics.utah.edu/content/labs/pcr/
Complete the following questions to act as a summary in your books.
1. What is the purpose of the Polymerase Chain Reaction?
2. Briefly describe how the Polymerase Chain Reaction works (temperatures,
names etc are NOT needed).
3. How has the development of the Polymerase Chain Reaction improved the
technique of DNA fingerprinting?
The Polymerase Chain Reaction
PCR can clone (or amplify) DNA samples as small as a single molecule. The
polymerase chain reaction is simply DNA replication in a test tube. If a length
of DNA is mixed with the four nucleotides (A, T, C and G) and the enzyme DNA
polymerase in a test tube, then the DNA will be replicated many times. The
details are shown in this diagram:
1. Start with a sample of the DNA to be amplified, and add the four nucleotides
(sugar, phosphate and base) and the enzyme DNA polymerase (sticks the new
nucleotides to the old nucleotides).
2. The two strands of the DNA double helix are separated by heating to 95°C
for two minutes. This breaks the hydrogen bonds.
3. The DNA must be cooled to 40°C to allow new nucleotides to be added to the
original DNA strand..
4. Each original DNA molecule has now been replicated to form two molecules.
5. The cycle is repeated from step 2 and each time the number of DNA
molecules doubles. This is why it is called a chain reaction, since the number of
molecules increases exponentially, like an explosive chain reaction. Typically
PCR is run for 20-30 cycles.
PCR can be completely automated, so in a few hours a tiny sample of DNA can be
amplified millions of times with little effort. The product can be used for
further studies, such as cloning and electrophoresis. Because PCR can use such
small samples it can be used in forensic medicine (with DNA taken from samples
of blood, hair or semen), and can even be used to copy DNA from mummified
human bodies, extinct woolly mammoths, or from an insect that's been encased
in amber since the Jurassic period. One problem of PCR is having a pure enough
sample of DNA to start with. Any contaminant DNA will also be amplified, and
this can cause problems, for example in court cases.
The following silly links are only if you have time 
“The PCR Song”
http://www.youtube.com/watch?v=7uafUVNkuzg
“Enzyme – DNA – PCR song”
http://www.youtube.com/watch?v=CQEaX3MiDow
4.4.2 Electrophoresis
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4.4.2 State that, in gel electrophoresis, fragments of DNA move in an electric
field and are separated according to their size.
Orange book  pg. 164
Green book  pg. 65
WS2: Gel Electrophoresis
To do:
View the animations – details of the process are NOT needed:
http://www.dnalc.org/resources/animations/gelelectrophoresis.html
http://learn.genetics.utah.edu/content/labs/gel/
http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis
.html
Read the information provided below.
In your green books, explain how the DNA fragments are separated.
This is a form of chromatography used to separate different pieces of DNA on
the basis of their length. It might typically be used to separate restriction
fragments (produced by the use of restriction enzymes on DNA). The DNA
samples are placed into wells at one end of a thin slab of gel and covered in a
buffer solution (allows the electricity to flow). An electric current is passed
through the gel. Each nucleotide in a molecule of DNA contains a negativelycharged phosphate group, so DNA is attracted to the anode (the positive
electrode). The molecules have to diffuse through the gel, and smaller lengths
of DNA move faster than larger lengths, which are retarded by the gel. So the
smaller the length of the DNA molecule, the further down the gel it will move in
a given time. At the end of the run the current is turned off.
Unfortunately the DNA on the gel cannot be seen, so it must be visualised.
There are three common methods for doing this:
The gel can be stained with a chemical that specifically stains DNA. The DNA
shows up as blue bands. This method is simple but not very sensitive.
The DNA samples at the beginning can be radiolabelled. Photographic film is
placed on top of the finished gel in the dark, and the DNA shows up as dark
bands on the film. This method is extremely sensitive.
The DNA fragments at the beginning can be labelled with a fluorescent
molecule. The DNA fragments show up as coloured lights when the finished gel
is illuminated with invisible ultraviolet light.
4.4.3 DNA Profiling
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4.4.3 State that gel electrophoresis of DNA is used in DNA profiling.
Orange book  pg. 164
Green book  pg. 65
WS3: DNA Profiling using PCR
To do:
Go through the following website in the order stated below:
“DNA Profiling”: http://www.dnai.org/d/index.html
 Human Identification (bottom of screen)


Profiling (top of screen)
1st Circle – DNA Variations and Fingerprints
A – DNA Variations (watch animation)
V – Tandem Repeats (listen to audio explanation of Tandem Repeats)
Return
Circle – The First DNA fingerprints
A – The First DNA “fingerprints”
No need to look at any other sections just yet.


Read the section below “Which sections of DNA are used for profiling?”
1. Explain in your own words why exons are no use for profiling.
2. Describe the sections that are used for profiling and why they can be used.
Read the section below “Profiling – the technique”
Explain the role of Electrophoresis in profiling.
Which sections of DNA are used for Profiling?
About 90% of human genome has no known function. The non-coding regions of
DNA – introns are removed before translation.
Exons that code for a protein vary little, since any change in the structure of an
essential protein is likely to be harmful.
Introns vary greatly in a population and are the basis for DNA profiling.
Intron DNA consists of regions called minisatellites – where there is great
variability from one individual to another.
Minisatellite – short sequences of 20-100 nucleotides called variable number
tandem repeats (VNTRs).
Each chromosome has many VNTRs and the number of repeats differs from
person to person and so does the length of the VNTR region.
Each individual has a unique profile.
Variation in fragment length depends on:


the number of repeats
number of bases in each repeated sequence.
E.g. CGCGCGCGCGCG (6 repeats of 2 bases) this will have the same fragment
length as 3 repeats of 4 bases.
Profiling - The Technique
** You do NOT need to learn the technique.
You just need to understand where gel electrophoresis fits in.
Extraction of the DNA from a sample.
Using chemicals and enzymes the DNA can be removed from a sample of tissue
and then purified.
DNA cut up.
Using restriction enzymes the DNA is digested. The restriction enzymes do not
attack any site with a repeated sequence and so the repeats are left intact.
Fragments separated by gel electrophoresis.
The DNA would have been labeled in some way and will show up as dark bands on
the gel.
The spacing of these and is unique to the individual.
4.4.4 Uses of Profiling
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4.4.4 Describe the application of DNA profiling to determine paternity and also
in forensic investigations.
Orange book  pg. 164
Green book  pg. 65
WS4: DNA Profiling
WS5 : Manual DNA Sequencing
To do:
Read the information below.
In your green books make a list of application of DNA profiling.
Explain how DNA profiling can be used to determine paternity and also in
forensic investigations.
Applications/ Uses
DNA profiling can be used in paternity suits when the identity of someone’s
biological father must be known for legal reasons.
Cases of incest where a child has been born  can be used to prove the accused
is genetically more similar to a child than if the father were a non-relative.
At a crime scene, forensic specialists can collect samples such as blood or
semen which contain DNA. Gel electrophoresis is used to compare the collected
DNA with that of suspects. If they match, the suspect has a lot of explaining to
do. A match can confirm the guilt of a suspect. If the samples do not match it
can also be used to clear someone who is claiming to be innocent.
Criminal cases are sometimes reopened many years after a judgement in order
to consider new DNA profiling results. In the United States, this has lead to
the liberation of many individuals who had been wrongly sent to jail for crimes
they did not commit.
In Europe it can be used to establish if a person has relatives in the country
they are trying to enter (for immigration purposes).
Captive breeding of endangered species. When numbers are low used to prevent
inbreeding by making it possible to select non-related animals.
In studies of ecosystems, DNA samples taken from animals can be used to
establish better understanding of social relationships, migrating patterns and
nesting habits. In addition, the study of DNA in the biosphere has given new
credibility to the ideas of evolution: DNA evidence can often reinforce previous
evidence of common ancestry based on anatomical similarities between species.
Advantages over blood testing.
Blood groups are not unique; they can be used to eliminate suspects not to
convict. DNA profiling can give a positive identification – it is unique.
Any tissue containing cells can be used e.g. skin, hair, bone can all serve as a
DNA source
4.4.5 Analysing Profiles
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4.4.5 Analyse DNA profiles to draw conclusions about paternity or forensic
investigations.
Orange book  pg. 164
Green book  pg. 66-67
To do:
The following website gives an example of a paternity test and also examples of
forensic investigations.
http://www.dnai.org/d/index.html
Go through the following website in the order stated below:
 Human Identification (bottom of screen)
 Family (top of screen)
 Murder
 Innocence
For each of the three cases:
1. Read through all the information.
2. Try the comparison – match the DNA fragments yourself.
3. In your own words summarise how DNA profiling was used each of the three
cases.
Exam Questions:
1. The following is a DNA gel. The results are from a single probe showing a
DNA profile for a man, a woman and their four children. Which fragment of
DNA is the smallest?
2. The following is a DNA gel. The results are from a single probe showing a
DNA profile for a man, a woman and their four children.
Which child is least likely to be the biological offspring of the father?
4.4.6 Human Genome Project
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4.4.6 Outline three outcomes of the sequencing of the complete human genome.
Orange book  pg. 158-159
Green book  pg. 67
WS6: The Human Genome Project
The website below is the official Human Genome Project website.
http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml
The following all require the same login:
Username: Biologyhelp
Password: Ilovebiology
“The Human Genome Project”
http://www.yteach.ie/page.php/resources/view_all?id=restriction_endonuclease
_nitrogenous_base_nucleotide_base_chromosomal_aberration_mitotic_division
_amnion_denaturation_sequencing_cytosine_thymine_uracil_denaturation_gene
_t_page_21&from=search
“Sequencing the Human Genome”
http://www.yteach.ie/page.php/resources/view_all?id=restriction_endonuclease
_nitrogenous_base_nucleotide_base_chromosomal_aberration_mitotic_division
_amnion_denaturation_sequencing_cytosine_thymine_uracil_denaturation_gene
_t_page_22&from=search
4
To do:
Have a browse through the official Human Genome Project website.
View the clip: “Introduction”
http://www.genome.gov/25019885
Read the corresponding page in the green text book and the information below.
Complete the objective: ”Outline three outcomes of the sequencing of the
complete human genome”.
General Information
The Human Genome Project was an international co-operative venture which set
out to sequence the complete human genome. Because the genome of an
organism is a catalogue of all the bases it possesses, the HGP hoped to
determine the order of all the bases A, T, C and G in human DNA. In 2003, the
HGP announced that it had succeeded in achieving its goal. Now, scientists are
working on deciphering which sequences represent genes and which genes do
what. The human genome can be thought of as a map which can be used to show
the locus of any gene on any one of the 23 pairs of chromosomes.
As you have seen, some diseases are sex linked, so it is relatively easy to
determine which chromosome the gene responsible for the disease is found on;
often the locus is on the X-chromosome. In traits which show no sex linkage, it
is difficult to know which of the 22 other chromosomes carries the gene. With
genome libraries of genetic diseases, doctors can find out exactly where to look
if they think one of their patients might posses a disease-carrying allele.
Another advantageous use of the HGP is the production of new medications.
This idea involves several steps:
 Find beneficial molecules which are produced naturally in healthy people
 Find out which gene controls the synthesis of a desirable molecule
 Copy that gene and use it as instructions to synthesize the molecule in a
laboratory.
 Distribute the beneficial molecule as a new medical treatment.
This is not science fiction; genetic engineering firms are finding such genes
regularly.
In addition, by comparing the genetic make-up of populations around the world,
countless details could be revealed about ancestries and how humans have
migrated and mixed their genes with other populations over time.
4.4.7 Genetic Code
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4.4.7 State that, when genes are transferred between species, the amino acid
sequence of polypeptides translated from them is unchanged because the
genetic code is universal.
Orange book  pg. 161
Green book  pg. 67
To do:
First a recap on the genetic code below.
Username: Biologyhelp
Password: Ilovebiology
“Characteristics of the genetic code”
http://www.yteach.ie/page.php/resources/view_all?id=termination_site_gene_c
odon_operator_operon_promoter_polymerase_binds_polymerase_dna_rna_tran
scription_translation_gene_gene_expression_polymerase_nucleosome_uracil_m
rna_triplet_code_anticodon_t_page_7&from=search
In your own words, in your green books, explain the significant to the genetic
code being universal.
Recap on the Genetic Code
All living organisms use the same bases:
This means that base sequences can be transferred from one organism to
another without changing their function.
So …
We could take the gene for healthy insulin production from a human and insert
it into a bacterial plasmid – and the bacteria will then be able to produce human
insulin.
4.4.8 Transgenesis
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4.4.8 Outline a basic technique used for gene transfer involving plasmids, a host
cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and
DNA ligase.
Orange book  pg. 161
Green book  pg. 68-69
WS7 : Restriction Enzymes
WS8 : Ligation
WS9: Gene Cloning Using Plasmids
WS10 : Transgenic Organisms
Introduction - **Do NOT learn – this is simply to set the scene:
Since the discovery of the hormone insulin in 1921, diabetic patients, whose
elevated sugar levels are due to impaired insulin production, have been treated
with insulin derived from the pancreas glands of slaughtered animals. The
hormone, produced and secreted by the beta cells of the pancreas' islets of
Langerhans, regulates the use and storage of food, particularly carbohydrates
Although bovine (cow) and porcine (pig) insulin are similar to human insulin, their
composition is slightly different. Consequently, a number of patients' immune
systems produce antibodies against it, neutralising its actions and resulting in
inflammatory responses at injection sites. Added to these adverse effects of
bovine and porcine insulin, were fears of long term complications ensuing from
the regular injection of a foreign substance, as well as a projected decline in
the production of animal derived insulin. These factors led researchers to
consider synthesising Human insulin by inserting the insulin gene into a suitable
vector, the E. coli bacterial cell, to produce insulin that is chemically identical
to its naturally produced counterpart. This has been achieved using Recombinant
DNA technology. This method is a more reliable and sustainable method than
extracting and purifying the abattoir by-product.
To do:
In no more than a couple of sentences (in your books) explain the benefits of
producing insulin from genetically modified bacteria compared to how it was
previously obtained from animals.
The Process of Transgenesis
The following website give you an overview of the entire process before we
break it down. Go through the webpage to get an overview before moving on.
http://www.abpischools.org.uk/page/modules/diabetes_16plus/diabetes8.cfm?c
ositenavigation_alltopic=1
1. Identifying the human insulin gene
The human insulin gene was isolated, cloned and sequenced in the 1970s, and so
it became possible to insert this gene into bacteria, who could then produce
human insulin in large amounts.
We know which gene codes for insulin.
2. Inserting the DNA into a plasmid vector using restriction enzymes and
DNA ligase
Restriction Enzymes
View the animations:
“Restriction Endonucleases”
http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.html
“DNA Restriction”
http://www.dnalc.org/resources/animations/restriction.html
“Restriction Enzymes”
Username: Biologyhelp
Password: Ilovebiology
http://www.yteach.ie/page.php/resources/view_all?id=hydrogen_bonds_conjuga
tion_transformation_ligase_clone_replication_transcription_vector_plasmids_r
etrovirus_nucleotides_dna_pcr_hybridization_polymerase_exons_introns_t_pa
ge_3&from=search
Read the section below on restriction enzymes:
These are enzymes that cut DNA at specific sites. They are properly called
restriction endonucleases because they cut phosphodiester (Between sugar and
phosphate) bonds in the middle of the polynucleotide chain. Some restriction
enzymes cut straight across both chains, forming blunt ends, but most enzymes
make a staggered cut in the two strands, forming ‘sticky ends’.
The cut ends are “sticky” because they have short stretches of single-stranded
DNA with complementary sequences. These sticky ends will stick (or anneal) to
another piece of DNA by complementary base pairing, but only if they have both
been cut with the same restriction enzyme. Restriction enzymes are highly
specific, and will only cut DNA at specific base sequences, 4-8 base pairs long,
called recognition sequences.
In your books summarise the role of restriction enzymes to include:



specificity
bonds broken
sticky ends
DNA Ligase
Watch the animations:
“Steps in cloning a gene”
http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapter14/animation_quiz_1.html
Username: Biologyhelp
Password: Ilovebiology
“Basic Tools in Genetic Engineering”
http://www.yteach.ie/page.php/resources/view_all?id=hydrogen_bonds_conjuga
tion_transformation_ligase_clone_replication_transcription_vector_plasmids_r
etrovirus_nucleotides_dna_pcr_hybridization_polymerase_exons_introns_t&fr
om=search
Read through section below:
This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It
is commonly used in genetic engineering to do the reverse of a restriction
enzyme, i.e. to join together complementary restriction fragments.
The sticky ends allow two complementary restriction fragments to anneal, but
only by weak hydrogen bonds, which can quite easily be broken, say by gentle
heating. The backbone is still incomplete.
DNA ligase completes the DNA backbone by forming covalent phosphodiester
bonds. Restriction enzymes and DNA ligase can therefore be used together to
join lengths of DNA from different sources.
In your books – explain, in a couple of sentences, the role of DNA ligase in
transgenesis.
Vectors
In biology a vector is something that carries things between species. For
example the mosquito is a disease vector because it carries the malaria parasite
into humans. In genetic engineering a vector is a length of DNA that carries
the gene we want into a host cell. A vector is needed because a length of DNA
containing a gene on its own won’t actually do anything inside a host cell. Since
it is not part of the cell’s normal genome it won’t be replicated when the cell
divides, it won’t be expressed, and in fact it will probably be broken down pretty
quickly. A vector gets round these problems by having these properties:




It is big enough to hold the gene we want.
It is circular (or more accurately a closed loop), so that it is less likely to be
broken down (particularly in prokaryotic cells where DNA is always circular).
It contains control sequences, such as a replication origin and a transcription
promoter, so that the gene will be replicated, expressed, or incorporated
into the cell’s normal genome.
It contains marker genes, so that cells containing the vector can be
identified.
Plasmids
View the animation:
“Construction of a plasmid vector”
http://highered.mcgrawhill.com/sites/0072552980/student_view0/chapter10/animation_quiz_1.html
Username: Biologyhelp
Password: Ilovebiology
View the animation:
“Features of a good plasmid vector”
http://www.yteach.ie/page.php/resources/view_all?id=amplification_cloning_ins
ulin_phospholipid_plasmid_polymerase_primer_restriction_endonuclease_plasmi
d_single_stranded_electrophoresis_denaturation_primers_nucleotides_polymer
ase_matrix_bioreactors_gene_vectors_sticky_blunt_ends_dna_purification_t_
page_7&from=search
1. Why do plasmid vectors need to be small?
2. Which “genetic tool” will be used to cut the plasmid?
3. Explain the purpose of choosing plasmids which contain antibiotic resistant
genes.
View the animation: “Use of plasmids in gene cloning”
http://www.yteach.ie/page.php/resources/view_all?id=amplification_gene_cloni
ng_insulin_phospholipid_plasmid_polymerase_primer_restriction_endonuclease
_plasmid_dna_single_stranded_dna_electrophoresis_denaturation_primers_nuc
leotides_page_2&from=search
1. What is a gene vector?
2. What is the first stage of gene cloning?
3. Why is the gene inserted into a plasmid?
4. Into which microorganism are the plasmids introduced?
5. Due to the addition of the plasmid containing the gene, what is the bacterial
cell capable of producing?
Read the section below on plasmids.
Plasmids are by far the most common kind of vector, so we shall look at how
they are used in some detail. Plasmids are short circular bits of DNA found
naturally in bacterial cells. A typical plasmid contains 3-5 genes and there are
usually around 10 copies of a plasmid in a bacterial cell. Plasmids are copied
separately from the main bacterial DNA when the cell divides, so the plasmid
genes are passed on to all daughter cells. They are also used naturally for
exchange of genes between bacterial cells (the nearest they get to sex), so
bacterial cells will readily take up a plasmid. Because they are so small, they are
easy to handle in a test tube, and foreign genes can quite easily be incorporated
into them using restriction enzymes and DNA ligase.
The diagram below shows how DNA fragments can be incorporated into a
plasmid using restriction and ligase enzymes.
The foreign DNA anneals with the plasmid and is joined covalently by DNA
ligase to form a hybrid vector (in other words a mixture or hybrid of bacterial
and foreign DNA).
Make sure you understand this diagram
Use the diagram above to help you draw and annotate your own diagram to show
the process of transgenesis that has been covered so far, so you should include:
 finding and cutting out the desired gene
 Cutting the plasmid


Sticking the desired gene into the plasmid
Formation of the recombinant plasmid.
3. Inserting the plasmid vector into the host bacterium
The plasmid must now be introduced into a bacterial cell, which will allow the
vector to multiply (clone itself and the foreign donor DNA it contains). The
bacterium commonly used is Escherichia coli (E. coli) a normal inhabitant of the
human gut and was chosen for this task because a great deal is known about its
genetics and because it grows rapidly with a doubling time of 30 minutes. A
mutant form of E. coli was specially developed for genetic engineering. This
form can only survive in special laboratory conditions. Therefore if it escapes
with foreign genes inserted, it cannot infect humans.
The process of adding new DNA to a bacterial cell is called transformation.
4. Cloning the bacteria and harvesting the human insulin
The bacterial cells need nutrients in order to grow, divide, and live. While they
live, the bacterial cell processes turn on the gene for human insulin and the
insulin is produced in the cell. When the bacterial cells reproduce by dividing,
the human insulin gene is also reproduced in the newly created cells.
Human insulin protein molecules produced by bacteria are gathered and purified.
Millions of people with diabetes now take human insulin produced by bacteria or
yeast (biosynthetic insulin) that is genetically compatible with their bodies, just
like the perfect insulin produced naturally in your body.
Username: Biologyhelp
Password: Ilovebiology
View the animation: “Biotechnology Past and Present - Biofermenters”
http://www.yteach.ie/page.php/resources/view_all?id=asepsis_dna_recombinati
on_dna_insulin_somatostatin_transgenic_organism_somatotrophin_fermentatio
n_pasteurization_malting_milling_whirling_brewing_maturation_filtration&from
=search
1. What containers are needed for the large scale production of Biological
molecules such as the protein insulin?
2. What conditions must be maintained?
View the animation: “Insulin producing bacteria”
http://www.bbc.co.uk/schools/gcsebitesize/science/edexcel/genes/genesrev3.s
html
View the animation: “Use of bioreactors in industrial scale drug production”
http://www.yteach.ie/page.php/resources/view_all?id=amplification_gene_cloni
ng_insulin_phospholipid_plasmid_polymerase_primer_restriction_endonuclease
_plasmid_dna_single_stranded_dna_electrophoresis_denaturation_primers_nuc
leotides&from=search
1. What is the role of the biotechnologists.
2. What role do the engineers play?
3. List the necessary conditions for optimum bacterial growth.
4. Explain the role of the propellers/ paddles in the bioreactor/ fermenter.
Questions
Question One (8)
Recombinant DNA technique (transgenesis) has enabled researchers to insert
DNA from one organism into the genome of bacteria for later multiplication.
The numbered diagrams on the following page show the sequence of events in
the process of gene cloning.
(a) Match the numbers of the diagram with the descriptions below. (The first
answer has been done for you). (4)
 A selected restriction enzyme cuts the donor DNA and the bacterial DNA do
that they that the same sticky ends. = 3
 The plasmids with the recombinant DNA are reincorporated into bacteria.
 The donor DNA is joined by a ligase to produce recombinant DNA molecules.






Recombinant bacteria are selected out to produce large numbers of
recombinant plasmids.
Donor cell DNA
The restriction enzyme is used again to cut the segments of donor DNA
from the plasmids.
Bacterial cell DNA.
Donor DNA is incorporated into the bacterial plasmid.
Recombinant DNA plasmids multiply inside the bacteria.
(b) Refer to the following and give the numbers of the two ‘sticky ends’
resulting from a cut by the same restriction enzyme. (2)
1. GAA
2. GCCTGGC
CTTAAG
4. CTGCAG
GAC
ACCG
5.
GAA
GGACTT
3. AAGCCT
TTC
6. AAGCTT
GAA
(c) State TWO reasons why an experimenter might want to insert some donor
DNA into a bacterial plasmid. (2)
Question Two (14)
Read the following extract carefully, then answer the questions which follow.
Volunteers are being recruited to eat raw potatoes in the first human trials of a
vaccine grown in genetically engineered potatoes. Researchers hope that people
who eat the potatoes will be protected from common gut infections.
The team first tried out the technique in tobacco plants. They took a strain of
Escherichia coli bacteria that causes food poisoning and identified that toxin as
a protein molecule. The toxin binds to receptor molecules on the cell surface
membrane of the gut cells of its victim. The researchers then used a modified
bacterium called Agrobacterium tumefaciens. Under normal circumstances,
these bacteria transfer plasmids into plant cells causing the plant to
manufacture the nutrients the bacteria need. In the modified bacteria,
however, the gene for producing the E. coli toxin had been inserted into the
plasmid DNA.
Once inside the tobacco cells, the foreign DNA becomes incorporated into the
tobacco chromosomes. These genetically engineered tobacco plants were grown
and propagated asexually by taking cuttings. These cuttings all contained the
gene for, and produced, the E. coli toxin.
Proof of success came when the tobacco leaves were mashed up and fed to mice.
Within days the mice started producing antibodies specific to the E. coli toxin.
The team then produced genetically engineered potatoes and fed these to mice
with similar results.
One problem with growing potatoes to produce vaccines is that the potatoes are
usually cooked before being eaten. Bananas, which are usually eaten raw, might
prove to be a better option.
(a) Use the information in this passage to help explain what is meant by:
(i) recombinant DNA (2)
(ii) a vector (2)
(b) Explain why:
(i) the tobacco cuttings all contained the gene for producing the toxin. (2)
(ii) Only some of the plants grown from the seeds of the genetically engineered
tobacco plants would be expected to contain the gene. (2)
(c) Explain why it is thought that bananas might be a better option than
potatoes for producing the vaccine. (2)
(d) What is a plasmid? (1)
(e) The gene for the toxin may be isolated and put into a plasmid from
Agrobacterium tumefaciens. Describe in detail the part played by restriction
enzymes in these processes. (3)
4.4.9 Uses of GMOs
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4.4.9 State two examples of the current uses of genetically modified crops or
animals.
Orange book  pg. 161
Green book  pg. 69
WS11 :What is Genetic Modification”
WS12 : Edible Vaccines
WS13 : Using Recombinant Bacteria
WS14 : Genetically Modified Plants
To do:
Read the two sections below: “Transgenic Plants” and “Transgenic Animals”.
Read pg. 69 in the Green Text book and make SHORT notes on the examples
given
In you books, state two example from the plant list (pick ones you think you will
remember) and the one example from the animal list.
Username: Biologyhelp
Password: Ilovebiology
www.yteach.ie
If you search the term “genetic engineering” you will find numerous animations.
They are all really short 1-2 mins and worth going through.
The following are relevant:
Traditional farming techniques and genetic engineering
Genetic engineering methods in agriculture
Legislation and the use of genetic engineering
Genetically Modified Foods
The simplest kinds of GM food is one in which an undesirable gene is removed.
In some cases, another more desirable gene is put in its place but in other
instances, only the introduction of a new gene is needed, no DNA has been
removed.
Whichever technique is applied, the end result is either that the organism no
longer shows the undesired trait or that it shows one which genetic engineers
want.
Transgenic Plants
The first commercial example of a GM food was the ‘Flavr Savr’ tomato. It was
first sold in the US in 1994 and has been genetically modified to delay the
ripening and rotting process so that is would stay fresher longer.
Another species of tomato was modified by a bioengineering company to make it
more tolerant to higher levels of salt in the soil. This makes it easier to grow in
certain regions of high salinity. One of the claims of the biotech industry is
that GM foods will help solve the problem of world hunger by allowing farmers
to grow foods in various otherwise unsuitable conditions. Critics point out that
the problem of hunger in the world is one of food distribution, not food
production.
Another plant of potential interest to the developing world is a genetically
modified rice plant which has been engineered to produce beta carotene in the
rice grains. The aim is that the people who eat this rice will not have
deficiencies in vitamin A (the body used beta carotene to form vitamin A). the
presence of beta carotene give a yellow colour  “Golden Rice”
Other crop examples include:
Bt Gene in Crops
Bt gene within maize makes a protein, which is toxic to insects feeding on the
maize.
Potatoes Modified to Increase Starch Content
They contain less water and absorb less oil when fried - healthier.
Antifreeze Gene in Crops
An antifreeze gene from Arctic flounder has been introduced to fruit and veg
to prevent frost damage.
Transgenic Animals
One way of genetically engineering an animal is to get to produce a substance
which can be used in medical treatment. Consider the problem faced by some
people with haemophilia  a blood condition in which their blood does not clot
because they lack a protein called factor IX. If such people can be supplied with
factor IX, their problem will be solved. The least expensive way of producing
large amounts of factor IX is to use transgenic sheep. If a gene which codes
for the production of factor IX is associated with the genetic information for
milk production in a female sheep, she will produce that protein in her milk.
4.4.10 Pros and Cons of GMOs
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4.4.10 Discuss the potential benefits and possible harmful effects of one
example of genetic modification.
Orange book  pg. 161
Green book  pg. 70
WS15 : Ethics of GMO Technology
As an introduction, watch the Youtube clip: “GM foods and You”
http://www.youtube.com/watch?v=B8p7M0WF_7A
 The information in the green book is good for this section so make sure you
read through it.
Is genetic engineering a good or bad thing?
Genetic engineering raises many profound social and ethical questions. As you
read through the ideas below, think about which ones you agree with and can you
justify your opinions?
Benefits, promises and hopes for the future
 GM crops will help farmers by improving food production
 GM crops which produce their own pest-control substances will be beneficial
to the environment because fewer chemical pesticides will be needed.
Reduction in use of pesticides leads to less contamination of ground water
around farmland, less harm to non-target species. Financial benefit not
having to pay for pesticides and a ‘greener’ image.
 Using GMOs to produce rare proteins for medications or vaccines could be,
in the long run, less costly and produce less pollution than synthesizing such
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proteins in laboratories.
Farmers can be more in control of what crops or livestock they produce.
There is always some randomness in breeding; genetic modification makes
the process less of a gamble. It is also much quicker than selective breeding.
The traits agricultural scientists are incorporating into our crops through
genetic engineering are the same as those traits that have been
incorporated by selective breeding e.g. improved nutritional content, delayed
ripening, resistance to disease caused by bacteria, fungi and viruses, better
taste, ability to withstand harsh environmental conditions, resistance to
pests such as insects and weeds.

The multinational companies who make GM plants claim that they will enable
farmers in developing nations to help reduce hunger by using pest-resistant
crops of GM plant which require less water.
As of January 2000 in over 30 countries, 10000 field tests of over 100 species
have been carried out. Over 100 million acres of genetically modified foods
have been grown – there have yet to be any unanticipated adverse environmental
consequences.
Harmful effects, dangers and fears
 No-one knows the long-term effects of GMOs in the wild. Efforts to keep
GM plants under control in well-defined areas have failed and pollen from GM
crops has escaped to neighbouring field. Genes from GM plants could be





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integrated into wild species giving them an unnatural advantage over other
species and an ability to take over the habitat.
There is a danger that the genes could cross species. It has been proves
possible in laboratories, so there is a possibility in nature too. Again, no one
knows the consequences of genes crossing species.
BT-crops which produce toxins to kill insects could be harmful to humans
because, unlike chemical pesticides which are only applied to the outer
surface, the toxins are found throughout the plant.
There are risks for allergies: if someone is not allergic to natural tomatoes,
but is allergic to GM tomatoes, they will need to know which ones they are
eating. But there is no difference in the outward appearance of the fruit and
food labelling is not always clear.
Critics are worried that large portions of the human food supply will be the
property of a small number of corporations
High-tech solutions are not necessarily better than simpler solutions. Crop
production could be increase by teaching farmers how to use water and
natural pest-control systems more efficiently
A proliferation of genetically modified organisms may lead to a decrease in
biodiversity.
4.4.11 Clone
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4.4.11 Define clone.
Orange book  pg. 165
Green book  pg. 70
To do:
Do exactly as it says in the objective  - but write it in your green books.
Clones and Cloning
The definition of a clone is a group of genetically identical organisms or a group
of cells artificially derived from a single parent. In either case, the resulting
cells or organisms were made using laboratory techniques. In farming, clones
have been made for decades by regenerating plant material or by allowing an invitro fertilized egg to divide to make copies of itself.
4.4.12 Cloning
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4.4.12 Outline a technique for cloning using differentiated animal cells.
Orange book  pg. 165
Green book  pg. 71-72
WS16: Cloning by Nuclear Transfer
To do:
“Click and Clone”
http://learn.genetics.utah.edu/content/tech/cloning/clickandclone/
“Try cloning a dog using the same method to create Dolly”
http://www.vtaide.com/png/cloning.htm
Draw and annotate a diagram to show the process in nuclear transfer cloning.
Cloning
Cloning is of great commercial importance, as brewers, pharmaceutical
companies, farmers and plant growers all want to be able to reproduce “good”
organisms exactly.
Natural methods of asexual reproduction are often quite suitable for some
organisms (such as yeast, potatoes and strawberries), but many important plants
and animals do not reproduce asexually, so artificial methods have to be used.
Until recently, cloning was only possible using genetic information from an egg
cell. Fertilised eggs are not differentiated (specialized) yet. After dividing
many times, some cells will specialize into muscle cells, others into nerve cells,
others into skin cells and so on until a foetus forms. For a long time, it was
thought that once a cell has gone through differentiation, it cannot be used to
make a clone. But then along came Dolly …
Cloning using a differentiated animal cell
Somatic Cell Cloning (or Nuclear Transfer)
Until recently it was thought impossible to grow a new animal from the somatic
cells of an existing animal (in contrast to plants). However, techniques have
gradually been developed to do this, first with frogs in the 1970s, and most
recently with sheep (the famous “Dolly”) in 1996.
1. From the original donor sheep to be cloned, a somatic cell (non-gametic cell)
from the udder was collected and cultured. The nucleus was removed from a
cultured cell.
2. An unfertilized egg was collected from another sheep and its nucleus was
removed.
3. The somatic udder cell was fused with the unfertilised egg cell which had
had its nucleus removed.
4. This combination of a diploid nucleus in an unfertilised egg cell was a bit like
a zygote, and sure enough it developed into an embryo in vitro.
5. The embryo was implanted into the uterus of a surrogate mother, and
developed into an apparently normal sheep, Dolly.
It took hundreds of attempts to achieve success with Dolly, but once the
technique is improved it will be possible to combine this technique with embryo
cloning to make many clones of an adult animal. Dolly’s “mother” was just an
ordinary sheep, but in the future prize animals (or genetically engineered ones)
could be cloned in this way.
4.4.13 Ethics
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4.4.13 Discuss the ethical issues of therapeutic cloning in humans.
Orange book  pg. 165
Green book  pg. 72
Read the information in the green book and below.
In your books, write a paragraph summarizing some of the ethical issues
surrounding therapeutic cloning in humans.
Ethical issues surrounding therapeutic cloning
Since therapeutic cloning starts with the production of human embryos, it
raises fundamental issues of right and wrong. Is it ethically acceptable to
generate a new human embryo for the sole purpose of medical research? In
nature, embryos are created only for reproduction and many people believe that
using them for experiments is unnatural and wrong.
However, the use of embryonic stem cells has lead to major breakthroughs in
the understanding of human biology. What was once pure fiction is coming closer
and closer to becoming an everyday reality thanks to stem cell research:
 Growing skin to repair a serious burn
 Growing new heart muscle to repair an ailing heart
 Growing new kidney tissue to rebuild a failing kidney.
With very rare exceptions, the vast majority of researchers and medical
professionals are against the idea of reproductive cloning in humans. However,
there is a growing popularity for the pursuit of therapeutic cloning since the
promises of stem cell research are so enticing.
Reproductive cloning is performed with the express intent of creating another
organism. This organism is the exact duplicate of one that already exists or has
existed in the past. Cloning of plants, animals, and humans falls into the class of
reproductive cloning.
Therapeutic cloning is performed, not to produce another organism, but to
harvest embryonic stem cells for use in medical treatments. Embryonic stem
cells are those cells found inside of developing embryos. They can be used to
produce a number of different cells including tissue, muscle, and organ cells