MODULE 6

GENE TECHNOLOGY
You need to understand 4 techniques.
1. Polymerase chain reaction (PCR)
2. Gel electrophoresis
3. use of DNA Probes
4. DNA Sequencing.
 Polymerase chain reaction (PCR)

A method to amplify/clone a small section of

DNA
Uses of PCR

Amplify DNA samples from:

• Tissue samples from crime scene / suspect

• Pathogen genomes to identify disease
• A donor of a gene needed for genetic
engineering
• Any samples on which DNA sequencing will
take place
 Polymerase chain reaction (PCR)
Make drawings and notes of the stages of the polymerase chain
reaction
TIP – there are 3 main stages (the 3 temperatures used)

Key words

Taq Polymerase Complementary 55oC

hydrogen bonds anneal 94-96oC Primer

Nucleotides 72oC repeat
Exponential Optimum
1. 96oC – break H bonds. Double helix
unwinds
2. 55oC – primers (10-20 bases) anneal to
complementary sequence at target strand
3. 72oC – Taq polymerase binds to double
strand at primer. Optimum temperature.
Adds free nucleotides to make copy
4. Repeat many times. Exponential growth
of DNA strand
 PCR - points to remember
• DNA is made of antiparallel strands e.g.
5’ AGGTCA 3’
3’ TCCAGT 5’
• DNA strands have a 5’ (prime) and 3’ (prime)
end
• New strand grows only from 3’ end
• Complementary base pairs (A-T and G-C)
 Advantages of PCR
• Quicker – 30 cycles takes few hours
• V little equipment used
• V little labour needed – cycles are
automated
• V little space required.
 How is PCR different to DNA
replication in a cell?
• PCR needs a primer to start replication
• PCR Uses heating and cooling to separate and
bind strands – and not the enzyme DNA
Helicase
• PCR only replicates short sequences of DNA, a
cell copies a whole genome
Fill in sheet to explain how DNA replicates
Gel Electrophoresis

electro referring to the energy of

electricity and Phoresis which come
from the Greek language meaning "to
carry across.“

This is used in DNA

fingerprinting/profiling (to identify
criminals)
 Gel Electrophoresis
E. Mix DNA with Restriction Endonucleases
B. DNA Cut into different lengths at restriction sites
A. Put into Agar and a buffer solution
D. Apply electric current
C. DNA moves through gel to positive terminus
H. Smallest fragments move quickest
G. DNA separates into bands of equal lengths
F. Hybridisation: Add radioactive/ fluorescent probe
and expose to X ray film/ UV light
I. DNA fingerprint image is now transferred
 Electrophoresis of proteins
Separates proteins of different mass/size in
a mixture An immunoglobin
chain is
overproduced in
the myeloma and
more is secreted in
the urine and found
in the blood serum.
Notice it travels
quickest because it
is small
Separates by Separation techniques
relative solubility / Separates by size /
interaction with length of DNA
stationary phase strand
(adsorption)

Only separates
Can separate Separates charged particles
charges and non - molecules from a
charged mixture to identify Electro-
Uses a buffer
TLC molecules or
No Buffer solution compare them
phoresis
Uses
– use a solvent fluorescence/radio
activity
Uses dyes to
make molecules
visible Automated

TLC not
automated
 Gene
Is identical in
These have variable short
each person
tandem repeats
Coding region Non Coding regions
 Explain why only selected
sections of non-coding DNA
are used when profiling a
human.
• most genes are the same so
• coding sequences do not have unique
profiles
• non-coding DNA contains variable
numbers of short tandem repeats
 GENE PROBE
• A short single stranded piece of DNA (50-80
nucleotides) complementary to a gene section
of DNA. The probe can be identified by using:
a) radioactive marker: (32P makes the
phosphoryl groups in strand. This emits beta
radiation which is detected on photographic
film)
b) Fluorescent marker: emits a colour when
exposed to UV light
Gene Probes
 Uses
• Can be used to locate a gene wanted for
genetic engineering
• To identify the same gene in a number of
different species’ genomes
• To identify the presence or absence of an
allele for a particular genetic disease e.g.
To find the mutant CFTR allele that
causes cystic fibrosis in an embryo
 Compare probes and primers

Similarities Differences
• Both anneal to • Probes are longer (about
complementary DNA 50-80 not 10-20)
sequences • Primers –binding site for
enzyme (T___ p______)
• Both single stranded • Probes used to identify
DNA Sequences/genes
 Revision
A cheek swab contains 10 cells. How might
you use these to identify the presence of the
cystic fibrosis allele?
A cheek swab contains 10 cells. How might you use these to identify the
presence of the cystic fibrosis allele?
1. PCR: 96oC – break H bonds. Double helix unwinds,
55oC – primers (10-20bp) anneal to complementary sequence at
target strand,
72oC – Taq polymerase binds to double strand at primer. Optimum
temperature. Adds free nucleotides to make copy, Repeat many times.
Exponential growth of DNA strand
2. Gel Electrophoresis - Mix DNA with Restriction Endonucleases
DNA Cut into different lengths at restriction sites
Put into Agar and a buffer solution
Apply electric current
DNA moves through gel to positive terminus
Smallest fragments move quickest
DNA separates into bands of equal lengths
3. Probe: Add radioactive/ fluorescent probe. 50-80 bases. Single
strand. This has complementary sequence to cystic fibrosis allele. Mix
with sample. Hybridisation. Expose to X ray film/ UV light–
Question
Chromatography is a process that
separates substances in a mixture.
Molecules dissolve in a solvent. Those
molecules that are most soluble travel
the furthest along filter paper. Those
that are lease soluble travel a shorter
distance up filter paper. Explain how gel
electrophoresis is similar and different
to this process.
 DNA Sequencing
LO
Describe how to sequence a gene
 DNA Sequencing
This explains how the HGP sequenced the
order of bases in each gene in the human
genome.
The genome (or plasmid from a gene library)
needs to be cut into smaller fragments using
restriction endonuclease enzymes
Then DNA sequencing is used to read the
genome. The dideoxy chain termination
method was developed in 1977 by Fred
Sanger
https://www.youtube.com/watch?v=FvHRio1y
yhQ
 The terminating base in each
sequence is coloured. As it passes
through a laser it is recognised:
 5 8

6 4

1 7

2 3
 5 8

6 4

1 7

2 3
The human genome is 3 billion base pairs long

If this sequencing method can only sequence

fragments of DNA that are 600 base pairs

a) Calculate the minimum number of fragments

that would be needed to be sequenced to read the
human genome

b) If each sequencing method took 2 hours to

complete what’s the minimum number of days it
would take one machine to read the human genome
= 3 billion = 3 000 000 000
600 600

= 5 000 000 fragments

= 5000 000 x 2hr = 10 000 000hr

In days :

= 10 000 000 = 416666.7 = 416667 days

24

(that’s 1141 years !!)

In reality there are many more fragments
sequenced. To work out the order of the
fragments overlapping sequences are
needed
a) the principles of DNA sequencing and the
development of new DNA sequencing
techniques To include the rapid
advancements of the techniques used in
sequencing, which have increased the
speed of sequencing and allowed whole
genome sequencing e.g. high-throughput
sequencing. HSW7
http://www.sumanasinc.com/webcontent/animations/content/highthroughput2.html
High-throughput sequencing
The speed of sequencing has increased
and allowed whole genome sequencing
using new techniques e.g. high-throughput
sequencing. This allows millions of
fragments to be sequenced at the same
time and the base sequence is read in real
time, as new nucleotides are added by
DNA polymerase.

How was this process different to Sanger

sequencing?
 Double bubble

Gene
PCR
Sequencing
 EVALUATE QUESTIONS
A scientists wanted to find the most suitable method to sequence the DNA of
new emerging viruses containing 15 genes. They used a 100 base pair
fragment of DNA cloned using PCR and sequenced the6fragment 3 times
using each process A and B
A summary of the findings are below: 5

4
Sequencing Mean run Cost per read Number
method time to (£) of errors 3
sequence per 100
100 bases bases 2

A 1.52 0.05 1

B 6.10 0.20 0
Method A Method B

The scientists stated that method A was preferable over method B

Evaluate the scientist's conclusion

Supports conclusion

• A faster run time / 4 times faster

• Lower cost / 4 times cheaper

Against Conclusion

• Higher mean error rate in A

• Error bars do not overlap

• Unit of time not stated

• Only 3 (low) fragments in sample size.
• Not sufficient to statistical analysis – anomalies have
bigger impact on mean
• Only 100 base fragment tested / more bases would be
analysed in viral DNA

Other details of method not provided

 https://www.youtube.com/watch
?v=u8bsCiq6hvM

3-6 minutes pros

After 8 cons

https://www.youtube.com/watch?v=ml9Qdq
tHHk8
AGCTATCGATAAATGCTAGCT
GGTACTTCATCCCACGTGCGC
ATGCCAAACGTCGGACATCGA
TCTTTAGCATCGACCATCGAC
ATGACAACAGTCGATTCGATA
CGATCGGCCCTAGCAGACGAC
ACACAGCTCGACCGTGCGATC
GACACAGCTCGCGACATCGCG
ACCGCTGACACCGCTGAGTTG
GAACAGCTGAACAACAACACA
GGACACAGGTGTGGTGTGCAC
CGTACATAGAGATCCAGCTGA
GATCGACAGCTGACATCGGTG
GACACAACAGCTGACAGTCGC
GACCTCTCTTCTCCTCTCGGA
 Comparative gene mapping
Comparing base sequences of the same
gene between individuals of the same or
of different species
Reasons for
comparative
gene
mapping
 Comparing genomes

How can we determine who is our closest

relative (with whom do we share the most
recent common ancestor)?
What’s this?

Do you remember Cytochrome C Oxidase from year 12?

 Molecular Evolution: comparing genomes

Human DNA sequence for a gene (for example Cyctochrome C Oxidase

in mt DNA)
3’ AGGTCAGCACATGGTATAGCGCGACATCTCC 5’

Same DNA sequence in 2 other species.

A: 3’ AGGTGCCCACATGCTATAGCTTGACATCTCC 5’

B: 3’ ATATCACCACCCGGTATTATGCGAGATCTAC 5’
Which species do you think is a closer relative to Homo sapiens?

This means they have a more recent common ancestor

 Common ancestor of A,
Common ancestor of A B and Homo sapien
and Homo sapien

Species B
Homo sapiens Species A
 Common ancestor of A,
Common ancestor of A B and Homo sapien
and Homo sapien

Species B
Homo sapiens Species A
By comparing base sequences of modern human DNA and Neanderthal DNA it can be
proven that there are some similarities which suggest here was interbreeding between
the 2 species they also shared 99.7% DNA with humans
 Each species
has a
cytochrome c
oxidase gene
with a different
sequence. This is
a method to
identify a new
species

When the time since each pair of organisms presumably diverged

is plotted against the number of nucleotide differences, what does
the resultant straight line suggest about the cytochrome c gene?
The 2 species synthesise the same protein,
however, due to millions of years sine a
common ancestor the genes have different
base sequences. Explain how this is possible

• DNA code is degenerate

• More than one codon codes for each
amino acid
• Silent mutations will not alter amino acid

• Silent mutations can also occur in the non-

coding introns of a gene
How do we know which
flu has migrated to
Britain this year?
TAC GGT TTA CTC TCA ATT

If this is the DNA code, what is the amino acid

code?
TAC GGT TTA CTC TCA ATT
AUG CCA AAU GAG AGU UAA

MET PRO ASN GLU SER STOP

Fake News!!
Reasons for
comparative
gene
mapping
 Show
evolutionary
Deduce relationships
primary Use in
structure of synthetic
protein (and
predict tertiary biology
/3D structure)

Reasons for
comparative
Identify genes
gene
we share with mapping
other Identify
organisms disease cause
and what is allele for
does genetic
disease
Compare
pathogenic
and non
pathogenic
strain of
bacteria.
Comparative gene mapping
1. Analysing genome of pathogens; comparing outbreaks
and identifying strains and identifying regions of DNA that
could be the ‘targets of drugs
2. Identifying new or different species - look for differences
in a particular gene – cytochrome C oxidase in mtDNA)
3. Phylogeny – compare evolutionary relationships between
species by analysing the number of differences
(mutations). More differences suggest a more distant
common ancestor
4. Work out the amino acid sequences (primary structure)
of proteins using the gene - this can prove difficult as DNA
contains introns and exons however only the exons code
for protein
5. Synthetic biology : genetic engineering, make a new
gene from free bases OR making a whole genome from
free bases and constructing a new organism: Cynthia
6. Identifying disease causing alleles/sequences
1. Use of computers and software tools for understanding
biological data. E.g. comparing large sets of DNA sequences,
predicting protein tertiary structure (interactions between R
groups)
Bioinformatics
2. This deals with the incidence, distribution, and possible control
of diseases and other factors relating to health. Comparing
pathogen DNA sequences can locate sites of outbreak
Epidemiology
3. Comparing DNA sequences to identify time of most recent
common ancestor (MRCA) and therefore classify organisms
Phylogeny
4. Construction of novel artificial pathways, organisms or devices,
or the redesign of existing natural biological systems

Synthetic biology
 GENETIC ENGINEERING
• LO
Write a sequence for Genetic Engineering
including
i) Isolate the gene
ii) Using a vector
iii) Introduce into a host cell

• http://www.abpischools.org.uk/page/modul
es/diabetes_16plus/diabetes8.cfm?age=A
ge%20Range%2016-19&subject=Biology
restriction
enzymes

‘sticky end’
‘sticky end’

ligase enzyme
 Genetic engineering
3 steps;
Isolate the gene
Use a vector
Insert to new organism
 Isolate the gene
1. Use Restriction Enzymes e.g. Human Growth
Factor
2. Use mRNA e.g. Insulin
3. Use the primary structure of the protein
 Isolate the gene
e.g. Human Growth Factor
1. Add r______ e_______ to DNA sample
2. Each enzyme cuts double stranded DNA at a
s_______ s________
3. It cuts each strand (hydrolysis of the
p_____________ bond ) at a different point
4. This leaves unpaired b_____ : “Sticky ends”
5. These are available join other strands of DNA as
they form H______ b______ with c_____________
b______
6. Use a g______ p_______ to identify desired gene
from DNA Sample.
7. Use PCR to amplify desired gene
Hydrolysis of the phosphodiester bonds
restriction
enzymes

‘sticky end’
‘sticky end’

ligase enzyme
 2. Getting the insulin gene….
Uses the mRNA that codes for the insulin
protein and turns this back into double
stranded DNA
 To isolate the insulin gene using mRNA
1. Remove m_______ from cytoplasm of cell that
produces desired protein e.g. beta cells in
pancreas
2. Use R________ Transcriptase to convert
mRNA to single stranded _____
3. Use _____ ____________ to form double
stranded DNA
4. This is the gene for the desired protein
5. Add nucleotides to ends to form s______
e______
Insulin UACGUUAUC
mRNA Reverse
transcriptase

Insulin gene: ATGCAATAG

cDNA DNA polymerase

ATGCAATAG
Double
stranded TACGTTATC
DNA

Stick ends ATGCAATAGGGG

added CCCTACGTTATC
 Insert into Vector – The Plasmid
• A vector is used to insert gene into bacteria
1. Remove p______ from bacteria – by centrifuging
2. Cut open using same r________ e_____ used to
cut out gene
3. Creates complementary s_____ e_____
4. Mix plasmid and human gene
5. Sticky ends pair up (anneal)
6. DNA ligase links d__________ –p________
backbone
7. Makes Recombinant Plasmid
8. Some plasmids contain the gene, some simply
rejoin without the gene
Vectors in GE

ANIMALS

HUMANS

PLANTS

BACTERIA
 Transformation: Plasmid
inserted into bacteria
• Electroporation, pulse of electricity applied to
bacteria, makes bacteria cell wall, plasma membrane
(and nuclear membrane in eukaryotes) more
permeable
• Can also use Ca2+ ions and ‘heat shock treatment’
• About 1% bacteria take up a recombinant plasmid –
this is called a transformed bacteria

Hi
I’m Optimus
Grime
1. Isolate the gene

2. Vector

3. Inserting into organism

 `

Electroporation
 Genetic Marker
A Genetic Marker identifies transformed
bacteria e.g. Using antibiotic resistance
genes or fluorescent genes in the plasmid
 To identify the transformed bacteria...
1. Use a plasmid with 2 types of ________ resistance
e.g. ________ and __________
2. Insert human gene within the __________ resistance
gene
3. Insert plasmid into ________ e.g. electroporation
4. Grow bacteria on agar with __________
5. Only bacteria with a _______ will grow
6. Using muslin – transfer impression of colonies onto
agar containing _________
7. Those colonies that do not grow on ____________
but do grow on _______ are the transformed
bacteria. This is called Replica Plating
 Genetic Marker

Problems:
Antibiotic resistant genes could escape
into wild bacterial populations. These
bacteria would not be killed by antibiotic
medicine and could cause more serious
infections.

Bacteria can pass on antibiotic resistance

genes to other populations by conjugation
 Key words Test
1.Primer
2.Negative
3.Gene probe
4.DNA Ligase
5.Reverse transcriptase
6.Di-deoxy nucleotide
7.Genome
8.Recombinant DNA
9.Transformed bacteria
GM Ethics
1. Insect resistance in genetically modified soya,
2. Genetically modified pathogens for research
3. ‘pharming’ i.e. genetically modified animals to
produce pharmaceuticals
4. Issues relating to patenting and technology
transfer e.g. making genetically modified seed
available to poor farmers
Figure 1. (A) bean leaf beetle, (B) wireworm, Photo courtesy of Frank Peairs, Colorado
State University, Bugwood.org., (C) seedcorn maggot, (D) white grub, (E) cutworm,
Photo courtesy of Clemson University-USDA Cooperative Extension Slide Series,
Bugwood.org., (F) soybean aphid
Can genetically modify so pathogenic virus is
altered to act as a non virulent vaccine
 Philippine farmers have
called for a halt to field
trials after very poor
results, large debts, and a
concern for their native rice
seeds.

What is golden rice?

GM Ethics
1. Insect resistance in genetically modified soya
Less crop eaten – higher yield – less pesticide
used – increased biodiversity
Large selection pressure – insect becomes
resistance quickly – to slow this non GM soy
planted with GM Soya

Pro and a con for each one

GM Ethics
2 Genetically modified pathogens for research
To act as a non virulent vaccine
Used as a pathogen for biological warfare

Pro and a con for each one

GM Ethics
3. ‘pharming’ i.e. genetically modified animals to
produce pharmaceuticals
Mammals are able to make eukaryotic proteins
(using golgi) in large quantities, easily harvested
from milk e.g. AAT made for emphysema
Potential harm to animal / religious objections

Pro and a con for each one

GM Ethics
4. Issues relating to patenting and technology
transfer e.g. making genetically modified seed
available to poor farmers

Companies make money

Expensive, need to bought each year as
they are sterile. Exploit poor farmers in
climates that benefit from GM e.g.
drought resistance

Pro and a con for each one

CAR T Cell Therapy
 GENE THERAPY
1. Definition
The insertion of a functional gene into
the human genome that can make a
functional, therapeutic protein to relieve
symptoms of disease
 GENE THERAPY
2 Vectors –
Liposome – vesicle with pb. Contains new gene
(e.g. in plasmid). Fuses with target cell . E.g. by
endocytosis and DNA enters cell.
Virus – remove viral DNA and insert new gene
– Virus binds to target cell and inject the new
gene
 GENE THERAPY
1. Somatic gene therapy – Gene inserted
into body cell, change not passed onto
offspring, may need repeating
2. Germline Gene Therapy – Gene inserted
into gametes or embryo, all cells in
human have new gene, banned
3. state 3 Uses – Treat genetic disease /
treat cancer cell / modify stem cell
 GENE THERAPY
State 3 Uses –
Treat genetic disease
Treat cancer cell
Modify stem cell
 Gene therapy

Somatic Germline
 GENE THERAPY

The insertion of healthy genes into body

cells to replace disease-causing alleles

Gene therapy is being developed to treat cystic fibrosis

 healthy gene cut out from DNA liposomes
and put into a liposome

human chromosome
liposomes
put into
inhaler
 healthy gene cut out from DNA viral DNA
and inserted into virus removed
to stop it
causing
disease
human chromosome virus

Modified virus then cloned

(grown in large numbers)

Each new virus receives a

copy of the healthy
human gene
The viruses are put in an
inhaler and the patient
breathes them in
The viruses move into the
cells lining the lungs, taking
the healthy human gene with correct
them amount
of mucus

lung cell
 GENE THERAPY
State 3 Uses –
Treat genetic disease
Treat cancer cell
Modify stem cell
 Cancer cells
1. Insert gene into
cancer cells
2. This genes codes for
protein (e.g. Cell
Surface antigen)
3. Immune system Is this germ cell
identifies them as
or somatic cell
non-self and can
destroy the cells gene therapy?
 SCID: Severe Combined Immunodeficiency
A more permanent gene therapy inserts a new gene into an
adult stem cell.
SCID is Genetic recessive inheritance – Defective gene for
enzyme ADA
Toxins in body builds up inside T lymphocytes and destroys
the cell – Patient has very poor immune system
CURE
1. Genetically modify retrovirus – insert gene for ADA
2. Remove Bone Marrow
3. Virus infects Bone Marrow (ex vivo) – ADA gene taken up by
cells – non reproductive cloning
4. Transgenic cells placed back into bone marrow and express
ADA gene
This could replace need to for Bone Marrow transplants
1. Genetic manipulation could be
passed on to patient’s offspring
5. Allele delivered to spermatozoa,
ovum or embryo – more straight
forward delivery

2.Need technique to deliver 6. Viruses used to deliver gene to

gene to target cell’s location. Or target tissue. However host immune
specific cells are removed and system may not accept virus vector on
then replaced (called ex vivo second or third treatment
therapy)

3. Unethical to manipulate 7. Genetic manipulation is restricted

embryos. Damage to embryo may to the patient’s cells only
not be known until organism fully
developed

4. All cells in the body will have 8. Treatment is short lived and
the replaced functional allele needs to be repeated regularly
 Which one is gene therapy?
A. The insertion of healthy genes into sex
cells (gametes) to replace disease-
causing alleles

B. The insertion of healthy genes into an

embryo to replace disease-causing
alleles

C. The insertion of healthy genes into body

cells to replace disease-causing alleles
Jan 2013 f215
Essay
Evaluate Genetic engineering in
organisms

(Bacteria, plants, humans, animals)

For each state pros, cons and examples

Due Wednesday

Tip – look up the advantages of using

bacteria in technology
Below is a list of cells in the body

A.Somatic skin cell

B.Bone marrow Stem cell
C.Ovum (gamete)
D.Skin Stem Cell

1. Which cell could you add a gene to in order to grow

new skin long term?
2. Which 2 cells may be able to treat sickle cell
anaemia permanently?
3. Which cell would require repeat treatments of gene
therapy?
4. Which cell could you add a gene so that the gene
would be in every genome in an organism?