IB Genetics Topic 4:
Genetic Engineering
Topics in Genetic Engineering
• Polymerase Chain Reaction (PCR)
• Gel electrophoresis
• DNA profiling
• The genome project
• Gene transfer
• Genetic modification in plants and animals
• Cloning
We will be brief!
• The Blog has videos and games for you to
cement your understanding of each topic!
• Virtual labs are located on Utah genetics
Key techniques in DNA identification
Polymerase Chain
reaction (PCR)
• Takes a tiny quantity of
DNA and copies all the
nucleic acids to make
millions of copies of the
DNA
• Only a portion of DNA is
amplified, not the
whole genome…
Gel electrophoresis
• This technique separates
fragments of DNA in an
attempt to identify its
origin
• Used for DNA
‘fingerprinting’ (profiling)
• Used for many diagnostic
procedures
What do we do with this DNA?
• All sorts of forensic and genetic tests…
The DNA sequences amplified by PCR and
matched by gel electrophoresis are used
for:
• Forensic identification - crime
(CSI….)
• Paternity suits
• Diagnosing diseases and
genetic disorders – genotype
analysis
• Detection of bacteria and viruses in
the environment
• The Genome project: determination
of the entire human genetic code
• Evolutionary research
4.4.1: The Polymerase Chain Reaction
• Kary Mullis, 1984,
Nobel Prize 1993
• ‘Reviewers and
Biographers often
latch on to his
enjoyment of drugs,
womanising and
surfing to paint him
as some sort of cool
rock star
rebel…but…’
What does PCR do?
• It mimics the process of
DNA replication
• It quickly amplifies a tiny
sample of DNA into
millions of identical
copies
• This produces enough
DNA to allow further
analysis and study.
How does PCR work?
First, you need to extract DNA from
cells.
Next, PCR requires:
• Custom-made DNA primers
• (TAQ) DNA polymerase
• Nucleotides for complementary
base pairing
• HEATING and COOLING:
Thermocycler machine
PCR can produce 100 billion copies of
the DNA sample in a few hours
Let’s do it!
• PCR the movie
• PCR the cartoon
• PCR the game
Gel electrophoresis
• Used to separate fragments of DNA and match
against ‘known’ or ‘test’ samples
• Forensic identification - crime (CSI….)
• Paternity suits
• Diagnosing diseases and genetic disorders –
genotype analysis
• Detection of bacteria and viruses in the environment
4.4.2: Gel electrophoresis
1.
2.
3.
4.
5.
6.
Commonly the DNA sample is first amplified
by PCR
The DNA sample is chopped into fragments
using restriction enzymes
Gel electrophoresis separates fragments of
DNA according to their size
DNA samples are placed in a gel, fluorescent
marker added (tag attached to a triplet), and
an electric charge is applied to push
fragments along
The shortest fragments travel furthest
through the gel, while the longest (heaviest)
remain closer to their origin
This lets us ‘match’ the sample against
another DNA sample or standard
Gel electrophoresis step by step
• animation 1
• Animation 2
• and the real thing....in 90 seconds...
Let’s do it!
• Virtual Lab 1
• Virtual lab
DNA profiling and forensic analysis
• An introduction
4.4.3: DNA profiling
HOW?
APPLICATIONS
• DNA sample (blood, semen,
cheek swab)
• Sample is amplified
by
PCR, then matched against
known samples using gel
electrophoresis
• We look for standard tandem
repeating DNA: highly repetitive
sequences from non-coding
region of DNA
• STR’s are unique to individuals
Numerous techniques for
DNA profiling:
• Criminal forensics
• Paternity suits
• Ecology – identifying
relationships in
migrating birds, whales
etc
• Evolutionary genetics
Famous examples of DNA profiling
• The innocence project
• The famous blue dress
• Identifying the Mona
Lisa
• Identifying the
Romanovs
DNA profiling: paternity and forensics
• The fluorescent banding
pattern shows up for each
DNA fragment and can be
compared with test/ known
sample
• Usually 13 ‘STR’ sites are
used to establish paternity
or identity
• PATERNITY: bands
will be shared with
relatives
• CRIME SCENE:
‘perfect match’
Past paper questions
How can fragments of DNA be separated?
A. Using polymerase chain reaction (PCR)
B. Using gel electrophoresis
C. Using gene transfer
D. Using gene cloning
(Total 1 mark)
What could be achieved by DNA profiling using gel electrophoresis?
A. The chromosome number of an organism could be counted.
B. It could be shown that human tissue found at the site of a crime did not come
from a person suspected of having committed the crime.
C. A karyotype could be produced.
D. Extinct species of living organisms could be brought back to life.
(Total 1 mark)
Past paper questions
A new allele that provides herbicide resistance is identified in soybean plants. The allele is
dominant.
Which of the following would be carried out in a herbicide-resistant plant to find out if it is
homozygous or heterozygous for the gene?
A. Gel electrophoresis
B. Karyotyping
C. Test cross
D. DNA profiling
(Total 1 mark)
A small amount of a suspect’s DNA is obtained from a crime scene. What techniques would be used to
carry out DNA profiling?
A.
Gel electrophoresis and paternity testing
B.
Paternity testing and the polymerase chain reaction (PCR)
C.
Polymerase chain reaction (PCR) and gel electrophoresis
D.
Test crossing and pedigree analysis
(Total 1 mark)
Past paper questions
What type of enzyme could be used to cut a DNA molecule as indicated by the dotted
line on the diagram below?
A. DNA ligase
B. DNA polymerase
C. Helicase
D. Restriction enzyme
(Total 1 mark)
Which process is used in polymerase chain reaction (PCR)?
A. Transcription
B. Translation
C. Replication
D. Mutation
(Total 1 mark)
4.4.6: The Human Genome project
• We’re all the same!
• We’re all different!
• Welcome to the human genome
Started in 1990, first draft published in 2003,
ongoing refinement…
Amazing international effort
• The BBC tell us about the genome 10 years on
• Hank tells us some surprises about the human genome
project
• ..so what has the human genome project achieved, so
far?
The brave new world of genomics
GENOMICS
– analysis of
the
structure
and function
of
genomes…
Key outcomes of the Human Genome
project
• We identified the number (30,000) and loci of all the genes
of our genome
1. The Bio-informatic and genomic industries: Genetic
databases and the bio-informatics industry
2. Human health: Medical diagnostics, treatments,
pharmacogenomics, gene therapy
• We identified many new proteins and their functions
3. Evolutionary genetics and evolutionary history
We compared human DNA with that of other species
4. Transgenics: move production of beneficial proteins from
one species to another, design novel proteins
Bio-informatics: The HGP and human
health (1)
• 2 out of 3 of us will die
of a genetically related
disease
• Many (some say all)
diseases have a genetic
cause
• Genetic report cards
can identify ‘potential
risk’ of future illnesses
Bio-informatics: Do you want to know
your genome?
• Do you want to
know your genome?
• Are you sure?
The human genome and health:
medical advances (1): Gene therapy
• Find defective genes
and ‘fix them’
• Insert a healthy normal
gene or gene product
(lipid, protein)to
achieve normal function
• Most effective for single
gene disorders: cystic
fibrosis, Sickle Cell
disease,
The human genome and health: medical
advances (1): Pharmacogenomics
Uses information about
your genetic make-up to
determine the drug, and
drug doses, that will
work best for you
Currently used for
treatment of:
• HIV
• Breast cancer
• Colon cancer
• Mental illness
more info here...
3. Using the HGP to understand human
evolution
By sequencing and databasing
genes, we can see similarities and
differences between species
• The closer the genome match,
the closer their evolutionary
history
• Human Chromosome 2 came
from fusion of two great ape
chromosomes
• Karl Miller on human evolution
• The time-tree of evolution
Tracing human migrations using
mitochondrial DNA (SNP’s)
Ethics and the human genome
How companies are
patenting your genetic
code
Ethical Issues surrounding the human
(and other) genomes
1. Shared nature and ownership of genetic
information
2. Limitations of genetic testing
3. Gender selection?
4. Gene patenting?
5. Genetic discrimination?
6. Forensic databases
7. Limits of cloning and genetically modified
organisms (plants and animals)
4.4.8: GENETIC ENGINEERING,
(MODIFICATION, TRANSGENICS)
BIOTECHNOLOGY
The basic premise for genetic
engineering…
The genetic code is universal
• All living things share the same code
• Each codon codes for the same animo acid,
regardless of the species
• Genes can be transferred from species to
species, to produce the same product
We can cut, copy and paste DNA between
species
Transgenic organism
An organism which contains genetically
altered material
Synonyms: Genetically engineered; genetically
modified
Gene transfer 101: the basics of cutting,
copying, pasting and cloning genes
• An introduction
• Step-by-step
How does genetic engineering work?
1. Cut out desired DNA
using restriction
endonuclease
2. Prepare plasmid using
the same restriction
enzyme
3. Insert DNA into
plasmid using DNA
ligase
4. Insert plasmid (vector)
into host cell
Applications of Genetic Engineering
• Gene Therapy – treatment of SCIDs, cystic
fibrosis, ?Huntingdon’s Disease?...
• ‘Pharming’: Transgenic organisms used to
produce human insulin, Human factor IX,
growth hormone
• Vaccines – hepatitis B vaccine
• Diagnostic tests and targeted drugs
• Xenotransplantation – production of
genetically modified organs in pigs
Genetic Engineering: Making Human
insulin
• E. Coli make human insulin
Genetic Engineering: Making Factor IX
in sheep…
1997: Factor IX isolated and purified from sheep’s milk
to treat one hereditary form of haemophilia (Haemophilia B,
Christmas Disease)
Polly and Molly the Factor IX sheep
Gene therapy
Gene therapy
• Identify beneficial molecules which are
produced in healthy people
• Identify the gene which produces the
desirable molecule
• Copy that gene and use it as a recipe to
synthesise the molecule in a laboratory (or in
another organism!)
• Distribute the beneficial molecule as a new
treatment
The human genome and new medicine
(1): Gene therapy
• Find defective genes
and ‘fix them’
• Insert a healthy normal
gene or gene product
(lipid, protein)to
achieve normal function
• Most effective for single
gene disorders: cystic
fibrosis, Sickle Cell
disease,
• gene therapy in depth
• A lucrative business...
Applied gene transfer: Gene Therapy
Gene therapy for treatment of Severe Combined
Immunodeficiency Disease
(2)
• Gene comparison slider
Transgenic (GM) plants
Conferred Trait
Organism
Genetic change
Herbicide resistance
Soybean
‘Roundup’: Glysophate
resistance
Flavr Savr
Tomato
Switch off gene for
ripening – delays natural
softening, improves
flavour
Insect Resistance
Bt Corn
Resistance to European
corn borer introduced by
addition of toxin from
Bacillus Thuringiensis
Salt-resistance
Tomato
Extended range for tomato
production
Beta carotene
‘Golden rice’
Protection against betacarotene (Vitamin A)
deficiency
Transgenic plants: The controversies…
The controversies….
Transgenic (GM) animals
Conferred Trait
Sheep
Genetic change
Fluorescence
(gfp gene)
Fish (pigs, cats)
Fluorescence
used as
biosensor for
other
transferred
genes
Factor IX
production
Sheep
Production of
Factor IX in
milk for
haemophilia
Milk quality
Cows
Milk equivalent
to human milk
Glowing animals?....what’s the big
deal?
Worth a Nobel prize….
Further Applications of Transgenic
Technology
• Vaccines (transgenic yeast produces vaccine
for Hepatitis B)
• Transgenic mice widely used for human
disease research
• Transgenic male mosquitoes carrying ‘lethal
gene’ used to reduce the incidence of Dengue
fever
Genetic modification: the PROs…
1. GM crops should improve yields,
quality and food security
2. Pest-resistant crops will reduce
the need for pesticides
3. GM can produce crops which
provide dietary supplements
such as retinol
4. GMO’s used to produce rare
proteins for medicine or vaccines
will be cheaper and less polluting
than conventional methods
5. GM allows farmers greater
control for selective breeding of
crops and animals
Genetic modification: the CONs…
• No one knows the long-term effects of GMO’s in the
biosphere PRECAUTIONARY PRINCIPLE
• Genes from GM crops are easily integrated into the wild
type crop
• Genes may be able to cross species
• Crops which produce toxins to kill insects could prove toxic
to humans
• Large portions of the human food supply may fall under the
control of a small number of companies
• High-tech agricultural ones are not necessarily better than
simpler solutions
• A proliferation of GMO’s could drastically reduce natural
biodiversity
Where do you stand?
4.4.11: CLONING
What is a clone?
• A group of genetically identical organisms
• A group of cells artificially derived from a
single parent
Natural Clones
• Asexual reproduction:
bacteria, yeasts,
protozoa
• Vegetative propagation
in plants
• Monozygotic (identical)
twins
4.4.12: ‘Outline a
technique for cloning
using differentiated
animal cells’
Utah Cloning Site
4.4.13: THERAPEUTIC
CLONING: Somatic cell
nuclear transfer
Therapeutic cloning
Reproductive versus therapeutic
cloning
• Both ‘therapeutic cloning’ and ‘reproductive
cloning’ refer to somatic cell nuclear transfer
• In therapeutic cloning, the newly formed embryo is
used as a source of embryonic stem cells for
research/therapy
• In reproductive cloning, the newly formed embryo
is transferred to a female and develops into an
organism
• Reproductive cloning has been used to clone
agricultural species, recently extinct species and
recently primates and domestic pets.
• Reproductive coining is illegal in most countries
Why clone?- advantages of therapeutic
cloning
1. Medical advances:- stem cells for
research, ‘pharming’ of
genetically engineered animals
for production of Factor IX etc,
source of embryonic stem cells
2. Reviving recently extinct species
3. Reproducing a recently
deceased pet
4. Producing babies???
Risks/Downsides of therapeutic
cloning
1. High failure rate: successful
nuclear transfer occurs only 0.1
– 3 % of the time
2. Problems during later
development (e.g. Large
Offspring Syndrome’)
3. Abormal gene expression
patterns
4. Telomeric differences
Learn more detail here
Therapeutic cloning (SCNT) versus IPS
stem cells
• IPS cells are derived from
somatic adult cells, which are
reprogrammed an embryonic
cell-like state
• Reprogramming: 3 – 4 genes
for transcription factors are
transferred to the adult cels
using a viral vector
• However they differ in many
genes from ‘natural embryonic
stem cells (271 )
• Different types of cell give rise
to slightly different IPS stem
cells
• No sperm, no uterus, no
pregnancy – but a developing
embryo
• Cloned SCNT cells produce
‘cloned cells’ which could be
re-imlpanted into the donor
with no risk of immune
rejection
• SNCT produces embryonic
cells into which disease genes
can be inserted, for research
into diseases including cancer
and Alzheimers Disease
More information here
Ethical issues surrounding therapeutic
cloning
• Therapeutic cloning allows
production of embryonic
stem cells from
differentiated cells
• These embryonic stem cells
have great potential for a
patient to replace their own
damaged cells - skin, heart,
kidney, brain, etc etc.
• A promising technique of
stem cell production
• These embryonic stem cells
have the potential to
become a human being (so
called reproductive cloning)
• Reproductive cloning of
humans is illegal in most
countries
Therapeutic cloning is
aimed NOT at making
people, but at CURING
people
Therapeutic cloning: The controversies
1. New potential therapies
for disease
2. Production of immunocompatible tissue for
self-transplantation
3. Advances in medical
research
4. Cost-benefit firmly in
favour of therapeutic
cloning
1. Manipulation and
destruction of human
embryos is wrong
2. Reproductive cloning
becomes more likely
3. We will create a global
market for women’s
eggs