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Topic 4.4
Using the technique called
polymerase chain reaction (PCR),
researchers are able to create vast
quantities of DNA identical to trace
samples. This process is also known
as DNA amplification.
Many procedures in DNA technology
require substantial amounts of DNA
to work with, for example;
Sometimes DNA samples
can be hard to obtain:
A crime scene
(body tissue samples)
A single viral particle
(from an infection)
DNA sequencing
DNA profiling/fingerprinting
Gene cloning
Transformation
Making artificial genes
Fragments of DNA from
a long extinct animal
Separate Strands
The laboratory process
called the polymerase
chain reaction or PCR
involves the following
steps 1-3 each cycle:
Separate the target DNA
strands by heating at 98°C for 5
minutes
Add Reaction Mix
Add primers (short RNA strands
that provide a starting sequence
for DNA replication),
nucleotides (A, T, G and C) and
DNA polymerase enzyme.
Incubate
Cool to 60°C and incubate for a few
minutes. During this time, primers
attach to single-stranded DNA. DNA
polymerase synthesises
complementary strands.
Repeat for about 25
cycles
Repeat cycle of heating
and cooling until enough
copies of the target DNA
have been produced.
Although only three
cycles of replication
are shown here,
following cycles
replicate DNA at an
exponential rate and
can make literally
billions of copies in
only a few hours.
The process of PCR
is detailed in the
following slide
sequence
of steps 1-5.
Original DNA
Sample
Cycle 1
Cycle 2
Cycle 3
PCR
cycles
No. of target
DNA strands
1
2
2
4
3
8
4
16
5
32
6
64
7
128
8
256
9
512
10
1024
11
2048
12
4096
13
8192
14
16 384
15
32 768
16
65 536
17
131 072
18
262 144
19
524 288
20
1 048 576
21
2 097 152
22
4 194 304
23
8 388 608
24
16 777 216
25
33 554 432
A DNA sample called the
target DNA is obtained
DNA is denatured (DNA strands
are separated) by heating the
sample for 5 minutes at 98C
Primers (short strands of mRNA)
are annealed (bonded) to the DNA
Primer annealed
McGraw-Hill PCR
animation
Media showcase
animation
PCR song
PCR song 2
Nucleotides
The sample is cooled to 60°C.
A thermally stable DNA
polymerase enzyme binds to
the primers on each side of the
exposed DNA strand.
This enzyme synthesises a
complementary strand of DNA
using free nucleotides.
After one cycle, there are now
two copies of the original
sample.
Nucleotides

PCR primers do not bind unless there is a
complementary sequence of nucleotides
◦
◦
◦
◦



A=T
T=A
G≡C
G≡C
One test for GM ingredients in food involves a
primer that will only bind to the GM DNA.
If GM DNA is present, the PCR process will
amplify the DNA
If no GM DNA is present, the PCR has no effect
Gel electrophoresis can be
used to separate large
molecules (including
nucleic acids or proteins)
on the basis of their size,
electric charge, and other
physical properties.
Sample
Wells
Cathode
Buffer
Plastic frame
DNA is split into fragments
using restriction enzymes
The DNA samples are
placed in wells and covered
with a buffer solution that
gradually dissolves them
into solution.
Gel
Anode
Buffer solution
Gel electrophoresis
demo lab
DNA has a negative charge
because the phosphate
groups are negatively
charged.
The DNA fragments in the
gel move through the gel
towards the positive
terminal of the electric field.
Smaller molecules move at a
faster rate through the gel;
longer fragments take
longer to work through the
small spaces in the gel.
Groups of DNA fragments
can be seen as bands on the
gel – usually seen with the
help of a dye.
Wells: Holes
created in the gel
with a comb.
negative
terminal
DNA
solutions:
Mixtures of
different sizes
of DNA
fragments are
loaded into
each well.
DNA
fragments:
The gel matrix
acts as a sieve
for the DNA
molecules.
Large
fragments
Small
fragments
positive
terminal
Tray: Contains the set
gel.
DNA markers:
A mixture of
DNA molecules
with known
molecular
weights. They
are used to
estimate the
sizes of the
DNA fragments
in the sample
lanes.
DNA profiling (DNA fingerprinting) is a technique for genetic analysis,
which identifies the variations found in the DNA of every individual.
The profile refers to the distinctive
pattern of fragments which is used to
identify an individual.
DNA profiling does not determine a
base sequence for a sample but merely
sorts variations in base sequences.
Only one in a billion (i.e. a thousand
million) persons is likely to have an
identical DNA profile, making it a useful
tool for forensic investigations and
paternity analysis
DNA profiling can be used for investigating:
the presence of a particular gene,
(such as cystic fibrosis) in a family.
genetic relatedness of different
organisms
e.g. checking on pedigree in
stock breeding programs.
e.g. checking that captive
populations of endangered
species are not inbred.
DNA profiling can be used for forensic purposes:
DNA fingerprints from tissue samples
can be used as evidence in the same
way traditional fingerprinting is used.
Which DNA fingerprint from the three
suspects matches that of the tissue
sample submitted as evidence?
Why would the DNA from the victim be
included in this test?
The DNA from the victim must be
excluded from the evidence.
In a paternity test, the DNA from the
mother must also be included to
exclude her contribution to the
banding patterns in the child’s profile.
We expect 100% match as
the cells left behind at the
scene are the perpetrator’s
cells
We expect 100% match as the
cells left behind at the scene are
the perpetrator’s cells
The overlapping bands between
victim and suspect indicate a
close genetic relationship
No. Without a stronger match,
the evidence is insufficient to
convict the suspect. He should be
released and a new suspect
found.
DNA evidence is being reviewed
in many wrongful convictions.
In this case, determine which man (1 or
2) is the biological father of the child.
Because the child inherits half its
genetic material from each
parent, any band that the child
has not inherited from his
mother, he must have inherited
from his father.
1. Refer to the
image on the
right
Which male (1 or 2) is
the father of the child?
Explain.
2. Refer to the
image on the left
Which suspect(1,2 or 3)
was present at the
crime scene?
Explain.
“DNA is better at proving
innocence than guilt.”

Discuss this statement
Textbook exercises:
Tiger book p164
Look at figure 7 and determine the culprit
Tiger book p167
Question 6
Outline three outcomes of the
sequencing of the complete human
genome
4.4.3
Completed in April 2003.
An international, collaborative effort to record
the entire base sequence of the human genome.
Also achieved the following:
Discovered the number and loci of all the genes (30k) in our
genome – further research in diagnostics, treatment and
pharmacology.
New proteins and their functions were discovered.
Comparisons between genomes of different species –
evolutionary history and links.
Bioinformatics was born – a high-tech way to collate and
access information from genetic databases.
Discussing the implications of the HGP
Scrubs – Huntington’s Disease
1. If you knew that a member of your family had a rare genetic disorder
and you could be tested for it quickly and easily, would you do it? Why?
Human Genome Project - Ethical, Legal, & Social Implications
2. If you were invited to share your genome with researchers in the hope
of finding cures for genetically-based illnesses, would you do it? Why?
Robert Cook-Deegan on Patenting Genes
3. Do you feel that gene patenting should be allowed? Why?
4.4.7 When genes are transferred between species, the
amino acid sequence of polypeptides translated from them
is unchanged because the genetic code is universal
All living things use the same bases (G.A.T.C!)
A particular codon will produce the same amino acid, regardless
of the species.
This means the sequence of amino acids in a polypeptide
remains unchanged.
This allows us to take a gene from one species and insert it into
the genome of another species.
A well-known example of this gene transfer process is the
production of human insulin by GM E. coli bacteria.
A potential treatment for haemophilia is the injection of human
clotting factors produced in the milk of GM sheep.
4.4.8 A basic technique for gene transfer involves plasmids,
a host cell, restriction enzymes and DNA ligase.
Naturally occurring bacterial enzymes are used as “molecular scalpels”
Allow genetic engineers to cut up DNA in a controlled way.
Restriction enzymes are used to cut DNA molecules at very precise sequences
of 4 to 8 base pairs called recognition sites.
Recognition Site
The restriction
enzyme EcoRI cuts
here
DN
A
Recognition Site
cut
GAATTC
GAATTC
CTTAAG
CTTAAG
cut
cut
It is possible to use
restriction enzymes
that cut leaving an
overhang; a so-called
“sticky end”.
A restriction enzyme cuts the doublestranded DNA molecule at its specific
recognition site
Fragment
GAAT T C
GAAT T C
C T TAA G
C T TAA G
Restriction enzyme: EcoRI
DNA cut in such a way
produces ends which
may only be joined to
AAT T C
other sticky ends with a
complementary base
G
DNA
from
sequence.
See steps 1-3
opposite:
Restriction
enzyme: EcoRI
another
source
G
C T TAA
Sticky end
The cuts produce a
DNA fragment with
two “sticky” ends
The two different fragments
cut by the same restriction
enzyme have identical sticky
ends and are able to join
together
When two fragments of DNA cut by the same
restriction enzyme come together, they can join by
base-pairing
Recognition Site
It is possible to use
restriction enzymes that
cut leaving no overhang; a
so-called “blunt end”.
C C CG G G
C C CG G G
G G GC C C
G G GC C C
DNA
cut
DNA cut in such a way is
able to be joined to any
other blunt end fragment,
but tends to be nonspecific because there are
no sticky ends as
recognition sites.
A special group of
enzymes can join
the pieces together
Recognition Site
Restriction enzyme
cut
cuts here
The cut by this type of restriction
enzyme leaves no overhang
CCC
GGG
GGG
CCC
GGG
CCC
CCC
GGG
DNA from another source
DNA fragments produced using restriction enzymes may be
reassembled by a process called ligation.
Pieces of DNA are joined together using an enzyme called DNA ligase.
DNA of different origins produced in this way is called recombinant DNA
because it is DNA that has been recombined from different sources.
Steps 1-3 are involved in creating a recombinant DNA plasmid:
Two pieces of DNA
are cut using the
same restriction
enzyme.
Plasmid
DNA
fragment
The two different DNA
fragments are attracted to
each other by weak hydrogen
Abonds.
ATTC
G Foreign DNA fragment
This other end of the
foreign DNA is
attracted to the
remaining sticky end of
the plasmid.
G
C T TAA
When the two matching “sticky ends” come together, they join by base pairing.
This process is called annealing.
This can allow DNA fragments from a different source, perhaps a plasmid, to be
joined to the DNA fragment.
The joined fragments will usually form either a linear molecule or a circular one, as
shown here for a plasmid.
Detail of Restriction Site
Plasmid
DNA
fragment
Restriction sites on the
fragments are
attracted by base
pairing only
Gap in DNA
molecule’s
‘backbone’
Foreign
DNA
fragment
The fragments of DNA are joined together by the enzyme DNA ligase,
producing a molecule of recombinant DNA.
These combined techniques of using restriction enzymes and ligation
are the basic tools of genetic engineering.
DNA ligase
Detail of Restriction Site
Recombinant
Plasmid DNA
Fragments
linked
permanently by
DNA ligase
No break in
DNA molecule
The fragments are able
to join together under
the influence of DNA
ligase.
A virus vector is used to insert the recombinant plasmid into the
genes of affected cells.
The virus is chosen or designed to target only those specific cells.
Severe Combined Immune Deficiency
can be treated this way
Gene therapy ‘reverses’
hereditary blindness
4.4.9 Give examples of the current uses of genetically
modified crops or animals
GMOs are already in circulation and have been
produced for many uses, including agricultural and
medical.
Golden rice: Enriched with beta-carotene, which is converted to
Vitamin A in the body. Can prevent malnutrition-related blindness in
developing countries.
Plant
Insect-resistant corn: Produces proteins which pests do not like,
examples therefore toxic pesticides are not needed on the farm.
Salt-resistant tomatoes: Can be grown in soil with a high saline
concentration.
Factor IX-producing sheep: Produce human clotting factors in their
milk , for the treatment of haemophilia.
Glowing pigs: Cells from these organisms are used to study
Animal transplants and grafts, and the final destinations of transplanted
examples cells in the host body.
Enviropig: Produce phytase in their saliva to convert insoluble
phytate into phosphate that is absorbed by the pig.
GM food
and you
4.4.10 Discuss the potential benefits and possible
harmful effects of one example of GM
The ethical debate over GMOs rages on, and as scientists we must
always bear in mind the precautionary principle:
“If an action is potentially harmful, the burden of proof of safety
lies with those who propose to take the action.”
Benefits
Increased yields of crops and faster breeding cycles
Crops can be grown in harsher environmental conditions
Reduced need for pesticides which can harm human and
environmental health through biomagnification
Nutrient-enriched crops in areas of high food pressures
or famine
Potential
harms
Potential genetic pollution of organic crops through
fertilisation by pollen of GM crops
Unknown health risks of some crops
Fear of monopoly-like behaviour as farmers need to buy
expensive seeds annually
Potential hybridisation of related species
4.4.11 Define
clone
A group of genetically identical organisms or groups of cells derived
from a single parent cell
Monozygotic (one zygote) twins are naturally occuring clones.
Why do they not look identical?... Epigenetics has the answer
Asexual reproduction (i.e. in bacteria) is an example of cloning
Cloning via binary fission
Taking plant cuttings and growing a new plant is also cloning.
As is plants producing bulbs and runners
4.4.12 Outline a technique for cloning using differentiated
animal cells
Cloning cell cultures using nuclear transfer:
1.
2.
3.
4.
5.
Remove a differentiated diploid nucleus from the
individual to be cloned
Enucleate a donor egg cell
Insert the diploid nucleus into the enucleated egg
cell
Stimulate it to divide and grow
Collect cells for therapeutic purposes, such as
creating skin tissue for burn patients
Reproductive cloning using nuclear transfer:
1. Remove the differentiated diploid nucleus from
the individual to be cloned
2. Enucleate the donor egg cell
3. Insert the diploid nucleus into the enucleated
egg cell
4. Insert into a surrogate mother and gestate
5. The newborn will be genetically identical to the
donor nucleus parent
Reproductive cloning
The first mammal cloned was Dolly the sheep
Human cloning is illegal around the world
There will be no: “Mini-me” as in ‘Austin
Powers’ (a clone to take over the world) or the
characters played by Ewen McGregor and
Scarlett Johansen in ‘The Island’ (spare parts
“just in case”).
Therapeutic cloning is less controversial
It has the potential to treat (using stem cells)
degenerative diseases like Parkinson’s disease
and Multiple Sclerosis.
Does involve producing an embryo that could
grow to full term (same as an IVF embryo) –
but then it would be cloning!
Dolly and her birth
mom.
A black faced ewe
cannot have a white
faced baby – so they
must not be
genetically related.
In 2008, a team led by DR Viviane Tabar extracted skin cells
from the tails of mice with Parkinson’s disease.
They removed the nucleus from each cell and implanted them
into egg cells from which the nuclei had been removed.
The resulting cells, which were genetically identical to the
donor mice, developed as embryos and produced stem cells
that could differentiate into dopamine neurons – the type that
are missing in Parkinson’s disease.
The team injected these stem cells into the affected regions of
the brains of the donor mice and found that there was a
marked improvement in the symptoms of Parkinson’s disease.
Is it acceptable to create embryos as
a source of stem cells for treatments
that reduce suffering in a child or
adult, but result in the death of the
embryo?
Nucleus is removed
Nucleus is removed
4.4.13 Discuss the ethical issues of therapeutic cloning in
humans
Possible benefits
Arguments against
Rejection risk reduced in
transplants
Religious objections to “playing
God” by creating what many
consider to be human life
No need to wait for human donor
to die to give organs
Some success stories already
reported in therapeutic cloning
UN recommendations against
reproductive cloning has no been
ratified by all countries – possible
risk of a race to create the first
human clone