3. Recap of 15.1
• That the cells in our bodies are highly specialised.
• They have specific functions to perform in different areas of
the body, and have structures that reflect these functions.
Essentially, what are all structures in cells made of?
PROTEIN
In order to produce these molecules, what process did we
establish had to occur?
GENE EXPRESSION
Gene expression is just a fancy way of saying.....
‘some DNA is used to produce protein’.
4. Here is a
totipotent cell
It was taken from
an embryo
Imagine it was taken from a very
simple mammal, with only a single
homologous chromosome pair
That DNA in those c’somes contains
genes (instructions) to make any
cell type from any organ.
heart cell gene intestinal cell gene brain cell gene
The lineage that the stem cell takes, depends on which genes within
it, are expressed.
If genes related to heart structure are expressed, the stem cell will
become a heart cell.
Once a stem cell differentiates, it can never go back to being
totipotent.
6. Gene Expression
You know that the basics of gene expression is that:
1. Transcription has to occur.
2. Pre-mRNA has to be spliced.
3. Translation has to occur.
It’s all well and good knowing the process of getting from gene
to protein product, but how is this process regulated?
Does it ‘just happen’?
But what decides when this
happens and at which section
of DNA?!
7. Transcription Factors
Genes don’t just start to transcribe themselves spontaneously.
If that was the case, cells in your pancreas would produce adrenaline,
and cells in testicles would begin to release oestrogen!
Your body contains regulatory proteins called TRANSCRIPTION
FACTORS.
Transcription
factors are a
protein complex,
with different
subunits.
DNA Binding Site
Receptor
Transcription
Factor
Hormone
Binding Site
8. How do the Transcription Factors Work?
• The gene that codes for the required protein, is stimulated by
a specific transcription factor.
• There are millions of transcription factors and each one has a
DNA binding site that is specific to a certain gene.
• When it binds to the correct region of DNA, transcription
begins.
• This would then produce mRNA, which would then be
translated into a protein.
What about when the gene doesn’t
need to be expressed? How could you
stop transcription factors from
stimulating DNA?
9. There are 2 possibilities if you think about it....
....maybe not
1.
2.
Inhibitor
Molecule
When a gene is not being
expressed, the DNA
binding site on its
complimentary
transcription factor is
BLOCKED.
This inhibitor stops the
transcription factor from
binding to DNA, thus
blocking transcription
from occurring.
11. There are 2 Mechanisms of Hormone Action
• The first mechanism involves protein hormones (such as
insulin) and molecules called second messengers.
• Transcription and translation though, are regulated via the
other hormone mechanism, which involves lipid-soluble
hormones (such as oestrogen).
Protein Hormones
Act via Second
Messengers
e.g. Insulin
Lipid-Soluble
Hormones
Act Directly
e.g. Oestrogen
12. Hormones like oestrogen can switch on a gene
and start transcription.
They do this by binding to their receptor on the
transcription factor.
This changes the transcription factors shape,
and thus releases the inhibitor molecule.
The transcription factor can then bind to DNA,
starting up the process of transcription.
15. siRNA
• Gene expression can be prevented by breaking down mRNA
before it is translated into a protein.
• To do this, small molecules of double-stranded RNA called
siRNA are essential (small interfering RNA).
• Large double-stranded molecules are cut into siRNA by
enzymes.
• The siRNA splits into single-stranded molecules, of which one,
associated with a different enzyme.
• The siRNA guides this enzyme to an mRNA molecule.
• Once there, the enzyme cuts the mRNA into small sections.
• This renders the mRNA useless, as transcription cannot occur.
16. siRNA inhibits translation of mRNA and turns genes
OFF
Enzyme 1 breaks
up dsRNA making
siRNA molecules
Enzyme 2
combines with
one of the two
molecules of
siRNA
Complimentary
base pairing
between siRNA and
target mRNA
Enzyme 2 cuts mRNA
into small sections
stopping it from
being translated
17. Uses of siRNA
1. It could be used to identify the role of genes in a biological
pathway. By using siRNA to block certain genes, you could
observe what effects occur. This could then tell you what the
role of the blocked gene is.
2. Some diseases are genetic and are caused by the expression
of certain genes. If these genes could be blocked by siRNA,
it may be possible to prevent the diseases caused by them.
19. Genetic Engineering
• Genetic engineering is a rapidly advancing field of
Biology.
• We can now manipulate, alter and even transfer genes
from one organism to another.
• The ability to do these things has proved invaluable in the
industrial and medical sectors.
20. Helping Humans
• Many human diseases are caused by the inability of the
body to produce certain protein products.
• These proteins of course, are the products of a gene.
• This gene may be faulty, preventing the correct
expression of the gene.
• There are now ways of isolating a gene, cloning it, and
then transferring it into microorganisms.
• The microorganisms then act as ‘factories’ where the
gene product (the desired protein) is continuously
manufactured.
An example: The production of Insulin
21. Genetically Modified Organisms
• When a certain gene is introduced into the DNA of another
organism (such as a bacterial cell), it is then called
recombinant DNA.
• The resulting organism is known as a genetically modified
organism (GMO).
1. Isolation
of the DNA fragments
that have the gene for
the desired protein.
2. Insertion
of the DNA fragment
into a vector.
3. Transformation
...Inserting the vector
into a suitable host
(such as a bacterial cell)
4. Indentification
of host cells that have
taken up the gene,
using gene markers
5. Growth/cloning
of the population of
host cells
The process of making a protein using DNA
technology
In this lesson, we will
cover ‘Step 1’
(isolation) in detail
22. Isolation of a gene
• There are two ways of isolating a gene:
1. Using Reverse Transcriptase
This method uses an enzyme that ‘works backwards’. It can
produce DNA from mRNA.
1. In a healthy individual, the desired protein is being
manufactured in specific cells of the body
2. It follows that these cells will contain large quantities of the
relevant mRNA for that protein.
3. If reverse transcriptase is added, it can make DNA from this
RNA.
4. It does so, by producing complementary DNA (cDNA).
(see next slide)
23. mRNA template
for the hormone,
vasopressin
A U G C U
T A C G A
1. You isolate the mRNA that has
been transcribed from the
gene you are interested in.
2. Reverse transcriptase is used
to synthesis a complimentary
DNA (cDNA) strand, to the
mRNA molecule.
3. Our old friend DNA
Polymerase (from translation)
can then synthesise the other
strand of DNA from free
nucleotides.
The Hypothalamus produces
a hormone called
vasopressin
A T G C T
You now have the actual gene
that codes for your protein!
You can produce it in vast
quantities and then insert them
into plasmids!
24. Isolation of a gene
• The 2nd method of isolating a gene:
Using Restriction Endonucleases
Restriction endonucleases are enzymes that cut DNA at specific
base sequences (recognition sequences). These enzymes can be
used to cut out a desired gene from the rest of the genome.
Cutting DNA with a restriction enzyme can have two results.
Some restriction
endonuclease
produce ‘blunt ends’
Some restriction
endonuclease
produce ‘sticky ends’
25. Summary Question
In the following passage replace each number with the most appropriate word or
words.
Where the DNA of two different organisms is combined,
the product is known as _____ DNA. One method of
producing DNA fragments is to make DNA from RNA using
an enzyme called _____. This enzyme initially forms a
single strand of DNA called _____ DNA. To form the other
strand requires an enzyme called _____. Another method
of producing DNA fragments is to use enzymes called
_____, which cut up DNA. Some of these leave fragments
with straight edges, called _____ ends. Others leave ends
with uneven edges, called _____ ends. If the sequence of
bases on one of these uneven ends is GAATTC, then the
sequence on the other end, if read in the same direction,
will be _____
27. DNA Probes
• DNA probes are simple, short and single-stranded sections of
DNA.
• They will bind to complementary sections of other DNA
strands.
• Due to being labelled in some way, they make this ‘other
DNA’ easily identifiable.
Labelling with radioactivity Labelling with fluorescence
28. Remember that probes can be used as an
easy method of screening (detecting) for
mutated genes.
But also remember that the probe needs to
be complementary to the mutated gene.
So this means, that to produce a probe, you
first need to sequence your gene.
How do we sequence genes?
29. Meet Frederick Sanger...
• Biochemist
• Cambridge University
• English
• Two Nobel Prizes
• Still Alive
Sanger’s work in the 1970’s, which
earned him his second Nobel Prize,
involved the sequencing of DNA.
His method used modified nucleotides that do now allow
another nucleotide to join after them in a sequence.
Sanger Sequencing Method
30. • The method is based on the premature ending of DNA
synthesis.
• If modified nucleotides are used during DNA synthesis, the
process can be halted.
G
T C
A A C T A A C T G G A A T C
Introducing Sanger Sequencing
T T
A G
G A T C
T A T G G G G
T T T T
A A A
C C C
What normally happens during DNA synthesis...
What happens if you modify a nucleotide...
G
T C
A A C T A A C T G G A A T C
You call these modified
nucleotides, TERMINATORS
31. • In Sanger Sequencing, four different terminators are used (A,
C, T and G).
• Due to this, four different reactions are run.
What you need...
In each reaction, you have the following:
The DNA being sequenced.
A mixture of ‘normal’ nucleotides (A, T, C, G)
One type of terminator nucleotide.
A primer.
DNA Polymerase.
G
T
C
A C
A
C
32. G
T
C
A C
A
C
G
T
C
A C
A
G
G
T
C
A C
A
A
G
T
C
A C
A
T
Remember that each tube probably contains millions of copies of the DNA
template, countless nucleotides, and a good supply of the specific terminator
nucleotide.
Due to this, you get a variety of ‘partially completed’ DNA strands, because they
have been ‘terminated’ at different points.
33. G
T
C
A C
A
A
• Lets take the example of the tube with an adenine terminator
So what happens in each tube?
Now let’s imagine this is the sequence of the
unknown DNA strand:
C C G T C T A G C A C T C A A G C T C T
What are the possible terminated
sequences going to be when the
reaction is over?
G G C A
G G C A G A
G G C A G A T C G T G A
G G C A G A T C G T G A G T T C G A
G G C A G A T C G T G A G T T C G A G A
Because there are both ‘normal’ and
‘terminator’ nucleotides in the mixture,
there is a chance that either is placed as the
next base
34. Remember that this is happening in four test-
tubes, each with a different type of
terminator nucleotide.
DNA fragments in each of the four tubes are
going to be of varying lengths.
Now the lengths of DNA need to be
separated, so that we can see why we went
through all of this trouble...
36. • When you’ve got a mess of DNA, especially DNA strands of varying
lengths, you can separate them out using this technique.
• The whole process relies on the fact that the phosphates in the
backbone of DNA, are negatively charged.
Gel Electrophoresis
• DNA fragments are placed in
wells at the top of an agar gel.
• An electric current is applied
over it.
• Agar is actually a ‘mesh’, which
resists the movement of the
DNA fragments through it.
• The DNA moves towards the
positive electrode, but at
different rates.
• Small sections get there
quicker.
37. • The fragments produced during the reactions can be separated using
gel electrophoresis.
• The smallest fragments will move furthest along the gel in a fixed
period of time.
• Due to being radioactively labelled, we can see where the DNA
fragments end up, by placing photographic film over the gel, after the
run.
Back to Sanger Sequencing
Terminator
C
Terminator
A
Terminator
T
Terminator
G
38. • Nowadays, DNA sequencing is automated, using computers.
• Nucleotides are fluorescently labelled with dyes.
• Everything occurs in only a single tube.
• And the separation can occur in one lane during gel
electrophoresis.
Automated Sequencing
Equipment used during
the Human Genome Project.
41. The importance of ‘sticky ends’.
• Last lesson, we discussed sticky ends that are left after the
action of restriction endonucleases.
• These are highly important in genetic engineering, for the fact
that they leave exposed bases – this is due to the staggered
nature of the cut.
• Due to the complementary base-pairing rules of DNA, sticky
ends on one gene, will pair up with sticky ends of another bit
of DNA that has also been cut by the same restriction
endonuclease.
42. KEY:
Gene from Human
Gene from E.coli
This gene (from a human) can be cut
with a restriction enzymesuch as EcoRI
Sticky End
If this section of
DNA from E.coli
is also cut with
EcoRI, a
complimentary
sticky end is
produced.
This is a section of DNA from E.coli.
Sticky End
If these two ‘cut’ pieces of DNA are mixed,
recombinant DNA has been produced.
43. Once the bases have paired, they form their
usual weak hydrogen bonds between each
other.
The only thing left to do, is form the link
between the sugar-phosphate backbones, and
this is done by the enzyme, DNA Ligase.
44. Inserting genes into Plasmids
• The real-life application of what we have just learnt, occurs
when geneticists insert an animal or plant gene into plasmids.
• Plasmids are small loops of DNA which are found in addition
to the large circular chromosome that bacterial cells possess.
• By inserting our chosen gene into a plasmid, the plasmid acts
as a ‘carrier’, or vector, which we can then introduce back into
a bacterial cell.
DNA coding for a desired
protein
Restriction Endonuclease
A plasmid
Restriction Endonuclease
As the DNA fragment was cut out using
the same restriction endonuclease as
used to cut the plasmid open, they have
complimentary sticky ends.
Remember, that DNA
Ligase would once again be
used to bond the sugar-
phosphate backbones.
This is ‘Step 2’ (insertion) in the process
of making a protein using gene
technology
45. Introducing our recombinant plasmids into host cells
• Introducing recombinant plasmids into bacterial cells is called
transformation.
• This is done by mixing the plasmids with the cells in a
medium containing calcium ions.
• The calcium ions make the bacterial cells permeable, allowing
the plasmids to pass through, into the cell.
Calcium ion medium
However, only a few bacterial cells
(approx 1%) will actually take up
the plasmids.
For this reason, we need to
identify which ones have been
successful. This is done with gene
markers.
This is ‘Step 3’ (transformation) of producing a
protein by DNA technology
46. Using Gene Markers to identify successful host cells...
• There are a number of different ways of using gene markers
to identify whether a gene has been taken up by bacterial
cells.
• They all involve using a second, separate gene on the
plasmid. This second gene is easily identifiable for one reason
or another....
• It may be resistant to an antibiotic
• It may make a fluorescent protein that is easily seen
• It may produce an enzyme whose action can be identified
Each of the 3 above mechanisms are actual methods of employing
gene markers to identify bacterial cells that have taken up
plasmids... They will be discussed on the next slides...
47. 1. Antibiotic-Resistance Markers
• Many bacteria contain antibiotic resistance genes in their plasmids.
Some in fact, can have two genes for resistance to two different
antibiotics, in the same plasmid.
Gene for
resistance to
ampicillin
Gene for
resistance to
tetracycline
Any bacterial cell posessing this
plasmid, would be resistant to both of
the antibiotics, ampicillin and
tetracycline.
But what if we cut right in the middle
of the tetracycline-resistance gene
(with a restriction endonuclease), and
insert a gene of our own interest?
Bacteria with this plasmid
would only be resistant to
ampicillin, not
tetracycline.
How is this of any
advantage to us?
48. First, the recombinant plasmids are
introduced into bacterial host cells
(transformation)
49. The bacteria is grown on agar
treated with ampicillin
Colonies are
allowed to grow,
but will only do so
if they are resistant
to ampicillin – i.e.
Bacteria that took
up the plasmid.
A replica plate is now made. This is when you literally press the agar of one
Petri-dish, onto the agar of a new Petri-dish, transferring bacterial cells from
each colony onto the new agar.
This agar
however,
has been
treated
with
tetracycline
Colonies are allowed to
develop
?
There is a missing
colony, which has
lost resistance to
tetracycline.
This must be a
colony containing
cells which have
taken up the
plasmid!
50. 2. Fluorescent Markers
• This is a more recent method of finding out whether bacteria have
taken up the desired plasmids.
• All you have to do is first insert your gene of interest (such as insulin) into a gene
for a fluorescent protein.
• Then insert this insulin/fluorescence hybrid gene into a plasmid vector.
• Then transfer the plasmids into bacterial cells!
• Any cells that successfully took up plasmids, will be glowing on your Petri-dish!
Throughout nature, there are organisms such as
jellyfish, that produce fluorescent proteins.
These are proteins, which obviously have their
own genes, which of course can be isolated and
then introduced into bacterial cells via vectors.
The range of natural fluorescent proteins can be
seen on this Petri-dish. These are colonies of
bacteria that are expressing the fluorescence
genes!
51. 3. Enzyme Markers
• This method involves inserting your gene of interest (e.g. Insulin),
into a gene that codes for an enzyme such as lactase.
• There is a particular substrate that is usually colourless, but turns
blue when lactase acts upon it.
• If you insert you chosen gene into the gene that makes lactase, you
will inactivate the lactase gene.
• If you now grow bacterial cells on an agar medium containing the
colourless substrate, any colonies that have taken up the plasmid,
will not be able to change its colour to blue.
• Any colourless spots will indicate to you, which cells have been
transformed.
The more boring of the 3 methods, but still important.
53. Criminals – Watch Out!
• Scientists can now identify criminals from the
smallest bit of DNA.
• Wherever you go, you leave a trail of DNA behind.
• We consistently shed hairs and flakes of skin.
• It is in these hair and skin cells that DNA is found!
• You DNA is your GENETIC FINGER PRINT!
54. Polymerase Chain Reaction(PCR)
• PCR is an method by which DNA can be
replicated in the lab.
• It can be used to create millions of copies of
DNA in just a few hours.
• It is essential in forensic science as very small
samples of DNA are difficult to analyse.
• This process amplifies DNA, so that it can be
analysed.
55. What do you need?
1. RNA primers provide the starting sequence
for DNA replication. They also stop the two
DNA strands from joining together.
2. DNA nucleotides containing the bases
adenine, guanine, cytosine and thymine.
3. Enzyme DNA polymerase.
56. The Stages of PCR
Strand Separation
DNA heated at 95°C for
5mins
Binding of Primers
Mixture cooled to
40°C
C
Mix with Primers
(RNA strands)
DNA Synthesis
Mixture heated to
70°C
(optimum temp. for
DNA polymerase)
REPEAT
CYCLING
Mix with
Free Nucleotides
DNA Polymerase
With every cycle the amount
of DNA doubles
57. A A G G T C A C T T
T T C C A G T G A A
The Double Stranded DNA Molecule
Heat to 950C to separate the DNA strands
58. A A G G T C A C T T
T T C C A G T G A A
C T T
C T T
DNA Strand
DNA Strand
RNA Primers
Cool to 400C to allow primers to bind (anneal) to DNA
59. A A G G T C A C T T
T T C C A G T G A A
T T C C A G T
G T C A C T T
G C C
A A G
G
G
G
G
Free DNA
nucleotides
Original DNA strand
Original DNA strand
DNA Polymerase
Free DNA nucleotides
Primer
Primer
Nucleotides join on
Nucleotides join on
Mix with DNA polymerase and free nucleotides and heat to 700C
60. Advantages
• It is a very rapid process
• Does not require living cells
• Is useful when we want to introduce a gene
into another organism
61. Disadvantages
These are also the advantages of In vivo cloning:
• There is risk of contamination by unwanted
DNA
• It cannot cut out specific genes
• Does not produce transformed bacteria
62. Summary Questions
• In the polymerase chain reaction, what are the
‘primers’?
• What is the role of these primers?
• Why are two different primers required?
• When DNA strands are separated in the PCR,
what type of bond is broken?
• It is important in the PCR that the fragments of
DNA used are not contaminated with any other
biological material. Suggest a reason why.
64. What used to take a thousand years, now takes weeks
• The animals that farmers keep today, have been selectively
bred over thousands of years.
• Cows used today for milk and meat production, do not look
anything like the wild animals they are descended from.
• Humans have unwittingly, manipulated the genetics of
various animals and plants over the last few millennia.
Humans have only very recently,
realised the great power of DNA
technology.
It is possible to manipulate genes in
many ways, inserting them back into a
genome, and then seeing new
phenotypes emerge.
What used to take many generations to
occur, can now happen in a few weeks
66. Genetic Modification
• Every species has a genome, and every individual has its
genotype.
• It is possible to alter the genotype of an organism by
transferring genes between:
There are many advantages to humans by doing either.
Individuals of the
same species
Individuals of
different species
67. Benefits to Humans
Developing pest and disease
resistant crops
Cultivating microorganisms
for the production of
medicines
Production of the active
ingredients of vaccines
Developing crops that are
resistant to extreme
weather, such as drought
and flooding.
Developing crops that are
resistant to herbicides
Increasing the yield and
nutritional value of
animals/plants
69. Three types of substances are produced using genetically modified BACTERIA...
ANTIBIOTICS
Mainly bacteria are used, but fungi are useful in some circumstances.
These organisms naturally produce antibiotics, but ca be genetically
modified to produce them in much larger quantities.
HORMONES
By far the most common substance produced by genetically modified
organisms. Bacteria can have the gene for a hormone inserted into
them, so that they produce the protein product.
ENZYMES
These are required for food and beverage production. The brewing
industry requires amylase to break down starch, producing glucose as
an energy source for yeast.
71. Three types of substances are produced using genetically modified PLANTS...
TOMATOES
When tomatoes ripen, they become soft, which is a problem during
transportation. This softening is caused by a gene in the tomato plant
genome. If a complementary gene is inserted into the plant, it can
block the translation of the softening gene.
HERBICIDE-RESISTANT CROPS
Some plants have a gene introduced that cause them to produce a
substance that blocks the action of weedkillers. When weedkillers
are applied, competing weeds are killed, leaving the crop plant
unaffected.
DISEASE-RESISTANT CROPS
Every year, rice crops are devastated by the RBSDV virus. Rice crops
have been developed though, that can withstand infection by
particular viruses.
72. PEST-RESISTANT CROPS
Some plants have had genes introduced, that cause them to produce
toxins which are insecticidal. These block respiratory pathways and
induce paralysis in many insect species that feed on them.
PLASTIC PRODUCING PLANTS
Many plants naturally produce a kind of latex in their stems. By
introducing a particular gene, some plants can be cultivated to
produce a more useful type of substance, that has properties similar
to plastic.
Asclepias syriaca is a species of
milkweed, that releases a sticky
latex when its stem is pierced.
74. Three types of substances are produced using genetically modified ANIMALS.
Sometimes genes from animals that are resistant to a certain
disease are transferred to animals that have no natural
resistance. This process is utilised in situations where domestic
animals can be made more economic, by helping to reduce the
cost of food production.
Growth hormones genes can also be added to animals such as
fish and sheep. In the case of salmon, they can grow up to 30
times as big, at 10 times the usual rate.
Genes for rare and expensive proteins can be inserted into
animals such as goats, so that the protein is produced in the
animal’s milk.
75. The case of anti-thrombin
• Anti-thrombin is an anticoagulant, which prevents blood
from clotting.
• There is an inherited disorder which affects once of the
alleles that codes for anti-thrombin. These individuals are at
risk from blood-clots.
• Vast quantities of anti-thrombin can be produced in goats
milk.
76. Mature eggs
removed from
female and
fertilised by sperm
Normal anti-thrombin
gene from human is
inserted next to milk
protein gene in
fertilised eggs
These transformed
eggs are transplanted
into uterus of female
goats
The resulting goats with
anti-thrombin gene are
crossbred, to give a
herd that produces
protein in milk.
Anti-thrombin is
extracted from milk,
purified and
administered to humans.
77. Summary Question
• State one advantage to humans of genetically
modified tomatoes.
• Suggest one benefit and one possible
disadvantage of using genetically modified,
herbicide-resistant crop plants together with
the relevant herbicide. Explain your answer.
• Why is insulin produced by recombinant DNA
technology better than insulin extracted from
animals?
