Download 3.5.8 Gene Cloning technologies allow study and alteration of gene

3.5.8 Gene Cloning technologies
allow study and alteration of gene
function in order to better
understand organism function and
to design new industrial and
medical processes
Genetic engineering
• Can allow genes to be manipulated, altered and
transferred from organism to organism
• Why might this be useful?
• One use has been to produce human chemicals
such as insulin.
• When the DNA is introduced into a new
organism and combined with its own it is known
as recombinant DNA and the organism is known
as a Genetically modified organism (GMO)
The process of making proteins using
DNA technology:
• Isolation – getting some desired DNA
• Insertion – putting the DNA into a vector
• Transformation – inserting the vector into a
host
• Identification – making sure that the host has
taken up the DNA
• Growth/Cloning – getting a large population of
host cells
Isolation - How to get some DNA
fragments!
• If the amino acid sequence of the desired
protein is known, the DNA code can be worked
out and the DNA made in the lab by stringing
together the correct order of nucleotides.
Note:Many proteins are extremely large,
therefore this would be a tedious process.
• Conversion of mRNA to cDNA, using reverse
transcriptase.
• Cutting DNA at specific palindromic recognition
sequences using restriction endonucleases.
Conversion of mRNA to cDNA,
using reverse transcriptase.
• Activity:
– Now that you know
the correct sequence
complete the cut and
stick worksheet to
put the synthesis of
cDNA into the
correct order
– Add any extra details
about the process
which is occurring
e.g. splicing and the
information on page
247
Interesting fact
• The technology for
producing cDNA is
actually used by
HIV
Cutting DNA at specific
palindromic recognition sequences
using restriction endonucleases.
• A restriction enzyme (or restriction
endonucluease) is an enzyme that cuts
double-stranded or single stranded DNA
at specific recognition nucleotide
sequences known as restriction sites,
which are usually 4 – 6 base long
Cutting DNA at specific palindromic
recognition sequences using restriction
endonucleases – Continued
• Such enzymes, in bacteria and archea, are
thought to have evolved to provide a defence
mechanism against invading viruses. Inside a
bacterial host, the restriction enzymes
selectively cut up foreign DNA in a process
called restriction; host DNA is methylated by a
modification enzyme (a methylase) to protect it
from the restriction enzyme’s activity.
• To cut the DNA, a restriction enzyme makes two
incisions, once through each sugar-phosphate
backbone (i.e. each strand) of the DNA double
helix.
Blunt or sticky!
Blunt or sticky!
• Sometimes a straight cut occurs this is known
as a blunt end.
• Sometimes a staggered cut occurs, which
leaves and uneven cut in which the DNA strand
has exposed unpaired bases – known as a sticky
end.
• If you read the unpaired bases each from left
to right they are opposites of one another, i.e
they are a palindrome.
But how do you know where to find
the desired gene in the first place?
• Using a genetic probe – You know the DNA
base sequence of the gene for the desired
protein so a section of base sequence can be
radioactively labelled.
• This section of DNA with the correct base
sequence is called a probe.
• The DNA is "unzipped" so that it becomes
single stranded and a probe would anneal
(attach) if there were complementary bases.
• The probe is added and sticks to the correct
complementary fragment. The correct
fragment can now be identified, as it is
radioactive.
Now is time to create lots of copies
of the isolated DNA
• There are two ways to get lots of clones
of the DNA sequences which has been
isolated:
– In vivo – cloning by transfering the DNA into
a host cell using a vector and the host copying
the DNA.
– In vitro – using polymerase chain reaction
(PCR)
In vivo gene cloning using vector
What type of organism would make a good
host?
1. Grows fast.
2. Is easily manipulated.
3. Has a simple chromosome (prokaryotic
cells do not have a nuclear envelope).
4. Contains naturally occurring vectors (see
later).
• A good option therefore is to use yeasts
or bacteria.
How to get the DNA into the Host –
use a vector!
• A vector is a carrier DNA molecule into which
the desired gene can be inserted.
• Most commonly, this vector is a plasmid. This
is a small, extra-chromosomal, circular piece of
DNA often found in bacteria in addition to
their functional DNA.
The plasmid • The plasmids are modified so that they have two
or more genes for resistance to antibiotics.
• They should also contain a sequence that can be
recognised by the same restriction enzyme used to
cut the fragments. This enables sticky ends to be
complementary – why do you think this would be
useful?
• The site that is cut should be in one of the genes
for antibiotic resistance.
• A Plasmid:
Importance of Sticky Ends
• Using pages 249 –
250 explain the
importance of
sticky ends – use
diagrams to help
you.
Step 1 – Cut the plasmid and the
Desired DNA
• Cut the genome with a restriction enzyme (RE)
and mix with the plasmid that has also been
cut with the same R.E so that the sticky ends
of the fragments and the plasmid are
complementary.
• Hopefully, some fragments will insert into the
plasmid DNA before either segment joins with
itself.
• The join is made permanent by DNA ligase
The fragments are added to the
plasmids with these possible outcomes:
1. Plasmid rejoins, tetracycline gene now intact.
2. Fragment joins with plasmid. Tetracycline
resistance gene is interrupted; the fragment
does not contain the desired gene.
The fragments are added to the
plasmids with these possible outcomes:
3. Fragment joins with plasmid. Tetracycline
resistance gene is interrupted; the fragment
does contain the desired gene.
4. The fragment joins with itself. .
Now it can be introduced into the
host
• Transformation – re-introducing plasmids to
bacterial cells
• Mix the bacterial cells together with the
plasmids and some calcium ions.
• Calcium ions and changes in temperature make
the cell membrane of the bacteria permeable
and allow the plasmid to pass through.
• Only about 1% will have taken up the correct
plasmid.
Identifying the transgenic bacteria – with
the introduced gene in the correct place!
• The bacteria are transferred to a plate containing
the antibiotic ampicillin.
• Those bacteria that have taken up any plasmid will
be resistant to the antibiotic so will survive and
form colonies.
• Those that have not taken up the plasmid will not
be resistant and die
Is this enough to make
sure that the gene is
present?
What would you do
next?
Use the genetic marker –
Antibiotic resistance
• These colonies are then
replicated onto plates
containing the antibiotic
tetracycline.
• Those bacteria with
recombinant plasmids will not
survive because the fragment
has disrupted the gene for
resistance.
• The 2 plates are compared and
those colonies resistant to
ampicillin but not to
tetracycline can be identified.
All these colonies contain
recombinant plasmids.
Can you see any
problems with this
process?
Pg - 252
Other markers
• A fluorescent protein – The gene can be
inserted into the green fluorescent protein
gene, this means that the bacterial which
cannot glow in the dark have not taken up the
plasmid.
• An enzymes marker – lactase enzyme, it can
turn a colourless substrate blue – this means if
grown on the substrate those that have not
taken up the plasmid with the gene inserted
into the lactase enzyme gene they will turn it
blue.
– Benefit – quicker because you do not need to carry
out replica plating because the colonies you need
are not killed.
In vitro – using polymerase chain
reaction (PCR)
• PCR – a rapid efficient method of copying
fragments of DNA
• It required the following:
– DNA fragments
– DNA polymerase – it’s extracted from bacteria
which live in hot springs – useful because it will
not denature at hot temperatures
– Primers
– Nucleotides
– Thermocyler – a computer controlled machine that
varies temperature precisely over a period of time
In vitro – using polymerase chain
reaction (PCR)
How the PCR works:
• There are three steps, repeated for up to 40 cycles in an automatic
cycle, which heats and cools the reaction mixture very rapidly.
1. Separation of DNA - The DNA strand is heated to 95°C, to denature it
and open the strands, forming single strands..
2. Annealing of primers - at 55°C. During this process the primers are
jiggled around by molecular collisions (Brownian motion). Ionic bonds are
formed and broken between the single strands of primer and the
template. In the areas where more exact fits are made, the bonds last
longer, allowing the DNA polymerase enzyme to start copying the
template. (The heat stable TaqDNA polymerase comes from a
thermophylic bacterium found in hot springs.) The primers also prevent
the two orignal strands from joinging
Note: Very pure DNA building blocks dCTP,dATP,dGTP and dTTP (one for
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of the four nucleotide bases) are in the machine at the start.
3. Sythesis of DNA - Extension at 72°C. This is the optimum
temperature for the DNA polymerase. Here the bases complementary
to the template are coupled to the primer on the 3’ side.
Note: Because both strands are copied during the PCR process, the rate of
increase is exponential.
Activities
• Using page 255
• Task 1 – Draw out figure 2 to aid with
your understanding of PCR
• Task 2 – Using the information on the
page draw up a table summarizing the
advantages of in vivo and in vitro cloning
of DNA
Using recombinant DNA technology
• Your task –
– You have been asked to write an article for Biological
science review about how recombinant DNA technology
can be used and the ethical, moral and social issues
related to its use.
– Due date – 20/04/10 – Remember you miss you lessons
next week so this has to reflect the time allowed.
– Length – at least 3 – 4 sides of types work with
images.
– Bibliography of resources used must be included.
– Content – must cover use in micro-organisms, plants
and animals.
– Your book is a good start point but it is only a start
point!