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

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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
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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)

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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

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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.
• NoteMany 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.

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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

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Interesting fact

• The technology for producing cDNA is actually
  used by HIV

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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

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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
  enzymes 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.

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Blunt or sticky!
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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.

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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.

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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)

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In vivo gene cloning using vector

• What type of organism would make a good host?
• Grows fast.
• Is easily manipulated.
• Has a simple chromosome (prokaryotic cells do not
  have a nuclear envelope).
• Contains naturally occurring vectors (see later).
  
• A good option therefore is to use yeasts or
  bacteria.

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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.

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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

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Importance of Sticky Ends

• Using pages 249 250 explain the importance of
  sticky ends use diagrams to help you.

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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

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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.

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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. .

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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.

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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?
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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
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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.

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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 its 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

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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.
• Separation of DNA - The DNA strand is heated to
  95C, to denature it and open the strands,
  forming single strands..
• Annealing of primers - at 55C. 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 each of the four
  nucleotide bases) are in the machine at the
  start.
• 3. Sythesis of DNA - Extension at 72C. 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.

Click me!
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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

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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!