Recombinant DNA Technology

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Recombinant DNA Technology

• BTEC3301

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Recombinant DNA Technology

• Recombinant DNA technology procedures by which
  DNA from different species can be isolated, cut
  and spliced together -- new "recombinant "
  molecules are then multiplied in quantity in
  populations of rapidly dividing cells (e.g.
  bacteria, yeast).

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Recombinant DNA Technology

• The term gene cloning, recombinant DNA technology
  and genetic engineering may seems similar,
  however they are different techniques in
  Biotechnology and they are interrelated

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Recombinant DNA Technology

• Human gene therapy, genetically-engineered crop
  plants and transgenic mice have become possible
  because of the powerful techniques developed to
  manipulate nucleic acids and proteins.

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Recombinant DNA Technology

• In the early 1970s it became possible to isolate
  a specific piece of DNA out of the millions of
  base pairs in a typical genome.

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Recombinant DNA Technology

• Currently it is relatively easy to cut out a
  specific piece of DNA, produce a large number of
  copies , determine its nucleotide sequence,
  slightly alter it and then as a final step
  transfer it back into cell in.

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Recombinant DNA Technology

• Recombinant DNA technology is based on a number
  of important things
• Bacteria contain extrachromosomal molecules of
  DNA called plasmids which are circular.

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Recombinant DNA Technology

• Bacteria also produce enzymes called restriction
  endonucleases that cut DNA molecules at specific
  places into many smaller fragments called
  restriction fragments.

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Recombinant DNA Technology

• Restriction Enzymes and plasmid
• There are many different kinds of restriction
  endonucleases
• Each nuclei cuts DNA at a specific site defined
  by a sequence of bases in the DNA called a
  recognition site

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Recombinant DNA Technology

• Restriction Enzymes and plasmid
• A restriction enzyme cuts only double-helical
  segments that contain a particular sequence, and
  it makes its incisions only within that
  sequence--known as a "recognition sequence".

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Recombinant DNA Technology

• Restriction Enzymes and plasmid
• Sticky end and blunt end are the two possible
  configurations resulting from the breaking of
  double-stranded DNA

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Recombinant DNA Technology

• Restriction Enzymes and plasmid
• If two complementary strands of DNA are of equal
  length, then they will terminate in a blunt end,
  as in the following example
• 5'-CpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpA
  pGpT-3'
• 3'-GpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApT
  pCpA-5'

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Recombinant DNA Technology

• Restriction Enzymes and plasmid
• However, if one strand extends beyond the
  complementary region, then the DNA is said to
  possess an overhang
• 5'-ApTpCpTpGpApCpT-3'
• 3'-TpApGpApCpTpGpApCpTpApCpG-5'

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Recombinant DNA Technology

• Restriction Enzymes and plasmid
• If another DNA fragment exists with a
  complementary overhang, then these two overhangs
  will tend to associate with each other and each
  strand is said to possess a sticky end

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Recombinant DNA Technology

• Restriction Enzymes and plasmid
• 5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT
  -3'
• 3'-TpApGpApCpTpGpApCpTpApCpGp
  CpApTpApCpGpA-5'
• Becomes
• 5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3'
• 3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5'

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Recombinant DNA Technology

• Restriction Enzymes and plasmid
• Restriction Enzymes are primarily found in
  bacteria and are given abbreviations based on
  genus and species of the bacteria.
• One of the first restriction enzymes to be
  isolated was from EcoRI
• EcoRI is so named because it was isolated from
  Escherichia coli strain called RY13.

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Recombinant DNA Technology

• Digestion of DNA by EcoRI to produce cohesive
  ends ( Fig. 3.1)

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Recombinant DNA Technology

• Creating recombinant DNA
• The first Recombinant DNA molecules were made by
  Paul Berg at Stanford University in 1972.
• In 1973 Herbert Boyer and Stanley Cohen created
  the first recombinant DNA organisms.

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Recombinant DNA Technology

• Creating Recombinant DNA (Fig 3.2)

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Recombinant DNA Technology

• Reading materials Summary of Recombinant DNA
  technology process
• Recombinant DNA technology requires DNA
  extraction, purification, and fragmentation.
• Fragmentation of DNA is done by specific
  'restriction' enzymes and is followed by sorting
  and isolation of fragments containing a
  particular gene.

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Recombinant DNA Technology

• Summary of Recombinant DNA technology process
• This portion of the DNA is then coupled to a
  carrier molecule.
• The hybrid DNA is introduced into a chosen cell
  for reproduction and synthesis.

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Recombinant DNA Technology

• Transformation and Antibiotic Selection
• Transformation is the genetic alteration of a
  cell resulting from the introduction, uptake and
  expression of foreign DNA.

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Recombinant DNA Technology

• Transformation and Antibiotic Selection
• There are more aggressive techniques for
  inserting foreign DNA into eukaryotic cells. For
  example, through electroporation.
• Electroporation involves applying a brief
  (milliseconds) pulse high voltage electricity to
  create tiny holes in the bacterial cell wall that
  allows DNA to enter.

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Recombinant DNA Technology

• Plasmids and Antibiotic resistance
• Plasmids were discovered in the late sixties, and
  it was quickly realized that they could be used
  to amplify a gene of interest.
• A plasmid containing resistance to an antibiotic
  (usually ampicillin) or Tetracycline, is used as
  a vector.

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Recombinant DNA Technology

• The gene of interest (resistant to Ampicillin) is
  inserted into the vector plasmid and this newly
  constructed plasmid is then put into E. coli that
  is sensitive to ampicillin.( Text bkPg 58)
• The bacteria are then spread over a plate that
  contains ampicillin.

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Recombinant DNA Technology

• Plasmids and Antibiotic resistance
• The ampicillin provides a selective pressure
  because only bacteria that have acquired the
  plasmid can grow on the plate.
• Those bacteria which do not acquire the plasmid
  with the inserted gene of interest will die.

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Recombinant DNA Technology

• Plasmids and Antibiotic resistance
• As long as the bacteria grow in ampicillin, it
  will need the plasmid to survive and it will
  continually replicate it, along with the gene of
  interest that has been inserted to the plasmid .

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Recombinant DNA Technology

• Fig 3.3 (a).
• Selecting a Gene in a plasmid and Antibiotic
  selection.

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Recombinant DNA Technology

• Assignment For above procedure,
• Read Transformation of Bacterial cells and
  Antibiotic selection pg 61.

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Recombinant DNA Technology

• Human Gene cloning
• Once inside a bacterium, the plasmid containing
  the human cDNA can multiply to yield several
  dozen replicas.

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Recombinant DNA Technology
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Recombinant DNA Technology

• Reading materials
• Summary of Recombinant DNA and Cloning (Fig.
  below)
• Isolation of two kinds of DNA
• Treatment of plasmid and foreign DNA with the
  same restriction enzyme
• Mixture of foreign DNA with plasmids

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Recombinant DNA Technology

• Addition of DNA ligase
• Introduction of recombinant plasmid into
  bacterial cells
• Production of multiple gene copies by gene cloning

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Recombinant DNA Technology

• Summary of Recombinant DNA and Cloning (Fig.)

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Recombinant DNA Technology

• This segment is "glued" into place using an
  enzyme called DNA ligase.
• The result is an edited, or recombinant, DNA
  molecule.

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Recombinant DNA Technology

• When this recombinant plasmid DNA is inserted
  into E. coli, the cell will be able to process
  the instructions to assemble the amino acids for
  insulin production.

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Recombinant DNA Technology

• More importantly, the new instructions are passed
  along to the next generation of E. coli cells in
  the process known as gene cloning.
• Assignment Human gene cloning pg 63

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Recombinant DNA Technology

• Fig Inserting a DNA sample into a Plasmid

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Recombinant DNA Technology

• References
• http//en.wikipedia.org/wiki/Restriction_enzyme
• http//web.mit.edu/esgbio/www/rdna/cloning.html
• http//faculty.plattsburgh.edu/donald.slish/Transf
  ormation.html