BIOTECHNOLOGY -intentional manipulation of genetic material of an organism

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BIOTECHNOLOGY-intentional manipulation of
genetic material of an organism

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Deoxyribonucleic Acid (DNA)

• determines the characteristics of all living
  organisms.
• occurs in most cells of all organisms
• composed of four different nucleotides in
  different combinations
• each cell in the human body contains more than 3
  BILLION letters

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Four bases Adenine Thymine Guanine Cytosine
2 bonds
3 bonds

• Sugar and phosphate backbone

• Double helix structure
• (two spirals around each other)

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• The only difference between living organisms is
  the amount and order of the four nucleotide
  bases.
• 
  

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• Genome the entire sequence of DNA
• Gene the part of the code that corresponds to a
  protein
• genes can be transferred from one organism to
  another

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BIOTECHNOLOGYThe intentional manipulation of
genetic material of an organism

• Why would we want to do this?

• To study cellular processes of an organism
• E.g. Glowing gene from jellyfish to tobacco plant

• To give one organism the trait(s) of another
• E.g. Anti-freeze from fish blood into
  strawberries to survive through early frosts

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• Part 1 Manipulating Bacteria
• The Making of a Plasmid

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Plasmid - a small circular piece of
extra-chromosomal bacterial DNA, able to replicate

• bacteria exchange these plasmids to share DNA
• E.g. antibiotic resistance genes

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• Since plasmid is made of DNA it can code for
  genes, ex. antibiotic resistance, and can carry
  specific sequences of DNA

• Specific DNA sequences can be recognized by
  enzymes called restriction endonucleases

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Restriction Endonucleases/Restriction Enzymes

• enzymes that are able to cut double-stranded DNA
  into fragments at specific recognition sites in
  DNA sequences
• Ex. EcoRI
• 5-GAATTC-3
• 3-CTTAAG-5

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• Restriction enzymes can create sticky ends or
  blunt ends

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• Sticky Ends
• fragment end of a DNA molecule with a short
  single-stranded overhang

• Blunt Ends
• fragment end of a DNA molecule with no overhang

Once made, the ends can be re-joined together by
other enzymes ("enzyme glue")
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To Make a Recombinant Plasmid
Insert

1. Cut the plasmid and the insert with the same
   restriction endonuclease to make complementary
   sticky ends.

• Combine the sticky ends using ligase.
• ligase enzyme used to join DNA together

3. Introduce the recombinant plasmid into
bacteria.
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Making a Recombinant Plasmid
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Bacterial Transformation

• introduction of foreign DNA into a bacterial
  cell

• plasmid is used as a vector, a vehicle by which
  DNA can be introduced into host cell

phospholipid bilayer
Ca2 ions
plasmid
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Following transformation bacteria are grown in
medium with antibiotic
Only the bacteria that have the plasmid (and
therefore the antibiotic resistance) will survive.
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Example plasmid
Origin of Replication

• where the plasmid starts to duplicate itself

• the specific sequence MUST NOT be cut by
  restriction endonucleases or it wont be able
  to replicate

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Part 2 Where do we get our insert sequence?

• From someone elses DNA
• ex. fish gene in strawberries,
• jellyfish gene in plants

• Make it!

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• In order to do these things, we need a way to
  make many copies of the genes we want

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Using Bacteria as Production Factories

• easy to grow
• no ethical issues
• small genome
• easy to manipulate

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Making an insert Polymerase Chain Reaction
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Common uses of biotechnology

• Making "stuff
• proteins, enzymes, medication, etc. can be
  produced by engineered bacteria!
• Food can be altered to have new traits
• Cloning (therapeutic and reproductive)
• Genetic screening
• crime cases, relationship, genetic screening,
  etc.
• 3. Gene Therapy

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Therapeutic cloning

• used to produce tissue that is identical to the
  donor, to prevent rejection

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Reproductive Cloning

• creates an organism with the same genetic
  material (DNA) as the original organism an
  EXACT COPY of the donor

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Dolly the Sheep

• the first cloned sheep

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Ex. RFLP Restriction Fragment Length Polymorphism

• Comparison of different lengths of DNA fragments
  produced by restriction enzymes to determine
  genetic differences between individuals

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• Gene therapy
• desired gene is inserted into cell's nucleus
  using a retrovirus as a carrier
• defective gene replaced by functional gene

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• Ex. ADA deficiency
• adenosine deaminase deficiency
• little immunity with low chances of recovery
• - the T-cells of a four-year-old were removed,
  modified and re-inserted to fix her immune system