BIOTECHNOLOGY

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BIOTECHNOLOGY

• IB BIOLOGY

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Recombinant DNA

• Defined
• DNA that contains DNA from other sources
• Transfer from one part of DNA molecule to another
• Transfer from one csome to another
• Transfer from one organism to another!

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Can occur artificially

• RECOMBINANT DNA TECHNOLOGY

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What is Recombinant DNA Technology?

• 1974
• Overview
• Select and isolate specific sequence of DNA in
  organism of choice (ie human gene)
• Incorporate selected DNA sequence into DNA of a
  vector organism
• Grow and amplify resulting recombinant DNA in a
  host that reproduces quickly

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Overview
Recombinant bacterium
Host cell grown in culture to form a clone of
cells containing the cloned gene of interest
Protein expressed by gene of interest
Gene of interest
Copies of gene
Protein harvested
Basic research and various applications
Basic research on gene
Basic research on protein
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic
waste
Protein dissolves blood clots in heart attack
therapy
Human growth hor- mone treats stunted growth
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The Specifics

• Requires use of
• RESTRICTION ENZYMES
• naturally occurring in bacteria (1960s)
• natural protection against viral infection

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How are Restriction Enzymes used in Recombinant
DNA Technology?

• THREE Parts
• Occurs in organism of choice
• Occurs in vector organism
• Occurs in host organism

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Part I

1. Restriction enzymes obtained from bacteria (ex
   EcoRI) and inserted into DNA of organism of
   choice!
2. Restriction Enzyme specifically cuts DNA of
   selected organism at specific recognition
   sequences of nucleotides.

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• Restriction Enzyme cuts across DNA double helix,
  producing restriction fragments with staggered
  ends extending beyond complementary strand
  (sticky ends)

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EcoRI
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LE 20-3
Restriction site
Using a restriction enzyme and DNA ligase to make
recombinant DNA
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DNA
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Restriction enzyme cuts the sugar-phosphate backbo
nes at each arrow.
Sticky end
DNA fragment from another source is added. Base
pairing of sticky ends produces various
combinations.
Fragment from different DNA molecule cut by
the same restriction enzyme
One possible combination
DNA ligase seals the strands.
Recombinant DNA molecule
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Part II

• THE MOST COMMON RECOMBINANT DNA VECTORS ARE
  PLASMIDS AND BACTERIOPHAGES!

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1. Treat plasmid with same restriction enzyme as was
   used to make the DNA restriction fragment in
   selected organism. (produces same sticky ends as
   carried by fragment)!
2. Mix the two strands of DNA (selected organism and
   vector) allowing for base pairing at sticky ends.

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1. Use DNA ligase to stabilize attachment
2. Recombinant Plasmid is now formed.
   CONGRATULATIONS.

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Part III

• IN ORDER TO BE EFFECTIVE, HOST ORGANISM SELECTED
  MUST BE CAPABLE OF REPRODUCING AT A RAPID RATE!
• why?

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1. Recombinant plasmid is introduced into a
   bacterial host organism by transformation.
2. Host organism reproduces, thereby multiplying the
   recombinant DNA plasmid!

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Genetically engineered Human insulin

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• http//www.abpischools.org.uk/res/coResourceImport
  /modules/hormones/en-flash/geneticeng.cfm

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Genetic Engineering Techniques

• Definition
• Technology that uses genetic and recombinant DNA
  methods to devise new combinations of genes to
  produce improved pharmaceutical and agricultural
  products.

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• Genes isolated from one organism can be modified
  and expressed in another organism.
• (E. coli, transgenic plants,
• transgenic animals)

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Transgenic Plants
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Transgenic Bacteria
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Genetically Modified Organisms
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C L O N I N G

• Process which genetically identical cells and/or
  individuals are created.
• Gene Cloning
• recombinant DNA injected into highly
  reproductive host organism (E. coli)
• see notes
• Organismal Cloning
• where the controversy arises

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How do you clone an individual?

1. Haploid nucleus in ovum is removed from organism
   A and replaced with diploid nucleus from somatic
   cell taken from organism B.
2. Ovum (with diploid nucleus) is reinserted into
   uterus of Organism C (surrogate mother), where it
   will develop to term.

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1. Organism born from altered ovum is genetically
   identical to organism A.

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Successful Vertebrate Cloning Experiments

• Amphibians (1970s)
• Mammals
• Sheep (DOLLY)
• Mice
• Cattle
• Pigs
• Cats (CC)
• Humans????

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DollyJuly 5, 1996 Feb 14, 2003

• RIP

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Polymerase Chain Reaction

• PCR
• Developed 1983
• Kerry Mullis

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What does PCR do?

• Amplifies small sample of DNA millions of times
  in a few hours rather than cloning an entire
  cell!
• Can generate 1million copies of DNA for every 20
  cycles of PCR.

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The PCR Process

• DNA polymerase is used to replicate the targeted
  DNA sequence.
• Replicate ? heat to separate?Replicate

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Uses for PCR

• Fossil Analysis

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• Criminal Cases

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Gel Electrophoresis

• DNA fragments of different lengths are separated
  as they diffuse through a gel under the influence
  of an electrical field.
• DNA is negatively charged (phosphate groups)
  they move toward the positive electrode!
• Shorter fragments move further through the gel
  than longer fragments! (WHY?)

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USES of Gel Electrophoresis

• Criminal Court Cases
• Determination of evolutionary relationships
  between closely related species.

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DNA Fingerprinting

• Comparing the DNA from a known individual to the
  DNA from an unknown sample
• Uses Criminology
• Paternity Tests

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How does DNA Fingerprinting Work?

• When Restriction Fragments between individuals of
  the same species are compared, the fragments
  differ in length due to polymorphisms (slight
  differences in sequences).
• Fragments are known as RFLPs (Restriction
  Fragment Length Polymorphisms)
• By using gel electrophoresis, RFLPs from
  different individuals can be compared to
  determine relatedness!

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• GENETIC SCREENING
• GENE THERAPY

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