Biotechnology

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Biotechnology

• Manipulation of Life for Knowledge and Application

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What is Biotechnology

• The broad definition of biotechnology is simply
  the industrial use of living organisms (or parts
  of living organisms) to produce foods, drugs, or
  other products.
• The oldest biotechnologies include fermentation
  and plant and animal hybridization.
• The newest biotechnologies range from protein
  separation technologies to genomics and
  combinational chemistry.

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A sampler of fields that fall under
biotechnology's broad umbrella would include

• bacteriology
• biochemical engineering
• bioinformatics
• bioprocessing
• cell biology
• chromatography
• computational mathematical modeling
• developmental and molecular genetics

• DNA  technologies
• electrophoresis
• embryology
• immunology
• materials science
• microbiology
• nucleic acid chemistry
• protein engineering
• virology

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Areas of Focus

• Procedures
• Restriction Digest, Gel Electrophoresis, PCR,
  Transformation
• Applications
• DNA profiling, Genetic Screening, GMOs
• (ex. Crops), Cloning
• Ethics
• Just because we can, does not mean we should

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The Big Picture

• Several procedures are used together in various
  ways to accomplish several different applications

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Summary of Procedures

• Restriction Digest
• Cutting DNA into fragments with enzymes
• Polymerase Chain Reaction PCR
• Amplification (copying) of DNA or RNA
• Gel Electrophoresis
• Separation of DNA or RNA fragments based on
  length
• Transformation
• Placement of DNA fragment into a plasmid and
  insertion into cell

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Restriction Digest Reaction

• To a tube add
• Sample DNA
• Restriction enzymes
• Water
• Buffer solution (ph, salt specific to enzyme)
• Incubate at 37C for 2 hours or overnight

Original Strand
Possible fragment lengths from partial digestion
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Restriction Enzyme

• A protein that recognizes and severs the double
  helix at a specific DNA sequence.
• Hundreds of different enzymes have been
  identified, isolated, and can be purchased for
  lab use

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• Restriction Enzymes are Endonucleases, meaning
  they cut DNA within the sequence
• (as opposed to an Exonuclease which chews DNA at
  the ends)

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Each enzyme recognizes and cuts a different DNA
sequence
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Next Steps

• Cut DNA can be
• amplified using PCR
• separated using electrophoresis to generate a DNA
  fingerprint / profile
• ligated (combined) with a plasmid for
  transformation to make a GMO

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Polymerase Chain Reaction - PCR

• From a single fragment of DNA, more than a
• billion identical copies can be synthesized in a
• few hours.
• Especially useful
• if DNA sample
• is small
• Basically this is
• artificial DNA replication

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The Taq DNA polymerase

• PCR uses the special properties of a DNA
  polymerase discovered in the bacteria,Thermus
  aquaticus
• The Taq polymerase is optimally active at 72C
• It can copy 1kb of DNA in 30 seconds

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The Cycling Reactions

• There are three steps in a PCR, which are
  repeated for 30 or 40 cycles.
• Denaturation 95C separating double helix
• Annealing 55C short DNA primers bind
• Elongation 72C DNA is replicated, beginning
  at primer location

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PCR Thermocycler
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• DNA Primers Taq Polymerase nucleotides
  many new DNA copies

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PCR works exponentially.. 2, 4, 8, 16, 32, 64
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PCR Video
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Next Steps

• Amplified (copied) DNA fragments can be
• separated using gel electrophoresis (used for DNA
  profiling/fingerprinting/screening)
• ligated into plasmids for transformation (used to
  make GMOs)

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Gel electrophoresis

• Process
• DNA and RNA have an overall negative charge due
  to the phosphate groups.
• If placed in an electric field the nucleic acids
  will move toward the positive pole of the field.
  
• In a gel matrix, smaller fragments will move
  faster than larger fragments.
• The DNA or RNA is separated by the length of the
  molecules

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1) DNA and RNA have an overall negative charge
due to the phosphate groups.
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2) If placed in an electric field the nucleic
acids will move toward the positive pole of the
field
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3) In a gel matrix, smaller fragments will move
faster than larger fragments
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4) The DNA or RNA is separated by the length of
the molecules
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Gel Electrophoresis Animation
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Transformation

• Process
• Digested and amplified DNA is inserted into a
  plasmid
• Cells are induced to uptake plasmids
• Within cells the inserted genes are expressed
• Resulting proteins will affect phenotype of
  organism

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Plasmids

• Plasmids are small rings of DNA
• Discovered in nature and now made artificially
• Used to transfer genes into cells
• Must have origins of replication and known
  multiple restriction enzyme sites

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Ligation

1. Plasmid and selected DNA fragment are cut with
   same restriction enzyme
2. Incubated together with DNA ligase
3. DNA is inserted into plasmid

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Bacterial Transformation

• 4) Cells are induced to uptake plasmid
• The gene can now be expressed, conferring its
  trait upon the cell
• Makes a GMO

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Applications

• DNA Profiling
• Crime scene investigation, paternity cases
• Genetically Modified Food
• Pesticide resistant corn, vitamin enriched rice,
• Genetic Screening
• Predicting future health issues by looking at
  genes
• Cloning
• Creating genetically identical organisms

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DNA Profiling

• Uses DNA fingerprints generated by
  electrophoresis of restriction fragment length
  polymorphisms (RFLPs)
• Most crime agencies have a DNA fingerprint
  database
• Some countries suggest all citizens be
  fingerprinted
• Can easily be done at birth

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Genetic Screening

• Uses PCR analysis of known genetic sequences to
  identify presence and type of specific gene
• Used to identify pre-disposition to diseases
• Can be used on the unborn
• Can be used to identify genes unrelated to disease

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Genetically Modified Organisms

• Uses plasmid transformation to insert genes into
  whole organisms
• Used in agriculture to generate toxin resistance
  and promote growth
• Can potentially be used to mix a variety of genes
  into any type of organism

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Cloning

• Through somatic cell nuclear transfer it is
  possible to develop an organism with the
  identical genome of a pre-existing organism
• Many types of organism, from bacteria, to plants,
  to humans, have been cloned
• Cloned humans have not yet been raised passed a
  few cells

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Ethics

• To what degree should we meddle with the power
  of life, a power which we know so very little of,
  but which we are entirely dependent upon?
• - some guy
• One day we will unlock the door that leads to
  the body immortal, but will you step through it?
• - the same guy