Biotechnology adapted from a PPT by Miss Price

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Biotechnologyadapted from a PPT by Miss Price
Guess the lambs name
DOLLY!--THIS IS A PHOTO OF THE FAMOUS CLONED SHEEP
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Biotechnology for AP

• Have on hand
• Plasmid Tangles
• Gel Electrophoresis Boxes
• Micropipet

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Biotechnologyadapted from a PPT by Miss Price
Guess the lambs name
DOLLY!--THIS IS A PHOTO OF THE FAMOUS CLONED SHEEP
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Biotechnology

• Use of living systems to help make usable
  products.
• Not a new idea
• Food production- Wine, beer, bread rising using
  yeast. Pickles and yogurt use bacteria.

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Technologies Addressed in this Showplease write
these on first slide

• - Recombinant DNA/ Transformation
• Cloning
• DNA fingerprinting (RFLP analysis)
• Southern Blotting
• Bioinformatics
• Microarray
• Other Tools

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Goals of Applied Genetics

1. Help humans create crops that can be frost or
   pesticide resistant ex potatoes we eat
2. Use transgenic organisms to help medical
   researchers model human physiology for testing
   (ex mice w human-like disease)
3. Help industry to create bacteria to break down
   pollutants into harmless products
4. Pharmaceutical companies use recombinant DNA to
   cheaply produce human hormones (insulin) and
   other proteins
5. Help solve crimes and determine familial
   relationships

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Selective breeding

• Selecting organisms with the most desirable
  traits
• Requires time and several generations to produce
  offspring with the desired trait
• Ex
• Short vs long haired cats
• Milk production in cattle
• Disease resistant foods
• Bacteria that break down oil

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Recombinant DNA Technology

• Genetic Engineering
• AKA bacterial transformation or gene cloning
• Uses a bacterial host because of fast
  reproduction and a circular DNA vehicle to hold
  the foreign DNAplasmid
• We will do this with a glow gene
• Organisms containing recombinant DNA or foreign
  DNA are known as transgenic

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Steps involved (pg 399)

1. select the desired gene(s) to be inserted into
   the organism and a bacterial host containing a
   plasmid (vehicle to hold the desired gene)
2. cut specific DNA molecules into fragments with
   special (restriction) enzymes
3. splice (rejoin) the fragments (gene and plasmid)
   together in the desired combination
4. introduce or insert the new DNA into a living
   cell for replication (cell division)

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Vector to transfer DNA
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Examples of genes

• Insulin
• Factor X blood clotting factor
• ???? You decide

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Restriction Enzymes

• Used to cut a DNA molecule at a specific
  nucleotide sequence
• Produces one of two types of DNA fragments
• Sticky ends (palindrome)
• Blunt ends

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Sticky Ends (palindrome)
Blunt ends
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Notice how plasmid is cut once to open it. When
gene is inserted, it remains intact.
Vector to transfer DNA
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Vectors

• Vector in nature, an organism that can transmit
  DNA to another organism, often an infection
• Biotechnology uses this ability to transfer
  desired genetic information to a host cell.

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Selecting for Transformed Bacteria

• Only a small percentage of the bacteria will be
  transformed.
• To select for these bacteria, you add an
  antibiotic resistance gene when you add the human
  gene.
• Now, grow the bacteria on a plate with
  antibiotic. The only bacteria that can grow on a
  petri dish are the transformants. The other
  bacteria are killed.

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These will die when grown On antibiotic petri
dish
These will survive when grown On antibiotic petri
dish
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Esherichia coli

• For our transformation lab, we use E. coli.
• We use the common, beneficial strain that all of
  us harbor in our intestines.
• We do not use 0157 H7the strain that causes
  disease.

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Gene Libraries

• Store DNA sequences for biotech applications
• May use plasmid or phage
• (Phage virus that infects bacteria and looks
  like a spaceship)
• May contain entire genome or only DNA used in
  gene expression.
• Expressed DNA is called cDNA (complementary DNA)
  and is made from mRNA with the enzyme...

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Reverse Transcriptase!!!!
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Gene Cloning

• Used to produce genetically identical copies of a
  cell, tissue, organ, and/or organism
• Needed to produce multiple copies of the desired
  DNA

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Gene Cloning

• What was the name of the first cloned cat?
• COPY CAT!!!!
• ITS TRUE

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Cloning Applications

• Currently
• Plants are cloned to produce a large number of
  genetically identical plants in a short amount of
  time
• Future?
• Clone productive and healthy animals to increase
  yield for farmers and to grow organs for
  transplants

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How it works.
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Nucleus is extracted with a syringe
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Cloned Glowing Cats
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Gene Therapy

• The insertion of normal genes into human cells to
  correct genetic disorders like cystic fibrosis.

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PCR

• PAGE 404

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DNA Fingerprinting(RFLP analysis) pg 405

1. Obtain a small sample of DNA
2. Make millions of copies using PCR (polymerase
   chain reaction) technique
3. Cleave (cut) DNA with restriction enzymes
4. Separate DNA fragments using gel electrophoresis
   and compare
5. Each humans DNA will have some unique pieces
   because each of our DNA is unique

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Restriction Fragment Length Polymorphism (RFLP)

• RFLP Each individual..
• ...has different numbers of restriction sites
• ...different of base pairs between restriction
  sites
• Gel electrophoresis is used to create a DNA
  fingerprint of these unique sizes.
• Small amounts of DNA are loaded into wells in the
  gel.
• An electric current pushes the small pieces of
  DNA farther down the gel than the larger pieces.

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RFLP

• DNA patterns are compared to known patterns.
• Used for forensics, blood samples, and paternity
  tests (children get half their bands from mom and
  half from dad)
• Used to identify diseases
• Used to sequence DNA base pairs (older method)

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In this simpified model, how many bands would
come from the top DNA strand? How many from the
bottom one.
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Running a gel
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How DNA moves
Step 1 Pour gel with comb for wells, not
unlike a jello mold.
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Step 2 Pipette DNA into wells. DNA has been
cut with restriction enzymes.
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Step 3 Run an electric current to watch DNA
migrate. Small strands, fewer base pairs,
travel further.
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Step 4 Notice similarities and differences in
banding patterns.
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Practice Loading Wells withMicropipette

• There are two types, fixed volume and
• variable volume.
• Must be used with a tip (usually sterile).
• To fill
• Press plunger to first stop.
• Immerse in loading dye.
• Release plunger
• Hover above the well, and dispense dye by
  pressing plunger all the way.
• The dye will sink into the well.

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The Human Genome Project

• There are approximately 25,000 genes on the 46
  human chromosomes (far fewer than expected)
• Human Genome Project- an international effort to
  completely map and sequence human chromosomes
  (April 2003)

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Southern Blotting (pg 407)

• Purpose to use a cloned gene to probe for the
  same gene in another sample.
• Named for Edward M. Southern-
• Western and Northern Blots play on that name
• These have slightly different procedure
• Western involves protein rather than DNA

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Southern Blotting

• Unknown DNA is run on a gel.
• DNA bands are blotted onto special paper.
• Paper is flooded with labeled complementary DNA

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Southern Blotting

• Hybridization Single stranded DNA probe binds to
  any complementary DNA on paper, and the rest is
  washed off
• Bands that are hybridized are radioactive and can
  be visualized.
• (Similar to microarray, but done on paper instead
  of glass slide).
• http//www.accessexcellence.org/RC/VL/GG/ecb/south
  ern_blotting.html

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Uses of Radioactive Probes

• Biochemists use radioactive probes to find things
  such as
• Genes
• Proteins
• Enzymes
• Receptors on membranes
• Antigens (by using radioactive antibodies)

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• Labeled glucose is used in brain PET scans.
• Glucose labeled with F-18 is injected into blood.
• Can monitor where in the brain the glucose is
  being used.

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Bioinformatics

• This area of study uses genetic material (or any
  biological material) to gather information.
• Such as

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Bioinformatics

• Whether a gene is similar to a previously
  sequenced gene.
• Give clues about a cells or genes function
• Whether a specific gene is correlated with a
  specific disease, such as which genes are
  prevalent in cancer cells.
• Whether a certain drug can benefit or harm a
  patient based on the patients genotype.

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Applications of Biotechnology

• Gene therapy (pg 418)
• Herpes and Hepatitis Vaccines (use protein coat
  gene introduced with vaccinia as vector).
• Using AAV (adeno-associated virus) to introduce
  correct version of gene
• Cystic fibrosis, hemophilia, some cancers

• Transformation (pg 399)
• Add a gene to a plasmid
• Get bacteria to make products
• Insulin
• Plants resistant to
• frost, disease, herbicides
• Clone tissues, organs, organisms

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Applications of Biotechnolgy

• DNA fingerprinting
• ( pg 405)
• RFLP analysis
• Identification of suspects, paternity, forensics
• Sequence Genes
• Identify diseases

• Microarray (pg 411)
• Determines which genes are expressed in which
  cells
• Compare cancer cell with non-cancer cells
• Customized medicine, determine which med works
  best for patient

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Sharing Lab Materials

• Tray with loops, pipets, sterile tubes, etc
• 3 shared ice
• 1 shared warm water bath
• 1 shared vial of plasmid

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Transformation Sterile Procedure

• Open the sterile petri dish as briefly as
  possible
• Do not talk (or breathe) while it is opened.
• Hold the lid, DO NOT place it on the desk.
• Sterile loops should be opened from the end
  opposite the loop.
• All materials must be placed in WASTE container.
• Agar is like jello. DO NOT DIG INTO THE AGAR.
• Gently sweep across the top to lift bacteria.
• Label petri dish with masking tape around
  perimeter

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Transformation Lab Notes

• USE THE INSTRUCTIONS ON YOUR DESKTHESE ARE
  SPECIFIC TO THIS KIT
• I NEED SOMEONE WHOSE MAIN JOB THE FIRST HALF IS
  TO KEEPT HE WATER BATH AT EXACTLY 420C
• DO NOT TOUCH T HE GLASS BEADS!!
• PUT THEM IN THE WASTE BEAKER WHEN FINISHED
• ALL MATERIALS ARE STERILE. USE AS PER DIRECTIONS
  AND PUT IT IN THE WASTE BEAKER
• BETTER TO USE SEVERAL SMALL COLONIES RATHER THAN
  A CLUMP OR A STREAK
• Step 10C Door Side of Room Negative (-) Control
• Other Side of Room Positive ()
  Control

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• BETTER TO USE SEVERAL SMALL COLONIES RATHER THAN
  A CLUMP OR A STREAK
• ONLY USE A NEW PIPET WHEN IT SAYS USING A
  STERILE PIPET
• So for 4, you use the same pipet the whole
  time.

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Gather individual colonies, not streaks
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Credits

• This Power Point was provided by Abby Price and
  adapted by Andrea Wise, Providence High School,
  2007