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Biotechnology adapted from a PPT by Miss Price

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Biotechnology Guess the lamb s name DOLLY!--THIS IS A PHOTO OF THE FAMOUS CLONED SHEEP – PowerPoint PPT presentation

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Title: Biotechnology adapted from a PPT by Miss Price


1
Biotechnology adapted from a PPT by Miss Price
Guess the lambs name
DOLLY!--THIS IS A PHOTO OF THE FAMOUS CLONED SHEEP
2
Biotechnology for AP
  • Have on hand
  • Plasmid Tangles
  • Gel Electrophoresis Boxes
  • Micropipet

3
Biotechnology adapted from a PPT by Miss Price
Guess the lambs name
DOLLY!--THIS IS A PHOTO OF THE FAMOUS CLONED SHEEP
4
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.

5
Technologies Addressed in this Show please write
these on first slide
  • - Recombinant DNA/ Transformation
  • Cloning
  • DNA fingerprinting (RFLP analysis)
  • Southern Blotting
  • Bioinformatics
  • Microarray
  • Other Tools

6
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

7
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

8
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

9
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)

10
Vector to transfer DNA
11
Examples of genes
  • Insulin
  • Factor X blood clotting factor
  • ???? You decide

12
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

13
Sticky Ends (palindrome)
Blunt ends
14
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15
Notice how plasmid is cut once to open it. When
gene is inserted, it remains intact.
Vector to transfer DNA
16
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.

17
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.

18
These will die when grown On antibiotic petri
dish
These will survive when grown On antibiotic petri
dish
19
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.

20
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...

21
Reverse Transcriptase!!!!
22
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

23
Gene Cloning
  • What was the name of the first cloned cat?
  • COPY CAT!!!!
  • ITS TRUE

24
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

25
How it works.
26
Nucleus is extracted with a syringe
27
Cloned Glowing Cats
28
Gene Therapy
  • The insertion of normal genes into human cells to
    correct genetic disorders like cystic fibrosis.

29
PCR
  • PAGE 404

30
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

31
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.

32
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)

33
In this simpified model, how many bands would
come from the top DNA strand? How many from the
bottom one.
34
Running a gel
35
How DNA moves
Step 1 Pour gel with comb for wells, not
unlike a jello mold.
36
Step 2 Pipette DNA into wells. DNA has been
cut with restriction enzymes.
37
Step 3 Run an electric current to watch DNA
migrate. Small strands, fewer base pairs,
travel further.
38
Step 4 Notice similarities and differences in
banding patterns.
39
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40
Practice Loading Wells with Micropipette
  • 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.

41
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)

42
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

43
Southern Blotting
  • Unknown DNA is run on a gel.
  • DNA bands are blotted onto special paper.
  • Paper is flooded with labeled complementary DNA

44
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

45
Uses of Radioactive Probes
  • Biochemists use radioactive probes to find things
    such as
  • Genes
  • Proteins
  • Enzymes
  • Receptors on membranes
  • Antigens (by using radioactive antibodies)

46
  • 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.

47
Bioinformatics
  • This area of study uses genetic material (or any
    biological material) to gather information.
  • Such as

48
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.

49
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

50
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

51
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52
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53
Sharing Lab Materials
  • Tray with loops, pipets, sterile tubes, etc
  • 3 shared ice
  • 1 shared warm water bath
  • 1 shared vial of plasmid

54
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

55
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

56
  • 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.

57
Gather individual colonies, not streaks
58
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59
Credits
  • This Power Point was provided by Abby Price and
    adapted by Andrea Wise, Providence High School,
    2007
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