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Chapter 13: DNA Technology


Chapter 13: DNA Technology Gene Therapy Treatment of a genetic disorder (like cystic fibrous) by correcting a defective gene that causes a deficiency of an enzyme. – PowerPoint PPT presentation

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Title: Chapter 13: DNA Technology

Chapter 13 DNA Technology
With all our knowledge of DNA and genes, is there
any way to manipulate DNA?
  • Genetic Engineering
  • form of applied genetics in which
    genes/DNA are manipulated
  • Ex. Genetic engineers believe they can improve
    the foods we eat. Tomatoes are sensitive to
    frost. This shortens their growing season. Fish,
    on the other hand, survive in very cold water.
    Scientists identified a particular gene which
    enables a flounder to resist cold and used the
    technology of genetic engineering to insert this
    'anti-freeze' gene into a tomato.

DNA Technology
  • Science involved in the ability to manipulate
  • Purpose
  • Treat diseases (Cystic fibrosis)
  • Treat genetic disorders (hemophilia, diabetes)
  • Improve food crops (better tasting veggies,
    longer shelf life, fungus resistance)
  • Improve human life in general (vaccines)

How is it all done?
  • Recombinant DNA Technology
  • Steps in the process                     -I.
    Isolation of DNA extraction
  • - II. Copying DNA with
    PCR                    -III. Cutting DNA with
    restriction enzymes                   
    -IV. Comparing DNA with gel eletrophoreisis
  • OR V. use as
    vectors in recombinant DNA

I. Isolate DNA.
  • Remove tissue from organism
  • (You will do this in your DNA extraction lab)
  • Store at 4C

PCR Polymerase Chain Reaction A genetic copy
  • The polymerase chain reaction (PCR) is a rapid
    way of amplifying (duplicating) specific DNA
    sequences from a small sample of DNA.

II. Copy DNA - PCR
  • Polymerase Chain Reaction
  • Uses DNA sample, DNA polymerase from T. aquaticus
    (hot springs)
  • Think of this process as a molecular
  • copying machine

III. Cutting DNA - Restriction enzymes
Enzymes that can cut at particular locations in
the DNA - DNA engineering today is totally
dependent on restriction enzymes -Restriction
enzymes are endonucleases - Think of them as
molecular scissors
What are restriction enzymes?
  • Bacterial enzymes used to cut DNA Different
    bacterial strains express different restriction
  • The names of restriction enzymes are derived from
    the name of the bacterial strain they are
    isolated from
  • Cut (hydrolyze) DNA into defined and REPRODUCIBLE
  • Basic tools of gene cloning

Names of restriction endonucleases
  • Titles of restriction enzymes are derived from
    the first letter of the genus the first two
    letters of the species of organism from which
    they were isolated.
  • EcoRI -  from Escherichia coli
  • BamHI - from Bacillus amyloliquefaciens
  • HindIII - from Haemophilus influenzae
  • PstI -  from Providencia stuartii
  • Sau3AI - from Staphylococcus aureus
  • AvaI -  from Anabaena variabilis

Source microorganism Enzyme Recognition Site Ends produced
Arthrobacter luteus Alu I AG?CT Blunt
Bacillus amyloiquefaciens H Bam HI G?GATCC Sticky
Escherichia coli Eco RI G?AATTC Sticky
Haemophilus gallinarum Hga I GACGC(N)5? Sticky
Haemophilus infulenzae Hind III A?AGCTT Sticky
Providencia stuartii 164 Pst I CTGCA?G Sticky
Nocardia otitiscaviaruns Not I GC?GGCCGC Sticky
Staphylococcus aureus 3A Sau 3A ?GATC Sticky
Serratia marcesans Sma I CCC?GGG Blunt
Thermus aquaticus Taq I T?CGA Sticky
Restriction enzymes recognize a specific short
nucleotide sequence
  • For example, EcoRI recognizes the sequence
  • 5- G/ A A T T C -3'
  • 3'- C T T A A /G -5'

Examples of restriction enzymes and the sequences
they cleave
  • Palindromes same base pairing forward and

Lets try some cutting
  • Using this piece of DNA, cut it with Eco RI

What results is

Sticky end - tails of DNA easily bind to other
DNA strands
Sticky end
Blunt Sticky ends
  • Sticky ends Creates an overhang. BamH1
  • Blunts- Enzymes that cut at precisely opposite
    sites without overhangs. SmaI is an example of an
    enzyme that generates blunt ends

IV. Gel Electrophoreisis
  • V. Analysis of DNA
  • DNA fingerprinting
  • Banding pattern of the fragments of cut DNA on a
    special gel medium (agarose)

Purpose for DNA fingerprinting
  • Comparing banding patterns to determine
    hereditary relationships between people
  • Comparing banding patterns of 2 different species
    to determine evolutionary relationship
  • Compare samples of blood or tissue for forensic
    purposes (who done it?)

  • Very accurate method of accessing DNA 99.99
  • Odds 1 in 1,000,000,000
  • Does not work with identical twins
  • Use strands of DNA that have a lot of VNTRs

How is it done?
  • VNTR analysis variable number tandem repeats -
    we each have non-coding segments on our DNA.
    Fragment lengths varies with each person
  • Extract DNA sample from blood or tissues
  • Cut DNA using restriction enzymes. Separate
    fragments by gel electrophoresis separates DNA
    fragments by the of base pairs (length of the
    fragment) and charge
  • Place DNA sample into wells in the agarose gel
    molecular sieve
  • Run a current through the gel. The DNA
    (negatively charged) will migrate from (-) to
    ()5.The larger fragments will not migrate that
    far. The small fragments will go the furthest
  • 5. Stain gel and bands in a dye or use a
    radioactive probe to analyze the banding

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Now that you have the desired gene or piece of
DNA, what do you do with it?
  • V. Recombinant DNA
  • Transfer of isolated gene to another organism
    with the purpose of having the organism transfer
    the gene to another.
  • Use bacterial plasmids

  1. The same restriction enzyme used to cut the
    desired gene, is used to splice the plasmid
  2. Donor gene (desired gene) is then spliced or
    annealed into the plasmid
  3. Plasmid is then returned to bacterium and
    reproduces with donor gene in it.
  4. Bacterium with donor gene can transfer donor
    genes to organisms it infects.

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How this works to help humans
  • Looking at the gene that produces insulin for the
    treatment of diabetes
  • Isolate insulin gene from a healthy human
  • Using a restriction enzyme, cut out insulin
    producing gene
  • Cut bacterial plasmids with same restriction
  • Introduce human insulin producing gene to
    bacterial plasmid
  • Bacterial plasmid takes up gene - recombinant DNA
  • Bacterial plasmid reproduces and starts
    expressing insulin produce gene
  • Insulin is produced and harvested from bacteria.

  • What has been produced is Recombinant DNA - DNA
    with genes from other organisms
  • Transgenic organisms have introduced DNA from
    another species in them

Practical Use of DNA technology
  • Pharmaceutical products insulin, HBCF (human
    blood clotting factor)
  • Genetically engineered vaccines to combat viral
    infections (pathogenic disease causing) your
    body recognizes foreign proteins, produces
    antibodies. Introduced viral proteins will
    trigger an immune response and the production of
  • Altering viral genomes makes them no longer
    pathogenic now a vaccine

  • Increasing agricultural yields
  • New strains of plants GMO Genetically
    Modified organism. Try this one!!
  • Insect resistant plants Insert gene that
    digests larvae when larvae try to eat the plant
    Not always specific to harmful species!!
    Monarch problem
  • Disease resistance Fungal resistance in
    tomatoes, corn, soybean
  • Herbicide resistance - Round Up wont harm the
    good plants, only the bad plants (weeds)
    cheaper and less labor extensive than weeding
  • Getting genes from Nitrogen fixing bacteria
    inserted into plants fix their own nitrogen (a
    must for plants) in N poor soils
  • Salt tolerant plants can grow plants where high
    concentrations of salt in the air or soil

  • Improve quality of produce
  • - Slow down the ripening process ship when
    unripened, to market when ripe
  • - Enhance color of produce
  • - Reduce hairs or fuzz on produce
  • - Increase flavor

Why GM Foods?
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Safety and Environmental Issues
  • All food products are regulated by the
  • Food and Drug Administration FDA
  • Natl Institutes of Health Recombinant DNA
    Advisory Committee and the Department of
    Agriculture (USDA)
  • Environmental Protection Agency (EPA)
  • All set standards for safety procedures and
    require permits and labeling (not in US though).
    Look for a 8 before the product code. 84011 GMO
  • Problem with transgenic foods is that an
    introduced gene may produce a protein that
    someone may be sensitive to. FDA does not require
    that on a label

Gene Therapy
  • Treatment of a genetic disorder (like cystic
    fibrous) by correcting a defective gene that
    causes a deficiency of an enzyme.
  • Nasal spray that carries normal enzyme gene. Body
    makes enzyme and patient breathes normally.
    Regular treatments necessary
  • Has not been proven to be successful in the long

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