DNA Technology

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DNA Technology

• and Biotechnology

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Biotechnology refers to the technology used to
manipulate DNA

• Procedures are often referred to as Genetic
  Engineering
• DNA is the genetic material of ALL living things
• All organisms use the same genetic code
• Genes from one organism can be transcribed and
  translated when put into another organism

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Examples...

• Human genes (and other animals) are routinely put
  into Bacteria in order to synthesize products for
  medical treatments and commercial use i.e. human
  insulin, human growth hormone, vaccines

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Recombined DNA

• Recombinant DNA refers to DNA from two
  different sources being combined
• Individuals receiving genes from other species
  are called Transgenic

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Vectors

• Vector anything used to transfer a gene into a
  host cell
• Plasmids and Viruses are the most common vectors
  used

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Whats the Difference?

• Virus (Protein coat DNA or RNA core) takes
  over the host cell machinery by incorporating
  itself into the host DNA to make more virus
  particles
• Plasmid a small ring of DNA, incorporates itself
  into the host DNA, cloned each time the host DNA
  is reproduced
• A vector must be capable of self replicating
  inside a cell!

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Tools of the Trade

• Restriction Enzymes discovered in 1970s in
  bacteria
• Used naturally in bacteria as a defense mechanism
  against viruses (bacteriophages)
• Cut DNA at specific base sequences
• Example EcoR1 always cuts DNA at GAATTC as
  shown

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Other restriction enzymes cut at different sites,
some examples are listed below

• Enzyme Cutting Sites
• Bam HI GGATCC
• Hae III GGCC
• Pst 1 CTGCAG
• Hind III AAGCTT

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Sticky Ends

• Sticky Ends Recombinant DNA Fragments of DNA
  that have been cut using restriction enzymes have
  unpaired nucleotides at the ends called Sticky
  Ends

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• Sticky ends have complementary bases , so they
  could rejoin
• If the vector and the gene to be cloned are both
  cut with the same restriction enzyme, they will
  both have complementary sticky ends
• After cutting, 2 DNA samples are mixed
• Result Recombinant DNA
• The enzyme DNA ligase seals the fragments
  together

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Genomic Libraries

• Genomic Libraries collection of all the genes
  that an organism possesses
• Bacteria or viruses can be used to store
  fragments of DNA from another species

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PCR

• Polymerase Chain Reaction (PCR) used to make
  numerous copies of small pieces of DNA
• Requires primers, DNA polymerase, nucleotides

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How Does PCR Work?

• 1. The DNA is heated to about 95O C to separate
  the 2 strands of the double helix
• 2. After the strands are separated, the DNA is
  cooled to about 50OC and the primers attach
• 3. Temp. is then raised to 70OC so the polymerase
  will attach and copy the strand

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• 4. The DNA Replication process repeats itself as
  the solution is heated and cooled at regular
  intervals

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DNA Fingerprints

• DNA Fingerprinting (RFLP Analysis) banding
  pattern produced on a gel representing the
  accumulation of DNA fragments of various sizes

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Gene Products and the Uses of Genetic Engineering

• E. coli used to produce insulin
• Mammalian cells used to produce proteins such as
  hormones
• Plant cells can be engineered with new properties
  (insect resistance)
• Bovine Growth Hormone (BGH) used to increase milk
  production in cows by about 10
• Animal viruses can be engineered to carry a gene
  for a pathogens surface protein so the virus can
  be used as a vaccine

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Gene Products and the Uses of Genetic Engineering

• Techniques were used to map the human genome
  through the Human Genome Project
• Could provide tools for diagnosis and possible
  repair of genetic diseases
• Gene therapy could be used to cure genetic
  diseases by replacing defective or missing genes

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Social and Ethical Issues OfGenetic Engineering

• Harmful organisms may be accidentally produced
• Organisms that are released into the environment
  may be engineered with genes that will eventually
  kill them
• There is currently little legislation on the use
  of genetic screening and the use of information
  produced by screening could result in
  discrimination

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Social and Ethical Issues OfGenetic Engineering

• This technology is increasing the ability to
  diagnose genetic diseases pre-natally, adding new
  complexity to the abortion controversy
• Ethical questions have been raised over whether
  we should modify the genes of humans
• Genetic screening and gene therapy are expensive
  and may be unavailable to the poor

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Social and Ethical Issues OfGenetic Engineering

• Biological weapons could be created using
  biotechnology
• The debate over stem cells would fall into this
  category as well as cloning limited legislation
  already exists

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DNA Fingerprinting

• Facts
• There are over 10 trillion cells in your body
• Every cell has the same DNA inside
• There are over 3 billion base pairs in your DNA
• Your DNA is spread over 46 chromosomes
• Your DNA is unique to you! (Unless you have an
  identical twin)

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How to make a DNA fingerprint

• 1. Pour restriction enzymes into DNA
• 2. Separate DNA fragments using an agarose gel
  (small pieces will move farther) using an
  electrical current
• 3. Add probes for certain DNA Markers
• 4. Create an x-ray film of your results called
  an Autoradiograph

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What do you end up with?

• A film with bands on it that represent the
  different fragment lengths of DNA.
• The bands show where the probes attached
  themselves to the DNA.

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What are DNA Fingerprints Used For?

• Criminal Cases (Murder, Rape, Etc.)
• Paternity cases (Who's yo daddy?)
• Free wrongly incarcerated inmates
• Create DNA Profiles
• Construct Genetic Databases (Convicted felons)

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History

• 1996 Dr. Ian Wilmut - first to successfully clone
  an adult mammal
• Dolly the sheep born 1996, died (euthanized)
  Feb. 14, 2003
• Named after Dolly Parton (donor cell was taken
  from an adult mammary gland)

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How the Process Works

• 1. Take a donor cell from an adult.
• 2. Starve the cell of nutrients and it will
  become dormant.
• 3. Remove an unfertilized egg cell from a
  female.
• 4. Replace the nucleus of the unfertilized egg
  cell with the nucleus of the adult donor cell.

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How the Process Works

• 5. Trick the egg cell into thinking that it
  was fertilized naturally (electric shock works
  well).
• 6. The egg will now grow and divide (mitosis) to
  form an embryo.
• 7. Implant the dividing egg cell into a
  surrogate mother.

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How the Process Works

• 8. Wait out the normal gestation period.
• 9. At the completion of the pregnancy, the
  organism that is born will have the same DNA as
  the original donor.

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Problems So Far / What weve Learned

• Dolly only lived 7 years, less than 1/2 the
  normal life expectancy
• Dolly developed early onset arthritis
• Successful cloning has a high failure rate
• Telomere found at the tips of chromosomes

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Significance of the Telomere

• It gets shorter every time the DNA replicates
  before Mitosis
• Acts as a Biological Clock
• When the telomere is gone the cell no longer
  divides
• An enzyme called telomerase is responsible for
  the eating away of the telomere

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Possible Medical Applications

• Organ and tissue transplants
• Taking a specialized cell and turning it into any
  tissue or organ you want
• Genetically resistant foods (Bananas)

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Stem Cells

• What are human embryonic stem cells?Stem cells
  are cells that have the remarkable potential to
  develop into many different cell types in the
  body.
• The two categories of stem cells include
  Embryonic and Adult Stem Cells and possibly a
  third, cord-blood-derived embryonic-like stem
  cells (CBEs).

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Stem Cell Treatment

• Medical researchers believe that stem cell
  research has the potential to change the face of
  human disease.
• Medical researchers anticipate being able to use
  technologies derived from stem cell research to
  treat Cancer, Parkinsons Disease, Spinal Cord
  Injuries, and muscle damage.

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The Controversy

• There exists a widespread controversy over stem
  cell research that emanates from the techniques
  used in the creation and usage of stem cells.
  Embryonic Stem Cell research is particularly
  controversial because, with the present state of
  technology, starting a Stem cell line requires
  the destruction of a human embryo and/or
  therapeutic cloning.

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