Gene Technologies

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Gene Technologies
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Analyzing Genes

• Multiple technologies exist to analyze genes of
  individuals or even populations, including
• Gel electrophoresis
• DNA microarrays

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Gel Electrophoresis

• Electrophoresis can be used to analyze DNA, RNA,
  or proteins.
• The polarity of these molecules causes them to
  move a particular direction through a gel when an
  electrical current is passed through it.
• The size of the DNA, RNA, or protein fragments
  affects the rate of movement.

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The properties of DNA allow it to be extracted
easily from living tissue. DNA is soluble in
water, so cells are ground up in water first. DNA
is insoluble in alcohol, so a layer of alcohol
can be floated on top of the solution and DNA
precipitates out of solution at the water-alcohol
interface.
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The crude DNA extraction also contains fragments
of proteins and other molecules, so it has to be
purified. Once purified, it can be cut with
enzymes to remove just the segments of interest.
Then the DNA fragments are put in a PCR machine
with free nucleotides and DNA polymerase. Cycles
of heat (to unzip DNA but not denature the
enzyme) and cool (to allow DNA to zip together)
cause multiple copies of the DNA to be made.
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The purified and amplified DNA fragments are
loaded into a gel, usually made either of agar (a
carbohydrate) or polyacrylamide (a synthetic
molecule). The gel is covered with a buffer
solution and an electrical current is run through
the gel. The DNA fragments migrate down the gel,
which is later stained or put under UV light to
show DNA bands.
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DNA Microarrays

• Electrophoresis can usually only test a few genes
  or individuals at a time.
• New DNA microarrays (gene chips) can test for
  thousands of genes at once.

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Microarrays take advantage of the base-pairing
rule. Small DNA fragments can be manufactured to
match genes of interest and printed directly on a
silicon chip.
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DNA or RNA may extracted from the organism of
interest DNA to analyze all genes present, RNA
if the researcher is interested in what genes are
being expressed. The extracted material is tagged
with fluorescing dyes, loaded on the chip, and
allowed to hybridized with the probes. The chip
is scanned to see which spots on the array glow,
and in what color.
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The results may tell a researcher which genes are
being expressed in different cells. For example,
cancerous cells may be compared with noncancerous
cells to indentify which genes are on and which
are off, in search of which genes cause a cell
to turn cancerous.
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Changing Genes?

• Some gene technologies involve changing the genes
  of an individual.
• Were going to look at some of the science and
  consider some of the risks and benefits of these
  technologies.

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Harnessing Nature

• Gene-altering technologies often harness the
  ability of certain microbes to insert or alter
  genes of other organisms.

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Bacteria already do gene therapy on one another.
(b) Transformation with DNA fragment
(c) Transformation with plasmid
bacterial chromosome
bacterial chromosome
DNA fragments
plasmid
Plasmid replicates in cytoplasm.
DNA fragment is incorporated into chromosome.
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Viruses are expert at inserting their genes into
living hosts.
virus
viral DNA
2 Virus enters host cell.
host cell
3 Virus releases its DNA into host cell some
viral DNA (red) may be incorporated into the host
cells DNA (blue).
host cell DNA
1 Virus attaches to susceptible host cell.
viral DNA
Viral proteins
hybrid virus
6 Host cell bursts, releasing newly assembled
viruses. When hybrid viruses infect a second
cell, they may transfer genes from the first cell
to the second cell.
4 Viral genes encode synthesis of viral proteins
and viral gene replication. Some host cell
DNA may attach to replicated viral DNA (red/blue).
5 New viruses assemble host cell DNA is
carried by hybrid viruses.
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Gene Gun

• Another method of inserting genes into cells is
  the biolistic particle deliver system, or gene
  gun.
• The gene gun uses compressed air to shoot
  microscopic particles coated with DNA into cells.

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Gene gun technology
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Gene Therapy

• Gene therapy may provide ways to treat
  single-gene genetic disorders.
• Gene therapy takes advantage of viruses as
  vectors for inserting good genes into cells
  that have broken genes.

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• Ashanti deSilva was one of the first people to
  undergo gene therapy.
• Ashi was born with ADA deficiency. She is missing
  an enzyme critical for her immune system.

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• The enzyme was necessary for white blood cell
  function. Researchers used a virus that
  parasitizes white blood cells to insert a
  functional gene for the ADA enzyme
• The virus inserted the gene in Ashis white blood
  cells, where it successfully produced the ADA
  enzyme

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• White blood cells live a few months, so Ashi has
  to return for frequent treatments. If her bone
  marrow cells could be treated, she might be
  cured.
• With gene therapy, Ashi lives a healthy,
  productive life.

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• However, there are problems and ethics involved
  in gene therapy.
• The therapy is only useful for single-gene
  disorders where the affected tissue is local and
  easily reached.
• In one study on treating cystic fibrosis with
  gene therapy, a good gene was inserted into a
  cold virus. A volunteer who was dosed with the
  cold virus had a violent immune system reaction
  and died. The study was halted until a safe dose
  could be established.

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GMOs

• For thousands of years, farmers have been
  changing the genetics of their crops through
  selective breeding. In the 18th century, after
  the discovery of pollination, hybridization was
  added as a means of improving crops.
• Only recently have humans been able to change the
  genetics of crops and livestock by deliberately
  inserting new genes to make Genetically Modified
  Organisms (GMOs).

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DNA including Bt gene
Ti Plasmid
Cut both with the same restriction enzyme.
Genes are prepared for insertion into a DNA
plasmid from bacteria, which will be used to
insert the gene into a plant cell.
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Mix Bt gene and plasmid add DNA ligase to seal
DNA.
The enzyme ligase is used to seal the trans gene
into the bacterial plasmid.
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Transform Agrobacterium tumefaciens with
recombinant plasmid.
A. tumefaciens
bacterial chromosome
plasmids
Plasmids are applied to a culture of bacteria
that are known to infect plant cells.
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Infect plant cell with transgenic bacterium.
plant chromosomes
A. tumefaciens
plant cell
The bacteria attacks a plant cell and attempts to
insert its own DNA. It inserts the trans plasmid
instead.
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plant chromosomes
A. tumefaciens
plant cell
Insert Bt gene into plant chromosome.
Bt gene
If all goes well, the gene will be inserted into
the plants DNA and will be expressed in the
plant.
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Transgenic plant cells are treated with hormones
to grow new plants, and plants are tested for
expression of the new gene.
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• Successful crops that have been developed by
  genetic modification
• Herbicide-resistant crops
• Crops that produce substances toxic to insect
  pests but not to people
• Bananas that contain vaccine proteins, to
  vaccinate children in developing nations against
  cholera
• Goats have been developed that produce
  pharmaceutical proteins in their milk.

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GMOs and ethics

• Producing GMO crops is expensive, and companies
  expect return on their investment. Companies
  patent the genome of their crops to protect their
  investment.
• Consider the following worst case scenarios
  (based on actual events) and the ethical issues
  involved.

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• A farmer in Canada grows non-GMO corn. He saves
  seed from each crop and plants it the next year.
  Technicians from a large corporation remove corn
  from his land without his knowledge, test it, and
  find that his corn has cross-pollinated with
  their patented GMO corn, which another farmer
  several miles away is growing. The corporation
  sues the farmer for pirating their crop. The
  farmer also loses his organic status for
  producing corn that is genetically modified.

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• A large corporation develops a GMO rice that has
  more protein and vitamins than ordinary rice.
  They give some to rice farmers in India. These
  farmers normally save seeds each year and replant
  them. After their first harvest, the corporation
  tells the farmers that they must buy seeds next
  year, because harvested seeds will be sterile.
  The farmers protest. They say they cant make a
  living if they have to buy seeds each year, and
  they should have been told this to begin with.