Genetic Technologies

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Genetic Technologies

• New applications, new ethical issues

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DNA Fingerprinting
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DNA Fingerprinting

• Used in a wide range of areas, from forensics to
  medicine to taxonomy, to analyze DNA.
• Researchers pick out areas of interest in DNA,
  and often use junk DNA because it tends to have
  more mutations than genes, so has greater
  differences from person to person.
• DNA fingerprinting can also be used to analyze
  genes to determine a persons genotype for a
  known genetic disorder.

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• Researchers have identified short tandem repeats
  (STRs) that vary widely between individuals. DNA
  fingerprinting usually focuses on these STRs.

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The samples are then loaded into a gel (usually
agarose or polyacrylamide)
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Different fragments have different molecular
sizes, so move at different rates through the
gel. Because they are polar molecules, they move
in response to the electrical field across the
gel.
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At this point, the gel could be stained,
photographed, and discarded. For finer analysis,
such as locating specific segments of interest
the gel may be placed on a nylon fiber pad, and
the DNA fragments driven into the pad using a
downward-directed electrical current.
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Labeled DNA probes designed to bind to segments
of interest are loaded onto the pad. These may be
tagged with radioactive phosphorous or
fluorescent dye.
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Probes stick to the segments of interest, such as
known STRs or specific forms of a disease-causing
allele.
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(Why do some of these people have one band, while
others have two?)
The resulting DNA fingerprint can then be
analyzed by the experts. The segment above, for
example, shows DNA from the same region for 13
different people. It could be used to determine
who was at a crime scene, or identify a childs
real parents.
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Microarrays
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Microarrays

• DNA fingerprinting is useful for testing a few
  genes or loci at a time.
• Microarrays, however, can analyze thousands of
  genes, proteins, or other molecules all at once.
• Microarrays are used to determine which genes in
  a cell are being expressed, and to analyze
  gene-gene interactions.

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Microarrays

• Microarrays are arrays of tens of thousands of
  artificial DNA probes arranged on a small glass
  plate.
• Each probe can be designed to bind to a
  particular segment of interest on a particular
  gene or DNA locus.

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2. mRNA is extracted from the subject. (Why
mRNA?)
1. The microarray is designed with probes that
will bind to specific loci.
3. mRNA is used to synthesize cDNA, which is
tagged with fluorescent dyes.
4. The cDNA is applied to the microarray and
allowed to hybridize with the probes.
6. A machine scans the microarray with red and
green laser light, making the probes fluoresce,
and photographs the results.
5. The microarray is washed to remove cDNA that
did not stick.
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The results are analyzed to determine which genes
are being expressed in specific cells, and which
are turned off. The differing brightness of the
fluorescence also tells researchers how much mRNA
is being generated by each of the genes that is
active. Researchers might, for example, compare
gene expression in normal cells and cancerous
cells to see which genes are active in cancer
cells that are not in normal cells, and which
genes are supressed in cancer cells.
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Gene Therapy
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• Ashanti deSilva was one of the first patients to
  undergo gene therapy.
• Ashi was born with ADA deficiency, a genetic
  condition in which she is missing an enzyme
  critical for the immune system.
• Because this is a single-gene trait, it was a
  good candidate for gene therapy.

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• The enzyme was missing from Ashis white blood
  cells. Doctors inserted a good copy of the ADA
  gene into a virus known to parasitize white blood
  cells.
• The virus successfully inserted the gene into the
  cells, where it began producing the enzyme.

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• However, white blood cells only live a few
  months. Ashi had to return several times a year
  for a new treatment.
• The goal for ADA therapy is to treat the bone
  marrow stem cells that give rise to all blood
  cells.

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• In spite of having to return for frequent
  treatments, Ashi today lives a healthy,
  productive life. Without gene therapy, she would
  have no immune system and might have died of what
  would be a minor illness for anyone else.

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Genetically Modified Organisms
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Recombinant DNA

• If gene therapy can fix genes in people, why
  not insert helpful genes into organisms?
• Recombinant DNA technology allows researchers to
  take a gene from one organism and insert it into
  another. This has been done most successfully
  with plants to give them resistance to disease,
  pests, or herbicides.

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• Recombinant DNA is also used in bio-pharming,
  in which genes for medically therapeutic proteins
  are inserted into plants or into milk-producing
  animals. The proteins can then be purified from
  the plant tissue or milk for use in medical
  treatments. Bio-pharming may also produce fruits
  that produce proteins found in specific vaccines,
  making edible vaccines that could be grown in
  third-world countries.
• However, altering the genes of organisms,
  especially those used for foods, remains highly
  controversial.

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• Creating a transgenic organism begins with
  locating the desired gene and creating a
  trans-gene. The DNA segment includes the desired
  gene and may include some marker gene that will
  be expressed in the phenotype, showing the gene
  has been incorporated.
• Plant cells can be grown in Petri dishes, and the
  cells treated with the gene.
• The trans gene may be inserted by a so-called
  Gene Gun that shoots small gold pellets, coated
  with the genes, into the cells.
• Virus vectors may be used, since they already
  have the machinery to insert genes into a cell.
• Bacteria that attack plants are also used.

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Here, a gene is prepared for insertion into a DNA
plasmid from a bacteria, which will be used to
insert the gene into a plant cell.
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The enzyme ligase seals the ends of the trans
gene into the bacterial plasmid.
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Plasmids are applied to a culture of bacteria
that are known to infect plant cells.
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This particular bacteria attacks by inserting
plasmids into the plant host cell. Now it inserts
the plasmid containing the trans gene.
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If all goes well, some of the cells will
incorporate the trans gene into their own DNA,
where it will be expressed.
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The transgenic cells are treated with plant
hormones to grow new plants, and the plants are
tested for the expression of the gene.
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Cloning
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Twins out of time

• Lots of myths exist about cloning. Clones are
  not
• Mindless zombies slaves raised for organ harvest
  later.
• Instant identical copies of yourself with all
  your memories.
• Clones made by nuclear transfer are genetically
  identical to the cell donor. They are the donors
  twin, delayed by time.

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Natural Clones

• Identical twins are natural clones, created by
  the complete division of a fertilized egg.
• Plants clone themselves when they produce shoots,
  runners, or other structures that take root and
  live independently.
• Some simple animals clone themselves by budding,
  a form of asexual reproduction.

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Hello, Dolly

• Dolly the sheep was the first mammal produced by
  nuclear transfer cloning.
• This process involves removing an intactcell of
  an adult and inserting it into an egg cell from
  which the nucleus has been removed.
• If the egg can be stimulated to divide, it will
  grow into a normal embryo that can be implanted
  into a host animals uterus.

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Dolly began life as a single cell from one breed
of sheep, a white-faced Finn Dorset.
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An egg of a Scottish Blackface ewe was harvested
and its nucleus removed.
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The two cells were stimulated with an electrical
pulse to unite. This also stimulated mitosis. The
egg cell carried on with multiple cell divisions
as though it had been fertilized.
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The embryo was implanted into the uterus of a
Blackface ewe. Some months later, she gave birth
to the white-faced lab, Dolly.
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• Cloning as a way to produce livestock is
  impractical. Its far more expensive than
  natures way.
• However, owners of expensive and valuable
  animals, such a race horses, are interested in
  the technique, which raises a whole new set of
  ethical questions.
• One problem cells seem to know how old they are.
  Animals born from cloned cells are born with aged
  cells and dont live as long.

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• Many other genetic technologies exist, and new
  technologies will arise in the future.
• All genetic technologies raise ethical concerns
  about the organisms involved and their use.