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Genetic Engineering

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Title: Genetic Engineering Author: profile Last modified by: Charles Forstbauer Created Date: 2/8/2010 12:20:43 PM Document presentation format: On-screen Show – PowerPoint PPT presentation

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Title: Genetic Engineering


1
Genetic Engineering
  • Honors Biology

2
Vocabulary
  • Genetic Engineering
  • Recombinant DNA
  • Transgenic Organisms
  • Cloning
  • Gene Cloning
  • Gene Therapy
  • PCR
  • Gel Electrophoresis
  • DNA Fingerprinting
  • Gene Sequencing
  • Stem Cells

3
Cloning
  • Reproductive Cloning
  • Reproductive cloning is a technology used to
    generate an animal that has the same nuclear DNA
    as another currently or previously existing
    animal.
  • Dolly Died at age 6 suffering from lung cancer
    and crippling arthritis (Dorset sheep normally
    live 11-12 years)
  • Dolly's success is truly remarkable because it
    proved that the genetic material from a
    specialized adult cell, such as an udder cell
    programmed to express only those genes needed by
    udder cells, could be reprogrammed to generate an
    entire new organism. Some scientists believe that
    errors or incompleteness in the reprogramming
    process cause the high rates of death, deformity,
    and disability observed among animal clones.

4
Cloning cont.
  • Therapeutic Cloning
  • Therapeutic cloning, also called "embryo
    cloning," is the production of human embryos for
    use in research.
  • The goal of this process is not to create cloned
    human beings, but rather to harvest stem cells
    that can be used to study human development and
    to treat disease.
  • In November 2001, scientists from Advanced Cell
    Technologies (ACT), a biotechnology company in
    Massachusetts, announced that they had cloned the
    first human embryos for the purpose of advancing
    therapeutic research. To do this, they collected
    eggs from women's ovaries and then removed the
    genetic material from these eggs. A skin cell was
    inserted inside the enucleated egg to serve as a
    new nucleus. The egg began to divide after it was
    stimulated with a chemical. The results were
    limited in success. Although this process was
    carried out with eight eggs, only three began
    dividing, and only one was able to divide into
    six cells before stopping.

5
Recombinant DNA
  • Def DNA in which genes from 2 different sources
    are linked
  • Genetic engineering direct manipulation of
    genes for practical purposes
  • Biotechnology manipulation of organisms or their
    components to perform practical tasks or provide
    useful products

6
Bacterial plasmids in gene cloning
7
Gene Cloning
  • Restriction enzymes (endonucleases) in nature,
    these enzymes protect bacteria from intruding
    DNA they cut up the DNA (restriction) very
    specific
  • Restriction site recognition sequence for a
    particular restriction enzyme
  • Restriction fragments segments of DNA cut by
    restriction enzymes in a reproducable way
  • Sticky end short extensions of restriction
    fragments
  • DNA ligase enzyme that can join the sticky ends
    of DNA fragments
  • Cloning vector DNA molecule that can carry
    foreign DNA into a cell and replicate there
    (usually bacterial plasmids)

8
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9
  • http//www.sumanasinc.com/webcontent/anisamples/mo
    lecularbiology/plasmidcloning_fla.html

10
DNA Analysis Genomics
  • PCR (polymerase chain reaction)
  • Gel electrophoresis
  • DNA Fingerprint
  • DNA sequencing
  • Human genome project

11
Polymerase chain reaction (PCR)
  • Amplification of any piece of DNA without cells
    (in vitro)
  • Materials heat, DNA polymerase, nucleotides,
    single-stranded DNA primers
  • Applications fossils, forensics, prenatal
    diagnosis, etc.
  • http//www.sumanasinc.com/webcontent/anisamples/mo
    lecularbiology/pcr.html
  • http//bio-rad.cnpg.com/lsca/videos/ScientistsForB
    etterPCR/

12
DNA Analysis
  • Gel electrophoresis separates nucleic acids or
    proteins on the basis of size or electrical
    charge creating DNA bands of the same length

http//www.sumanasinc.com/webcontent/anisamples/ma
jorsbiology/gelelectrophoresis.html
13
Agarose gel electrophoresis
14
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15
Negative end (-)
Positive end ()
Larger DNA Fragments
Smaller DNA Fragments
16
After staining with ethidium bromide to visualize
DNA
Large DNA pieces
- pole
Small DNA pieces
pole
17
DNA Sequencing
  • Determination of nucleotide sequences (Sanger
    method, sequencing machine)
  • Genomics the study of genomes based on DNA
    sequences
  • Human Genome Project

18
Practical DNA Technology Uses
  • Diagnosis of disease
  • Human gene therapy
  • Pharmaceutical products (vaccines)
  • Forensics
  • Animal husbandry (transgenic organisms)
  • Genetic engineering in plants
  • Ethical concerns?

19
What is the current status of gene therapy
research?
  • The Food and Drug Administration (FDA) has not
    yet approved any human gene therapy product for
    sale. Current gene therapy is experimental and
    has not proven very successful in clinical
    trials. Little progress has been made since the
    first gene therapy clinical trial began in 1990.
    In 1999, gene therapy suffered a major setback
    with the death of 18-year-old Jesse Gelsinger.
    Jesse was participating in a gene therapy trial
    for ornithine transcarboxylase deficiency (OTCD).
    He died from multiple organ failures 4 days after
    starting the treatment. His death is believed to
    have been triggered by a severe immune response
    to the adenovirus carrier.
  • Another major blow came in January 2003, when the
    FDA placed a temporary halt on all gene therapy
    trials using retroviral vectors in blood stem
    cells. FDA took this action after it learned that
    a second child treated in a French gene therapy
    trial had developed a leukemia-like condition.
    Both this child and another who had developed a
    similar condition in August 2002 had been
    successfully treated by gene therapy for X-linked
    severe combined immunodeficiency disease
    (X-SCID), also known as "bubble baby syndrome."
  • FDA's Biological Response Modifiers Advisory
    Committee (BRMAC) met at the end of February 2003
    to discuss possible measures that could allow a
    number of retroviral gene therapy trials for
    treatment of life-threatening diseases to proceed
    with appropriate safeguards. In April of 2003 the
    FDA eased the ban on gene therapy trials using
    retroviral vectors in blood stem cells.

20
What factors have kept gene therapy from becoming
an effective treatment for genetic disease?
  • Short-lived nature of gene therapy - Before gene
    therapy can become a permanent cure for any
    condition, the therapeutic DNA introduced into
    target cells must remain functional and the cells
    containing the therapeutic DNA must be long-lived
    and stable. Problems with integrating therapeutic
    DNA into the genome and the rapidly dividing
    nature of many cells prevent gene therapy from
    achieving any long-term benefits. Patients will
    have to undergo multiple rounds of gene
    therapy.
  • Immune response - Anytime a foreign object is
    introduced into human tissues, the immune system
    is designed to attack the invader. The risk of
    stimulating the immune system in a way that
    reduces gene therapy effectiveness is always a
    potential risk. Furthermore, the immune system's
    enhanced response to invaders it has seen before
    makes it difficult for gene therapy to be
    repeated in patients.
  • Problems with viral vectors - Viruses, while the
    carrier of choice in most gene therapy studies,
    present a variety of potential problems to the
    patient --toxicity, immune and inflammatory
    responses, and gene control and targeting issues.
    In addition, there is always the fear that the
    viral vector, once inside the patient, may
    recover its ability to cause disease.
  • Multigene disorders - Conditions or disorders
    that arise from mutations in a single gene are
    the best candidates for gene therapy.
    Unfortunately, some the most commonly occurring
    disorders, such as heart disease, high blood
    pressure, Alzheimer's disease, arthritis, and
    diabetes, are caused by the combined effects of
    variations in many genes. Multigene or
    multifactorial disorders such as these would be
    especially difficult to treat effectively using
    gene therapy. For more information on different
    types of genetic disease,

21
DNA Fingerprinting
  • Involves several techniques to analyze and
    compare DNA from separate sources
  • DNA is taken from one or more of these sources,
    chemically cut into segments and the segments are
    sorted by length in a gel using electrophoresis
  • Segments contain specific sequences of repeating
    base pairs that are variable from person to
    person (VNTR)
  • Segments are tagged radioactively and form a
    visual pattern on X-ray film
  • These DNA fingerprints can be compared to DNA
    found as evidence, DNA of suspects and DNA of
    victims

22
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23
The Basics of the Procedure
  • DNA is extracted
  • A chemical process called polymerase chain
    reaction (PCR) uses enzymes to amplify the amount
    of DNA
  • Sections of DNA where there are repeats are cut
    in order to determine the number of repeats
    present
  • The fragments are put on an electric field that
    sorts them by size (gel electrophoresis)
  • The fragments are then placed onto a nylon
    membrane where they are treated with radioactive
    probes

24
  • The probes stick to some DNA fragments but not to
    others, due to complimentary base pairing
  • A piece of X-ray film is put on top and a spot is
    produced on the film where the probes stick
  • Using a ruler, scientists measure the position of
    the spots on the film and produce a set of
    numbers
  • The odds of two individuals having the same
    pattern are any where from thousands to one to
    trillions to one, depending on what type of
    analysis is used.

25
VNTR Specifics
  • In the US, 13 STR (single tandem repeats) loci
    have been selected for forensic typing and
    inclusion in CODIS (Combined DNA Index System).
  • The average random match probability when all 13
    are typed is less than 1 in a trillion among
    unrelated individuals!!!
  • Because each of the loci used in forensic DNA
    typing is on a different chromosome, they are
    each inherited independently of one another.
  • This allows forensic scientists to calculate the
    frequency of any given DNA profile by multiplying
    each individual allele frequency together.

26
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27
Downfalls of DNA
  • DNA is useful, but there are some downfalls
  • Contamination of evidence is possible can occur
    in lab through the air
  • Transfer of Evidence from pieces of evidence is
    also possible because uneducated crime scene
    processors often mix pieces of evidence that may
    contain DNA together in the same package (coffee
    cups, doorknobs, telephones, etc.)
  • Testing not always conclusive need to use more
    loci when creating a DNA fingerprint.
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