Genetic Engineering HS

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Genetic Engineering
Honors Biology
Vocabulary
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Genetic Engineering
Recombinant DNA
Transgenic Organisms
Cloning
Gene Cloning
Gene Therapy
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PCR
Gel Electrophoresis
DNA Fingerprinting
Gene Sequencing
Stem Cells
Cloning
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Reproductive Cloning
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Reproductive cloning is a technology used to generate an animal
that has the same nuclear DNA as another currently or previously
existing animal.
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Dolly – Died at age 6 suffering from lung cancer and crippling
arthritis (Dorset sheep normally live 11-12 years)
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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.
Cloning cont.
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Therapeutic Cloning
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Therapeutic cloning, also called "embryo cloning," is the production of
human embryos for use in research.
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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.
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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.
Recombinant DNA
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Def: DNA in which genes from 2
different sources are linked
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Genetic engineering: direct
manipulation of genes for
practical purposes
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Biotechnology: manipulation of
organisms or their components to
perform practical tasks or
provide useful products
Bacterial plasmids in gene cloning
Gene Cloning
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Restriction enzymes (endonucleases): in
nature, these enzymes protect bacteria from
intruding DNA; they cut up the DNA
(restriction); very specific
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Restriction site: recognition sequence for a
particular restriction enzyme
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Restriction fragments: segments of DNA cut
by restriction enzymes in a reproducable way
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Sticky end: short extensions of restriction
fragments
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DNA ligase: enzyme that can join the sticky
ends of DNA fragments
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Cloning vector: DNA molecule that can carry
foreign DNA into a cell and replicate there
(usually bacterial plasmids)
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http://www.sumanasinc.com/webcontent/anis
amples/molecularbiology/plasmidcloning_fl
a.html
DNA Analysis & Genomics
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PCR (polymerase chain
reaction)
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Gel electrophoresis
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DNA Fingerprint
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DNA sequencing
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Human genome project
Polymerase chain reaction (PCR)
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Amplification of any piece of DNA
without cells (in vitro)
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Materials: heat, DNA polymerase,
nucleotides, single-stranded DNA
primers
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Applications: fossils, forensics,
prenatal diagnosis, etc.
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http://www.sumanasinc.com/webcont
ent/anisamples/molecularbiology/pcr.
html
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http://biorad.cnpg.com/lsca/videos/ScientistsF
DNA Analysis
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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/majorsbiolog
y/gelelectrophoresis.html
Agarose gel electrophoresis
Negative end
(-)
Positive end
(+)
Larger DNA Fragments
Smaller DNA Fragments
After staining with ethidium bromide
to visualize DNA…
- pole
+ pole
Large DNA pieces
Small DNA pieces
DNA Sequencing
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Determination of nucleotide
sequences (Sanger method,
sequencing machine)
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Genomics: the study of
genomes based on DNA
sequences
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Human Genome Project
Practical DNA Technology Uses
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Diagnosis of disease
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Human gene therapy
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Pharmaceutical products (vaccines)
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Forensics
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Animal husbandry (transgenic
organisms)
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Genetic engineering in plants
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Ethical concerns?
What is the current status of gene
therapy research?
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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.
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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."
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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.
What factors have kept gene therapy from becoming an effective
treatment for genetic disease?
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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.
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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.
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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.
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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,
DNA Fingerprinting
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Involves several techniques to analyze and compare DNA
from separate sources
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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
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These DNA fingerprints can be compared to DNA found as
evidence, DNA of suspects and DNA of victims
The Basics of the Procedure
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DNA is extracted
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A chemical process called polymerase chain reaction (PCR) uses
enzymes to amplify the amount of DNA
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Sections of DNA where there are repeats are cut in order to
determine the number of repeats present
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The fragments are put on an electric field that sorts them by size
(gel electrophoresis)
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The fragments are then placed onto a nylon membrane where
they are treated with radioactive probes
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The probes stick to some DNA fragments but not to
others, due to complimentary base pairing
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A piece of X-ray film is put on top and a spot is produced
on the film where the probes stick
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Using a ruler, scientists measure the position of the spots
on the film and produce a set of numbers
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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.
VNTR Specifics
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In the US, 13 STR (single tandem repeats) loci have been selected for
forensic typing and inclusion in CODIS (Combined DNA Index System).
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The average random match probability when all 13 are typed is less than
1 in a trillion among unrelated individuals!!!
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Because each of the loci used in forensic DNA typing is on a different
chromosome, they are each inherited independently of one another.
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This allows forensic scientists to calculate the frequency of any given
DNA profile by multiplying each individual allele frequency together.
Downfalls of DNA
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DNA is useful, but there are some downfalls:
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Contamination of evidence is possible – can occur in
lab through the air
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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.)
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Testing not always conclusive – need to use more loci
when creating a DNA fingerprint.
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