Download Is the process of manipulating genes and genomes Biotechnology

Genetic Engineering
-Is the process of manipulating genes and
genomes
Biotechnology
-Is the process of manipulating organisms or
their components for the purpose of making
useful products
Restriction Enzymes
-Are used to cut strands of DNA at specific
locations (called restriction sites). They are
derived from bacteria
-When a DNA molecule is cut by restriction
enzymes, the result will always be a set of
restriction fragments which will have at least one
single-stranded end, called a sticky-end
-Sticky ends can form hydrogen bonds with
complementary single-stranded-pieces of DNA
these unions can be sealed with the enzyme DNA
ligase
-Is DNA that has been artificially made, using
DNA from different sources and often different
species. An example is the introduction of a
human gene into an E coli bacterium
Recombinant DNA
Gene Cloning
-Is the process by which scientists can produce
multiple copies of specific segments of DNA they
can then work with in the lab
Plasmid
-Extra small circular double stranded pieces of
FNA found in viral, yeast, or bacterial cells used
extensively in Biotechnology and Recombinant
DNA
-Serve as a “vehicle” for transporting genes from
one organism into another (transformation) so
multiple copies can be made (gene cloning)
-Gene cloning yields multiple copies of a gene or
other DNA segment
-The cloning of genes generally occurs in five
steps
-Identify and isolate the gene of interest and a
cloning vector. The vector is the plasmid (usually
bacterial) that will carry the DNA sequence to be
cloned
Step One of Gene Cloning
Step Two of Gene Cloning
-Cut both the gene of interest and the vector
with the same restriction enzyme. This gives the
plasmid and the human gene matching sticky
ends
Step Three of Gene Cloning
-Join the two pieces of DNA from recombinant
plasmids by mixing the plasmids with the DNA
fragments. The human DNA fragments can be
sealed into the plasmid using DNA ligase
Step Four of Gene Cloning
-Get the plasmid vector carrying the gene of
interest into a host cell. The plasmids are taken
up by bacterium by transformation
Step Five of Gene Cloning
-Select for cells that have been transformed. The
bacterial cells carrying the clones must be
identified or selected
-This can be done by linking the gene to an
antibiotic resistance gene or a reporter gene
such as green fluorescent protein.
-In the transformation lab, we will use an
ampicillin-resistant plasmid. Any bacterial cells
that did not pick up the plasmid by
transformation will be killed when grown on agar
with the antibiotic ampicillin
-Insulin Production
-Human Growth Hormone Production
-Production of other proteins that make crops
resistant to pests
Application of Gene Cloning
Comments about Recombinant Plasmid
Another Biotechnology Tool We Use is PCR
-Gene can’t be above certain size or a plasmid
won’t work
-mRNA must not need splicing to remove introns
because we are placing it directly into a cell to be
expressed (bacteria can’t remove introns)
-But if we still want a bacterial cell to make a
human protein that does need mRNA processing,
we can make cDNA copy of the gene and insert
that instead
-cDNA is created using reverse transcriptase to
turn a processed mRNA coding for a certain
protein back into a DNA to insert into the
bacterial plasmid
-PCR (polymerase chain rxn) is a method used to
greatly amplify a particular piece of DNA without
the use of cell
-PCR is used to amplify DNA when the source is
impure or scanty (as it would be at a crime
scene)
-DNA technology allows us to study the
sequence, expression, and function of a gene
Gel Electrophoresis
-Is a lab technique that is used to separate
macromolecules, primarily DNA and proteins, on
the basis of their size and charge with the use of
an electrical current
-In separating DNA, the negative charges on
phosphates in the molecule cause DNA to move
more toward the positive pole
-The gel allows smaller molecules to move more
easily than larger fragments of DNA. The DNA
fragments are separated by size
Forensic Applications
-Links suspect bodily to the crime scene, but
doesn’t prove they committed the crime
-Results take much longer then on TV shows
-Analysis of old evidence is reversing some
sentences
Cloning organisms may lead to production of
stem cells for research and other applications
Stem Cells
Practical Application of DNA Technology:
Diagnosis of Disease
Practical Application of DNA Technology: Gene
Therapy
-In animal cloning the nucleus of an egg is
removed and replaced with the diploid nucleus
of a body cell, a process termed nuclear
transplantation
-The ability of a body cell to successfully form a
clone decreases with embryonic development
and cell differentiation
-The major goal of most animal cloning is
reproduction, but not for humans
-In humans, the major goal is the production of
stem cells
-Can both reproduce itself indefinitely and under
the proper conditions, produce other specialized
cells. Stem cells have enormous potential for
medical applications
-If the sequence of a particular virus’s DNA or
RNA is known, PCR can be used to amplify
patient’s blood samples to detect even small
traces of the virus
-Different alleles have different DNA sequences
--These different sequences can be found using
restriction enzymes that yield different lengths
of DNA fragments, or restriction fragment length
polymorphisms (RFLP)
-The difference in banding patterns after
electrophoresis allows for diagnosis of the
disease, or even a carrier of the disease
-The alteration of an afflicted individual’s genes.
Gene therapy holds a great potential for treating
disorders traceable to a single defective gene,
such a cystic fibrosis
Practical Application of DNA Technology: The
Production of Pharmaceuticals
-Gene splicing and cloning can be used to
produce large amounts of particular proteins in
the lab
Practical Application of DNA Technology:
Forensic Applications
-DNA samples taken from the blood, skin cells, or
hair of alleged criminal suspects cen be amplified
with PCR and compared to DNA collected from
the crime scene DNA fingerprints
(electrophoretic bands that are unique to each
individual) can be compared and used to identify
persons at that crime scene
Practical Application of DNA Technology:
Environmental Cleanup
-Scientists engineer metabolic capabilities into
microorganisms, which are then used to treat
environmental problems, such as removing
heavy metals from toxic mining sites or
engineering bacteria to digest oil
Practical Application of DNA Technology:
Agricultural Applications
-Certain genes that produce desirable traits have
been inserted into crop plants to increase their
productivity and efficiency. We have genetically
engineered rice to contain vitamin A, saving
about 60,000 children in Asia per year from
death due to this deficiency
-An organism that has acquired by artificial
means one or more genes from another species
or variety is termed a genetically modified
organism (GM organism)