Download Chapter 12 - Biotechnology

Biotechnology
What Is Biotechnology?
• Biotechnology refers to technology used to manipulate DNA. The
procedures are often referred to as genetic engineering.
• DNA is the genetic material of all living organisms and all organisms
use the same genetic code. Genes from one kind of organism can
be transcribed and translated when put into another kind of
organism.
• For example, human and other genes are routinely put into bacteria
in order to synthesize products for medical treatment and
commercial use. Human insulin, human growth hormone, and
vaccines are produced by bacteria.
• Recombinant DNA refers to DNA from two different
sources. Individuals that receive genes from other species are
transgenic.
Tools of the Trade …
• Viruses
Structure
Reproduction
viral DNA  mRNA  protein
viral RNA  cDNA  mRNA  protein
Recombinant DNA Technology
Vectors
• Vectors are DNA used to transfer genes into a
host cell.
• A vector must be capable of self-replicating
inside a cell.
• Marker genes can be used to determine if the
gene has been taken up.
SEE AMPICILLIN
Recombinant DNA Technology
Plasmids
• The host bacterium takes up the plasmid,
which includes the foreign gene.
• When the bacteria reproduces, the plasmids
are also reproduced. The gene is cloned.
• Shuttle vectors are plasmids that are capable
of existing in several different species. They
are useful when transferring genes to
multicellular organisms.
Recombinant DNA Technology
Viruses
• Viruses are the vectors of choice for animal
cells.
• They can accept larger amounts of DNA than
plasmids.
• When the virus reproduces within the animal
cell, it also reproduces the foreign gene that it
carries. The gene is therefore cloned.
• The DNA of some retroviruses becomes
integrated into the host chromosome.
Recombinant DNA Technology
Restriction enzymes
• Restriction enzymes were discovered in bacteria. Bacteria use them as a
defense mechanism to cut up the DNA of viruses or other bacteria.
• Hundreds of different restriction enzymes have been isolated. Each one
cuts DNA at a specific base sequence. For example, EcoRI always cuts DNA
at GAATTC as indicated below.
Other examples
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Enzyme Cutting Site
Bam HI GGATCC
Hae III
GGCC
Pst I
CTGCAG
Hinf I
GATC
Sticky Ends
If the vector and the gene to be cloned are both cut with the same
restriction enzyme, they will both have complimentary sticky ends.
BLUNT ENDS??
Making Recombinant DNA
• COMPLETE THE RECOMBINANT DNA ACTIVITY
Genomic Libraries
• A genome is all of the genes in a particular
organism. Bacteria or virus vectors can be
used to store fragments of the DNA from a
species.
• The DNA is cut up into fragments and the
different fragments are inserted into bacteria
or viruses. The collection of bacteria or viruses
is called a genomic library.
Other Methods of Delivering DNA
• Electroporation involves using an electric current to create
pores in the cell wall and plasma membrane for DNA to enter.
• It is difficult to create transgenic plants because the cell wall
prevents entry of DNA. One solution is to remove the cell wall.
These cells (called protoplasts) are then placed in a liquid with
foreign DNA. Electroporation is a technique that uses an
electric current to make small, temporary holes in the
membrane DNA can pass in.
• A gene gun propels small gold pellets coated with DNA. The
pellets penetrate the cell wall and plasma membrane and
enter the cell to deliver their DNA.
Polymerase Chain Reaction (PCR)
• The polymerase chain reaction can be used to
make many copies of small pieces of DNA.
Because techniques in biotechnology usually
require many copies of genes, PCR has
allowed much of the biotechnology
development that we have seen in recent
years.
Materials and Procedure
short chains of about 20 nucleotides
Complementary to DNA Gene
called Taq polymerase
from the thermophile Thermus aquaticus is
used because this species thrives at
temperatures that are near boiling.
Procedure
• 1) The DNA in question is heated to approximately 95
degrees C to separate the two strands of the double helix.
• 2) After the strands are separated, the DNA is cooled to about
50 degrees and the primers attach.
• The temperature is raised to approximately 70 degrees C. This
is the optimal temperature for Taq polymerase. The
polymerase attaches to and copies the strand.
The solution is then heated and cooled at regular intervals. Each
time it is heated and cooled, the DNA replication process repeats
itself.
DNA Fingerprinting (RFLP Analysis)
• In RFLP analysis, the DNA of an organism is cut up
into fragments using restriction enzymes. A large
number of short fragments of DNA will be produced.
• Electrophoresis is a technique used to separate the
DNA fragments according to their size. They are
placed on a sheet of gelatin and an electric current is
applied to the sheet. DNA is charged and will move in
an electric field toward the positive pole.
GEL-E LABS + RFLP ACTIVITIE
The smallest fragments will move the fastest because they are able
to move through the pores in the gelatin faster.
Bands will be produced on the gelatin where the fragments
accumulate.
The shortest fragments will accumulate near one end of the gelatin
and the longer,
slower-moving ones will remain near the other end.
CONT’D
The DNA bands must be stained to make them visible. Ethidium bromide-stained DNA
will fluoresce when illuminated with UV light.
PCR techniques are used to produce sufficient quantities of DNA for this technique.
Uses for Electrophoresis
• identification of diseased genes including
oncogenes,
• identification of viral infections,
• determining family relationships among
individuals,
• and identifying tissue found at a crime scene.
• Taxonomists can use this technique to explore
evolutionary relationships.
Gene Therapy
• Gene therapy uses technology to change the genetic composition of a cell.
• Ex vivo
• Ex vivo methods are done outside the organism. Cells
are removed, treated and returned to the individual.
• In vivo
• In vivo gene therapy treats cells in the individual
without removing them.
• Retroviruses can be used to introduce genes directly
into the body.
Example of ex vivo gene therapy
• This procedure has been used to treat severe combined
immunodeficiency syndrome (SCID). People with this disease
are susceptible to infections because their white blood cells
do not produce an enzyme needed by their immune systems.
This disease has been treated in two different ways. In a
short-term solution, the white blood cells were removed and
infected with a retrovirus that carried the needed gene. After
the cells were replaced, many of the cells contained the gene.
White blood cells, however, are short-lived and a long-term
solution is to apply this technique to the cells that produce
the white blood cells (called stem cells).
Products Made Using
Biotechnology
• Genes that code for the desired protein can be inserted into
plasmids and the plasmids are used to transform cells. Many
useful human proteins are now synthesized by transgenic
bacteria. Some of these are listed below.
• Human growth hormone is used to treat dwarfism. It
previously took the pituitary glands from over 50 cadavers to
make one dose.
• Human Insulin is used to treat diabetes. Insulin was
previously obtained from the pancreas of slaughtered cattle
and pigs. It sometimes caused allergic reactions.
CONT’D
• Tissue plasminogen activator dissolves blood clots in heart
attack victims.
• Clotting factor VIII will soon be available. Most cases of
hemophilia are due to the absence of this factor.
• Human lung surfactant is used in premature infants with
respiratory distress syndrome.
• Atrial natriuretic hormone can be used to treat hypertension.
• Bovine growth hormone (bGH) increases milk production in
cows by about 10%.
BESIDES BACTERIA …
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Vaccines
Transgenic plants
Transgenic Animals
Cloning Mammals
Safety and Ethical Issues?