Chapter 12 - Biotechnology

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22 Οκτ 2013 (πριν από 3 χρόνια και 9 μήνες)

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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


Enzyme

Cutting Site


Bam HI GGATCC


Hae III GGCC


Pst I CTGCAG


Hinf I GATC



Sticky Ends


BLUNT 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.

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

from the thermophile
Thermus aquaticus

is
used because this species thrives at
temperatures that are near boiling.

called Taq polymerase

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 …


Vaccines


Transgenic plants


Transgenic Animals


Cloning Mammals



Safety and Ethical Issues?