The Genetic Revolution
The Brave New World
Or not?
I. Introduction-Biotechnology
A. in general biotechnology
is the use or manipulation of
organisms to make useful
products
• 1. using yeast to have bread
rise
• 2 .using microbes to make
wine and cheese
• 3 selective breeding of dairy
cattle to produce larger
quantities of milk
B. Genetic engineering
• 1. a subcategory of
biotechnology
• 2. the direct manipulation of
genetic material for practical
purposes
• 3. a growingly important tool
in the field of genetic
engineering is recombinant
DNA
• 4. in recombinant DNA,
different sources of DNA can
be joined together to transfer
traits or qualities between
organisms
5. Recombinant DNA
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a.
allows scientists to produce
products in larger quantities than
were possible before
b.
since the chemistry of
inheritance is virtually the same in
all organisms studied
c.
human genes can be moved
to the bacterial world to produce
large quantities of desired product
d.
diabetics in the past had to
use bovine insulin to help control
their blood sugar levels
e.
this was a problem as the
patient could develop antibodies
against the bovine insulin
f.
not exactly a human proteinsome differences that would make
it less efficient
5. Human Growth Hormone
• a.
• b.
• c.
• d.
who was just arrested
in Australia
can be administered to
children showing
abnormally low growth
stimulate to reach
more normal size
used to be harvested
from human cadavers
• e. can see the potential for
abuse when these chemicals
become more available at a
lower cost
6. Other uses
• a. genes for resistance to
certain pathogens can be
moved from one plant variety
to another
• b. bacteria can be
genetically modified to perform
in ways that they never did
before-bacterial cleanup of oil
spills
• c. the transfer of nitrogen
fixation capabilities in plants
II. The tools of recombinant DNA
•
A.
Vectorsmechanisms to carry
genetic material from one
organism to another
1. Plasmids
• a. small circular pieces of DNA
found in bacterial cells that carry
extrachromosomal genes
• b. plasmids are circular, doublestranded DNA molecules capable of
autonomous replication within living
cells.
• c. although not essential for the
survival of their host, they may
encode a wide variety of genetic
determinants that increase survival in
adverse environmental conditions
• d. can be used to move genes from
one cell to another by transformation
2. electroporation
3. Viral vectors
B. Molecular Scissors-endonucleases
• 1. restriction endonucleases
are naturally occurring
enzymes found in bacterial
cells
• 2. they are a defense
against viral infection-if a
bacterium is attacked by a
virus-the bacterium can digest
the viral genetic material
3. Many different restriction enzymes
• a. the restriction enzymes
often cut at palindromic sites
• b. these are sequences that
read the same in the forward as
well as in the reverse direction
• c. the restriction enzymes that
cut in a staggered fashion are
most useful for recombinant
DNA
• d. they produce sticky ends
that can complimentary base
pair across species lines
e. importance of sticky ends
C. Molecular glue
• 1. this ability is resident in a molecule that we
have already talked about
• 2. ligase joined together the Okazaki fragments
of DNA replication
• 3. can be used to join the DNA from foreign
sources to form a chimera
III. Cloning
A. Notice characteristics of plasmid
• 1. amp R gene
• 2. lac Z gene
B. Isolate plasmid and gene of interest using the
same restriction enzyme
• 1. restriction sites widespread
• 2. do not recognize gene limits
• 3. indiscriminate cutters
C. Join with ligase
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1.
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3.
4.
again problems
plasmid can bind with plasmid
reclose with nothing added
two pieces of DNA can bind
D. Transform competent bacteria
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1. definition of transformation
2. competent means treated
Calcium chloride
3. bacteria are lac Z deficientcannot metabolize disaccharide
4. bacteria are not resistant to
ampicillin
5. Not all bacteria are
transformed
6. Not all bacteria transformed
have engineered plasmid
E. Cloning of cells and duplication of foreign
genes
• 1. Dilution of transformed cells-insures that each colony
is product of one original cell
• 2. plated onto growth media containing ampicilin and Xgal
• 3. ampicillin will kill bacteria that have not been
transformed
• 4. bacteria containing a plasmid with an intact lac-Z
gene will turn blue
• 5. this isolates clones that contain fragments of foreign
genes
F. Identifying clone of interest
• Use of a probe
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1.
2.
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4.
5.
transfer
denature
add probe
autoradiography
compare
IV. Issues and solutions
• A. Genomic libraries old way of doing
business
• B. Two sources of ????
• 1. sites of restriction action do not
discriminate between gene boundaries
• 2. genomic libraries contain introns
C. Solutions
• 1. cDNA library
• 2. reverse
transcriptase
• 3. original use
of enzyme
• 4. use in
biotechnology
Reverse transcriptase
III. PCR (Polymerase chain reaction)
1986
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A. Procedure
1. heat
2. primers
3. DNA
polymeraseThermus aquaticus
• 4. repeat
• 5. five minute
cycle
• 6. 20 cycles1,048,576 copies
B. Uses of PCR
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1.
2.
3.
4.
5.
6.
amplifies small amounts of DNA
DNA samples from wooly mammoth
crime scene analysis
embryonic samples
phylogenies
like xerox-may introduce contaminants
IV. Gel electrophoresis
• A. Principles
• RFLP’s
B. Uses
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1.
2.
3.
4.
5.
molecular biology
genetics,
microbiology
forensics,
biochemistry.
V. Southern Blotting
VI. Limitations of
biotechnology
• Operation
Cat Drop