DNA Technology & Genomics
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Chpt. 20
The use of recombinant DNA
technology has already impacted
your life in ways that you might
not expect.
How has recombinant DNA
affected your life?
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• The popular stonewashed denim look is actually achieved
by treating denim with cellulase enzymes which
partially break down the cotton fibers of the denim. This
gives stonewashed jeans their soft texture when compared
to regular jeans. Many different cellulase enzymes have
been discovered in microorganisms. Recombinant DNA
technology is used to clone the genes encoding these
enzymes so that large quantities of enzyme can be
produced and sold to textile manufacturers.
How has recombinant DNA
affected life?
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• Insulin from an animal source,
such as pigs has traditionally
been used to treat diabetics.
Insulin from these animals is
similar but not identical to
human insulin. Because of
this, many patients develop
allergic reactions. Recombinant
DNA tools have enabled
researchers to locate and
clone the gene for human
insulin, ensuring an ample
supply of insulin that does not
cause allergic reactions.
How do we do this?
• The Nobel Prize in Physiology or Medicine 1978.
• "for the discovery of restriction enzymes
and their application to problems of
molecular genetics"
How do we do this?
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Swiss
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American
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American
• The Nobel Prize in Physiology or Medicine 1978.
• "for the discovery of restriction enzymes and their
application to problems of molecular genetics"
WHAT… is a
RESTRICTION
ENZYME??
• Bacteria are under constant attack by
bacteriophages (viruses).
phage
phage
phage
phage
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phage
• To protect themselves, many types of
bacteria have developed a method to
chop up any foreign DNA,
such as that from attacking
phages.
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• These bacteria create endonucleases-(restriction enzymes)
• an enzyme that cuts DNA that enters the
bacterium via. the phage
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• . The endonucleases are termed
"restriction enzymes" because
they restrict the infection of
bacteria phages.
• restriction enzymes do not attack
their own bacterial DNA b/c they
have a gene that prevents the
r.e. from attaching to their
chromosomal DNA
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Restriction Enzymes
• Restriction enzymes
are enzymes isolated
from bacteria that
recognize specific
sequences in DNA
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• and then cut the DNA
to produce fragments
called restriction
fragments.
Restriction Enzymes
• Different restriction
enzymes recognize
and cut different
sequences of DNA.
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Restriction Enzymes
G AAT T C
C T TAA G
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So, how can we use
these?
Restriction Enzymes
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• If we are able to locate a eukaryotic
“gene of interest”. ex. Insulin gene
Restriction Enzymes
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• And that gene of interest is
“downstream” from a restriction site…
Restriction Enzymes
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• We are able to cut the gene of interest
out of a eukaryotic genome… and
“attach” it to the prokaryotic genome
Restriction Enzymes
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“glue”
together w/ LIGASE
Restriction Enzymes
• The “recombined” genome of the
prokaryote will now be placed
BACK INTO a prokaryote
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Restriction Enzymes
• The bacteria will produce insulin
right along with the replicating
bacteria!!
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Restriction Enzymes:
restriction site area where DNA is cut
usually only 4 - 6 bp’s long
-it is a palindrome
-some DNA molecules have
many of these specific sites…
some have none
Naming the R.E.’s ex. BamH I
B = genus Bacillus
am = species amyloliquefaciens
H = strain (kind)
I = order
order inwhich
this R.E. from this species
of bacterium was isolated
So, how does this
work?
Gene cloning via. a Bacteria
1) Get a plasmid/
find your “gene of
interest” in the
eukaryote~ cut both
with restriction
endonuclease
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Gene cloning via. a Bacteria
2) Using ligase,
combine “gene of
interest” into plasmid
RECOMBINANT!
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Gene cloning via. a Bacteria
3) Put
RECOMBINANT
plasmid back onto
bacterial cell
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TRANSFORMATION
Gene cloning via. a Bacteria
4) Grow transformed
host cell in culture~
forms many cloned
genes of interest
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Gene cloning via. a Bacteria
5) Protein
harvested
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Gene cloning via. a Bacteria
6) Gene
inserted into
other
organisms
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Gene cloning via. a Bacteria~ a bit more complex
lacZ gene codes for an enzyme that hydrolyzes
lactose & X-gal (a lactose mimic)
when X-gal (a lactose mimic) is hydrolyzed , a
blue product is formed
Notice there is a lacZ gene and right in the
middle is the restriction site!!
Gene cloning via. a Bacteria~ a bit more complex
Many eukaryotic genes get excised,
only one of those carries the gene of
interest
Gene cloning via. a Bacteria~ a bit more complex
add ligase
The thing is…many other recombinants will form~
think about how many sites the human DNA was
cut. How many carry the gene of interest… ONE
Gene cloning via. a Bacteria
The recombinant plasmids… are mixed
with bacteria that has a mutation in their
lacZ gene.
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Ampicillin w/ kill any bacteria
without the resistance gene
with lacZ gene intact ->
X-gal will cause the
bacteria to turn blue
So, how do you find
the colony with the
gene of interest?
Nucleic Acid Hybridization
Master plate
Solution
containing Radioactive
probe
single-stranded
DNA
Probe
DNA
Gene of
interest
Filter
Filter lifted
and flipped over
Hybridization
on filter
A special filter paper
is pressed against
the master plate,
transferring cells to
the bottom side of
the filter.
The filter is treated to
break open the cells
and denature their
DNA; the resulting
single-stranded DNA
molecules are treated
so that they stick to the
filter.
Colonies
containing
gene of
interest
Master plate
Single-stranded
DNA from cell
The filter is laid
under
photographic
film, allowing
any radioactive
areas to expose
the film
(autoradiograph
y).
After the
developed film is
flipped over, the
reference marks
on the film and
master plate are
aligned to locate
colonies carrying
the gene of
interest.
Nucleic Acid Hybridization
Master plate
Solution
containing Radioactive
probe
single-stranded
DNA
Probe
DNA
Gene of
interest
Filter
Filter lifted
and flipped over
Hybridization
on filter
A special filter paper
is pressed against
the master plate,
transferring cells to
the bottom side of
the filter.
The filter is treated to
break open the cells
and denature their
DNA; the resulting
single-stranded DNA
molecules are treated
so that they stick to the
filter.
Colonies
containing
gene of
interest
Master plate
Single-stranded
DNA from cell
The filter is laid
under
photographic
film, allowing
any radioactive
areas to expose
the film
(autoradiograph
y).
After the
developed film is
flipped over, the
reference marks
on the film and
master plate are
aligned to locate
colonies carrying
the gene of
interest.
Nucleic Acid Hybridization
Master plate
Solution
containing Radioactive
probe
single-stranded
DNA
Probe
DNA
Gene of
interest
Colonies
containing
gene of
interest
Master plate
Filter
Hybridization
on filter
A special filter paper
is pressed against
the master plate,
transferring cells to
the bottom side of
the filter.
The filter is treated to
break open the cells
and denature their
DNA; the resulting
single-stranded DNA
molecules are treated
so that they stick to the
filter.
Single-stranded
DNA from cell
After the
The filter is laid
developed film is
Single
stranded,
under
flipped over, the
photographic
radioactive probe added
reference marks
film, allowing
on the film and
any radioactive
master plate are
areas to expose
aligned to locate
the film
colonies carrying
(autoradiograph
the gene of
y).
interest.
Nucleic Acid Hybridization
Master plate
Solution
containing Radioactive
probe
single-stranded
DNA
Probe
DNA
Gene of
interest
Filter
Filter lifted
and flipped over
Hybridization
on filter
A special filter paper
is pressed against
the master plate,
transferring cells to
the bottom side of
the filter.
The filter is treated to
break open the cells
and denature their
DNA; the resulting
single-stranded DNA
molecules are treated
so that they stick to the
filter.
Colonies
containing
gene of
interest
Master plate
Single-stranded
DNA from cell
The filter is laid
under
photographic
film, allowing
any radioactive
areas to expose
the film
(autoradiograph
y).
After the
developed film is
flipped over, the
reference marks
on the film and
master plate are
aligned to locate
colonies carrying
the gene of
interest.
Nucleic Acid Hybridization
Master plate
Solution
containing Radioactive
probe
single-stranded
DNA
Probe
DNA
Gene of
interest
Filter
Filter lifted
and flipped over
Hybridization
on filter
A special filter paper
is pressed against
the master plate,
transferring cells to
the bottom side of
the filter.
The filter is treated to
break open the cells
and denature their
DNA; the resulting
single-stranded DNA
molecules are treated
so that they stick to the
filter.
Single-stranded
DNA from cell
The filter is laid
under
photographic
film, allowing
any radioactive
areas to expose
the film
(autoradiograph
y).
Colonies
containing
gene of
interest
Master plate
Colonies can now be
After the
isolated
developed film is
flipped over, the
reference marks
on the film and
master plate are
aligned to locate
colonies carrying
the gene of
interest.
Nucleic Acid Hybridization
Master plate
Solution
containing Radioactive
probe
single-stranded
DNA
Probe
DNA
Gene of
interest
Filter
Filter lifted
and flipped over
Hybridization
on filter
A special filter paper
is pressed against
the master plate,
transferring cells to
the bottom side of
the filter.
The filter is treated to
break open the cells
and denature their
DNA; the resulting
single-stranded DNA
molecules are treated
so that they stick to the
filter.
Colonies
containing
gene of
interest
Master plate
Single-stranded
DNA from cell
The filter is laid
under
photographic
film, allowing
any radioactive
areas to expose
the film
(autoradiograph
y).
After the
developed film is
flipped over, the
reference marks
on the film and
master plate are
aligned to locate
colonies carrying
the gene of
interest.
So, how do we
store these once we
find them?
Genomic Libraries
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http://plantandsoil.unl.edu/croptechnolo
gy2005/crop_tech/animationOut.cgi?ani
m_name=genecloning.swf
Would this take a LONG time???
What if I only have a little sample?
PCR - Polymerase Chain Reaction
A DNA sample, target DNA, is
obtained (crime scene?)
PCR - Polymerase Chain Reaction
DNA denatures by heating at
98oC for 5 minutes
PCR - Polymerase Chain Reaction
The sample is cooled to 60oC /
DNA PRIMERS are bonded to
each strand
PCR - Polymerase Chain Reaction
Free nucleotides, and the
enzyme DNA polymerase are
added / complementary strands
synthesized!
PCR - Polymerase Chain Reaction
There are now two copies of the
original strand
PCR - Polymerase Chain Reaction
Process repeated… now four
strands
PCR - Polymerase Chain Reaction
LOTS of strands in
very little time
PCR - Polymerase Chain Reaction
They key… heat-stable
DNA polymerase - this
comes from thermophillic
bacteria…
PCR - Polymerase Chain Reaction
They key… heat-stable
DNA polymerase - this
comes from thermo bacteria…
This does not denature in high
heat. Benefit - keep heating the
test tube up after each round.
PCR - Polymerase Chain Reaction
http://www.youtube.com/watch?v=ZmqqRPISg0g
http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html
"Beginning with a single molecule of the genetic
material DNA, the PCR can generate 100 billion
similar molecules in an afternoon. The reaction is
easy to execute. It requires no more than a test
tube, a few simple reagents and a source of heat.
The DNA sample that one wishes to copy can be
pure, or it can be a minute part of an extremely
complex mixture of biological materials. The DNA
may come from a hospital tissue specimen, from a
single human hair, from a drop of dried blood at
the scene of a crime, from the tissues of a
mummified brain or from a 40,000-year-old wooly
mammoth frozen in a glacier."
PCR - Polymerase Chain Reaction
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Dr. Kary Mullis awarded Nobel Prize 1993 for discovery of PCR
in 1983… Hobby was a Freshman @ Miami U. that day he
invented it!! Probably, I had no idea of this happening…Hmmm,
what will happen when YOU are Freshman that you wont realize
until later??
DNA Fingerprinting
• Murder. A body lies on the sidewalk near an
alley. It looks like the victim fought off her
attacker, leaving blood and tissue under her
fingernails. There is also a pool of blood on
the sidewalk next to the victim. Two suspects
have been picked up just a block away with
fresh scratches on them. Could one of them
be the killer?
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DNA Fingerprinting
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• The lead detective on this case will
be using DNA fingerprinting to
determine if one of the suspect's
DNA matches DNA found at the
crime scene.
Lets Practice …
DNA Fingerprinting
http://www.pbs.org/wgbh/nova/sheppard/analyze.html
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GEL ELECROPHORESIS
http://www.npr.org/templates/player/mediaPlayer.html?actio
n=1&t=1&islist=false&id=5534279&m=5534280
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GEL ELECROPHORESIS
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GEL ELECROPHORESIS
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How will we use restriction
enzymes?
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RFLP ~ Restriction Fragment
Length Polymorphisms
• Method for
detecting
minor
differences in
DNA structure
between
individuals.
RFLP ~ Restriction Fragment
Length Polymorphisms
• individuals
inherently have
differences in
their fragment
lengths b/c of
individual
insertions,
deletions etc.
RFLP ~ Restriction Fragment
Length Polymorphisms
• RFLP’s can be
used to
genetically tell
individuals apart.
• RFLP’s can also
show the genetic
relationship
between
individuals,
because children
inherit genetic
elements from
their parents.
RFLP ~ Restriction Fragment
Length Polymorphisms
1. Digest DNA with restrictive enzymes.
2. Separate pieces by Gel Electrophoresis
3. Identify sequences with identifying
probes.
RFLP ~ Restriction Fragment
Length Polymorphisms
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• converts a GAG codon (for Glu) to a GTG codon
for Val
• abolishes a sequence CTGAGG, recognized and
cut by one of the restriction enzymes.
RFLP ~ Restriction Fragment
Length Polymorphisms
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• converts a GAG codon (for Glu) to a GTG codon
for Val
• abolishes a sequence CTGAGG, recognized and
cut by one of the restriction enzymes.
Southern Blotting
• Makes RFLP fragments
that are different
IDENTIFYABLE.
• With just the gel
electro… you would not
really be able to identify
where the SPECIFIC
difference was!
Southern Blotting
• Move fragments from
the gel to a nylon sheet.
• Make a DNA fragment
that is complementary
to the “area of interest.
Southern Blotting
• Put a radioactive
PROBE on the DNA
fragment of interest
Southern Blotting
• Wash the membrane
“paper”
Southern Blotting
• Only your “area of interest”
shows up well!