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Chapter 20 - DNA Technology and Genomics
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms and transgenic organisms
Genetically modify organisms (GMO’s) and transgenic organisms
GMO’s at home:
Genetically modified organisms (GMO’s):
-Organisms whose genes have been altered using genetic
engineering techniques.
Transgenic organisms
- Most GMO’s are transgenic organisms… they have received
genes from a different organism.
Ex. A mouse is given a gene from a human. The mouse is a
transgenic GMO.
Trans- ; across (across species in this case)
Zebra danio
GloFish
1. Zebra danio was genetically engineered with a gene from sea coral that
causes the fish to glow in the presence of environmental toxins.
2. Gene was inserted into the embryo of the fish.
3. First GMO available as a pet.
Chapter 20 - DNA Technology and Genomics
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms (GMO’s) and transgenic organisms
Genetically modify organisms (GMO’s) and transgenic organisms
GMO’s in research:
GMO’s in research:
GFP (green fluorescent protein)
GFP Mice
1. Gene from a jellyfish (Aequorea victoria)
that codes for GFP was inserted into the
embryos of mice.
Aequorea victoria
(jellyfish, phylum cnidaria)
GFP (green fluorescent protein) – a reporter protein
1. GFP is used in cellular and molecular biology.
2. You can attach this protein to any other protein you want making it a
reporter protein.
- It “reports” to you where the protein is going since it emits green
light (similar to radioactivity in that sense)
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Chapter 20 - DNA Technology and Genomics
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms (GMO’s) and transgenic organisms
Genetically modify organisms and transgenic organisms
GMO’s in research:
GMO food:
Ex.
- GFP has been attached to a protein called MFD, which is found in
peroxisomes.
European Corn Borer Larva
Bt Corn
1. Corn plants containing Cry genes from a bacterium – Bacillus thurengensis.
- You can track any protein you want…in a single cell or an entire organism
2. The genes code for enzymes that produce a toxin (insecticide), Bt toxin,
which will kill European corn borer larvae, the most damaging insect to
corn in US and canada.
Chapter 20 - DNA Technology and Genomics
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms and transgenic organisms
Genetically modify organisms (GMO’s) and transgenic organisms
GMO food:
GMO food:
- Those little green dots are peroxisomes…
Bt Corn
European Corn Borer Larva
1. Corn plants containing Cry genes from a bacterium – Bacillus thurengensis.
Are these toxins safe for you to eat???
Ordinary rice
“Golden” rice
- “Golden” rice is genetically engineered with genes that code for
enzymes that make beta-carotene, a precursor to Vitamin A for countries
deficient in foods with Vit. A…
- This rice has never been used because of environmental concerns.
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Chapter 20 - DNA Technology and Genomics
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms and transgenic organisms
Genetically modify organisms and transgenic organisms
GMO medicine:
GMO medicine:
AAT Sheep
E. Coli with the human insulin gene
Genetically engineered sheep with the human gene for alpha-1-antitrypsin (AAT).
- Insulin is made using the bacterium E. coli.
AAT is extracted from their milk and used to treat humans deficient in AAT, which is one
cause of emphysema (a breathing disorder) in approximately 100,000 people in the
western world.
- The human gene coding for insulin is inserted into E. coli, which will then make insulin
for us (we will see how this is done shortly)…
Chapter 20 - DNA Technology and Genomics
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms and transgenic organisms
Genetically modify organisms and transgenic organisms
Conclusion
- We can basically move any gene(s) between
members of a species or between any species.
- We can also alter the genes to our liking (GFP
tagged proteins) before inserting them into
embryos.
Let’s look at some of the ways we genetically engineer
organisms starting with how we can take a human
insulin gene and put it into E. coli…
Is all of this genetic engineering positive, negative?
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
There are three methods by which bacteria take up DNA in nature.
1. Transformation
First we must understand
bacteria and how they take
up DNA…
Bacteria can take up a free piece of
bacterial DNA
Griffith
(it is more than just mutation that gives
certain species of bacteria their genetic
diversity)
Fig. 12.1A-C
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
There are three methods by which bacteria take up DNA in nature.
There are three methods by which bacteria take up DNA in nature.
2. Transduction
3. Conjugation
Bacteriophage is mistakenly packaged with
bacterial DNA. Injects this DNA into another
bacteria.
“Male” (F+) bacteria extend sex pili (long
tube) to “female” (F-) bacteria. Part of
chromosome is replicated and transferred.
Hershey and Chase
Fig. 12.1A-C
Fig. 12.1A-C
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Once the DNA is transferred, integration must occur:
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Fig. 12.1D
1. Transformation
Crossing over occurs (where do you think we got it from?) and the new
DNA is integrated in place of the original DNA, which is degraded.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
2. Transduction
3. Conjugation
We will focus mostly on transformation when we look at genetic engineering…
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Transformation in the lab:
Heat Shock Method in bacteria
1. Take bacteria in a tube (in solution)
2. Add the DNA you want it to take up into
the tube.
3. Let the tube chill on ice for a few minutes
1. Transformation
4. Then quickly heat the tube to 42°C (107°F) for 90 seconds.
- This will open up “holes” in the bacterial membrane for the DNA
to slip in.
5. Cool on ice for 10 minutes…done
The bacterium now has the DNA…simple.
Bacteria can have more than just a single
circular chromosome…
(They may have little circular extra-chromosomal DNA called Plasmids)
extra-chromosomal = outside of the chromosome like extra-terrestrial
means coming from outside Earth (E. T.)
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
mcs
Plasmid
The majority of the DNA above is chromosomal, but you can see the small
circular pieces not part of the chromosome…plasmids.
Fig. 12.2C
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
- Small, circular piece of DNA distinct from bacterial chromosome
- has own origin of replication (ori)
- Carries assorted genes
- called vectors when used in genetic engineering…
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
mcs
Vectors
- We have engineered plasmids to contain clusters of restriction
sites called polylinker regions or multiple cloning site (mcs) where
we can easily insert the gene of our choice.
- We have also engineered these plasmids to contain an antibiotic
resistance.
mcs
Vector Summary
1. Ori (origin of replication)
2. MCS for inserting gene of choice
3. Antibiotic resistance gene like ampr (ampicillin resistance) for
selecting positive transformants.
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
An actual vector: pET16b
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Recall how a bacterium defends itself when a bacteriophage
injects its DNA into a bacterium…
mcs
Amp resistance
ori
The bacterium has enzymes called restriction enzymes that
attempt to cut up the bacteriophage DNA before it can take
over the cell. Different species have evolved different
restriction enzymes…
Aside: Why do these enzymes not cut the bacterial chromosome?
The bacterial chromosome is methylated (modified by adding –CH3
groups so the enzymes can’t bind to it)
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Restriction enzymes
Restriction enzymes
1. molecular DNA scissors (enzymes that cut DNA)
Ex. EcoRI
2. Different restriction enzymes cut different sequences.
3. Scientists have isolated hundreds of different restriction enzymes from
many different bacteria – EcoRI, BamHI, NcoI, etc…
Notice anything interesting about this sequence?
- It is palindromic, read the same way forward and backward on
each strand.
- Majority of restriction sites are palindromic…
Fig. 12.4
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Restriction enzymes
Restriction enzymes
Ex. EcoRI
Ex. EcoRI
EcoRI
EcoRI
Notice that is doesn’t cut straight through like paper scissors. The enzyme cuts each
strand on the 3’ side of G generating single-strand regions called sticky ends.
Why do you think we call them sticky ends?
Because they can base pair to a complementary sticky end…they are “sticky”. If it cut
straight through then it could not base pair.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Restriction enzymes
Subcloning
More examples of
restriction enzymes
Now that we understand transformation, plasmids and restriction
enzymes, we are ready to take the next step and learn how to take a
gene from an organism of choice (ex. Human insulin) and put it into
a bacterium so that the bacterium can make the polypeptide (ex.
insulin) for us. This process is called subcloning.
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
General overview of
gene cloning
Subcloning
Ex. EcoRI
(aka subcloning)
Restriction site engineered into the polylinker (mcs) region
plasmid (vector)
Let’s look at how we do
this…
Fig. 12.3
Now imagine this restriction site was engineered into a plasmid (now called a vector) as
shown above.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Subcloning
Ex. EcoRI
Ex. EcoRI
BamHI
What happens if you treat it with the restriction enzyme BamHI?
Nothing, BamHI does not cut that sequence.
EcoRI
What happens if you treat it with the restriction enzyme EcoRI?
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Subcloning
Ex. EcoRI
EcoRI
1. You can isolate the DNA from the organism of interest, which has the
gene you want to put into the vector. You will likely do this using PCR
(polymerase chain reaction), a technique we will discuss later on.
EcoRI cuts the vector leaving two sticky ends… Now what?
We need to insert our gene of choice into the plasmid.
Fig. 12.3
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Subcloning
Zoom in
…CGATTAGAATCCCGCC
…GCTAATCTTAGGGCGG
What do we need to do?
Insulin gene
Zoom in
CGGATTGAATCCCGAA…
GCCTAACTTAGGGCTT…
…CGATTAGAATTCCGCC
…GCTAATCTTAAGGCGG
Insulin gene
CGGATTGAATTCCGAA…
GCCTAACTTAAGGCTT…
2. Cut the gene with the same restriction enzyme that you cut the
plasmid/vector with to get complementary sticky ends.
Fig. 12.3
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Subcloning
Zoom in
…CGATTAG
…GCTAATCTTAA
AATTCCGCC
GGCGG
Insulin gene
CGGATTG
GCCTAACTTAA
+
AATTCCGAA…
GGCTT…
AATTCCGCC
GGCGG
Insulin gene
CGGATTG
GCCTAACTTAA
What now?
3. Mix countless numbers of cut vector with countless numbers of cut
gene…what should happen?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Subcloning
DNA ligase
Use DNA ligase + ATP to ligate the strands together
The sticky ends should base pair (the two pieces anneal = base pair to each other).
However, you still have gaps between the nucleotides in each strand…
what should we do?
Every enzyme/protein we discover is a new tool for scientists to use in the lab to
manipulate DNA. DNA ligase was discovered when investigating DNA replication, but
now we use it as “glue” when subcloning genes into vectors.
Now what should we do with this vector containing our gene or interest?
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Subcloning
X 1,000,000’s
Put it into bacteria like E. coli by
transformation using the heat-shock
method.
X 1,000,000’s
Transformation efficiency if LOW:
(very few of the bacteria will receive a vector…)
How can we identify the ones that do get a vector?
Combine millions of vector with millions of E.coli and heat shock…
One can also use electroporation (forming pores using electricity) to transform as opposed to heat shocking. It involves
sending electricity through the vector/bacterial solution, which induces temporary holes in the bacterial membrane for
vector to enter. This is also routinely used to “transform” eukaryotic cells. I will put a slide of this later on.
Remember that antibiotic resistance gene in the plasmid?
If you take these billions of bacteria and spread them on an agar plate (a sort of
nutrient rich jello) containing antibiotic, only those that have the vecor (have the
resistance gene) will grow...see next slide for agar plate
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Subcloning
Transformation efficiency if LOW:
Bacterial colonies (yellow dots)
(very few of the bacteria will receive a vector…)
Agar Plates:
•  This agar plate (right) contains the antibiotic
ampicillin.
•  The yellow dots you see are called colonies.
• Each colony is a group of millions of bacteria
that are essentially genetically identical
(clones) because…
• Each colony arose from a single ampicillin
resistant bacterium spread onto the plate the night
before
Millions of bacteria were put on this plate…how many were transformed with a vector?
Count the colonies, only about 100 or so.
-DNA means no vector transformed
+DNA means vector was transformed
LB = luria broth = bacterial food in agar
AMP = ampicillin
Don’t worry about ARA
Here you can see that when no vector (-DNA)
was used as a control in the transformation,
the bacteria grew only on the LB plate, not
the plate with ampicillin. What you see on
the LB plate is known as a “lawn” of
bacteria , which means that so many grew
that all the colonies mix together and just
When vector was used (+DNA) bacteria that
cover the plate.
got the vector could grow on the AMP plates
and colonies are observed.
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Subcloning
X 1,000,000’s
Reminder: The vector has an origin of
replication, and will be replicated by DNA
polymerase during binary fission.
1. We can take the bacteria after many round of binary fission and isolate the plasmid/
vector, and take back the gene. In essence, the bacteria replicated it for us…
Now our gene is inside the bacteria. How does this help us?
2. Or we can have the bacterium make the protein for us and then we can take the
protein and use it.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Review Slide
Restriction digest not 100% and Ligation efficiency is LOW:
lig
est
dig
on
i
t
ic
str
Re
We have a few more problems
1. Restriction digests are not 100%
2. Ligations have very low efficienty
+
Insert ligates
Gene insert
lig
Cut plasmid
Res
tric
ti
Plasmid
(1,000,000’s in a small volume
of aqueous solution in tiny
Eppendorf tube)
Fig. 12.3
on
ati
on
dig
est
ati
on
Plasmid
self-ligates
Uncut plasmid
Only a fraction will get cut
After ligation, you have a mix of
plasmid where most of it does not
contain your insert. How can we
identify the ones that do?
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Ligation efficiency is LOW
Ligation efficiency is LOW
The plasmid can be engineered to have a lacZ gene within the polylinker
(mcs) as shown above.
How does this help?
If the gene gets inserted, the lacZ gene will be non-functional. If the gene is not inserted
then lacZ will be fine.
I still don’t see how this helps…
First, remember what lacZ does…It codes for the enzyme beta-galactosidase, which
hydrolyzes lactose to glucose and galactose.
We have designed a small molecule called X-gal, which resembles lactose and is
hydrolyzed by beta-galactosidase as shown above.
So What?
X-gal is clear (no color, absorbs no light), while the molecule that forms after hydrolysis is blue…
OK, but how does this help you determine which bacteria contain plasmid with insert?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Ligation efficiency is LOW
Ligation efficiency is LOW
First, the bacteria we use have
a natural mutation in their
genomic lacZ gene. Basically,
they do not have lac Z.
BLUE-WHITE SCREENING:
After transformation and overnight growth on agar plates containing X-gal, some
colonies will turn blue meaning they have a working beta-galactosidase enzyme and
therefore do not contain your gene. Those that remain white do not have the functional
enzyme and therefore must have your gene.
Why do the agar plates
contain the antibiotic
ampicillin (Amp)?
Remember that bacteria that
do not pick up plasmid cannot
grow and therefore your are
selecting for only those that
have been transformed.
Summary: Two selection criteria:
1. Ampicillin resistance showing presence of plasmid
2. White colony showing presence of insert.
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Review Slide
Review Slide
What is the problem with this if we
were subcloning a eukaryotic gene?
INTRONS!! If you take a eukaryotic
gene and insert it straight into a
vector, the introns are still there and
bacteria cannot splice out introns.
Why can’t they splice out introns?
Because they do not have introns!
Fig. 12.3
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Let the eukaryotic cell take out the
introns for you…
Instead of taking the gene from the
eukaryotic cell, take the processed
mRNA.
How do we fix this eukaryotic gene problem?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Make cDNA (complementary
DNA) from the mRNA:
1. Isolate mRNA
2. use reverse transcriptase to make a
dsDNA copy
3. cut with restriction enzyme
and ligate into a vector
But this leads to another problem, we can’t put RNA into a DNA plasmid…
Fig. 12.7
Advantages to cDNA
1. No introns
2. No junk DNA
Fig. 12.7
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Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Summary
1. Isolate plasmid
2. Isolate gene of interest (straight from genome if
bacterial or via mRNA if eukaryotic)
3. Cut both with same restriction enzyme
4. Mix together to allow sticky ends to ANNEAL
forming recombinant DNA
5. Ligate using DNA ligase
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Conclusion
We can make any protein we want or more of any
gene (gene cloning) by putting it into a plasmid
and transforming a bacterium.
6. Transform bacteria with vector (plasmid)
7. Bacteria will express (make) the protein and divide
making more copies of the gene (gene cloning)
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
There is another, more efficient way of
making more of any gene or DNA
segment we want…using a method
called:
PCR (Polymerase Chain Reaction)
Technique used to amplify (make more of) a specific piece of DNA.
Can be a gene or any other segment. It is essentially DNA
replication in a test tube with a twist…
http://www.maxanim.com/genetics/PCR/PCR.htm
http://www.youtube.com/watch?v=x5yPkxCLads
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Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
PCR
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
A crime has been committed and you
have a suspect as well as a tiny bit of
DNA sample from the scene of the crime.
What do you do?
Combine template DNA, DNA primers
flanking the target region, Taq
polymerase, deoxynucloside
triphates
1. Denaturing
2. Annealing
3. Extending
The first thing you do is PCR the DNA to make more copies of it…
Let’s assume you go ahead and do this…now what?
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Fig. 12.11A
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
You have a suspect. What should you do?
**Everyone’s DNA has a slightly different
sequence (every 1 in 1000 bases is
different), so we all have different
restriction site patterns.
The PCR amplified portion of this person
has two restriction sites.
How many restriction fragments (DNA
pieces) would there be after cutting with
the restriction enzyme? three
Use PCR to amplify the same segment of the subjects DNA and cut
it with the same restriction enzyme.
Amplified section of the DNA
from the crime scene
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Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Crime scene DNA
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Crime scene DNA
Suspect DNA
Fig. 12.11A
Suspect DNA
Restriction fragment length polymorphisms
(RFLP’s = “rif lips”)
How many restriction fragments will the
suspects DNA yield? two
The differences in restriction sites found
on homologous chromosomes giving rise
to different numbers and lengths of
restiriction fragments.
The suspect has a different allele with a
mutation in the first restriction site. The
restriction enzyme will not cut this
sequence.
Conclusion:
The suspect did not commit the crime.
Amplified section of the DNA
from the crime scene
Amplified section of the same
DNA segment from the suspect.
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Amplified section of the DNA
from the crime scene
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
Fig. 12.11A
This is great, but you can’t see DNA
restriction fragments directly so how will
we actually count the fragments?
Amplified section of the same
DNA segment from the suspect.
cathode
How can we OBSERVE the DNA
restriction fragments?
anode
This technique allows one to separate DNA fragments by size and view
the DNA fragments.
Amplified section of the DNA
from the crime scene
Amplified section of the same
DNA segment from the suspect.
Fig. 12.10
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Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
As you saw in the video, the researcher
put a chemical called ethidium bromide
(shown below)into the gel solution.
This compound binds to DNA (right,
below) and fluoresces when hit with UV
light thus allowing us to see where the
DNA is located in the gel (right, above).
Fig. 12.10
http://www.youtube.com/watch?v=QEG8dz7cbnY
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
cathode
Gel Electrophoresis
cathode
Gel (like jell-o)
anode
The gel is made of either agarose or polyacrylamide. It has tiny,
microscopic pores that DNA can fit through.
Fig. 12.10
Gel (like jell-o)
anode
The DNA sample is loaded in the wells at the top of the gel. One
sample per well.
Fig. 12.10
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Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
cathode
Gel Electrophoresis
cathode
Electricity (electrons flow
from top of gel by the samples
to the bottom of the gel)
anode
anode
Electricity is then run through the gel. Why do you think the negative
end is on the sample side and the positive end is on the other end of
the gel?
DNA is negative because the phosphates are negative. The negative
Fig. 12.10 electrons moving down push (repel) the DNA down with them.
Which will move faster through the micro-porous gel, the longer DNA
fragments or the shorter DNA fragments?
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
The small fragments (fewer nucleotides) will move more easily through the gel and hence go
faster than the large ones. Therefore, gel electrophoresis separates DNA fragments by SIZE.
Gel Electrophoresis
Gel Electrophoresis
cathode
cathode
anode
anode
This is all great, but we still can’t physically see the DNA…
The gel is soaked with a a compound called ethidium bromide, which
sticks to DNA and lights up when you hit the gel with UV light…
Fig. 12.10
Fig. 12.10
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Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
AIM: What are some of the
other tools of DNA technology?
Gel Electrophoresis
Virtual Lab
You are not observing the DNA move. You are seeing a blue dye added to
the sample move through the gel. You cannot see the DNA until you put
the gel under a UV lamp as discussed before.
http://www.youtube.com/watch?v=Wwgs-FjvWlw&feature=related
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
(http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/virgel.html)
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Draw what the gel would
look like for the restriction
digest of the criminal and
the suspect.
Amplified section of the DNA
from the crime scene
Amplified section of the same
DNA segment from the suspect.
Criminal’s
DNA fingerprint
criminal
suspect
Suspect’s
DNA fingerprint
Fig. 12.11A
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Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Can also be used to determine:
2. Diseases resulting from DNA
changes that alter restriction
sites.
Can also be used to determine:
1. Paternity (allele 1 from child, allele
2 amplified from suspected father).
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Review:
1. Use PCR to get more of the desired DNA
2. Digest DNA with restriction enzymes
3. Run restriction fragments on a gel (gel
electrophoresis)
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Question: You have been given two DNA samples that have
gone through PCR. Both samples are of the same DNA
segment with a size of 1kb (1 kilobase = 1000bp). Sample
1 has four restriction sites at 100bp, 300bp, 350bp, and
700bp. The second piece has the same sites in addition to a
fifth site at 725bp. Draw how the gel should look for these
two pieces.
4. Compare fragments
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Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
100bp
300bp 350bp
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
700bp
-
Sample 1
Sample
1
Segments of DNA:
Five segments in total 50bp, 100bp, 200bp, 300bp, 350bp
350bp
300bp
e100bp
300bp 350bp
700bp
Sample
2
350bp
275bp
200bp
200bp
100bp
100bp
50bp
50bp
Do not forget to
label the charges
on the gel and
show the flow of
electrons (the
current).
Sample 2
Six segments in total 25bp, 50bp, 100bp, 200bp, 275bp, 350bp
725bp
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
+
25bp
Chapter 20 - DNA Technology and Genomics
NEW AIM: Making transgenic organisms.
Gel electrophoresis can be done using proteins as well. In
this case the gel is made of polyacrylamide and the proteins
are coated with negatively charged molecules called SDS
since they are not always negative like DNA. It is a little
more complicated, but not much…
“Pharm” animals
Fig. 12.16
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Chapter 20 - DNA Technology and Genomics
Chapter 20 - DNA Technology and Genomics
AIM: Making transgenic organisms.
AIM: Making transgenic organisms.
Transforming plants:
Transgenic mice have been
invaluable tools:
We have the ability to add or take away
any gene we want from mice to observe
the affect of that gene.
Two methods are available to do this:
1. Transform embryonic stem cells
2. Inject desired gene into male nulceus
after fertilization, but before fusion of
nuclei occurs
by electroporation
An infectious soil bacterium, Agrobacterium tumefaciens, contains a plasmid known as Ti plasmid that naturally
integrates a section of its plasmid into the plant’s DNA.
We have isolated the plasmid, rendered it non-infectious, and put desired genes into the part of the plasmid that gets
incorporated known as the T DNA.
Fig. 12.18AB
Chapter 20 - DNA Technology and Genomics
AIM: Making transgenic organisms.
Transgenic mice have been invaluable tools:
Chapter 20 - DNA
Technology and Genomics
AIM: Making transgenic
organisms.
Gene Therapy
- Replacing a defective gene
with a normal gene.
An example:
Normal mice cannot be infected with polio virus. They lack the cell-surface molecule that, in
humans, serves as the receptor for the virus. So normal mice cannot serve as an
inexpensive, easily-manipulated model for studying the disease. However, transgenic mice
expressing the human gene for the polio virus receptor
* can be infected by polio virus and even
* develop paralysis and other pathological changes characteristic of the disease in
humans.
Fig. 12.19
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