Download Chapter 20

Chapter 20 - DNA Technology and Genomics
Genetically modify organisms and transgenic organisms
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)
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms (GMO’s) and transgenic organisms
GMO’s at home:
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
Genetically modify organisms (GMO’s) and transgenic organisms
GMO’s in research:
GFP (green fluorescent prote
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)
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms (GMO’s) and transgenic organisms
GMO’s in research:
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)
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms (GMO’s) and transgenic organisms
GMO’s in research:
Ex.
- GFP has been attached to a protein called MFD, which is
found in peroxisomes.
- Those little green dots are peroxisomes…
- You can track any protein you want…in a single cell or an entire
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms and transgenic organisms
GMO food:
Bt Corn
European Corn Borer Larv
1. Corn plants containing Cry genes from a bacterium – Bacillus
thurengensis.
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
Genetically modify organisms and transgenic organisms
GMO food:
Bt Corn
European Corn Borer Larv
1. Corn plants containing Cry genes from a bacterium – Bacillus
thurengensis.
Are these toxins safe for you to eat???
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms (GMO’s) and transgenic organisms
GMO food:
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
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms and transgenic organisms
GMO medicine:
AAT Sheep
Genetically engineered sheep with the human gene for alpha-1antitrypsin (AAT).
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.
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms and transgenic organisms
GMO medicine:
E. Coli with the human insulin gen
- Insulin is made using the bacterium E. coli.
- 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
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.
Is all of this genetic engineering positive,
negative?
Chapter 20 - DNA Technology and Genomics
Genetically modify organisms and transgenic organisms
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…
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
First we must
understand bacteria
and how they take up
DNA…
(it is more than just mutation that
gives certain species of bacteria their
genetic diversity)
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 n
1. Transformation
Bacteria can take up a free piece of
bacterial DNA
Griffith
Fig. 12.1A-C
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 n
2. Transduction
Bacteriophage is mistakenly packaged
with bacterial DNA. Injects this DNA
into another bacteria.
Hershey and Chase
Fig. 12.1A-C
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 n
3. Conjugation
“Male” (F+) bacteria extend sex pili
(long tube) to “female” (F-) bacteria.
Part of chromosome is replicated and
transferred.
Fig. 12.1A-C
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Once the DNA is transferred, integration must occur:
Fig. 12.1D
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?
1. Transformation 2. Transduction
3. Conjugation
We will focus mostly on transformation when we look at genetic
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
1. Transformation
minutes
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.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
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 extraterrestrial means coming from outside Earth (E. T.)
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
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?
mcs
Plasmid
- 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
- We have also engineered these plasmids to contain an
choice.
antibiotic resistance.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
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.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
An actual vector: pET16b
mcs
Amp resistance
ori
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…
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?
Restriction enzymes
1. molecular DNA scissors (enzymes that cut DNA)
2. Different restriction enzymes cut different sequences.
3. Scientists have isolated hundreds of different restriction
enzymes from many different bacteria – EcoRI, BamHI, NcoI,
etc…
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Restriction enzymes
Ex. EcoRI
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
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Restriction enzymes
Ex. 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
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Restriction enzymes
Ex. EcoRI
EcoRI
Why do you think we call them sticky ends?
Because they can base pair to a complementary sticky end…they are “sticky”.
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?
Restriction enzymes
More examples of
restriction enzymes
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
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.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
General overview of
gene cloning
(aka subcloning)
Let’s look at how we do
this…
Fig. 12.3
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Ex. EcoRI
Restriction site engineered into the polylinker (mcs) region
plasmid (vector)
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?
Subcloning
Ex. EcoRI
BamHI
What happens if you treat it with the restriction enzyme BamHI?
Nothing, BamHI does not cut that sequence.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Ex. EcoRI
EcoRI
What happens if you treat it with the restriction enzyme EcoRI?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Ex. EcoRI
EcoRI
EcoRI cuts the vector leaving two sticky ends…
Now what?
We need to insert our gene of choice into the plasmid.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
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.
Fig. 12.3
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Zoom in
…CGATTAGAATCCCGCC Insulin gene
…GCTAATCTTAGGGCGG
CGGATTGAATCCCGAA…
GCCTAACTTAGGGCTT…
What do we need to do?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Zoom in
…CGATTAGAATTCCGCC Insulin gene
…GCTAATCTTAAGGCGG
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
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
Zoom in
…CGATTAG
AATTCCGAA…
…GCTAATCTTAA
What now?
AATTCCGCCInsulin gene
GGCGG
CGGATTG
GCCTAACTTAA
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
+
AATTCCGCC Insulin gene
GGCGG
CGG
GCC
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?
Subcloning
The sticky ends should base pair (the two pieces anneal = base pair to ea
However, you still have gaps between the nucleotides in each
strand…what should we do?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
DNA ligase
Use DNA ligase + ATP to ligate the strands together
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 inte
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
X 1,000,000’s
Put it into bacteria like E. coli by
transformation using the heatshock method.
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
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
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?
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?
Subcloning
Transformation efficiency if
LOW:
Bacterial colonies (yellow dots)
Agar
Plates:
(very
few of the bacteria will receive a vector…)
• 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.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
-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
When vector was used (+DNA) bacteria
colonies mix together and just cover the that got the vector could grow on the
plate.
AMP plates and colonies are observed.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
X 1,000,000’s
Reminder: The vector has an origin of
replication, and will be replicated by
DNA polymerase during binary fission.
Now our gene is inside the bacteria. How does this help us?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Subcloning
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…
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?
Review Slide
We have a few more problems
1. Restriction digests are not 100%
2. Ligations have very low efficienty
Fig. 12.3
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Restriction digest not 100% and Ligation efficiency
is LOW:
+
Insert ligates
Gene insert
Cut plasmid
Plasmid
self-ligates
Plasmid
(1,000,000’s in a small
volume of aqueous solution
in tiny Eppendorf tube)
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?
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
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.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Ligation efficiency is
LOW
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
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
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.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Ligation efficiency is
LOW
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.
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
Review Slide
Fig. 12.3
Chapter 20 - DNA Technology and Genomics
How can we use bacteria to manipulate DNA and protein?
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!
How do we fix this eukaryotic gene problem?
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.
But this leads to another problem, we can’t put RNA into a DNA
plasmid…
Fig. 12.7
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
Advantages to cDNA
1. No introns
2. No junk DNA
Fig. 12.7
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
6. Transform bacteria with vector (plasmid)
7. Bacteria will express (make) the protein and
divide making more copies of the gene (gene
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.
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
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
http://www.youtube.com/watch?v=x5yPkxCLads
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
PCR
Combine template DNA, DNA
primers flanking the target
region, Taq polymerase,
deoxynucloside triphates
1. Denaturing
2. Annealing
3. Extending
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?
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
**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
threeenzyme?
cutting with the restriction
Amplified section of the
DNA from the crime scene
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
You have a suspect. What should
you do?
Use PCR to amplify the same segment of the subjects DNA
and cut it with the same restriction enzyme.
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Crime scene DNA
Suspect DNA
Fig. 12.11A
How many restriction fragments will
two
the suspects DNA yield?
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
Amplified section of the
DNA from the crime scene same DNA segment from the
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Crime scene DNA
Suspect DNA
Restriction fragment length
polymorphisms (RFLP’s = “rif lips”)
The differences in restriction sites
found on homologous chromosomes
giving rise to different numbers and
lengths of restiriction fragments.
Amplified section of the
Amplified section of the
DNA from the crime scene same DNA segment from the
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Fig. 12.11A
This is great, but you can’t see DNA
restriction fragments directly so how
will we actually count the fragments?
How can we OBSERVE the
DNA restriction fragments?
Amplified section of the
Amplified section of the
DNA from the crime scene same DNA segment from the
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
cathode
anode
This technique allows one to separate DNA fragments by size
and view the DNA fragments.
Fig. 12.10
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
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?
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).
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
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
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
cathode
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
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
cathode
Electricity (electrons
flow from top of gel by the
samples to the bottom of
the gel)
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
gel?because the phosphates are negative. The
DNAofis the
negative
Fig. 12.10 negative electrons moving down push (repel) the DNA down with
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
cathode
anode
Which will move faster through the micro-porous gel, the
longer DNA fragments or the shorter DNA fragments?
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
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
cathode
anode
This is all great, but we still can’t physically see the DNA…
Fig. 12.10
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
cathode
anode
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
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Gel Electrophoresis
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
AIM: What are some of the
other tools of DNA technology?
Virtual Lab
(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
Amplified section of the
DNA from the crime scene same DNA segment from the
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
Criminal’s
DNA fingerprint
criminal
suspect
Suspect’s
DNA fingerprint
Fig. 12.11A
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
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?
Can also be used to
determine:
2. Diseases resulting from
DNA changes that alter
restriction sites.
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
2. Digest DNA with restriction enzymes
DNA
3. Run restriction fragments on a gel
(gel electrophoresis)
4. Compare fragments
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.
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
100bp
300bp350bp
700bp
Sample 1
Segments of DNA:
Five segments in total 50bp, 100bp, 200bp, 300bp, 350bp
100bp
300bp350bp
700bp
Sample 2
725bp
Six segments in total 25bp, 50bp, 100bp, 200bp, 275bp, 350bp
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
-
Sample
1
350bp
Sample
2
350bp
300bp
e
275bp
200bp
200bp
100bp
100bp
50bp
50bp
25bp
+
Do not forget
to label the
charges on the
gel and show
the flow of
electrons (the
current).
Chapter 20 - DNA Technology and Genomics
AIM: What are some other tools of DNA technology?
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…
Chapter 20 - DNA Technology and Genomics
NEW AIM: Making transgenic organisms.
“Pharm” animals
Fig. 12.16
Chapter 20 - DNA Technology and Genomics
AIM: Making transgenic organisms.
Transforming plants:
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:
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
Chapter 20 - DNA Technology and Genomics
AIM: Making transgenic organisms.
Transgenic mice have been invaluable tools:
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.
Chapter 20 - DNA
Technology and Genomics
AIM: Making transgenic
organisms.
Gene Therapy
- Replacing a defective gene
with a normal gene.
Fig. 12.19