Download Gel Electrophoresis!

What do you think Biotechnology is?
What are some applications of biotechnology?
What are the impacts and ethics of biotechnology?
(a) Tobacco plant expressing
a firefly gene
(b) Pig expressing a
jellyfish gene
Biotechnology
A little of this, a little of
that (C. 14.3, 15.2, 15.3)
Genetic Ethics Quiz
What would you do?
A biotechnology company has spliced
together genes from two different species
of fish to produce salmon that grow faster.
This allows the fish to be ready for purchase
more quickly, decreasing consumer costs and
increasing the availability of salmon.
Will you:
GMO Salmon
a)
b)
c)
Wait in line for the first genetically modified salmon. It
sounds super yummy!
Wait for further research to analyze the potential
environmental and health impacts of GMO salmon
Boycott any store willing to sell the salmon. Don’t trust what
isn’t natural!!
You’ve found out that the child you (or your
wife) carries has the gene for dwarfism. A
new therapy exists that may repair this
gene before the child is born. What do you
do?
a) Allow the child to be born with the
gene, and we will accept the child as is.
b) Attempt the new therapy to repair the
gene.
c) Terminate pregnancy.
New Hope for Gene Therapy...A Young
Boy's Fight Against Blindness
An agricultural company has found
a way to make tomatoes 50%
larger by splicing elephant genes
into the tomatoes. Will you:
a) Buy the tomatoes and have no problem
eating them
b) Probably not buy or eat the tomatoes.
c) Protest the company. It’s not right to fool
with mother nature!
Pet cloning is now available at the local mall.
You used to have a pet dog named Charlie,
but he died two years ago. Now you learn
that you can get a new Charlie by just
bringing in a sample of his hair. What do you
do?
a) Find a strand of Charlie’s hair as soon as
possible. Can’t wait to see Charlie again!
b) Let sleeping dogs lie. (Don’t clone
Charlie)
Your Dog has just Died and you want to save his cells for Cloning
Important! Please read below and act now!
If your dog has just passed then you must act quickly as in right
now! There is a 5 day window to successfully extract good quality
cells from your beloved friend before the cells begin to
deteriorate.
This means that once your dog has passed the body will begin to
quickly deteriorate and your chances of extracting quality cells
diminishes with every passing day.
Warning!!!
Do not place your dog in the Freezer. If you do there will be zero
chance of cloning your dog. You must wrap your dogs entire body
with wet bathing towels and place it in the Refridgerator to keep it
cool. Do this first and then call us right away. Time is of the
http://myfriendagain.com/
essence!
A company can now create test tube babies
according to parent specifications. The
company can make sure that your child has
all the traits you desire- hair color,
intelligence, athletic ability, etc… What do
you do?
a) Sign me up, I want my child to be perfect.
b) I’d rather let nature take its course.
c) We might adjust some things to make sure
the child is healthy (and has no genetic
abnormalities).
Cloning technology is perfected. A clone can
be made from adult tissue samples. You
learn that you need a kidney transplant, but
there are no donors available. A doctor
suggests that you make a clone of yourself,
so that the kidney would be a perfect match.
What do you do?
a) One is enough of me, I’ll wait for a donor.
b) Clone myself, two is better than one.
A government organization is proposing to
have all citizens of the US submit a sample
of their hair so that their DNA can be
scanned and kept on file. Each person’s
DNA would be kept in a national database
so that police could access the DNA when a
crime was committed. Do you?
a) Support this legislation
b) Oppose this legislation
c) Suggest the legislation be modified, only
people with criminal records should be
scanned.
An insurance company is requiring
individuals to get genetic testing performed
to determine whether they have a higher risk
of heart disease, cancer, or other diseases.
They are requiring that all people wanting
health insurance be tested. What do you
do?
a) Find another insurance company, that
information is none of their business.
b) Submit my DNA for a test, I’d like to know
anyway.
c) File a lawsuit against the company.
What is Biotechnology?
• Using cellular and biomolecular
processes in the production of
agricultural, medicinal, industrial, or
other products.
– Usually involves manipulation of DNA
What are some current
applications/uses for
biotech?
•
•
•
•
•
•
•
•
•
Transgenic organisms: plants,
animals
GMOs: genetically modified
organisms.
Reproductive Cloning
Stem Cell Research
(Therapeutic Cloning)
Forensics
Diagnosis of genetic disease
Human Gene Therapy
Pharmaceutical Products
Evolutionary relationships
How does Biotechnology Work?
Scientists must be able to manipulate
DNA to serve various purposes.
• Manipulation may include:
-
Gene identification
Cutting DNA
Amplifying/Copying DNA segments
Separating and visualizing DNA fragments
Inserting DNA into living cells
…..so how do we do this???
Cutting DNA!
• Restriction Enzymes:
– Enzymes that separate (“cut”) the DNA
backbone (sugar-phosphate bond) between very
specific DNA bases
• Each RE has its own recognition sequence
– Enable scientists to be very specific in the sequences they
cut (specificity)
– Uses: Disease diagnosis, DNA profiling, Transgenic
organisms, etc.
Diagnosis of Disease: a mutation within a recognition sequence can
be used to identify the presence of a disordered allele
How do we see DNA?
• DNA is colorless and tiny!
• Gel electrophoresis is a technique that uses electricity
and a sponge-like substrate to separate DNA fragments
by size
– DNA is negatively charged…moves towards positive electrical pole
– Large DNA fragments travel slower than small fragments
Steps to genetic marker analysis
using Gel Electrophoresis:
1. DNA is extracted from cells
2. DNA is cut into pieces using restriction enzymes
3. Digested DNA is put into a gel electrophoresis
apparatus
Video
Fig. 20-9a
TECHNIQUE
Mixture of
DNA molecules of
different
sizes
Power
source
Cathode +
– Anode
Gel
1
4. Electricity is applied.
DNA travels to + pole
• Smaller fragments
travel faster than
larger ones
Longer
Power
source
–
+
molecules
2
Shorter
molecules
5. Result is a banded pattern as DNA is sorted by charge & size
Another application of Biotechnology:
Forensic Profiling/DNA Fingerprinting using Gel
Electrophoresis
• Profiling: creating a unique DNA fingerprint/profile from
pieces of DNA
• GENETIC MARKERS: Single base differences and areas of
repeating bases; the regions of DNA that we use when
comparing DNA from 2 different people
How are the DNA fragments different??
• Humans are 99.9% genetically identical! But…there are
areas of DNA that differ from person to person (Genetic
Markers)
– Genetic markers can be single base differences
OR repeating sequences of DNA between active genes (i.e.
CCGCCGCCGCCG)
• Number of repeats for each locus varies from person to person =
polymorphic
• Restriction enzymes can cut DNA sequences that
flank the repeat regions
– When these regions are removed from the DNA, they
differ in length from one person to the other
•
Restriction fragments = pieces of DNA after cutting with restriction
enzymes = GENETIC MARKERS
DNA
GTA
GTA
GTA
GTA
GTA
GTA
GTA GTA
GTA
Cut with Restriction Enzyme
DNA
GTA
GTA
DNA
Cut with Restriction Enzyme
GTA
GTA
GTA
GTA
GTA
GTA
GTA
GTA
GTA GTA
GTA
DNA
Homologous
Chromosomes
DNA Profiling Review W-up
1.
2.
3.
4.
5.
6.
7.
8.
What percent of the DNA is identical from one human to the next?
What types of differences are found in human DNA?
How can we isolate/remove the variable regions from DNA?
Using the sequence ATGC, create a repeating DNA segment.
Pair up with a neighbor and write down the number of repeats for
both of your segments (i.e. 5,2)
Find a neighboring pair and write down their numbers.
Pretend that these numbers represent homologous chromosomes
from two parents…what are the possible combinations of their
offspring?
Create a DNA profile for both parents and one child by drawing a
“gel” and writing in bands to represent the restriction fragments.
Label the wells (mom, dad, child) and the (+) end of the gel.
Did OJ do it?
Read the short article regarding the OJ case.
Answer the questions in complete sentences
O. J. Simpson was a Hall of Fame football player
who became a running back for the Buffalo Bills
after completing a storied college career at
U.S.C. Other than his heroics on the field,
Simpson gained fame by starring in many major
motion pictures and in television commercials. In
June, 1994, Simpson was accused of murdering
his ex-wife, Nicole Brown Simpson, and her
companion, Ron Goldman.
At the trial which took place a year after the
deaths, DNA fingerprinting evidence was
presented for the first time in a major case.
The evidence was overwhelming and irrefutable,
yet the jury incomprehensibly came back with an
innocent verdict. It was apparent that the
prosecutorial attorneys made some errors in their
presentation of the DNA evidence. However,
subsequent cases have been very successfully
prosecuted in light of what was learned through
the Simpson trial.
(a) This photo shows Earl
Washington just before
his release in 2001,
after 17 years in prison.
In 1984 he was convicted
and sentenced to life in
prison for the 1982 rape
and murder of
Rebecca Williams.
Source of
sample
marker 1
marker 2
marker 3
Semen on victim
17, 19
13, 16
12, 12
Earl Washington
16, 18
14, 15
11, 12
Kenneth Tinsley
17, 19
13, 16
12, 12
(b) These and other genetic marker data by forensic scientists
(as part of The Innocence Project) exonerated Washington and
led Tinsley to plead guilty to the murder.
Scientists can artificially replicate DNA
• PCR = Polymerase Chain Reaction
– A method for amplifying small amounts of
specific segments of DNA
• Able to make lots of copies of a very specific
locus in the genome
• i.e. GFP gene in jellyfish
– The area of interest is identified and
isolated without the use of restriction
enzymes
An animated version of PCR
What are the ingredients for
DNA replication using PCR?
1. Original DNA
2. Heat (provided by a machine called a thermocycler)
– Denatures (splits) original, double stranded DNA; cools
for primers; warms for polymerase
3. Primers: Single stranded DNA that is complementary to
regions that flank the DNA of interest
– Allows for copying the DNA in that region only
4. DNA Polymerase: specifically heat resistant taq
polymerase
– base pairing to replicate original sequence
5. All 4 nucleotides (A, G, C, T)
– Remember, you’re copying DNA…so you need the pieces
to manufacture LOTS of new strands
STEPS TO PCR:
• 1. Heat to
Denature
(94°C)
• 2. Cool to
anneal primers
(54°C)
• 3.Elongation
(72°C)
– Adding new
nucleotides
using
polymerase
• 4. REPEAT
An animated version of PCR
Genetic Recombination
• Recombinant DNA: DNA from two different sources fused
together.
– Uses: gene cloning, transgenic organisms, pharmaceuticals, gene therapy
• Glowing Marmoset article
(a) Tobacco plant expressing
a firefly gene
(b) Pig expressing a
jellyfish gene
Creating Recombinant DNA
Requirements:
1. Gene of interest (Insulin, human growth hormone,
GFP, etc.)
2. Cloning Vector = object that will deliver the gene
of interest into a living cell
– Bacterial plasmid: small, circular DNA found outside
bacterial chromosome; inserts gene into prokaryotes
(bacteria)
– Virus: tissue and species specific; can insert genes into
plant, animal, or bacterial cells
3. Restriction enzymes (“Scissors”)
– Used to remove gene of interest from original genome
– Used to “open” vector so that gene of interest can be
inserted.
4. DNA ligase (“Glue”)
– attaches sugar phosphate backbones together to secure
gene of interest into vector
Use of Restriction Enzymes to Create “Sticky Ends”
Fig. 20-UN4
5
3
TCCATGAATTCTAAAGCGCTTATGAATTCACGGC
AGGTACTTAAGATTTCGCGAATACTTAAGTGCCG
Aardvark DNA
WHICH RESTRICTION
ENZYME SHOULD WE USE??
EcoRI
A
Plasmid
3
5
Fig. 20-UN7
Fig. 20-2a
Transformation: inserting recombinant DNA into a living cell
***Gives the cell the ability to make new proteins
Bacterium
1 Gene inserted into
Cell containing gene
of interest
plasmid
Bacterial
chromosome
Plasmid
Recombinant
DNA
Gene of
interest
2
2 Plasmid put into
bacterial cell
Recombinant
bacterium
DNA of
chromosome
Fig. 20-2b
Recombinant
bacterium
3 cell grown in culture
to form clones
containing the gene of interest
Protein expressed
by gene of interest
Gene of
Interest
Copies of gene
Protein harvested
4 Basic research and
Basic
research
on gene
Gene for pest
resistance
various applications
Gene alters
bacteria for cleaning
up waste
Protein dissolves
blood clots
Basic
research
on protein
Human growth hormone treats stunted
growth
Not all bacterial cells take up the
plasmid (become transformed)…
•
•
Ampicillin (Amp)
Resistance Gene
Some don’t encounter a plasmid
Others reject the plasmid because it increases
metabolic needs and decreases reproduction
The plasmid must be beneficial to a cell
while also allowing scientists to isolate those
with a plasmid and those without…
So, how do scientists isolate bacteria with
a plasmid & encourage bacteria to keep
the plasmid?
Answer:
Add a gene for antibiotic resistance to
plasmid
Grow the bacteria on antibiotic agar
• Those that didn’t absorb the plasmid will
die
• Those that did take the plasmid have to
keep it to survive
Recombination Warm-up 2/29
Plasmid
Bacteria with circular ________
1. Recombination requires ________
Gene
Interest
and eukaryotic ______
of ________.
Plasmid
2. Remove ________
from bacterium
Plasmid
Gene
3. Cut ________
and remove ________
of interest using
Restriction Enzyme
same ________
_________
Restriction Enzyme
4. Cutting with ___________
__________ creates
Sticky
________ ends
Plasmid
Gene
5. Mix cut _________
and __________
ligase
plasmid
6. Use ________
to glue gene into ____________
Plasmid
7. Mix recombinant ___________
with new bacteria
growing
8. Clone gene by ________
bacteria with plasmid on
nutrient agar
9. Isolate transformed bacteria using antibiotics (EXPLAIN
WHY ANTIBIOTIC RESISTANCE IS IMPORTANT IN
IDENTIFYING TRANSFORMED BACTERIA)
Why use antibiotic resistance?
Resistance gives bacteria a survival edge in antibiotic rich environments
Bacteria not exposed to plasmid
= no protein production
Bacteria get rid of
Bacteria w/
plasmid = no
plasmid = make
protein
desired protein
Two Applications of
Recombination…
Green Fluorescent Protein
Img Src: http://icbxs.ethz.ch/members/leu/jellyfish.gif ,
http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Jellyfish.jpeg
Img Src: http://www.biolum.org/
This sleeping baby monkey expresses GFP
with the gene for the mutant Huntington protein.
These animals are being used by researchers to study Huntington’s disease.
Credit: Anthony Chan, Emory University
And more neato pictures
brought to you by GFP:
Img Src: http://www.bio.umass.edu/microscopy/images/gfp.jpg
genetik.fu-berlin.de/ institut/en_GFP_fly3.jpg
Img Src:
http://www.mshri.on.ca/nagy/graphics/GFP%2
0mice.jpg
Img Src: http://www.computerra.ru/pubimages/73944.jpg
Img Src: http://www.antville.org/img/pop/gfp.jpg
Overall Goal of Lab Experiment

Use genetic engineering techniques to
insert the GFP gene into E. coli
GFP (Protein we want to
produce)
Amp Resistance
Plasmid
containing gene
of interest
Transformation Lab
1. What does it mean when a bacterial cell
is competent?
2. How will we make the E.coli competent?
3. What are the three important genes on
our recombinant plasmids?
4. How many microcentrifuge tubes will you
have? What goes in each tube?
5. How many agar plates will you have?
What will be in the agar?
Gene for
antibiotic
resistance
Pre-lab
1.
Identify the materials on
your desk: pipette,
microcentrifuge tube,
inoculating loop, agar plate
Step through the
procedure as a class
2.
-
Practice using 1mL pipette
by obtaining .25mL of water
from the beaker and
transferring it to the
microcentrifuge tube
= 0.25 ml
Pipette
Gene Therapy
The use of a vector (usually a virus) to insert a working
gene into a cell with a defective version of that gene
1. engineer virus to contain healthy gene
2. Infect patient’s bone marrow/stem cells in lab
3. Inject recombinant stem cells into patient’s bone
marrow.
– Still in its trial stages, but holds promise for treating
various genetic disorders
– Many setbacks including patient deaths due to severe
immune response to viral vectors
Even more biotechnology…
Cloning!!
• Clone: exact copy of the
original
• How do we clone genes?
– Recombinant DNA and
transformation
– PCR
How do we clone cells & tissues?
• Therapeutic cloning of stem
cells
– Stem cells (embryonic, adult, or iPS)
are coaxed into various cell types using
combinations of “ingredients”
– These cells can then be grown
together to form tissues
– Layering tissues in the correct pattern
will form cloned organs
Some current methods being
tested for engineering organs…
• Bio rubber scaffolding
• “washing” cadaver organs for
scaffolding
• Ink jet organs
How do we clone an
organism (aka
reproductive cloning)?
• Plants:
– Specialized cells can
dedifferentiate &
give rise to all the cell
types of a mature
plant = Clone
• Because of this, plant
cells are totipotent!
Reproductive cloning of animals
Mature animals do not have totipotent cells =
Specialized cells cannot develop into
new organisms
• Cloning is achieved through nuclear
transplantation replace the nucleus
of an unfertilized egg with a nucleus
from a specialized cell
= CLONE
Dolly: first successfully
cloned mammal via nuclear
transplantation
Cloning the Woolly Mammoth
Review Below
Biotechnology HW Packet
1. DNA Fingerprinting Webquest
2. Restriction Enzyme Practice
3. All Biotechnology Notes (pictures,
handwritten notes, Restriction
Enzymes and Recombination notes,
Cloning/Stem cells notes/pictures)
4. Biotechnology Review Worksheet
STOP 09-10
Some Vocabulary to Help
Understand the Science of Cloning
Specialized vs. Unspecialized Cells
• All cells have the same DNA
• All multicellular organisms begin as a group
of unspecialized cells = stem cells
– No particular function
• Differences in cell types arise during
development (embryo)
• Different cell types result from different
gene expression as an organism develops
Gel Electrophoresis is often followed by a
technique called Southern Blotting
Southern blotting attaches radioactive probes to the DNA so that it can
be seen using x-ray technology.
Why???
2 Reasons:
1. DNA digestion with Restriction enzymes produces LOTS of fragments
-
A probe will only attach to complementary fragments
Scientists select probes that complement fragments of interest (easier to find the
proverbial “needle in the haystack”)
2. DNA is colorless
-
X-ray allows visual image
Gel Electrophoresis and
Southern Blotting to find gene
of interest
Add
probes
(stick to
1-2
specific
places)
Transfer of DNA to a
nylon sheet
Voila: DNA F.P.
THE ENTIRE TECHNIQUE: GEL ELECTROPHORESIS & SOUTHERN BLOTTING
DNA + restriction enzyme
Restriction
fragments
I
II
III
Heavy
weight
Nitrocellulose
membrane (blot)
Gel
Sponge
I Normal II Sickle-cell III Heterozygote
-globin allele
allele
2 Gel electrophoresis
1 Preparation of restriction fragments
Paper
towels
Alkaline
solution
3 DNA transfer (blotting)
Radioactively labeled
probe for -globin gene
Southern Blotting animation
I
II III
Probe base-pairs
with fragments
Fragment from
sickle-cell
-globin allele
Nitrocellulose blot
Fragment from
normal -globin
allele
4 Hybridization with radioactive probe
I
II III
Film
over
blot
5 Probe detection
Cloning Warm-up
On a piece of paper
1. What determines what you are?
2. In your own words, define clone.
3. Describe what you know about the
science of cloning.
4. If you could clone any living
organism, would you? If not,
explain. If so, what would you
clone and why?
5. What are the ethical considerations
surrounding cloning?
Warm Up: Intro to DNA
Fingerprinting
Read about the first case in
the world that used DNA
evidence to convict and
sentence a man to death!
Answer the 3 questions on
the ½ sheet using
complete sentences…
1. Who is the likely suspect that
assaulted the woman in this case?
2. What are the odds that someone
else did it?
3. What do you think the bands
represent?
Steps for using DNA as forensic evidence
1. Collect the DNA:
• at the crime scene
• from the suspect
2. Analyze the DNA to create
DNA fingerprints (aka
DNA profiles)
3. Compare the profiles to
each other
- CODIS
How do we analyze the DNA
after it is cut?
Answer: Gel Electrophoresis!
A method of separating digested DNA
based on its charge and fragment sizes
• DNA is negatively charged
• Restriction enzymes can cut DNA in
very specific regions which creates
fragments of DNA
Recap: Using bacterial plasmids
allows us to:
1.
Mass produce useful
proteins
–
–
–
Insulin
HGH (Human Growth
Hormone)
TPA (Tissue Plasminogen
Activator)
Desired
Protein
TPA
HGH
2. Study/use gene itself
–
–
–
Genetically modify plants /
bacteria
Clone/make many gene
copies
Study cloned genes
GM pest
GM oil eating
Resistant plants bacteria
Gene Therapy Technique Slows Brain Disease ALD
Featured In Movie 'Lorenzo's Oil'
•Cartier and her colleagues took blood
stem cells from two 7-year-old boys
with ALD, infected the cells with a
virus carrying a correct copy of the
defective gene, then re-injected the
stem cells.
•The boys' symptoms stabilized within
14 months and have not worsened
since.
Remember the gene determines the amino acid
sequence and the amino acid sequence
determines the shape of the protein and the
shape of the protein determines its function!
•
•
http://www.sciencedaily.com/releases/200
http://www.newscientist.com/article/dn18139-boys-with-ald-bring-gene-therapy-in-from-cold.html9/11/091105143706.htm
Therapeutic Cloning:
Creating cells for
medical use, not to
develop an entire
organism (a.k.a.
Stem Cells)
Most common
sources of stem cells
for cloning:
- embryonic cells
- adult cells
New Advances in Stem Cells
• iPS = induced Pluripotent Stem Cells
(Pluripotent = ability to become any cells in
the body)
– Use viruses to insert developmental genes
into existing skin cells
• Genes “transform” skin cells into pluripotent
cells
• Importance:
– Utilize “own” cells for research
– Bypass embryonic stem cell controversy
Your 9 year old daughter has been battling
leukemia for many years and is not
responding to treatment. The doctors
suggest a new therapy where they would
infect your daughter with a deactivated
HIV strain that might cure her cancer.
What do you do?
a) Find another doctor, these medical professionals are
out of their mind.
b) Try the new treatment and hope that the HIV strain
cures the cancer and does not mutate into a disease
causing virus.
c) Do nothing, she has gone through enough treatments
already.
Fire with fire clip