Download DNA Fingerprinting

Biotechnology
1
Biotechnology
• The manipulation (as through genetic
engineering) of living organisms or their
components to produce useful usually
commercial products (as pest resistant crops,
new bacterial strains, or novel
pharmaceuticals)
• Examples: DNA Fingerprinting, Genetic
Engineering, Cloning, and Stem Cells
Biotechnology Topics
• DNA Fingerprinting
• Stem Cells
• Genetic Engineering to make Transgenic
organisms
• Cloning (both reproductive and
therapeutic)
DNA Fingerprinting Technique
• a technique used especially for identification (as
for forensic purposes) by extracting and
identifying the base-pair pattern in an individual's
DNA —called also DNA typing
• Uses:
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–
–
–
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help solve crimes
determine paternity
to locate gene segments that cause genetic diseases
to map the genetic material of humans
to engineer drought-resistant plants to produce
biological drugs from genetically altered cells.
Restriction
Enzymes
Section 13-2
Recognition
sequences
DNA
sequence
Restriction
enzyme EcoRI
cuts the DNA
into fragments.
Sticky end
Section 13-2
Figure 13-6 Gel
Electrophoresis
DNA plus
restriction
enzyme
Power
source
Longer
fragments
Shorter
fragments
Mixture of
DNA
fragments
Go to
Section:
Gel
Steps to DNA Fingerprinting
1. Extraction
DNA is extracted from cells by breaking down
the cell membrane, nuclear membrane, and
releasing DNA from histones (proteins that
keep DNA wound up).
Steps to DNA Fingerprinting
2. Amplification
Use of polymerase chain reaction (PCR) to
make copies of DNA; multiple copies of a
small amount of DNA make analysis possible
without destroying the evidence.
PCR
• Polymerase Chain Reaction: process in which
multiple copies of DNA are made from a very
small sample
Figure 13-8 PCR
DNA polymerase adds
complementary
strand
Section 13-2
DNA heated
to separate
strands
DNA
fragment to
be copied
PCR
cycles
1
Go to
Section:
DNA
copies
1
2
3
2
4
4
5 etc.
8
16 etc.
Steps to DNA Fingerprinting
3. Tandem Repeats are Identified
DNA is cut at specific locations by restriction
enzymes; segments are called restriction
fragments
Example: A restriction enzyme called HindIII
recognizes the sequence AACGTT and will make
a cut between A/A therefore all A/ACGTT will be
cut. These restriction fragments are tandem
repeats. Different people have a different
number of repeats.
Steps to DNA Fingerprinting
4. Electrophoresis
DNA is loaded into a machine that uses
electricity to move DNA from one side to the
other. Fragments are separated by size.
Smaller segments move faster than larges
segments
Electrophoresis & Paternity
• When electrophoresis is used for paternity
testing, a child should have ½ bands match
one parent and ½ bands match the other
parent.
• Read the caption by Figure 13-5 and look at the example.
Note: restriction enzymes work in both directions at the
same time. Now let’s practice using the DNA sequence
below. You will be EcoRI, the restriction enzyme that looks
for CTTAAG. When it finds this sequence, it cuts between the
last A and G. Use your pencil to show these cuts.
DNA Sample 1
C T T A A G C T T A A G C T T A A G A A G C T T A A G G G C
G AA T T C G AA T T C G AA T T C T T C G AA T T C C C G
DNA Sample 2
C T T A A G T A C T A C C T T A A G C T T A A G C T T A A G
G AA T T C A T G A T G GA AT T C G A A T T C G A A T T C
Practice, Practice, Practice!
• 1st- practice using the pipette
Pipette water with dye both 5
µL and 10 µL
• 2nd – practice loading the gel with
Load Gel with 25 µL
Gel Electrophoresis Lab
•
•
•
•
•
•
Load 6 Wells with DNA samples (A-F)
35 µL in each well
Treat equipment gingerly!
Run at 150 V
20 minute run
Afterward- gently lift gel tray out and slide gel
onto white laminated sheet. Record results in
lab (either take picture or draw immediately)
3. Refer to your cut DNA from question 2. How many pieces of DNA do you end up with?
Hint: The DNA in Figure 13-5 is cut into three pieces. These pieces are called DNA
Fragments.
DNA Sample 1: _________ DNA Sample 2: ___________
Separating DNA
1. Fill in the blanks to outline the steps for gel electrophoresis

A mixture of DNA ________________________ are places at one end of a porous gel.

An _________________ __________________ is applied to the gel.

When the power is turned on, DNA molecules (fragments) which are

__________________ charged move toward the _______________ end of the gel.
The ________________ fragments move faster.
Now let’s practice. I need you to copy the DNA fragments from the first page onto into
the space below. Leave some space between fragments where you cut. I know it will be
annoying to flip back and forth. Maybe you can ask for some help. Just do it!
DNA Sample 1
DNA Sample 2
Now I want you to count the base pairs in each fragment
(do not include the extra bases, only the paired bases)
and write that number under the fragment. Let’s load it
into the gel electrophoresis machine. In lane 1 will be
DNA Sample 1 and lane 2 will be DNA Sample 2. For each
sample, shade in the box beside the corresponding
number of base pairs. Remember, shorter segments
travel further.
Length of DNA Sample 1
Fragment
11
10
9
8
7
6
5
4
3
2
1
DNA Sample 2
Use the DNA Fingerprint below (which is just like yours but turned on its side) to determine which suspect’s
DNA matches the DNA found at the crime scene. By the way, a Ladder is a DNA standard of known fragment
lengths. It is used as a control for comparison.
Which suspect’s DNA is an exact match with the Crime Scene DNA? ____________________
Which suspect had DNA fragments that were smallest? _____________________
Which suspect had DNA fragments that were longest? ______________________
Sample Gel Results
Applications of Genetic Engineering
• Genetic Engineering involves transferring DNA
sequences (whole genes) from one organism
to another.
• One of the techniques used is called
Transformation (we will be doing this in class
next week)
• The result of genetic engineering is a
transgenic organism (Genetically Modified
Organism or GMO)
Transgenic or GMO
(Genetically Modified Organisms)
• Genetically Modified Microorganisms
• Genetically Modified Plants
• Genetically Modified Animals
Examples of Genetic Modification
Medical
Using bacteria to make
insulin for diabetic
humans.
1. Provides needed human 1. Expensive. Research to
proteins using transgenic develop takes a long time.
bacteria.
Foods
Corn that has been
genetically changed to
resist insects.
1. Farmers will not have to 1. We don’t know long
spray chemical insecticides
term effects of eating
on crops.
transgenic foods
2. People with allergies
cannot be sure if they
will have a reaction.
Animals
1. Provides easy treatment 1. Expensive. About 4
Sheep that include a
to humans who are missing
million dollars to
human gene which
essential proteins.
produce one transgenic
produces proteins that can
sheep
be used by humans as
2. Unsure of side effects.
medicine.
Genetic Transformation
The Genetic Creation of Glowing Bacteria
Genetic Transformation: Change caused by genes; inserting the
genes from one organism into another organism
Green Fluorescent Protein (GFP):
The protein created in the jellyfish when the gene for
bioluminescence (glowing) is expressed
Bioluminescence:
is the production and emission of light by a living organism
Antibiotic: drug that kills or prevents the growth of bacteria
Ampicillin: A type of antibiotic that destroys E.Coli under
normal conditions
pGLO: trade name for plasmid that contains recombinant DNA.
(contains GFP gene, Ampicillin Resistance gene)
You will recognize the flow chart below which represents protein synthesis (gene
expression). Complete flow chart #1 and then use this information to complete flow chart #2.
1. DNA → RNA → protein → Genetic Characteristic (Trait)
2. DNA → RNA →
GFP
→ bioluminescence (glowing)
Arabinose: Sugar that “turns on” the GFP gene
Materials
7 sterile loops
6 sterile pipettes
2 small collection tubes
1 floating foam
1 sharpie
Ice bath
Incubator (37^C)
1 petri dish (LB)
2 petri dishes (LB/Amp)
1 petri dish (LB/Amp/Ara)
antibacterial soap
masking tape
Warm water bath (42^C)
Safety
•Wash hands thoroughly before and after lab.
•Wash hands immediately after any handling of the bacteria.
•Report any spills to the teacher immediately.
•Dispose of all contaminated loops, pipettes, and collection tubes in the
designated trash cans.
•For additional information about E.Coli refer to the supplemental poster.
Plasmid:
Circular piece of DNA in a bacterial cell; can be
passed from one bacteria to another easily;
usually contains genes for traits beneficial to
survival.
Predictions
Four Petri dishes will be used to grow bacteria under different
conditions. There will be abbreviations on each Petri dish to help explain
the conditions under which the bacteria will be grown.
What does each abbreviation mean?
•LB: Luria Broth (bacteria food)
•Amp: Ampicillin (antibiotic that destroys E.Coli)
•Ara: Arabanose (sugar that turns GFP gene on)
•+pGLO: Contains GFP gene and Antibiotic Resistance Gene
•-pGLO: Does not contain “new” genes; regular bacteria
Petri Dish
+pGlo/LB/Amp
+pGlo/LB/Amp/Ara
-pGlo/LB/Amp
-pGLO/LB
Explanation of
Conditions (Petri Dish
Contents)
*GFP & Antibiotic
Resistance Gene
*Luria Broth
(bacteria food)
*Ampicillin
(antibiotic)
* GFP & Antibiotic
Resistance Gene
* Luria Broth
(bacteria food)
* Ampicillin
(antibiotic)
* Arabanose
* No new genes
* Luria Broth
(bacterial food)
* Ampicillin
(antibiotic)
* No new genes
* Luria Broth
(bacterial food)
Hypothesize growth on Petri
dishes
Observation of Petri dishes
after several days
Grow? Glow?
Grow? Glow?
Section 13-3
Figure 13-9 Making Recombinant DNA
Recombinant
DNA
Gene for
human growth
hormone
Human
Cell
Bacterial Cell
Sticky
ends
DNA
recombination
DNA
insertion
Bacterial
chromosome
Plasmid
Gene for human
growth
hormone
Bacterial cell for
containing gene
for human
growth hormone
Section 13-3
Figure 13-9 Making Recombinant DNA
Recombinant
DNA
Gene for
human growth
hormone
Human
Cell
Bacterial Cell
Sticky
ends
DNA
recombination
DNA
insertion
Bacterial
chromosome
Plasmid
Gene for human
growth
hormone
Bacterial cell for
containing gene
for human
growth hormone
February 28th 1953
The Eagle Pub in
Cambridge,
England
Tuesday
• Stem Cell Discussion
• Review for Biotechnology quiz
• Biotechnology Test Tomorrow
• GATTACA – rest of week
CLONING
SCIENCE FICTION OR REAL????
DO YOU KNOW A CLONE?
• The term clone simply means “genetically
identical”
• Yes, there are many examples of naturally
occurring biological clones
CLONING
• TWO TYPES OF CLONING
• FIRST: REPRODUCTIVE CLONING – DOLLY THE
SHEEP
• The world's first clone -- a tadpole -- was
actually created in 1952
• And at least a dozen different species have
been cloned since: everything from the
common cow to the endangered guar, a type
of wild ox.
Section 13-4
Figure 13-13 Cloning
of the First Mammal
A donor cell is
taken from a
sheep’s udder.
Donor
Nucleus
These two cells are
fused using an
electric shock.
Fused Cell
An egg cell is
taken from an Egg Cell
adult female
sheep.
The nucleus of
the egg cell is
removed.
The embryo develops
normally into a
lamb—Dolly
The fused cell
begins
dividing
normally.
Embryo
Cloned Lamb
Foster Mother
The embryo is
placed in the
uterus of a
foster mother.
USES
• In 2001 FDA requested livestock producers and
researchers to keep food from animal clones or
their offspring out of the food supply.
• Since then, FDA has conducted an intensive
evaluation that included examining the safety of
food from these animals and the risk to animal
health.
• January 2008, FDA has concluded that meat and
milk from cow, pig, and goat clones and the
offspring of any animal clones are as safe as food
we eat every day.
• Cloning has additional uses besides its ability
to help farmers breed consistently top-notch
burgers and bacon. Other potential
applications include the preservation of
species, biomedical research, drug and organ
production and even commercial ventures
that aim to keep little Fido (or at least a
convincing substitute) in the family forever.
THERAPEUTIC CLONING AND STEM
CELLS
• Cloning designed as therapy for a disease.
• In therapeutic cloning, the nucleus of a cell,
typically a skin cell, is inserted into a fertilized
egg whose nucleus has been removed.
• The nucleated egg begins to divide repeatedly
to form a blastocyst.
• Scientists then extract stem cells from the
blastocyst and use them to grow cells that are
a perfect genetic match for the patient. The
cells created via therapeutic cloning can then
be transplanted into the patient to treat a
disease from which the patient suffers
Types of Stem Cells:
Totipotent- can give
rise to any type of
tissue
Pluripotent – can give
rise to all types except
germ cell (reproductive
cells)
Multipotent- can give
rise to only a few cell
types
Stem Cell- A
cell that can
continuously
divide and
differentiate
into various
types of tissue
Population Genetics & DNA Databases
• Percentages of a population that have a certain allele (DNA
sequence) have been determined and entered into
databases; the greater the tested population the more
accurate the results
• These percentages are used to determine the probability of
an allele occurring
• Probability Rule is used to determine the probability of a
group of alleles occurring at the same time.
• Same probability rule as blood typing:
– Example: In United States:
• % Type O = 43%
• % Rh+ = 85%
• Probability of O+ = 36.55%
Sources of DNA
•
•
•
•
•
Saliva (envelope/bite wound/toothbrush)
Seminal Fluid
Skin
Hair Follicle
Coming Soon…Fingerprints!
DNA and Probes
• Specific Tandem Repeats can be identified after
electrophoresis by using radioactive probes (molecules
that attach to certain sequences)
Example: If trying to identify AAGCTTA then probe is a
synthetic sequence of TTCGAAT
• If probes contain fluorescent dyes, the tandem repeats
will glow under ultraviolet light
• If probes contain radioactive isotopes, x-ray film is used
to create an image of fingerprint called an
autoradiograph
Where’s the Cat: DNA Fingerprinting
Materials Used in DNA Fingerprinting
State the Function
Gel
Filters DNA (Restriction Fragments) by
size; smaller fragments go further
Restriction Enzymes
Cuts DNA into restriction fragments at
certain locations (example: Restriction
Enzyme HindIII cuts at AAGCTT
Electric Current
Pulls DNA through gel; DNA is negative so
it “runs to red”
VNTR
Repeating sequence (polymorphism);
Different number of VNTRs represent
different individuals
Radioactive Probes
Binds to specific DNA sequences after
electrophoresis so that we can see them
because DNA is transparent
DNA Fingerprinting: Practice
•
•
DNA Fingerprinting Pre-Lab
Practice with Tools
– Calibrate Pipette to 5µL
– Pipette 5µL of colored water onto white paper
– Calibrate Pipette to 10µL
– Pipette 10µL of colored water onto white paper
– Place Gel Tray into Electrophoresis Machine
– Cover Gel Tray with Buffer Solution
– Add Gel to Tray (wells on the negative end)
– Add more Buffer to just cover Gel
– Obtain 10µL of DNA sample and place into gel wells
•
•
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Crime Scene
Victim
Suspect 1
Suspect 2
Suspect 3
Suspect 4
– Plug Machine into Power Source (150V)