DNA Extraction
What is DNA?
• DNA (deoxyribonucleic acid) is one of four
major families of organic molecules in the
cell.
• DNA’s primary structure consists of a
sugar/phosphate backbone to which
nitrogenous bases are attached. The
bases are either Purine or Pyrimidine
ring’s.
• It serves as the “master copy” for most
information in a cell.
Sugar/Phosphate
Backbone
Purine Structures
Pyrimidine Structures
Discovery of DNA
• In the late nineteenth century, a German
biochemist discovered the long-chain
polymers of nucleotides were made up of
sugar, phosphate, and nitrogen bases.
• Determined that the sugar in nucleic acids
can be of two forms…ribose (RNA) or
deoxyribose (DNA).
• In 1943, American Oswald Avery proves
that DNA carries genetic information.
Discovery of DNA,
cont.
• In the early1950’s, Cambridge University
graduate student Francis Crick and
research fellow James Watson became
interested in the work of Linus Pauling.
• Watson and Crick were interested in
Pauling’s 1948 discovery that many
proteins take the shape of an alpha
helix…in other words, they spiraled like a
spring coil.
Discovery of DNA,
cont.
• At King’s College in London, DNA
pioneers Maurice Wilkins and Rosalind
Franklin were using x-ray diffraction
images to look at DNA.
• Also in 1950, biochemist Erwin Chargaff
found that the arrangement of nitrogen
bases in DNA varied widely, but the
amount of certain bases always occurred
in a one-to-one ratio.
Discovery of DNA,
cont.
• Chargaff’s and Pauling’s discoveries
would prove to be an important foundation
later for the description of DNA.
• In 1953, without the consent of Franklin,
Wilkins showed Franklin’s results to
Watson and Crick.
• Based on that information, Watson and
Crick suggested the DNA molecule was
made of two chains of nucleotides.
Discovery of DNA,
cont.
• Watson and Crick showed that each
strand of the DNA molecule was in a helix
configuration, but one was going up and
the other going down.
• They also showed that each strand was a
template for the other.
Discovery of DNA,
cont.
• Franklin passed away in 1958.
• Watson, Crick, and Wilkins were awarded
the Nobel Prize in 1962.
• The Nobel Prize is only awarded to living
persons.
• DNA’s discovery has been called the most
important biological work of the last 100
years.
DNA Extraction
• DNA can be extracted from any living
thing.
• Procedures for DNA extraction vary from
simple experiments that can be performed
at home, to extensive experiments that
require the use of a laboratory.
• The procedure used is outlined below.
DNA Extraction using
Wheat Germ
• The basic procedure is to lyse the wheat
germ cells rupturing the cell walls,
membranes and nuclear membrane if
there is one.
• Carefully read the instructions prior to
beginning the DNA extraction.
Materials
• The following materials/supplies are
needed to achieve DNA extraction:
• Raw Wheat Germ
• Warm water bath (50°- 60°C)
• Meat tenderizer
• Salt (NaCl)
• Sodium bicarbonate solution
• Liquid detergent such as Woolite™
Materials, cont.
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250mL beaker
Cheese cloth or coffee filter
Thermometer
Pipet
Scale
95% Ethanol or 100% Isopropanol alcohol
Centrifuge
Gloves
DNA Extraction
Procedure
• Prior to beginning the extraction, prepare
the warm water bath (50°- 60°C).
DNA Extraction
Procedure, cont.
• PUT ON GLOVES! Failure to wear gloves
will allow your DNA to contaminate the
DNA of the Wheat Germ!
• Place 45mL of tap water in a beaker and
place it in the warm water bath.
• Use the scale to measure 2g wheat germ.
• Sprinkle the wheat germ into the prewarmed water.
DNA Extraction
Procedure, cont.
• Record the exact temperature of the
water.
• The optimal temperature for DNA
extraction is 55°- 60°C.
• DO NOT ALLOW THE TEMPERATURE
TO RISE ABOVE 60°C.
• Add 2-5mL of detergent and stir gently
trying to create as few bubbles as
possible.
DNA Extraction
Procedure, cont.
• Continue to stir the mixture for an
additional 5 minutes remembering to keep
the temperature below 60°C.
• After 5 minutes, gently stir in 2g of meat
tenderizer.
• Add 5mL of sodium bicarbonate solution
and continue to stir and heat for an
additional 5-15 minutes.
DNA Extraction
Procedure, cont.
• Immediately cool in ice bath to room
temperature.
• Filter through cheesecloth or coffee filter.
DNA Extraction
Procedure, cont.
• After filtration, place approximately 5mL of
the filtrate to a test tube.
• Next, pipette 5mL ice-cold alcohol down
the side of the test-tube making sure the
alcohol doesn’t mix with the filtrate.
• 2 distinct layers should be visible.
• Spool the DNA from the filtrate.
DNA Extraction,
Additional Procedures
• 3 additional extraction procedures were
conducted.
• Each was carried out using the procedure
explained above with the following
exceptions:
• 1. No heat was used.
• 2. Heating time was reduced to 5
minutes.
• 3. Heating time was increased to 25
minutes.
Results of DNA Extraction
using Wheat Germ
• Results were inconclusive when DNA
extraction was attempted without the
benefit of heat.
• Heating the wheat germ as specified in
the original procedure (15 minutes)
proved to be the most successful.
• Extremely fragmented DNA occurred
when solution was heated for 25 minutes.
Results of DNA Extraction
using Wheat Germ
• DNA was easily extracted, but
isolating the DNA from the filtrate
proved to be the biggest obstacle.
• Sterile cotton swabs and sterile loops
were used with little success.
DNA Extraction with
Green Peas
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The following materials are needed:
Fresh or frozen Green peas
Blender
Liquid detergent, such as Woolite™
Coffee filter
Meat tenderizer
70-95% rubbing alcohol or ethyl alcohol
250mL beaker
Test tubes
DNA Extraction
Procedures
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Place the following items in a blender:
½ cup of green peas.
1/8 tsp of salt.
1 cup of cold water.
Blend on high for 15 seconds.
Strain through coffee filter into 250mL
beaker and add 2 tablespoons of liquid
detergent.
DNA Extraction
Procedures, cont.
• Allow mixture to sit for 5-10 minutes.
• Pour mixture into test tubes, add a small
pinch of meat tenderizer and stir gently.
• Gently pour an amount of alcohol equal to
the amount of filtrate into the test tubes.
• DNA will appear in the alcohol layer.
Results of DNA Extraction
using Green Peas
• All attempts to extract DNA using green
peas were unsuccessful.
• DNA extraction was never observed.
• Procedure altered in the following way:
• Peas were crushed instead of blended.
• Peas were blended less than 15 seconds.
• Peas were blended for 30 seconds.
• The mixture was heated for 15 minutes
and cooled to room temperature.
DNA Detectives Lab
• The DNA Detectives Lab uses restriction
enzymes to isolate specific fragments of
DNA.
• The exact number and size of fragments
produced by a specific restriction enzyme
vary from person to person.
• Restriction enzymes are used to “cut”
DNA molecules internally (endo) or on the
ends (exo).
DNA Detectives Lab,
cont.
• Hind III recognizes the sequence
AAGCTT and cuts the DNA at different
points of the strands resulting in a
staggered cut (endo).
• Pvu II cuts through both strands of DNA
resulting in a clean cut, leaving blunt ends
(exo).
DNA Detectives Lab,
cont.
• Examples of Restriction Enzymes:
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BamH I
EcoR I
Hae III
Hind III Pvu II - (used in the DNA Detectives Lab)
DNA Detectives Lab,
cont.
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Materials needed:
0.8% Agarose
5X TBE running buffer
Quickview DNA stain concentrate
Microfuge tubes
Staining trays
Loading dye
Lambda DNA/Hind III marker
DNA Detectives Lab
Materials, cont.
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4 suspect DNA samples
2 Pvu II restriction enzyme
2 Pvu II reaction buffer 10X
Electrophoresis chamber
Power supply
Micropipettes
Permanent marker
Distilled water
DNA Detectives Lab
Materials, cont.
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Microwave oven
37°C water bath
65°C water bath
Duct tape
Gloves
Ice bath for sample tubes
Petroleum ether for rinsing micropipette
Test tube rack
9-Volt Batteries
DNA Detectives Lab
Procedures
• Remove DNA sample kit from freezer and
allow samples to thaw.
• Label 5 microfuge tubes as follows:
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Crime scene
Suspect 1
Suspect 2
Suspect 3
Suspect 4
DNA Detectives Lab
Procedures, cont.
• Using a 10µl micropipette, pipette 10µl of
the Crime scene DNA into the microfuge
tube labeled “Crime scene.”
• Rinse the micropipette with petroleum
ether and add 2µl Pvu II reaction buffer
10X.
• Rinse the micropipette with petroleum
ether and add 2µl Pvu II restriction
enzyme.
• Repeat these steps for Suspects 1- 4
using the same procedure outlined above
DNA Detectives Lab
Procedures, cont.
• Incubate all tubes at 37°C for 1 hour.
• Cast Agarose Gel during this time if not
prepared ahead of time.
DNA Detectives Lab
Casting Agarose Gel
• Place bottle of 0.8% Agarose Gel in
microwave oven and microwave on high
at 30 second increments until gel is
melted. (usually one 30 second cycle)
• HANDLE WITH CAUTION…GEL IS
EXTREMELY HOT!
• Use duct tape as end dams for the gel
casting tray making sure tape securely
adheres to the tray.
DNA Detectives Lab
Casting Agarose Gel
• Insert the gel comb at the end of the gel
tray.
• Place the gel tray on the countertop and
add melted Agarose Gel until it’s just
below the bottom portion of the solid
comb.
• Allow the gel to cool and solidify which
takes approximately 30 minutes.
DNA Detectives Lab
Procedures, cont.
• Prepare the Dilute Stain while the gel is
cooling.
• Add 5ml of DNA stain concentrate to 95ml
warm (50-55°C) distilled water.
• Set solution aside, but maintain
temperature.
DNA Detectives Lab
Procedures, cont.
• After all tubes have incubated for 1 hour,
and the Agarose Gel has cooled, add 2µl
of loading dye to each tube.
• Incubate all tubes for 5 minutes at 65°C.
• This incubation period along with the
loading dye stops enzyme activity from
occurring.
• If enzyme activity is not stopped, the
enzymes will continue to degrade the DNA
causing smaller fragments.
DNA Detectives Lab
Procedures, cont.
• While the tubes are incubating at 65°C,
wear gloves and remove the duct tape
from the ends of the Agarose Gel tray.
• If gloves aren’t worn, your DNA could
contaminate the Agarose Gel leading to
false/inaccurate results.
• Place the gel tray into the chamber and
slowly add 350ml of 1X TBE running
buffer.
DNA Detectives Lab
Procedures, cont.
• Add buffer to one end of the chamber until
that chamber is filled.
• Repeat this procedure with the other
chamber.
• Once both chambers are filled, slowly
continue adding buffer until the Agarose
Gel is completely covered.
DNA Detectives Lab
Procedures, cont.
• Use a 50µl pipette and add the following
to the Agarose Gel:
– Lane 1: 10µl Lambda DNA/Hind III
– Lane 2: 15µl Crime Scene DNA
– Lane 3: 15µl Suspect 1 DNA
– Lane 4: 15µl Suspect 2 DNA
– Lane 5: 15µl Suspect 3 DNA
– Lane 6: 15µl Suspect 4 DNA
DNA Detectives Lab
Procedures, cont.
• When loading the DNA to the Agarose
Gel, make sure the DNA isn’t injected into
the gel.
• Once all wells are loaded, gently slide the
cover into the chamber.
• Connect the 9-volt batteries to the cover –
no more than 75-125 volts.
DNA Detectives Lab
Procedures, cont.
• Monitor the movement of the DNA
samples. Pay attention to Lane 1.
• Lane 1 contains Hind III marker which
serves as an indicator.
• Hind III has a lighter molecular weight
than the DNA samples. Therefore, it will
run through the gel faster and reach the
end of the gel before the DNA samples.
DNA Detectives Lab
Procedures, cont.
• Disconnect the power supply, and remove
the cover.
• Gently pour the running buffer out of the
chamber.
• Wear gloves and remove the gel tray from
the chamber.
• Carefully slide the gel into a staining tray
and add stain solution until it covers the
gel.
DNA Detectives Lab
Procedures, cont.
• Cover the tray and allow the gel to stain
for approximately 30-40 minutes.
• Once the gel has finished staining,
carefully pour the stain out of the tray
ensuring that the gel remains flat, and
doesn’t slide into the corner of the tray.
• Add distilled water to the tray using care
not to pour water directly onto the gel.
DNA Detectives Lab
Procedures, cont.
• For best results, allow the gel to “Destain”
overnight.
• You may also expedite the destaining
process by gently rocking the tray.
• This process will take approximately 30
minutes and several water changes.
DNA Detectives Lab
Results
• After performing all steps necessary to
isolate the DNA,
perform gel electrophoresis, and complete
the destaining process, the banding
patterns were reviewed.
• The banding patterns indicate……
DNA Detectives Lab
Results, cont.
• Suspect 3 committed the crime!!!
PCR
PCR, cont
• PCR (Polymerase Chain Reaction) is
essentially a system for cell-free DNA
replication.
• PCR is repetitious…
• It involves subjecting DNA to repeated
cycles of high temperature and lowtemperatures for 30 cycles.
• Subjecting DNA to high temperature
causes denaturation.
PCR, cont
• Exposure to low temperature produces a
copy of the DNA.
• The DNA template doubles with each
cycle amplifying the DNA quantities.
• After 30 cycles, one DNA molecule
becomes a billion copies.
PCR, cont
• In short…the double-stranded DNA is
separated at high temperature and
becomes a single strand…that strand is
then copied by DNA polymerase.
• The low temperature allows the DNA
primers to bind only to its complementary
sequences, e.g. A across from T, and G
across from C.
• Since each cycle produces a copy, the
amount of DNA doubles with each cycle.