DNA Extraction

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DNA Extraction

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What is DNA?

• DNA (deoxyribonucleic acid) is one of four major
  families of organic molecules in the cell.
• DNAs primary structure consists of a
  sugar/phosphate backbone to which nitrogenous
  bases are attached. The bases are either Purine
  or Pyrimidine rings.
• It serves as the master copy for most
  information in a cell.

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Sugar/Phosphate Backbone
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Purine Structures

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Pyrimidine Structures

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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 formsribose (RNA) or deoxyribose (DNA).
• In 1943, American Oswald Avery proves that DNA
  carries genetic information.

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Discovery of DNA, cont.

• In the early1950s, 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 Paulings
  1948 discovery that many proteins take the shape
  of an alpha helixin other words, they spiraled
  like a spring coil.

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Discovery of DNA, cont.

• At Kings 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.

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Discovery of DNA, cont.

• Chargaffs and Paulings discoveries would prove
  to be an important foundation later for the
  description of DNA.
• In 1953, without the consent of Franklin, Wilkins
  showed Franklins results to Watson and Crick.
• Based on that information, Watson and Crick
  suggested the DNA molecule was made of two chains
  of nucleotides.

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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.

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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.
• DNAs discovery has been called the most
  important biological work of the last 100 years.

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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.

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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.

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Materials

• The following materials/supplies are needed to
  achieve DNA extraction
• Raw Wheat Germ
• Warm water bath (50- 60C)
• Meat tenderizer
• Salt (NaCl)
• Sodium bicarbonate solution
• Liquid detergent such as Woolite

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Materials, cont.

• 250mL beaker
• Cheese cloth or coffee filter
• Thermometer
• Pipet
• Scale
• 95 Ethanol or 100 Isopropanol alcohol
• Centrifuge
• Gloves

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DNA Extraction Procedure

• Prior to beginning the extraction, prepare the
  warm water bath (50- 60C).

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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 pre- warmed
  water.

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DNA Extraction Procedure, cont.

• Record the exact temperature of the water.
• The optimal temperature for DNA extraction is
  55- 60C.
• DO NOT ALLOW THE TEMPERATURE TO RISE ABOVE 60C.
• Add 2-5mL of detergent and stir gently trying to
  create as few bubbles as possible.

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DNA Extraction Procedure, cont.

• Continue to stir the mixture for an additional 5
  minutes remembering to keep the temperature below
  60C.
• 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.

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DNA Extraction Procedure, cont.

• Immediately cool in ice bath to room temperature.
• Filter through cheesecloth or coffee filter.

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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 doesnt
  mix with the filtrate.
• 2 distinct layers should be visible.
• Spool the DNA from the filtrate.

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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.

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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.

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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.

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DNA Extraction with Green Peas

• 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

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DNA Extraction Procedures

• 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.

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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.

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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.

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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).

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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).

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DNA Detectives Lab, cont.

• Examples of Restriction Enzymes
• BamH I
• EcoR I
• Hae III
• Hind III -
• Pvu II - (used in the DNA Detectives Lab)

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DNA Detectives Lab, cont.

• Materials needed
• 0.8 Agarose
• 5X TBE running buffer
• Quickview DNA stain concentrate
• Microfuge tubes
• Staining trays
• Loading dye
• Lambda DNA/Hind III marker

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DNA Detectives Lab Materials, cont.

• 4 suspect DNA samples
• 2 Pvu II restriction enzyme
• 2 Pvu II reaction buffer 10X
• Electrophoresis chamber
• Power supply
• Micropipettes
• Permanent marker
• Distilled water

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DNA Detectives Lab Materials, cont.

• Microwave oven
• 37C water bath
• 65C water bath
• Duct tape
• Gloves
• Ice bath for sample tubes
• Petroleum ether for rinsing micropipette
• Test tube rack
• 9-Volt Batteries

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DNA Detectives Lab Procedures

• Remove DNA sample kit from freezer and allow
  samples to thaw.
• Label 5 microfuge tubes as follows
• Crime scene
• Suspect 1
• Suspect 2
• Suspect 3
• Suspect 4

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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

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DNA Detectives Lab Procedures, cont.

• Incubate all tubes at 37C for 1 hour.
• Cast Agarose Gel during this time if not prepared
  ahead of time.

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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 CAUTIONGEL IS EXTREMELY HOT!
• Use duct tape as end dams for the gel casting
  tray making sure tape securely adheres to the
  tray.

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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 its just below the
  bottom portion of the solid comb.
• Allow the gel to cool and solidify which takes
  approximately 30 minutes.

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DNA Detectives Lab Procedures, cont.

• Prepare the Dilute Stain while the gel is
  cooling.
• Add 5ml of DNA stain concentrate to 95ml warm
  (50-55C) distilled water.
• Set solution aside, but maintain temperature.

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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 65C.
• 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.

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DNA Detectives Lab Procedures, cont.

• While the tubes are incubating at 65C, wear
  gloves and remove the duct tape from the ends of
  the Agarose Gel tray.
• If gloves arent 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.

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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.

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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

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DNA Detectives Lab Procedures, cont.

• When loading the DNA to the Agarose Gel, make
  sure the DNA isnt 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.

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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.

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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.

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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 doesnt slide into the
  corner of the tray.
• Add distilled water to the tray using care not to
  pour water directly onto the gel.

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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.

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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

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DNA Detectives Lab Results, cont.

• Suspect 3 committed the crime!!!

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PCR

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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 low-temperatures for 30
  cycles.
• Subjecting DNA to high temperature causes
  denaturation.

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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.

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PCR, cont

• In shortthe double-stranded DNA is separated at
  high temperature and becomes a single strandthat
  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.