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					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.

•   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 pre-
  warmed 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
•   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
•   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:

•   BamH I
•   EcoR I
•   Hae III
•   Hind III -
•   Pvu II - (used in the DNA Detectives Lab)
              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
                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
                DNA Detectives Lab
                  Materials, cont.
•   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:

•   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 low-
  temperatures 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.

				
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posted:10/17/2011
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