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COMPARISON OF FOUR-, SIX-, AND EIGHT-bp CUTS IN CALF THYMUS DNA

Dawn M. Murray
1993 Woodrow Wilson Biology Institute


IINTRODUCTION

DNA Extraction:

The thymus is a gland that is very large in immature animals. It functions as part of the immune system so there are many white blood cells with large nuclei. Many thousands of thymus cells will be used for extraction so you will be combining thousands of nuclei. In this way, you should be able to see long, combined strands of DNA.

Restrictive enzymes are used to "cut" DNA. Scientifically called restrictive endonucleases, these enzymes cleave DNA at internal positions in a precise and predictable manner. This occurs because the nuclease has the ability to break the phosphodiester bonds that link adjacent nucleotides in DNA and RNA molecules. A phosphodiester bond is a bond in which a phosphate group joins adjacent carbons. A condensation reaction between adjacent nucleotides results in a phosphodiester bond between 3' and 5' carbons in DNA and RNA. Within or very near the recognition site, the restriction enzyme catalyzes a hydrolysis reaction that uses water to break a specific phosphodiester linkage on each strand of the DNA helix. This produces two DNA fragments. A phosphate group at the 5' end and a hydroxyl group at the 3' end.

Restriction enzymes are broken into three categories. These categories are called Type I, Type II, and Type III. Types I and II both restrict and modify. Modification (methylating) is a protection mechanism which occurs when the enzyme attacks and digests internal regions of the DNA of the invading organism but not that of the host. It protects the DNA from digestion by adding methyl groups to a nucleotide within the sequence recognized by the restrictive enzyme. Both of these types cut the DNA at sites a distance away from their recognition sequencing. Therefore, ATP (Adenosine Triphosphate) is needed to provide energy to move the enzyme along the molecule from the recognition site to the cutting site. Type I frequently cuts at large distances away from the recognition site. Type III enzymes cut nearer the recognition sequence at specific sites but these sites are unable to be predicted.

Type II restrictive enzymes are the most commonly used for three reasons which are listed below:

  1. Each type II enzyme completes only a restriction activity. The modification activity is carried out by a separate enzyme.

  2. Each type II enzyme cuts in a predictable and consistent manner and at a site within or adjacent to the recognition sequence.

  3. Type II enzymes do not need ATP. They use only magnesium ions (mg++) as cofactors.

A type II restriction enzyme will scan a DNA molecule, stopping only when it recognizes a specific sequence of nucleotides. A given restriction enzyme cuts all DNA in exactly the same fashion, regardless of whether the source is a bacterium, a plant or an animal.


EXPECTED OUTCOME

In this laboratory exercise, restriction enzymes will recognize a four-, six-, and eight-nucleotide sequence. Assuming that the four component nucleotides (A,C,T,G) are distributed randomly within a DNA molecule, then any four nucleotides will occur, on average, every 256 nucleotides, a six-nucleotide recognition site will occur every 4096 nucleotides, and an eight-nucleotide recognition site will occur every 65,536 nucleotides.

The following restrictive endonucleases will be used to cut Thymus DNA. The results will then be compared by viewing the DNA fragments on Agragose Gel Electrophoresis which will show how RNA and protein molecules are separated by molecular weight.

TARGET AGE:

High School Biology/all levels

CLASS TIME REQUIRED:

5 class periods

ORGANIZATION:

Groups of 4 to later break into 2 groups of 2.

MATERIALS USED:

  • centrifuge tubes
  • centrifuge
  • gel box
  • blender
  • pipettes
  • pH meter
  • staining boxes
  • test tubes
  • methylene blue
  • 1/2 pound calf thymus (commonly called "sweetbread" and is found at your local grocers or butchers)
  • SOLUTIONS:

    THYMUS DNA EXTRACTION - PREP BUFFER

  • 57 g table sugar
  • 3.1 g Epsom salts
  • distilled H20/500 ml.
  • pH 7.5 adjusted with1 N HCl/NaOH (add one drop at a time)
  • 6g Buffered aspirin (about 1 tablet)
  • SDS

  • liquid detergent and tap water to make 20% solution.(This is used to break down the cell membranes by emulsifying the lipids disrupting the polar interactions holding the cell together. SDS also deprotenizes the DNA by removing histones.)
  • NaCl

  • 9.2 g table salt
  • distilled water to make 250 ml

    95% Ethanol - ice cold(put in freezer night before in plastic bottle)

  • optional: EDTA (.72g EDTA, 250 ML H20, PH/7.5
  • RESTRICTION ENZYME DIGESTION:

  • Extracted DNA in volumes of 10ul and 20ul
  • 10x buffer (16ul NEB Buffer/8ul NEB 2 buffer)
  • 7ul of EcoRI, HaeIII, NotI enzymes
  • 35ul distilled water
  • Gel electrophoresis
  • 250ml Methylene Blue stain
  • 18ul loading dye
  • Distilled water

  • PROCEDURE: EXTRACTING DNA FROM THYMUS:

    DAY 1

    1. ISOLATE NUCLEI

      a. Mix 200 ml of prep buffer with 10 ml thymus. Puree in blender.

      b. Strain to remove large chunks of tissue - save liquid. This can be done by placing a piece of cheese cloth over a beaker and pouring solution through.

      c. Centrifuge 4 ml. of strained liquid to make a pellet of nuclei.

      d. Pour off liquid (supernatant) from top of pellet.

      e. Resuspend pellet in 2 ml of prep buffer. Stir with plastic pipette.

      f. Put 40 drops of resuspended pellet into a clean test tube for each lab group.

    2. LYSE NUCLEI

      a. Add 6 drops of SDS composite. This solubilizes proteins and disorients fats in the cell membrane.

      b. Add five drops of NaCl solution, one drop at a time, mixing gently after each drop.

    3. PRECIPITATE DNA

      a. Gently add 1ml of 95% iced Ethanol by pipetting down the side of the test tube. The alcohol will form an overlay. You should begin to see strings of DNA precipitating at the point of the overlay because all of the other components of the cell are soluble in the ethanol - only DNA is not.

      b. Spool the DNA where the ethanol and the DNA mixture meet. The DNA will look like gobs of white mucus. Wrap this substance around a glass pipette and lift out of the tube. Place into a centrifuge tube for later. Put in freezer.


    DAY 2

    The spooled thymus DNA will now be digested (cut) in 4bp, 6bp and 8bp se-quences using HaeIII, EcoRI, and NotI enzymes, respectively. The digestion products will be analyzed using gel electrophoresis.

    1. PURIFYING THE DNA

      A. Remove spooled DNA from freezer and thaw.

      B. Centrifuge for 30 seconds. Pour off supernatum.

      C. Add 1ml of pineapple juice (papaine). This will get rid of any foreign proteins on the DNA. Pour off after 2 minutes.

      D. Add 1ml of 100% ethanol. This will get rid of the papaine. Pour off after 2 minutes.

      E. Resuspend the DNA in TE for 30 minutes. Use same amount as the DNA in the tube. You will not see any change. The DNA is going into solution to be aliquoted from the top of the tube later on. Put in freezer.

    2. PREPARING THE GEL (break group into 2's-each prepares a gel)

      A. Follow directions for Agarose Gel Electrophoresis preparation to make your gel. This procedure will take about 30 minutes. Let gel set in TAE overnight.

      Direction for the gel preparation can be found in "Desktop Electrophoresis Lab: Moving Molecules".


    DAY 3

    1. PREPARING RESTRICTION ENZYME DIGESTS (thaw DNA)

      A. You will be digesting 20ul of resuspended purified thymus DNA with a buffer, an enzyme and distilled water. To prepare your digestion enzymes you will need to aliquot the proper amounts of each into a centrifuge tube.


      DNA                        10ul                           20ul
      10x buffer                 4ul                             4ul
      Enzyme                     2ul                             5ul
      H2O                        24ul                           40ul
      Total                      40ul                           40ul
      


      B. Label all of your tubes! You should complete 6 tubes. There will be enough digest in these 6 tubes for your group of four to separate into groups of two. Each group of two will complete their own electrophoresis gel.

      C. Place your centrifuge tubes in 37deg.C for 30 minutes where they will digest (cut).

  • LOADING SAMPLES INTO THE GEL WELLS

    A. You will load your samples into the wells in the following order. (Well numbers 1 and 8 will each be loaded with a different marker to be sure there is a good comparison to differentiate between the wide range of DNA fragment sizes found when using 4bp, 6bp and 8bp cutters). Load 20ul sample and 3ul loading dye in wells # 2 - 7.

    well #1: 123kb (load 10ul)
    well #2: EcoRI (10ul)
    well #3: EcoRI (20ul)
    well #4: HaeIII(10ul)
    well #5: HaeIII(20ul)
    well #6: NotI (10ul)
    well #7: NotI (20ul)
    well #8: 1kb (load 10ul)

    B. When all samples are loaded, close the lid on the gel box and attach the electrical leads. The black lead attaches to the side of the gel with the wells. The red attaches to the opposite side of the wells. When using a homemade electrophoresis gel box, the voltage is only 45V, therefore twice the amount of time will be needed for completion; about 45 minutes.


    DAY 4

  • STAINING THE DNA BANDS ON THE GEL

    A. Methylene Blue is a buffer which will intercalate into the DNA in order to visualize the DNA bands. Complete the following steps to insure proper staining.

    * USE GLOVES WHEN WORKING WITH METHYLENE BLUE STAIN

    Pour liquid out of gel box. Carefully slide gel into staining tray. Pour Methylene Blue stain into tray (not directly onto gel) to just cover the gel. Let stain for three hours. Pour off stain into sink - flush sink with running water. Add 250ml of distilled water to staining tray. Destain for 12-18 hours.(This time can be cut back if methylene blue is diluted.) Agitating on an orbital agitator will speed up destaining. Remove gel from the tray and wrap in plastic wrap, then place in the refrigerator. (Bands will remain clear for at least 3 weeks before fading since nuclear acids are not fixed.)


    DAY 5

  • COMPARISON OF DNA CUTS USING DIFFERENT RESTRICTION ENZYMES

    A. Place your gel on the overhead projector to view your DNA fragments showing the cuts which were made by each enzyme.

    B. Compare fragments from each of the different cutting enzymes. Record your results.


    References:

    Freyer, Greg A., and David A. Micklos. DNA SCIENCE. Cold Spring Harbor Laboratory Press, New York, 1990.


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