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Preparation of Genomic DNA from Hawaiian Bobtail Squid

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					           Protocol


Preparation of Genomic DNA from Hawaiian Bobtail Squid
(Euprymna scolopes) Tissue by Cesium Chloride Gradient
Centrifugation
Patricia N. Lee1,2, Margaret J. McFall-Ngai3, Patrick Callaerts4, and H. Gert de Couet1,5
1
  Department of Zoology, University of Hawaii at Manoa, Honolulu, HI 96822, USA
2
  Kewalo Marine Laboratory, Pacific Biosciences Research Center/University of Hawaii at Manoa, Honolulu, HI
96813, USA
3
  Department of Medical Microbiology, University of Wisconsin-Madison, Madison, WI 53706-1521, USA
4
  Laboratory of Developmental Genetics, K.U. Leuven and VIB, B-3000 Leuven, Belgium

5
    Corresponding author (couet@hawaii.edu).



INTRODUCTION

This procedure describes the extraction of genomic DNA from adult bobtail squid (Euprymna
scolopes) tissues by cesium chloride (CsCl) gradient centrifugation. There are numerous generic
methods and commercial kits for the preparation of genomic DNA based on proteolytic
digestion of chromatin components, followed by selective binding of nucleic acids to ion-
exchange affinity media, but many of these do not yield DNA that can be readily restricted. Also,
molluscan tissues contain mucopolysaccharides, which tend to copurify with DNA under certain
conditions. Although nucleic acids prepared this way can serve as a template for polymerase
chain reaction (PCR), other enzymatic modifications of nucleic acids are inhibited by these
contaminants. The method described here yields high-molecular-weight DNA that can be readily
restricted for Southern hybridization. The procedure uses brain tissue under the assumption that
its genome is unlikely to be rearranged in any way, has a high nucleic acid:protein ratio, and
avoids potential sources of enzymatic contaminants and parasites from the intestinal sac.
However, the method can be applied to other tissue sources and works well with other species.
The purification of DNA by gradient centrifugation is an established method based on the
specific buoyant density of double-stranded nucleic acids and the ability of CsCl solutions to
form a salt gradient in a centrifugal field. It can also be adapted to the purification of RNA,
which has a higher buoyant density than DNA. Unfortunately, this method is somewhat involved
and expensive and produces large amounts of ethidium bromide waste.


RELATED INFORMATION

For specific information on dealing with mucopolysaccharide contamination of DNA derived
from molluscan tissues, see Sokolov (2000). For additional information on the background,
husbandry, and potential uses of E. scolopes as model organisms, see The Hawaiian Bobtail
Squid (Euprymna scolopes): A Model to Study the Molecular Basis of Eukaryote-
Prokaryote Mutualism and the Development and Evolution of Morphological Novelties in
Cephalopods (Lee et al. 2009).


MATERIALS

Reagents

Agarose gel and other reagents for agarose gel electrophoresis

  CsCl, solid, analytical grade

The procedure requires at least 7 g of solid CsCl per gram of tissue.

  CsCl solution (50% [w/v], prepared in TE buffer at pH 7.5).

Prepare at least 10 mL to have enough to balance several ultracentrifuge tubes.

  Ethidium bromide (10 mg/mL)

Purchase a ready-made stock solution to avoid the dangers of handling solid ethidium bromide.

  Isoamyl alcohol, water-saturated

Restriction enzyme (common six-base cutter; see Step 28)

  Sarkosyl (30% [w/v])

Sarkosyl is a strong ionic detergent that readily solubilizes lipids and denatures proteins. It is
used in lieu of sodium dodecyl sulfate because of its solubility characteristics.

Squid, adult

  Squid tissue homogenization buffer

  TE buffer, pH 7.5

Equipment

Analytical balance

Centrifuge (e.g., Sorvall refrigerated superspeed centrifuge), equipped with an SS-34 fixed-angle
rotor

Cheesecloth
Container and/or absorbent paper (see Step 18)

Container, styrofoam

Dialysis tubing, sterilized

Store in TE buffer.

Dissection instruments

Equipment for agarose gel electrophoresis

Flask, Dewar

Glass rod or spatula

Gloves, ultralow temperature

Ice

  Liquid nitrogen

Mortar and pestle

Clean and sterilize before use.

Needles, 21-gauge

Petri dishes, large

Pipettes, graduated

Pipettes, transfer, large-bore

Plastic film or aluminum foil

Rotator, end-over-end

Safety goggles, UV-opaque

Scalpel, equipped with fresh blades

Spectrophotometer

Stand with boss head clamp
Syringes, disposable, 2-mL

Trays

Tube sealer

Tubes, centrifuge

Tubes, screw-cap, sterile, 5-10 mL

Tubes, ultracentrifuge

Ultracentrifuge, tabletop, equipped with vertical rotor (e.g., Beckman TLN-100 or equivalent)

The Beckmann TLN-100 rotor accepts eight tubes with a volume of 3.9 mL each. Other
centrifuges and rotor types can be used, but centrifugation times and rotor speeds can vary.
Consult your ultracentrifuge’s manual about DNA separations in CsCl gradients.

  Ultraviolet (UV) light, short wavelength, handheld

Handheld UV lights have a low-energy output and are therefore less likely to damage the DNA
than a conventional transilluminator. They also present fewer health and safety issues.

Waterbath preset to 40°C-60°C


METHOD

Tissue Homogenization

        1. Dissect the head capsule (or other tissue) of one or more adult squid. Weigh the tissue.
        Keep on ice.

        2. Pre-cool the mortar and pestle with liquid nitrogen in a styrofoam container.

        3. Freeze the tissue in the mortar with a small amount of liquid nitrogen. Allow the
        nitrogen to evaporate.

        4. Pound and grind the frozen tissue to a fine powder.
        Place plastic film or aluminum foil over the mortar while grinding to minimize
        condensation of atmospheric water, which can add considerably to the volume of the final
        homogenized sample. Keep mortar and tissue at ultralow temperature during the process
        by adding small amounts of liquid nitrogen.

        5. Add 6 mL of squid tissue homogenization buffer per each gram of tissue to the mortar.

        6. Thaw the tissue by warming the mortar and its contents to ~40°C-60°C in a water bath.
      7. Add 0.5 mL of 30% Sarkosyl. Carefully mix the homogenate with a glass rod or
      spatula.
      The solution will be highly viscous because the DNA has been stripped of chromosomal
      proteins. Be very gentle when handling the homogenate from this point on.

      8. Clear the homogenate by centrifugation at 10,000g for 10 min.
      Lipids will form a raft on the surface of the tube.

      9. Carefully decant the supernatant through cheesecloth to remove the lipids. Measure the
      volume of the filtrate with a graduated pipette.
      Discard the pelleted insoluble extracellular matrix components and pigments.

      10. Add 1.0 g of solid CsCl per milliliter of homogenate. Dissolve gently by end-over-end
      mixing in a Falcon centrifuge tube.
      Some insoluble material will form from precipitating proteins.

      11. Add 50 µL of 10 mg/mL ethidium bromide per milliliter of homogenate. Mix gently.

      12. Clear the solution again by centrifugation at 10,000g for 10 min. Discard the pellet.
      Material not removed at this stage will interfere with the DNA extraction later.

CsCl Gradient Centrifugation

      13. Load the homogenate into sealable ultracentrifuge tubes with a large-bore transfer
      pipette. If necessary, top off the tubes with CsCl solution of equal density. Seal the tubes.

      14. Centrifuge in a tabletop ultracentrifuge with a near-vertical rotor at 100,000 rpm for 3
      h at 18°C, with the brake inactivated.
      Centrifugation at lower temperatures can lead to salt precipitation, subsequent failure of
      the tube and damage to the rotor.

      15. Remove tubes from the rotor carefully to avoid mixing the gradient. Take samples to a
      darkened room.

      16. Clamp an ultracentrifuge tube to the stand. Observe the position of the main
      fluorescent band with a handheld UV light.
      Highly concentrated samples can yield a discernible band visible in daylight. Optimally,
      the main band should be roughly in the center of the tube. Additional minor bands might
      be visible, corresponding to satellite and mitochondrial DNA. There might also be a pellet
      of insoluble material that sinks to the bottom; it is important to avoid this material when
      aspirating the DNA.

      17. Make a small hole at the top of the tube by inserting a syringe needle.
      Air will draw through this hole as the DNA-containing material is removed from the
      center of the tube with a syringe.

      18. Carefully insert a 21-gauge hypodermic needle attached to a 2-mL disposable syringe
      slightly below the fluorescent band, with the beveled side of the needle up.
      Place a container and/or absorbent paper below the centrifuge tube when collecting the
      DNA to avoid contaminating the bench surface with ethidium bromide.

      19. Slowly draw liquid into the syringe while moving the needle side-to-side.
      Because of its viscosity, the fluorescent band will appear to be a single blob of material.

      20. Remove the needle from the syringe. Transfer the liquid to a small screw-cap tube.
      The procedure can be interrupted at this point. DNA in buffered CsCl is stable but should
      be protected from light as long as it still contains ethidium bromide.

      21. Perform a second gradient to increase further the purity of the DNA:
      Alternatively, extract the ethidium bromide and CsCl from the sample immediately (see
      Steps 22-25).
      i. Transfer the extracted DNA into fresh tubes. Dilute with CsCl solution to make enough
      for the desired number of sample tubes.

      ii. Add 50 µL of the ethidium bromide stock solution per milliliter of sample volume.

      iii. Seal the tubes according to the manufacturer’s instructions.

      iv. Repeat Steps 14-20.

Removal of Ethidium Bromide and CsCl

      22. Add approximately one-fourth of the sample volume of water-saturated isoamyl
      alcohol to the sample (from Step 20 or Step 21.iv). Mix gently end-over-end.
      The ethidium bromide will accumulate in the upper, organic phase. If nonsaturated
      isoamyl alcohol is used, the volume of the sample will decrease with each extraction step,
      ultimately leading to precipitation of salt.

      23. Remove the organic phase with a transfer pipette.

      24. Repeat Steps 22 and 23 at least four times until the organic phase appears colorless
      against a white background.
      Multiple extractions with a small volume are more efficient than fewer extraction steps
      with larger volumes of solvent.

      25. Transfer the DNA solution to a dialysis tube. Seal tightly to minimize the volume.
      Because the solution is close to saturation, water entering the dialysis tube during the
      next step will dilute the DNA.

      26. Dialyze the sample against four or more changes of TE buffer to remove salt.

      27. Measure the DNA concentration spectrophotometrically.

      28. Analyze an aliquot before and after restriction digestion with a common six-base
      cutter by agarose gel electrophoresis. Keep the following points in mind:
              Undigested DNA should have the appearance of a smear with the highest DNA
              amount near the sample well.

              Digested DNA will produce a haze of nucleic acid across all molecular weight
              ranges, with several faint background bands, which represent repetitive sequences.

              Large amounts of DNA near the front of the gel indicate excessive degradation.


       This method should yield DNA in the 80- to 150-kb range. It is advisable to perform a
       Southern blot with a cloned Euprymna sequence as a probe. Because this method
       produces large amounts of pure DNA sufficient for many experiments, it is worth
       investing the extra time and effort to ascertain the quality of the preparation.
       See Troubleshooting.

       29. Store DNA solutions at 4°C for extended periods.
       Avoid repeated freezing and thawing.


TROUBLESHOOTING

Problem: DNA appears degraded.

[Step 28]

Solution: The most likely reason for degradation is excessive mechanical shearing. The
homogenization procedure, developed initially for plant tissues and other specimens containing
mechanically resilient tissue, is based on the assumption that the subcellular structure (e.g.,
chromatin) remains relatively intact during homogenization at very low temperatures. Consider
the following:

       1. Semifrozen or previously frozen tissue can lead to degraded DNA because it might no
       longer exist as compact chromatin.

       2. Pipetting a DNA solution through narrow-bore pipettes or passage through syringe
       needles can cause degradation, as can vortexing or shaking.

       3. It might prove impractical to homogenize small tissue samples in the manner described
       here. Although other authors have described successful extractions based on Dounce and
       Potter-type homogenizers, it must be emphasized that these methods are based on
       shearing forces dependent on the clearance between the pestle and the wall of the
       homogenizer. In the presence of detergent, very little mechanical action is required to
       disrupt the cell structure and liberate the DNA.

       4. Do not ethanol-precipitate genomic DNA, and avoid repeated freeze-thawing.

Problem: DNA will not restrict completely under standard digestion conditions.
[Step 28]

Solution: Consider the following:

       1. Failure of DNA to restrict indicates persistent contamination, most likely with proteins.
       It is possible to combine this method with a phenol/chloroform extraction or one of the
       conventional purification methods based on protease digestion. However, two consecutive
       CsCl gradient separations usually are sufficient to produce DNA pure enough to allow
       both cloning manipulations and PCR amplification.

       2. Another potential source of problems is the tissue homogenization buffer, which
       contains high concentrations of EDTA. Although this chelating agent is included to
       inhibit nucleases during the extraction procedure, relatively small amounts can also
       effectively inhibit restriction enzymes used to analyze the DNA. Exhaustive dialysis
       against TE buffer should eliminate this problem.


DISCUSSION

Although the PCR method has revolutionized developmental biology by its simplicity and
robustness, genomic work still relies on the availability of sizeable cloned DNA fragments for
the construction of representative libraries. Both the average molecular weight of the nucleic acid
preparation and its restrictability are limiting factors during workflow. With experience, CsCl
gradient centrifugation yields quantities of DNA for these specific purposes with purity superior
to that obtainable with other techniques. It is, however, unsuitable for the construction of
bacterial artificial chromosome (BAC) libraries. Larger cephalopod species have reproductive
tissues large enough for DNA extraction; spermatophores, for example, can be recovered easily
and consist almost exclusively of gametes. Although E. scolopes has no known parasites or
commensals, the renal organs of many cephalopods are occupied by ciliates. The possibility of
minor contaminants from other eukaryote genomes should be taken into account as a possible
source of artifacts when preparing nucleic acids from whole animals.


REFERENCES

Lee PN, McFall-Ngai MJ, Callaerts P, de Couet HG. 2009. The Hawaiian bobtail squid
(Euprymna scolopes) A model to study the molecular basis of eukaryote-prokaryote mutualism
and the development and evolution of morphological novelties in cephalopods. Cold Spring
Harb Protoc (this issue). doi: 10.1101/pdb.emo135.[Abstract/Free Full Text]

Sokolov EP. 2000. An improved method for DNA isolation from mucopolysaccharide-rich
molluscan tissues. J Molluscan Stud 66: 573–575.[Abstract/Free Full Text]

				
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