Monday, June 1, 2009

DNA Extraction From Fresh Bone

You can extract nucleic acids, such as DNA, from bone samples in order to analyze gene expressions, to look for somatic mutations of tumors or other pathological tissue, or for genotyping archive material when other sources of DNA are not available. You can use several kits that have already provided by biotech companies. But, if you are extracting DNA from large number samples, you can use a homemade method as described here to be effective in cost.

There are four procedures that ascertain the successful extraction of nucleic acids from tissue:
  1. disrupting the tissue so that extraction reagents can reach the cells.
  2. disrupting the cell membranes so that nucleic acids are liberated.
  3. separation of the nucleic acid from other cellular components.
  4. precipitation and solubilization of the nucleic acid.

  1. DNA extraction buffer: Add 17.6 mL of 0.75 M sodium citrate, pH 7.0, 26.4 mL of 10% sodium lauryl sarkosyl, and 250 g of guanidinium isothiocyanate to 293 mL of distilled water and mix well. Add 7.2 microliter of beta-mercaptoethanol/mL of lysis buffer on the day of use

    All chemicals should be of molecular biology grade. The solutions can be stored at 4oC for up to 3 months.

  2. 0.5 M ETDA: Add 93.05 g of EDTA to 300 mL of distilled water and add 10 N NaOH to pH 8.0. Make up to 500 mL. Autoclave.

    Tris–EDTA: Add 1 mL of 1 M Tris to 200 microliters of 0.5 M EDTA. Make up to 100 mL with distilled water.
  3. 3 M Sodium acetate, pH 5.2: Add 401.8 g of sodium acetate to 800 mL of distilled water. Adjust pH to 5.1 with glacial acetic acid. Make up to 1 L with distilled water. Autoclave.
  4. General Reagents: Tris-saturated phenol pH 7.8–8.0 (Sigma), Chloroform, Isopropanol 100%, Ethanol.

  1. Collect the bone sample in a sterile container containing phosphate-buffered saline (PBS) and transport to the laboratory within 1–2 h.

    If the DNA extraction is not initiated immediately, freeze the sample at –20oC or below for later use.

  2. Place the bone tissue in a clean glass Petri dish. Using bone cutters or a strong sharp pair of scissors, isolate a piece of bone measuring about 1 cm3 and transfer to a clean 5-mL bijoux container.
  3. Add 1 mL of DNA extraction buffer and homogenize the tissue with the scissors until a slurry solution is obtained.
  4. Transfer 500 microliters aliquots of slurry into screw-capped conical-bottomed 1.5-mL Eppendorf tubes.
  5. Add one volume of Tris-saturated phenol, followed by one volume of chloroform per tube. Mix well by inverting the tubes a few times or by shaking. Do not vortex, because vortex-mixing causes long strands of DNA to shear.
  6. Centrifuge the tubes at 10,000 g for 20 min to separate the phases.
  7. Transfer the upper layer to a fresh centrifuge tube (taking note of the volume), being careful not to disturb the milky layer at the interface. Repeat steps 5–7 if the interface is disturbed.
  8. Add one volume of ice-cold isopropanol and 0.1 volumes of 3 M sodium acetate to the supernatant. Mix well and allow to stand for 15 min on ice.
  9. Centrifuge the tubes at 10,000g for 20 min to pellet the DNA.

    Orientate the Eppendorf tube so that you can identify where the DNA pellet lies. A pellet should be visible the bottom of the tube.

  10. Aspirate and discard the supernatant, taking care not to disturb the pellet. Wash the sample with 1.75 mL of ice-cold ethanol and centrifuge at 10,000g for 5 min. Aspirate and discard the supernatant and then repeat the wash.
  11. Dissolve the DNA pellets in 10–50 microliters of water or Tris–EDTA buffer (you can pool DNA from the same sample at this stage) and quantitate by spectrophotometry or with Hoechst 33258.

    Hoechst 33258 is a DNA-specific dye that can be used to quantitate DNA.

  12. Store the sample frozen at –20oC or below.

Kindly regards.

Reference: Number 15 on References