EXTRACTION OF DNA
DNA can be extracted from any blood or tissue sample. The quality and quantity of the DNA obtained will vary depending on the size, age, and cell count of the sample. As a rule, 3 ml of blood in EDTA will suffice. The DNA is extracted from all nucleated cells and is called genomic DNA.
In the nucleus, the DNA is tightly associated with many different proteins as chromatin. It is important to remove these as well as other cellular proteins to extract the DNA. This is achieved through the use of organic solvents or salt precipitation. An aqueous solution of DNA is obtained, from which the DNA is further purified by ethanol precipitation.
A number of DNA extraction kits are now commercially available. These can significantly reduce the amount of time required for DNA extraction, bypass the use of organic solvents, and provide good quality control of the reagents used. However, the use of kits in all aspects of molecular biology may inhibit the development of improvements.
Protocol: Extraction of Genomic DNA (The protocol described here is manual method)
Please note that for all the buffers and solutions,it is recommended that reagents of the highest grade available and double distilled deionised water are used throughout.
- NaCl 5 mol/l. Weigh 146.1 g of NaCl into a beaker and make up the volume to 500 ml with water; stir until dissolved.
- Tris-HCl, 1 mol/l, pH 8.5. Dissolve 60.5 g of Trizma base (tris[hydroxy]methylaminomethane) in 350 ml water; add concentrated HCl until the pH falls to 8.5; make up to 500 ml with water.
- Tris-HCl, 1 mol/l, pH 7.4. Prepare as for the above, but reduce the pH to 7.4 with HCl.
- NaOH, 5 mol/l. Add 200 g of NaOH to 800 ml water and stir until dissolved; make up to 1 litre with water.
- EDTA, 0.5 mol/l, pH 8.0. Weigh 93 g of EDTA disodium salt (dihydrate) and add to 400 ml of water; stir until most of it has dissolved. Add 0.5 mol/l NaOH until the pH rises to 8.0, when the rest of the solid should go into solution. Make up to 500 ml with water.
- Phosphate buffered saline (PBS), pH 7.3.
- Nonidet P-40 (NP40), 10%. Add 10 ml of NP40 to 90 ml water and mix well.
- Sodium dodecyl sulphate (SDS, lauryl sulphate), 20%. Weigh 100 g of SDS and add to 350 ml of water. Stir and heat to 65°C until it is in solution and top up to 500 ml with water.
Caution: SDS is a respiratory irritant. Wear a face mask and weigh out in a fume hood.
- PBS + 0.1% NP40. Add 5 ml of 10% NP40 to 495 ml of PBS.
- Ten times concentrated (×10) lysis buffer). Mix 60 ml of 5 mol/l NaCl, 20 ml of 0.5 mol/l EDTA, 10 ml of 1 mol/l Tris pH 7.4, and 10 ml of water to give 100 ml of 3 mol/l NaCl, 100 mmol/l EDTA, and 100 mmol/l Tris.
- Lysis solution. Prepare an appropriate amount of this solution fresh every time; for 50 ml weigh 21 g of urea (7 mol/l), add 5 ml of ×10 lysis buffer, and make up to a final volume of 50 ml with water.
- Chloroform/isoamyl alcohol (24:1). Add 20 ml of isoamyl alcohol to 480 ml of chloroform.
- Ethanol 70%. Add 30 ml of water to 70 ml of absolute ethanol.
- Tris EDTA (TE) (10 mmol/l Tris, 1 mmol/l EDTA). Add 5 ml of 1 mol/l Tris pH 7.4 and 1 ml of 0.5 mol/l Na2 EDTA to 494 ml water.
- TE equilibrated phenol. The condition of the phenol is crucial to the quality of the DNA obtained. DNA is soluble in acidic water-saturated phenol, and so it is necessary to equilibrate it to a neutral pH.
- Take 500 ml of water-saturated phenol. Prepare 500 ml of 0.5 mol/l Tris pH 8.5, add 150 ml of this to the phenol, and mix by inversion for 2–3 min. Leave to stand until the aqueous and organic phases have separated.
- Remove and discard the upper aqueous layer. Add another 125 ml of 0.5 mol/l Tris, mix, stand, remove the aqueous layer as before, and then repeat.
- To the remaining 100 ml of 0.5 mol/l Tris, add 400 ml of water to give 500 ml of 0.1 mol/l Tris. Add 150 ml of this to the phenol, and then mix, stand, and remove. Repeat two more times.
- To the remaining 50 ml of 0.1 mol/l Tris add 449 ml of water and 1 ml of 0.5 mol/l EDTA to give 500 ml of TE. Add, mix, stand, and remove this in three stages as before. The phenol will have reduced in volume during this procedure but is now TE equilibrated and ready for use.
- Freeze an anticoagulated blood sample at -20°C. EDTA is the preferred anticoagulant. It is convenient to collect the blood in a tube that can be centrifuged, such as a disposable plastic 25 ml universal container. Any sample size from 2 to 20 ml will be satisfactory. Blood can be shipped at room temperature to a reference laboratory, preferably within a few days of taking it. The sample can be stored at -20°C for several weeks; storage for longer periods is better at -80°C.
- Thaw the blood and centrifuge at 700 g for 15 min. Carefully pour off the supernatant. The pellet is hard to see at this stage and may be quite loose.
- Resuspend the pellet in 1–2 ml of PBS + 0.1% NP40 by mixing up and down in a wide-bore standard plastic transfer pipette. Top up the suspension to the original volume with PBS + 0.1% NP40.
- Centrifuge again at 700 g for 15 min and pour off the supernatant. If necessary, repeat until the pellet has lost most of its red colour.
- Add 2–3 drops of lysis solution. Break up the pellet into this solution using a nonwettable sterile stick (e.g., a plastic disposable bacterial inoculating loop) or a clean siliconised glass rod. The solution will become viscous. Make it as homogeneous as possible.
- Add successive 0.5 ml volumes of the lysis solution, mixing each time, until the viscosity is such that the solution can be pipetted up and down without difficulty. The final volume will depend on the size, nature, and quality of the blood sample. For 10 ml of freshly frozen normal blood, use 2–3 ml of lysis solution.
- Add 1/10 volume of 20% SDS. Mix gently with a transfer pipette and incubate at 37°C for a minimum of 15 min. The samples can be left overnight at this stage.
- Transfer the sample to a capped polypropylene tube. Add an equal volume of chloroform/isoamyl alcohol and an equal volume of phenol. Mix gently by inversion for 5 min. Centrifuge at 1300 g for 15 min.
- Transfer the upper aqueous phase to a new polypropylene tube. Leave behind the white protein interface and the organic phase. This may be difficult if the solution is too viscous, in which case further dilution with lysis solution is necessary.
- Repeat steps 8 and 9 at least once more and continue until the interface is clear. Add an equal volume of chloroform/isoamyl alcohol and mix gently by inversion for 5 min. Centrifuge as before and again transfer the aqueous phase to a universal or capped 10 ml tube.
- Add 2.5 volumes of absolute ethanol. Mix the solution by inverting the tube several times. The DNA should precipitate as a “cotton wool” ball. Using a micropipette tip, transfer the DNA to a microcentrifuge tube containing 1 ml of 70% ethanol.
- Centrifuge in a microcentrifuge at 12,000 g for 5 min. Pour off the residual ethanol, and remove all of this with a micropipette.
- Leave to dry on the bench for 10 min.
- Add 50–500 μl of TE depending on the size of the pellet. Aim to have a DNA concentration of approximately 0.5 mg/ml. Leave to resuspend for at least one night. Mix gently by flicking the tube; never vortex. The DNA can be stored for long periods at 4°C or frozen at -20° C.
Extraction of DNA from Other Sources
For the analysis of Ig and TCR gene rearrangements, it is necessary to enrich a peripheral blood sample for lymphocytes prior to DNA extraction. This is achieved through the separation of mononuclear cells on Ficoll/Hypaque (or Lymphoprep). After washing the cell pellet in PBS, lysis and DNA extraction can proceed as from step 5.
DNA is extracted from bone marrow aspirates in the same way as from peripheral blood, except that before freezing they are diluted in at least 5 volumes of PBS.
Tissue biopsies vary greatly in nature, size, and cell content and, as a result, so does the quality and quantity of DNA obtained from them. To obtain sufficient quantities of high molecular weight DNA for Southern blot analysis, the biopsy must be several mm3 in size and fresh frozen. If such a biopsy is available, sufficient cells can be obtained by mechanically disrupting it into PBS first by chopping it finely with a clean blade and then breaking it up with the blunt end of a 5 ml syringe plunger. The suspension is centrifuged to obtain a pellet, from which DNA is extracted as before (from step 5). For smaller biopsies, it is better to treat the sample with proteinase K prior to extraction, as follows:
- Place the tissue in 700 μl of 50 mmol/l Tris-HCl, pH 8.0, 100 mmol/l NaCl, 1% (w/v) SDS containing 100 μg/ml proteinase K.
- Cut up the tissue with a fine pair of scissors, and incubate at 50°C overnight.
- Proceed with phenol/chloroform extraction and DNA precipitation as from step 8 earlier.
Determining the DNA Concentration
Take 5 μl of the DNA solution and dilute into 245 μl of water. Mix well by vortexing. (This DNA is to be discarded and can therefore be treated in this way.) Read the absorbance (A) in a spectrometer at 260 nm against a water blank. An A of 1.0 is obtained from a solution of DNA at a concentration of 50 μg/ml. Therefore, multiply the A reading obtained by 2500 to get the concentration of the original DNA solution in μg/ml. The ratio of the A260 to the A280 gives an indication as to the purity of the DNA solution. This ratio should be in the range of 1.7–2.0.
(Source: Dacie and Lewis Practical Haematology, 10th Edition)