Extraction of DNA from Whole Blood (John M. S.

Bartlett and Anne White)

1. Introduction There are many differing protocols and a large number of commercially available kits used for the extraction of DNA from whole blood. This procedure is one we use routinely in both research and clinical service provision and is cheap and robust. It can also be applied to cell pellets from dispersed tissues or cell cultures (omitting the red blood lysis step. 2. Materials This method uses standard chemicals that can be obtained from any major supplier; we use Sigma. 1. Waterbath set at 65C. 2. Centrifuge tubes (15 mL; Falcon). 3. Microfuge (1.5 mL) tubes. 4. Tube roller/rotator. 5. Glass Pasteur pipets, heated to seal the end and curled to form a loop or hook for spooling DNA. 6. EDTA (0.5 M), pH 8.0: Add 146.1 g of anhydrous EDTA to 800 mL of distilled water. Adjust pH to 8.0 with NaOH pellets (this will require about 20 g). Make up to 1 L with distilled water. Autoclave at 15 p.s.i. for 15 min. 7. 1 M Tris-HCl, pH 7.6: Dissolve 121.1 g of Tris base in 800 mL of distilled water. Adjust pH with concentrated HCl (this requires about 60 mL). CAUTION: the addition of acid produces heat. Allow mixture to cool to room temperature before finally correcting pH. Make up to 1 L with distilled water. Autoclave at 15 p.s.i. for 15 min. 8. Reagent A: Red blood cell lysis: 0.01M Tris-HCl pH 7.4, 320 mM sucrose, 5 mM MgCl2, 1% Triton X 100. 9. Add 10 mL of 1 M Tris, 109.54 g of sucrose, 0.47 g of MgCl2, and 10 mL of Triton X-100 to 800 mL of distilled water. Adjust pH to 8.0, and make up to 1 L with distilled water. 1

Autoclave at 10 p.s.i. for 10 min (see Note 1). 10. Reagent B: Cell lysis: 0.4 M Tris-HCl, 150 mM NaCl, 0.06 M EDTA, 1% sodium dodecyl sulphate, pH 8.0. Take 400 mL of 1 M Tris (pH 7.6), 120 mL of 0.5 M EDTA (pH 8.0), 8.76 g of NaCl, and adjust pH to 8.0. Make up to 1 L with distilled water. Autoclave 15 min at 15. p.s.i. After autoclaving, add 10 g of sodium dodecyl sulphate. 11. 5 M sodium perchlorate: Dissolve 70 g of sodium perchlorate in 80 mL of distilled water. Make up to 100 mL. 12. TE Buffer, pH 7.6: Take 10 mL of 1 M Tris-HCl, pH 7.6, 2 mL of 0.5 M EDTA, and make up to 1 L with distilled water. Adjust pH to 7.6 and autoclave 15 min at 15. p.s.i. 13. Chloroform prechilled to 4C. 14. Ethanol (100%) prechilled to 4C. 3. Method 3.1. Blood Collection 1. Collect blood in either a heparin- or EDTA-containing Vacutainer by venipuncture (see Note 2). Store at room temperature and extract within the same working day. 3.2. DNA Extraction To extract DNA from cell cultures or disaggregated tissues, omit steps 1 through 3. 1. Place 3 mL of whole blood in a 15-mL falcon tube. 2. Add 12 mL of reagent A. 3. Mix on a rolling or rotating blood mixer for 4 min at room temperature. 4. Centrifuge at 3000g for 5 min at room temperature. 5. Discard supernatant without disturbing cell pellet. Remove remaining moisture by inverting the tube and blotting onto tissue paper. 6. Add 1 mL of reagent B and vortex briefly to resuspend the cell pellet. 7. Add 250 L of 5 M sodium perchlorate and mix by inverting tube several times. 8. Place tube in waterbath for 15 to 20 min at 65C. 9. Allow to cool to room temperature. 10. Add 2 mL of ice-cold chloroform. 11. Mix on a rolling or rotating mixer for 30 to 60 min (see Note 3). 2

12. Centrifuge at 2400g for 2 min. 13. Transfer upper phase into a clean falcon tube using a sterile pipet. 14. Add 2 to 3 mL of ice-cold ethanol and invert gently to allow DNA to precipitate (see Note 4). 15. Using a freshly prepared flamed Pasteur pipet spool the DNA onto the hooked end (see Note 5). 16. Transfer to a 1.5-mL Eppendorf tube and allow to air dry (see Note 6). 17. Resuspend in 200 L of TE buffer (see Notes 7 and 8). 4. Notes 1. Autoclaving sugars at high temperature can cause caramelization (browning), which degrades the sugars. 2. As will all body fluids, blood represents a potential biohazard. Care should be taken in all steps requiring handling of blood. If the subject is from a known high risk category (e.g., intravenous drug abusers) additional precautions may be required. 3. Rotation for less than 30 or over 60 min can reduce the DNA yield. 4. DNA should appear as a mucus-like strand in the solution phase. 5. Rotating the hooked end by rolling between thumb and forefinger usually works well. If the DNA adheres to the hook, break it off into the Eppendorf and resuspend the DNA before transferring to a fresh tube. 6. Ethanol will interfere with both measurements of DNA concentration and PCR reactions. However, overdrying the pellet will prolong the resuspension time. 7.The small amount of EDTA in TE will not affect PCR. We routinely use 1 L per PCR reaction without adverse affects. 8. DNA can be quantified and diluted to a working concentration at this point or simply use 1 L per PCR reaction; routinely, we expect 200 to 500 ng/L DNA to be the yield of this procedure.

Genomic DNA isolation from blood(easiest method):

The blood samples (stored at -70degC in EDTA vacutainer tubes ) are thawed, standard citrate buffer is added, mixed, and the tubes are centrifuged. The top portion of the supernatant is discarded and additional buffer is added, mixed, and again the tube is centrifuged. After the supernatant is discarded, the pellet is resuspended in a solution of SDS detergent and proteinase K, and the mixture is incubated at 55deg C for one hour. The sample then is phenol extracted once with a phenol/chloroform/isoamyl alcohol solution, and after centrifugation the aqueous layer is removed to a fresh microcentrifuge tube. The DNA is ethanol precipitated, resuspended in buffer, and then ethanol precipitated a second time. Once the pellet is dried, buffer is added and the DNA is resuspended by incubation at 55degC overnight, the genomic DNA solution is assayed by the polymerase chain reaction.

Protocol

1. Blood samples typically were obtained as 1 ml of whole blood stored in EDTA vacutainer tubes frozen at -70deg C.

2. Thaw the frozen samples, and to each 1 ml sample, add 0.8 ml 1X SSC buffer, and mix. Centrifuge for 1 minute at 12,000 rpm in a microcentrifuge.

3. Remove 1 ml of the supernatant and discard into disinfectant.

4. Add 1 ml of 1X SSC buffer, vortex, centrifuge as above for 1 minute, and remove all of the supernatant.

5. Add 375 ul of 0.2M NaOAc to each pellet and vortex briefly. Then add 25 ul of 10% SDS and 5 ul of proteinase K (20 mg/ml H2O) (Sigma P-0390), vortex briefly and incubate for 1 hour at 55degC.

6. Add 120 ul phenol/chloroform/isoamyl alcohol and vortex for 30 seconds. Centrifuge the sample for 2 minutes at 12,000 rpm in a microcentrifuge tube.

7. Carefully remove the aqueous layer to a new 1.5 ml microcentrifuge tube, add 1 ml of cold 100% ethanol, mix, and incubate for 15 minutes at -20deg C.

8. Centrifuge for 2 minutes at 12,000 rpm in a microcentrifuge. Decant the supernatant and drain.

9. Add 180 ul 10:1 TE buffer, vortex, and incubate at 55degC for 10 minutes.

10. Add 20 ul 2 M sodium acetate and mix. Add 500 ul of cold 100% ethanol, mix, and centrifuge for 1 minute at 12,000 rpm in a microcentrifuge.

11. Decant the supernatant and rinse the pellet with 1 ml of 80% ethanol. Centrifuge for 1 minute at 12,000 rpm in a microcentrifuge.

12. Decant the supernatant, and dry the pellet in a Speedy-Vac for 10 minutes (or until dry).

13. Resuspend the pellet by adding 200 ul of 10:1 TE buffer. Incubate overnight at 55degC, vortexing periodically to dissolve the genomic DNA. Store the samples at -20degC.