Extraction of DNA from Blood

The average WBC of an adult is 5 - 10 X 106 cells per ml of blood.  Therefore, the theoretical recovery of DNA per ul of blood is 30 - 60 ng. The PCR reactions call for on average 1 ng of DNA (single or double stranded). This is the equivalent of 1/20 of 1 ul of blood, or 350 sperm. Many of the commercially available kits  are sensitive below 1 ng of DNA (100-250 pg). 

Cell Lysis Buffer - Non-ionic detergent, Salt, Buffer, EDTA designed to lyse outer cell membrane of blood and epithelial cells, but will not break down nuclear membrane. EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations such as Mg2+. Mg2+is a cofactor for Dnase nucleases. If the Mg2+is bound up by EDTA, nucleases are inactivated.

Proteinase K - it is usual to remove most of the protein by digesting with proteolytic enzymes such as Pronase or proteinase K, which are active against a broad spectrum of native proteins, before extracting with organic solvents. Protienase K is approximately 10 fold more active on denatured protein. Proteins can be denatured by SDS or by heat.

Phenol - often means phenol equilibrated with buffer (such as TE) and containing 0.1% hydroxyquinoline and 0.2% b-mercaptoethanol (added as antioxidants. The hydoxquinoline also gives the phenol a yellow color,making it easier to identify the phases (layers). Chloroform - often means a 24:1 (v/v) mixture of chloroform and isoamyl alcohol. The isoamyl alcohol is added to help prevent foaming. The Phenol/Chloroform/Isoamyl Alcohol ratio is 25:24:1

•The four critical variables are the purity of the DNA, its molecular weight, its concentration, and the speed at which it is pelleted.

•DNA a concentrations as low as 20 ng/ml will form a precipitate that can be quantitatively recovered.

•Typically 2 volumes of ice cold ethanol are added to precipitate the DNA.

Very short DNA molecules (<200 bp) are precipitated inefficiently by ethanol.

The optimum pelleting conditions depend on the DNA concentration. Relatively vigorous microcentrifuge steps such as 15 minutes at or below room temperature at 12,000 rpm are designed to minimized the loss of DNA from samples with yields in the range of a few micrograms or less.

Solutes that may be trapped in the precipitate may be removed by washing the DNA pellet with a solution of 70% ethanol. To make certain that no DNA is lost during washing, add 70% ethanol until the tube is 2/3 full. Vortex briefly, and recentrifuge. After the 70% ethanol wash, the pellet does not adhere tightly to the wall of thetube, so great care must be taken when removing the supernatant.

Isopropanol (1 volume) may be used in place of ethanol (2 volumes) to precipitate DNA. Precipitation with isopropanol has the advantage that the volume of liquid to be centrifuged is smaller.

Isopropanol is less volatile than ethanol and it is more difficult to remove the last traces; moreover, solutes such sodium chloride are more easily coprecipitated with DNA when isopropanol is used.

TE Buffer - Tris-EDTA Buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA, or TE-4 which is 10 mM Tris, 0.1 mM EDTA. DNA is resuspended and stored in TE buffer. DNA must be stored in a slightly basis buffer to prevent depurination, and the EDTA chelates any Mg2+ helping to inactivate DNases.

DNA Extraction from Buccal Swabs

The buccal swab samples were suspended in 500 μl lysis buffer [10 mM Tris (pH 8.0), 10 mM EDTA, and 2.0% SDS], and 50 μl 10% SDS, followed by 5–10 μl 20 mg/ml proteinase K (Himedia, Mumbai, India), was added. The samples were incubated 1–3 h at 56°C until the tissue was totally dissolved. The DNA was then extracted from each sample with an equal volume of phenol:chloroform: isoamyl alcohol solution (25:24:1) and mixed gently by inverting the tubes for 3 min. The samples were then centrifuged (Eppendorf 5415R; Hamburg, Germany) for 10 min with 10,000 g (4°C), and the upper aqueous layer was transferred to a fresh, sterilized microcentrifuge tube. RNase A (10 μl of 10 mg/ml; Fermentas, Thermo Scientific, Germany) was added, and the solution was incubated at 37°C for 30 min. Equal volumes of chloroform:isoamyl alcohol solution were added and centrifuged (Eppendorf 5415R), again with 10,000 g (4°C) for 10 min. The upper aqueous layer was transferred to a sterilized microcentrifuge tube, and double the volume of chilled isopropanol (Merck, Whitehouse Station, NJ, USA) was added, along with one-tenth volume of 3 M sodium acetate, and chilled at −20°C for 1 h for precipitation. After 1 h, the sample was centrifuged (Eppendorf 5415R) at 10,000 g (4°C) for 10 min. After decanting the supernatant, 250 μl 70% ethanol (Merck) was added, and the pellet was dissolved; the mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant was decanted gently. The pellet was air-dried under laminar air flow, and the dried pellet was resuspended in 50 μl nuclease-free water or 1× 10 mM Tris-HCl, 1 mM EDTA, pH 7.6 (TE), buffer and frozen at −20°C or at −80°C for storage.

DNA Extraction from Hair Sample

DNA was isolated from hair shafts using modified versions of the microscopic glass-grinding and organic solvent extraction protocol.12–14 As these protocols expose the specimen to increased risks of contamination, the present study has replaced the tedious physical digestion method with a smooth chemical digestion method using dithiothreitol (DTT) (Hi-media) as it is a strong reducing agent with relatively high salt content and also an anionic detergent. Digestion buffer (500 μl; 10 mM Tris-HCl, 10 mM EDTA, 50 mM NaCl, 20% SDS, pH 7.5) was added to a 1.5-ml microcentrifuge tube, along with 40 μl of 1 M DTT (to a final concentration of ∼80 mM, 240 mM of sodium acetate, pH 5.2) and 15 μl of 10 mg/ml proteinase K (to a final concentration of ∼0.3 mg/ml; Himedia). Hair sample was added to this solution before vortexing and incubating for 2 h at 56°C. After 2 h of incubation, the sample tube was vortexed again, and an additional 40 μl of 1 M DTT and 15 μl of 10 mg/ml proteinase K were added, followed by gentle mixing and incubation at 60°C for 2 more h or until hair was dissolved completely.

The DNA was then extracted from each sample with an equal volume of phenol:chloroform: isoamyl alcohol solution (25:24:1) and mixed gently by inverting the tube for a few minutes. The samples were centrifuged (Eppendorf 5415R) for 10 min with 10,000 g (4°C), followed by transferring the upper aqueous layer into a fresh, sterilized microcentrifuge tube. RNaseA (10 μl of 10 mg/ml; Fermentas, Thermo Scientific) was added and kept for incubation at 37°C for 30 min. An equal volume of chloroform:isoamyl alcohol was added, and the tube was centrifuged (Eppendorf 5415R) again at10,000 g (4°C) for 10 min. The upper aqueous layer was transferred into a fresh, sterilized microcentrifuge tube before double the volume of chilled isopropanol and one-tenth volume of 3 M sodium acetate were added. The sample was chilled at −20°C for 1 h for the DNA precipitation to occur. The sample was centrifuged (Eppendorf 5415R) at 10,000 g (4°C) for 10 min. The supernatant was discarded, 250 μl 70% ethanol was added, and the pellet was tapped gently before further centrifugation (Eppendorf 5415R) at 10,000 rpm for 10 min. The supernatant was discarded, and the pellet was air-dried in a laminar air flow, resuspended in 50 μl nuclease-free water or 1× TE buffer, and frozen at −20°C or −80°C for storage.

DNA Extraction from Urine Sample

Frozen urine samples were thawed at room temperature and then placed immediately in ice before DNA isolation. The urine specimen was inverted or swirled in a specimen cup to create a homogenous suspension of cells. One milliliter of the specimen was transferred into an Eppendorf tube and centrifuged (Eppendorf 5415R) for 10 min at 10,000 g (4°C). The supernatant was removed, and a dry pellet containing cells was chilled at −20°C for 15 min. Lysis buffer (500 μl; 10 mM Tris, 1.2 mM EDTA, 10% SDS, pH 9.0) was added to the dry pellet, and the sample was vortexed to resuspend the pellet. Proteinase K (20 μl of 20 mg/ml; Himedia) was added, and the tube was incubated in a water bath (CW-30G; Jeio Tech, Seoul, Korea) at 56°C for 2 h. Sodium acetate (60 μl of 3 M) and 0.5 ml cold isopropanol were added, mixed, and chilled at −20°C for 1 h, followed by centrifugation at 10,000 g (at 4°C) for 20 min. The supernatant was discarded, 250 μl 70% ethanol was added, and the pellet was tapped gently, followed by centrifugation at 10,000 rpm for 10 min before the supernatant was discarded gently. The pellet was air-dried in a laminar air flow, and the dried pellet was resuspended in 50 μl nuclease-free water or 1× TE buffer and frozen at −20°C or −80°C for storage.

DNA Extraction from Blood Sample

Lymphocytes from whole blood were separated by lysing the red blood cells (RBCs) using a hypotonic buffer (ammonium bicarbonate and ammonium chloride; Himedia) with minimal lysing effect on lymphocytes. Three volumes of RBC lysis buffer was added to blood sample and mixed by vortexing and inverting thoroughly for 5 min and centrifuged (Eppendorf 5415R) at 20,00 g for 10 min. The supernatant was mostly discarded, leaving behind ∼1 ml to prevent loss of cells. To the pellet, 3 vol RBC lysis buffer was added, and vortexing, inverting, and centrifuging steps were repeated two to three times until a clear supernatant and a clean white pellet were obtained. After the final wash, the supernatant was discarded completely, and the pellet was resuspended in 500 μl PBS, followed by addition of 400 μl cell lysis buffer (10 mM Tris-HCl, 10 mM EDTA, 50 mM NaCl, 10% SDS, pH 7.5) and 10 μl proteinase K (10 mg/ml stock; Himedia). The sample was vortexed to dissolve the pellet completely and incubated for 2 h at 56°C in a water bath (CW-30G; Jeio Tech) for lysis. An equal volume of phenol (equilibrated with Tris, pH 8) was subsequently added to the tube and mixed well by inverting for 1 min. The tube was centrifuged at 10,000 g (at 4°C) for 10 min, and the aqueous upper layer was transferred to a fresh tube containing equal volumes (1:1) of phenol and chloroform:isoamyl alcohol (24:1). The tube was mixed by inverting for 1 min and centrifuged for 10 min at 10,000 g (at 4°C). The supernatant was then transferred to a fresh tube, and 10 μl of 10 mg/ml RNase A (Fermentas, Thermo Scientific) was added.

The sample was incubated at 37°C for 30 min before an equal volume of chloroform: isoamyl alcohol (24:1) was added and mixed by inverting the tube for 1 min and centrifuging at 10,000 g (at 4°C) for 10 min. The supernatant was transferred to a fresh tube, and twice the volume of absolute alcohol (Merck) was added and inverted gently a few times and chilled at −20°C, followed by centrifugation at 10,000 g at (4°C) for 20 min. The supernatant was discarded, 250 μl 70% ethanol was added, and the pellet was tapped gently, followed by centrifugation at 10,000 rpm for 10 min and decanting the supernatant gently. The pellet was air-dried in a laminar air flow, and the dried pellet was resuspended in 50 μl nuclease-free water or 1× TE buffer and frozen at −20°C or −80°C for storage.

Isolation of genomic DNA from frozen clotted whole blood using the QIAGEN® Genomic-tip 100/G

Procedure
1. Weigh tissue. For every 50 mg add 1 ml of Lysis Buffer. Homogenize briefly to disrupt the solid mass.
Lysis Buffer:
1 % Triton® X-100
600 mM Guanidine-HCl
10 mM EDTA
100 mM NaCl
10 mM Tris·Cl, pH 8.0
2. Add boiled RNase A to a final concentration of 20 μg/ml and incubate at 37°C for 1 h with gentle agitation.
3. Add Proteinase K to a final concentration of 1 mg/ml and incubate at 50°C for 2–3 h with gentle agitation.
4. Centrifuge sample for 10 minutes at 10–12,000 x g to pellet insoluble debris. Carefully transfer supernatant into a clean tube. Try to avoid carryover of particulate matter.
5. Pre-equilibrate a QIAGEN Genomic-tip 100/G with 3 ml of Buffer QBT.
6. Load the genomic sample onto the QIAGEN Genomic-tip 100/G.
7. Wash the QIAGEN Genomic-tip 100/G with 3 x 10 ml of Buffer QC.
8. Elute the ultrapure genomic DNA from the QIAGEN Genomic-tip 100/G with 5 ml of Buffer QF.
9. Add 0.7 volumes of room-temperature isopropanol to precipitate the genomic DNA. Invert gently several times to mix. A visible, fibrous genomic precipitate should form immediately.

10. Pellet DNA by centrifugation, or capture it on a Pasteur pipette which has been heated and bent into a hook at the end.
11. Wash genomic DNA with 3–5 ml of ice-cold 70% ethanol.
12. Briefly air-dry, and resuspend in an appropriate volume of TE.
Note: Be careful not to over-dry the DNA. Ten minutes under ambient conditions should suffice.