In this article we will discuss about the Extraction and Estimation of Deoxyribonucleic Acid (DNA).
Principle:
Extraction of DNA is accomplished by the rupturing of cell wall and nuclear membrane followed by deproteinization and precipitation of the nucleic acid using ethanol.
Materials:
a. Sample Material
b. Extraction Medium 0-15 M NaCl, 0-1 M Na2
c. Lysozyme solution, 10 mg/ml
d. 25% SDS Solution
e. 5M NaClO4
f. Chloroform: Isoamyl alcohol (24: 1)
g. 95% Ethanol
h. Saline Citrate (1x) 015 M NaCl, 0 015 M Trisodium Citrate and 1/10-fold concentrations)
i. 3 M Sodium acetate
j. Isopropanol
k. DNA standard (0-5 mg/ml)
l. Saline citrate (015 M NaCl, 0-15 M Na3 citrate) solution
m. Diphenylamine Reagent (Mix 5 g fresh or recrystallized diphenylamine, 500 ml glacial acetic acid and 13-75 ml conc. H2SO4 stable for 6 minutes at 2°C, warm to room temperature and switch to revux before use.
(A) DNA Extraction Procedure:
1. Grind 2 g of the sample material in 25 ml of extraction medium in a pre-chilled pestle and mortar.
2. Add 1 ml of lysozyme solution to the above suspension and incubate at 37°C for 30 min, shaking occasionally.
3. After the incubation, complete the lysis by adding 2 ml of SDS solution, heating this preparation for 10 min in a 60°C water bath, and finally cooling the solution to room temp, in a bath of tap water.
4. Add sufficient 5 M perchlorate solution to the lysed preparation to a final concentration of 1 M.
5. Add an equal volume of chloroform-isoamyl alcohol (24: 1) to the lysed preparation suspended in 1 M perchlorate and slowly shake (30-60 oscillations/min) in a tightly stoppered flask for 30 min at room temp.
6. Separate the resulting emulsion by centrifuging for 5 min at 10,000 g at room temperature.
7. After centrifugation, carefully pipette off the top clear aqueous phase from the coagulated protein emulsion at the interface.
8. Place the aqueous phase containing the nucleic acids in a beaker.
9. Gently stir the nucleic acid solution with a sterilized glass rod while slowly adding two volumes of 95% ethanol down the side of the beaker so that ethanol is layered over the viscous aqueous phase. Continue to gently stir the preparation to mix ethanol throughout the entire aqueous phase and spool all of the gelatinous, thread-like DNA rich precipitate on the glass rod.
10. Drain off excess fluid from the spooled crude DNA by pressing the rod against the wall of beaker until no further fluid can be squeezed from the spooled preparation.
(If the squeezing is not done sufficiently, the alcohol adhering to DNA will make it difficult to dissolve DNA.)
11. Dissolve the crude DNA on stirring the glass rod with its spool of material in 9 ml of dilute (1/10 fold) saline citrate in a test tube or small beaker. If any solubility difficulty is encountered, continue working the sample to obtain an even suspension.
12. To the even suspension, add 1 ml of 3 M sodium acetate, 1 mM EDTA, pH 7-0 solution, transfer the preparation to a 100 ml beaker, and gently swirl the sample while dripping in 5.4 ml of isopropanol.
13. If fibrous DNA is readily apparent, collect the DNA threads by stirring and spooling with a sterilized glass rod as before. If a gel-like preparation develops, add 0-5 mL more of isopropanol and stir to spool the DNA threads as before. Finally, remove excess fluid from the spooled DNA by pressing the sample against the walls of the beaker.
14. Wash the sample in test tubes containing, in turn, 10 ml of 70% ethanol and then 10 ml of 95% ethanol. Store the DNA in a stoppered tube (2°C refrigerator) as a spool submerged on the rod in 95% ethanol.
15. Remove alcohol from the spooled DNA by blotting with a clean piece of filter paper and then dissolve the DNA by stirring the glass rod in a test tube containing 9 ml of dilute (1/10 fold) saline citrate. When the DNA is dissolved add 1 ml of concentrated (10x) saline citrate solution to achieve approximately a standard (1x) saline citrate concentration. This solution can be stored at 2°C with a few drops of CHCI3.
(B) Estimation of DNA:
1. Prepare separate marked tubes containing 1 ml, 2 ml, 3 ml aliquots of the isolated DNA dissolved in standard saline citrate and similar aliquots of a 0-5 ml DNA/ml standard.
2. Make all sample tubes and a separate blank, up to 3 ml with H2O.
3. Add 6 ml of diphenylamine reagent to each tube and after mixing, heat the tubes in a boiling water bath for 10 min. Cool the tubes.
4. Read the absorbance of blue solution at 600 nm against blank.
5. Construct a standard gmph A600 (ordinate) versus quantity of DNA (abscissa) and then calculate the concentration of DNA dissolved in the saline citrate solution.