Molecular Biology. A Practical Guide - Velikov V.A. 2013

Isolation of Total DNA from Cells
Isolation of DNA from Bacteria

The Procedure of DNA isolation from Cells and Tissues is frequently the foundational (initial) step in studying living organisms at THE MOLECULAR LEVEL. All cellular biosyntheses and Catabolism depend directly on DNA, either on its own or through enzyme Proteins. The Cell must be disrupted by one method or another, and the chromosomal DNA purified from other cellular components. First and foremost, DNA must be separated from the proteins that make up Chromatin nucleoprotein complexes. At the same time, it is crucial to protect DNA from the action of Nucleases and preserve its integrity as much as possible, since long linear DNA molecules inevitably undergo fragmentation during isolation from the cell.

Methods for DNA isolation typically include the following steps:

1) cell lysis (or physical/mechanical disruption);

2) enzymatic degradation of proteins by proteinases and/or deproteinization of the cell lysate using phenol and chloroform; 3) centrifugation to remove denatured proteins and cell organelle fragments. Next, DNA is precipitated from the solution with ethanol, and following centrifugation, the pellet is dissolved in a buffer solution. RNA is also partially co-isolated with DNA and is subsequently removed using the enzyme RNase.

For cell lysis and Protein Denaturation, the detergent sodium dodecyl sulfate and the chaotropic agent guanidine isothiocyanate are frequently employed. A number of modern methods involve the sorption of DNA onto silica gel beads in the presence of chaotropic agents, followed by centrifugation and subsequent elution of DNA from the beads into solution. Some companies supply reagent kits for DNA isolation that utilize magnetic particles coated with silica SiO₂. Certain commercial kits rely on DNA sorption onto membranes or ion-exchange sorbents. The phenol-chloroform extraction method is considered the standard.

Quantitative and qualitative Assessment of the resulting DNA sample is carried out through a subsequent standard agarose gel Electrophoresis procedure (Topic 3) by visual comparison with samples of known concentration. Spectrophotometric determination provides a more precise characterization of the DNA preparation (Topic 2).

The isolation of total genomic DNA from bacterial cells using sodium dodecyl sulfate, proteinases, and phenol (Dhaese et al., 1979) is a fairly common approach. The method is simple and reliable, and like many others, it exists in several modifications.

Along with chromosomal DNA, plasmid and phage DNA are also isolated if present in the cell, as well as RNA, which is readily removed.

In this method, The Plasma Membrane of the bacterial cell is disrupted by the action of sodium dodecyl sulfate (SDS), one of the most widely used Surfactants. The integrity of the peptidoglycan layer, the so-called murein sacculus, is likewise compromised. All proteins, including those of the nucleoid, are removed by treating the bacterial lysate with phenol, which denatures proteins without affecting Nucleic Acids. Other cellular Organic compounds and low-molecular-weight substances are lost during DNA precipitation with ethanol because they remain in solution. The nucleic acid pellet (containing both deoxyribonucleic and Ribonucleic Acids) obtained by sample centrifugation is dissolved in a specialized DNA storage buffer, TE buffer. Its constituent chelating agent, ethylenediaminetetraacetic acid (EDTA), prevents cellular nucleases from attacking the DNA by binding the Mg²⁺ cations essential for their activity.

Materials and Equipment

Basic equipment for molecular biology research: microcentrifuge, large-capacity centrifuge, thermostatic orbital shaker, vortex mixer, incubator, power supply, electrophoresis chambers, UV transilluminator, dry block incubator for microcentrifuges, pH meter, DNA thermal cycler, electroporator, spectrophotometer, and micropipettes with disposable tips.

For practical laboratory work, the following equipment is also required: laminar flow hood, fume hood, drying oven, refrigerator, freezer, balance, Water distiller, hot plate, microwave oven, UV germicidal lamp, and laboratory plasticware and glassware.

Protocol 1.1 requires the enterobacterial strain Escherichia coli XL1-Blue (Stratagene^TM) or the soil bacterium Azospirillum brasilense Sp245. CHARACTERISTICS OF THE bacterial strains and Plasmids used in the practical exercises are provided in the Appendices.

Solutions

- 2YT Medium. Per 1 L: tryptone - 16 g; Yeast extract - 8 g; NaCl - 5 g. Bring to 1 L with distilled water, adjust pH to 7.0. To prepare solid medium, add Agar up to 1.5% prior to autoclaving.

- 5% SDS. A 5% solution of sodium dodecyl sulfate (lauryl sulfate, sarkosyl, N-laurylsarcosine) in TE buffer.

- Pronase. A 5 mg/mL solution in TE buffer.

- TE Buffer. 10 mM Tris-HCl, pH 8.0; 1 mM EDTA.

- 5 M NaCl. Dissolve 292.5 g of NaCl in 800 mL of water and bring the volume to 1 L.

- Phenol-Chloroform Mixture. Water-saturated phenol (saturated after distillation with 0.1 M Tris-HCl, pH 8.0) is mixed 1:1 with a previously prepared chloroform-isoamyl alcohol mixture.

- Chloroform-Isoamyl Alcohol Mixture. The substances are mixed in a 24:1 volume ratio.

Procedure

1. Using a single colony, inoculate E. coli XL1-Blue (or A.brasilense Sp245) Bacteria into a test tube containing 5 mL of liquid 2YT nutrient medium and culture overnight with aeration at 37°C (or 30°C, respectively)^a

2. Using a micropipette, transfer 1.5 mL of the culture into a 1.7 mL Eppendorf-type plastic microcentrifuge tube. Pellet the cells by centrifugation for 5 min at 10,000 rpm. Note that this is the most frequently used speed for a microcentrifuge and is set as the default unless another speed is specified.

3. Remove the supernatant (pour off the nutrient medium over the rim: do not worry, the pellet is dense). Add 300 µL of TE buffer to the pellet and resuspend the cells using a micropipette.

4. Add 100 µl of 5% SDS solution to the cell suspension and mix by gently inverting the tube 3 times.

5. Add 150 µl of pronase solution (5 mg/ml) and mix.

6. Incubate for 1 (0.5) h in a thermostat at 37°C. This results in cell lysis and Enzymatic Protein Hydrolysis.

7. Precipitate nucleic acids from the lysate using alcohol. To do this, add an equal volume of isopropanol (550 µl) to the tube and centrifuge for 10 minutes. (If necessary, the procedure can be paused after adding the alcohol; do not centrifuge the sample immediately, but place it in the refrigerator instead. DNA can be safely stored "under alcohol" for an extended period).

8. Following centrifugation, use a micropipette to remove any remaining liquid from the tube. Dissolve the nucleic acid pellet in 500 µl of TE buffer and perform deproteinization, i.e., purify the DNA from proteins (steps 9–12).

9. Add an equal volume (500 µl) of phenol-chloroform mixture to the solution and shake vigorously until a stable emulsion forms.

10. Separate the aqueous and organic phases by centrifuging for 5 min. Phenol remains at the bottom, while the aqueous phase containing the dissolved DNA is at the top. Phenol-denatured proteins dissociate from the DNA, lose their solubility, and collect at the phase boundary (interphase) during centrifugation. This effectively extracts the DNA from the DNP complex.

11. Carefully transfer the aqueous phase containing the DNA into a clean 1.7 ml microcentrifuge tube using a micropipette. When aspirating, take care not to disturb the interphase—the dense, whitish layer between the aqueous and organic phases that contains aggregates of denatured protein molecules ^b.

12. Perform another chloroform extraction of the DNA to remove residual phenol. To do this, add an equal volume of chloroform-isoamyl alcohol mixture to the sample tube, mix, and centrifuge for 3 min. Transfer the aqueous phase to a clean tube.

13. Estimate the volume of the recovered aqueous phase using a micropipette or the graduation marks on the tube, and add 1/25 of that volume of 5M NaCl to a final salt concentration of 0.2M. Then add 2.5 volumes of ice-cold (-20°C) ethanol to precipitate the DNA (specifically, its sodium salt). Leave at -20°C in a freezer for 1–2 h. DNA can be kept under alcohol for longer periods until the sample is needed.

14. Pellet the nucleic acids by centrifuging for 10 min at the microcentrifuge's maximum speed. Discard the supernatant and wash the pellet by adding 1 ml of ice-cold 70% ethanol, followed by another centrifugation for 3 min ^c.

15. Decant the 70% ethanol and invert the tubes on filter paper. Once all the liquid has drained, air-dry the nucleic acid pellet (DNA and RNA ^d) briefly until the smell of alcohol disappears ^e. Dissolve the pellet in 50 µl of TE buffer. For DNA electrophoresis (Topic 3), 5–10 µl of the sample is sufficient.

Notes

^a For information on bacterial cultivation, consult standard microbiology manuals. Some Background details are provided in this guide only where strictly necessary.

^b A certain portion of the aqueous phase is inevitably lost during this step, up to 1/5 of the total volume. In practice, it is better to sacrifice this volume now than to compromise the purity of the preparation later. Phenol is a toxic and irritating substance; Skin contact must be avoided, and work should be carried out using microscale volumes with appropriate safety precautions.

^c Decanting the 70% alcohol directly, without centrifugation, is also acceptable.

^d If necessary, RNA can be eliminated using RNase (see Practical Work 4.1). RNA contamination in the DNA preparation does not interfere with restriction Digestion or PCR.

^e Do not over-dry the pellet, as completely dehydrated DNA becomes virtually insoluble in water. Typically, pellets should be air-dried for no more than half an hour; drying under a stream of warm air or in a solid-state micro-thermostat takes only about 5 min.