Total cell DNA preparation and purification

In this article, I briefly describe the preparation and purification of total cell DNA.

Total DNA and its preparation

The complete set of genetic information is the genome of an organism. The genome is stored in long molecules of DNA, called chromosomes. Total cell DNA acts as a source for the genes to be cloned. To clone genes, first total cell DNA needs to be prepared. Total cell DNA may be prepared from the DNA of a bacterial culture, animal cells, plant, or other living organisms.

Preparing bacterial total cell DNA includes four steps (figure 1). The first step involves the growth and harvesting of a bacterial culture. In the second step, the cells are broken to release their contents. In the third step, the DNA obtained from the cell extract undergoes purification. The DNA undergoes the process of concentration in the final step.

The growth and harvesting of a bacterial culture

Bacteria can be grown in a liquid medium, which is also known as broth culture. The bacteria grow and divide efficiently by getting a balanced mixture of the required nutrients at a specific concentration. The medium M9 is a defined medium in which a mixture of inorganic nutrients, such as nitrogen, magnesium, and calcium with glucose (carbon and energy source) are present. The LB medium is a complex undefined medium in which two of the ingredients, tryptone, and yeast extract are complicated mixtures of unknown chemical compounds.

To grow a bacterial culture under precisely controlled conditions, a defined medium must be used. But, when the culture is grown only as a source of DNA, a complex medium can be used. To prepare a cell extract, the bacteria must be obtained in a very small volume. Then, harvesting is done by spinning the culture in a centrifuge. Low centrifugation speeds will pellet the bacteria at the bottom of the centrifuge tube, allowing the culture medium to be poured off.

Preparation of cell extract

In the second step of total cell DNA preparation, cell extract is prepared. Bacterial cells can be opened by physical methods and chemical methods. Bacterial cells most commonly undergo cell lysis by chemical methods. Cell lysis by chemical method is brought about by one chemical that attacks the cell wall while the other disrupts the cell membrane.

The enzyme lysozyme or a combination of lysozyme and ethylenediamine tetraacetate (EDTA) weakens the cell wall of bacteria. Lysozyme digests the polymeric compounds that give the cell wall its rigidity. EDTA helps in the removal of magnesium ions, which are essential for preserving the overall structure of the cell envelope. The cellular enzymes that could degrade DNA are also removed by EDTA. Sometimes a detergent such as sodium dodecyl sulfate (SDS) is added along with the chemicals because detergents aid the process of lysis by removing lipid molecules and thereby cause disruption of the cell membranes. The insoluble cell debris can be pelleted by centrifugation, (figure 2) leaving the cell extract as a supernatant.

Purification of DNA from a cell extract

Bacterial cell extract is purified from significant quantities of protein and RNA. DNA-associated proteins and other cellular proteins may be degraded with the addition of a protease. The protein is precipitated by the addition of a salt such as ammonium or sodium acetate. When the sample is vortexed with phenol-chloroform (1:1 ratio) and centrifuged, the precipitated proteins are left as a white coagulated mass at the interface between the aqueous and organic layers. (figure 3). With the help of a pipette, the aqueous solution of nucleic acids (DNA and RNA) is removed. The enzyme ribonuclease rapidly degrades the RNA molecules into ribonucleotide subunits.

Concentration of DNA samples

For DNA concentration, ethanol precipitation is the most frequently used method. Absolute ethanol, in the presence of a salt (only monovalent cations) efficiently precipitates polymeric nucleic acids at around a temperature of -20ºC. The ethanol with a thick solution of DNA can be layered on top of the sample, causing molecules to precipitate at the interface. The precipitate can be collected by centrifugation of the mixture of ethanol with a dilute solution and then re-dissolved in an appropriate volume of water.

Ultraviolet absorbance spectrophotometry aids in the accurate measurement of DNA concentrations. The amount of ultraviolet radiation absorbed by a solution of DNA is directly proportional to the amount of DNA in the sample. Absorbance is measured usually at 260nm. A pure sample of DNA indicates the ratio of absorbances at 260 and 280nm is 1.8 i.e., A₂₆₀/A₂₈₀ is 1.8. If the ratio is less than 1.8, it indicates the sample with a contamination of protein or phenol.

Preparation of DNA from plant and animal cells

DNA Preparation from bacterial cells has a disparity with plant and animal cells. The enzyme lysozyme, which degrades bacterial cell walls does not affect plant and animal cell walls. Most animal cells have no cell wall at all and can be lysed simply by treating them with detergent. The process of phenol extraction does not work for plant tissues, as they are enriched with carbohydrates. Thus, a detergent called cetyltrimethylammonium bromide(CTAB), which forms an insoluble complex with nucleic acids, is used. The addition of CTAB to a plant cell extract causes precipitation of the nucleic acid-CTAB complex, leaving carbohydrates, protein, and other contaminants in the supernatant. The precipitate is then collected by centrifugation and resuspended in 1M NaCl, which causes the complex to break down. Then, the RNA is removed by ribonuclease treatment.

Conclusion

Total cell DNA acts as a source for the genes to be cloned. To clone genes, first total cell DNA needs to be prepared. Total cell DNA may be prepared from the DNA of a bacterial culture, from animal cells, from a plant, or from any other living organism.

The preparation of total bacterial cell DNA starts with the growth and harvesting of a bacterial culture. This step is followed by the preparation of the cell extract. After the cell extraction is done, the concentration of the DNA sample is done with ethanol precipitation. Ultraviolet absorbance spectrophotometry aids in the accurate measurement of DNA concentrations.

DNA preparation from plant and animal cells differs from bacterial cells. The bacterial cell wall degrading enzyme lysozyme does not affect plant and animal cell walls. As most animal cells have no cell walls, they can be simply lysed with the addition of a detergent. The process of phenol extraction does not work for plant tissues, as they are enriched with carbohydrates. Thus, a detergent called cetyltrimethylammonium bromide(CTAB), which forms an insoluble complex with nucleic acids, is used.

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