What is DNA isolation?

DNA Discovery and Extraction

Deoxyribonucleic acid (DNA) was discovered in 1869 by the Swiss physician Friedrich Miescher, who studied white blood cells in pus obtained from a surgical clinic. Miescher found that when bandages that had been removed from the postoperative wounds of injured soldiers were washed in a saline solution, the cells on the bandages swelled into a gelatinous mass that consisted largely of DNA. Miescher had isolated a denatured form of DNA—that is, DNA not in the normal double-stranded conformation. After a series of experiments, Miescher concluded that the substance he had isolated originated in the nuclei of the blood cells; he first called the substance nuclein and later nucleic acid.

The first problem when extracting DNA is lysing, or breaking open, the cell. Bacteria, yeast, and plant cells usually have a thick cell wall protecting them, which makes lysis more difficult. Bacteria, such as Escherichia coli, are the easiest of these cells to open by a process called alkaline lysis, in which cells are treated with a solution of sodium hydroxide and detergent that degrades both the cell wall and the cell membrane. Yeast cells are often broken open with enzymes such as lysozyme that degrade cell walls or by using a “French press,” a piston in an enclosed chamber that forces cells open under high pressure. Plant tissue is usually mechanically broken into a fine cell suspension before extraction by grinding frozen tissue in a mortar and pestle. Once the suspension of cells is obtained, the tissue may be treated with a variety of enzymes to break down cell walls or with strong detergents, such as sodium lauryl sarcosine, that disrupt and dissolve both cell walls and cell membranes. Animal cells, such as white blood cells, do not have cell walls and can generally be opened by osmotic shock, the lysing of cells by moving them from a liquid environment with a high solute concentration to an environment with a very low solute concentration.

Isolation and Purification

Although lysis methods differ according to cell type, the process of DNA isolation and purification is more standardized. The isolation process may be imagined as a series of steps designed to remove either naturally occurring biological contaminants from the DNA or contaminants added by the scientist during the extraction process. The biological contaminants already present in cells are proteins, fat, and ribonucleic acid (RNA); additionally, plant cells have high levels of complex carbohydrates. Contaminants intentionally added by scientists may include salts and various chemicals.

After cells are lysed, a high-speed centrifugation is performed to form large-scale, insoluble cellular debris, such as membranes and organelles, into a pellet. The liquid extract remaining still contains dissolved proteins, RNA, and DNA. If salts are not already present in the extract, they are added; salt must be present later for the DNA to precipitate efficiently. Proteins must be removed from the extract since some not only degrade DNA but also inhibit enzymatic reactions with DNA that would be involved in further DNA manipulations used in cloning, for example. Proteins are precipitated by mixing the extract with a chemical called phenol. When phenol and the extract are mixed in a test tube, they separate into two parts like oil and water. If these fluids are centrifuged, precipitated proteins will actually collect between the two liquids at a spot called the interphase. The liquid layer containing the dissolved DNA is then drawn up and away from the precipitated protein.

The protein-free solution still contains DNA, RNA, salts, and traces of phenol dissolved into the extract. To remove the contaminating phenol, the extract is mixed with a chloroform/isoamyl alcohol solution (CIA). Again like oil and water, the DNA extract and CIA separate into two layers. If the two layers are mixed vigorously and separated by centrifugation, the phenol will move from the DNA extract into the CIA layer. At this point the extract—removed to a new test tube—contains RNA, DNA, and salt.

The extract is next mixed with 100 percent ethanol, inducing the DNA to precipitate out in long strands. The DNA strands may be isolated by either spooling the sticky DNA around a glass rod or by centrifugation. If spooled, the DNA is placed in a new test tube; if centrifuged, the liquid is decanted from the pellet of DNA. The precipitated DNA, with salt and RNA present, is still not pure. It is washed for a final time with 70 percent ethanol, which does not dissolve the DNA but forces salts present to go into solution. The DNA is then reisolated by spooling or centrifugation and dried to remove all traces of ethanol. At this point, only DNA and RNA are left; this mixture can be dissolved in a low-salt buffer containing the enzyme RNase, which degrades any RNA present, leaving pure DNA.

Technological advances have allowed deproteinization by the use of “spin columns” without the employment of toxic phenol. The raw DNA extract is placed on top of a column containing a chemical matrix that binds proteins but not DNA; the column is then centrifuged in a test tube. The raw extract passes through the chemical matrix and exits protein-free into the collection tube. These newer methods not only increase safety and reduce the production of toxic waste; they are also much faster.

For the isolation of DNA for cloning, DNA is typically broken into fragments using enzymes called restriction endonucleases. The fragment of interest is typically separated from other DNA using gel electrophoresis in an agarose gel. The DNA is stained with ethinium bromide, which permits visualization using UV light. The fragment of DNA is cut out of the agarose gel and purified using spin columns, which contain silica to which DNA binds in the presence of chaotropic salts. The chaotropic salt, such as guanidium chloride, denatures biomolecules by disrupting the shell of hydration around them. This allows a positively charged ion to form a salt bridge between the negatively charged silica and the negatively charged DNA backbone when the salt concentration is high. After the DNA is adsorbed to the silica surface, all other molecules pass through the column. The DNA is then washed with high salt and ethanol, and ultimately eluted
with low salt.

Plasmids are used as vectors to clone DNA of interest. Plasmids are extrachromosomal DNA that replicate independent of the chromosome and occur naturally in bacteria. To isolate plasmids independent of chromosomal bacterial DNA, holes are punctured in the bacterial cell wall by gently mixing a bacterial cell suspension with alkali, which is then neutralized. The holes that are generated are of a size that permits the plasmids to leak out of the cell while the chromosomal DNA remains trapped in the bacteria and is separated from the plasmid DNA and RNA by differential centrifugation. The cell debris forms a pellet, which is discarded. Proteins, RNA, and plasmid DNA are present in the supernatant. RNA is removed with RNAse. Plasmid DNA can be purified using either phenol/chloroform extraction and ethanol precipitation or silica column chromatography.

Key Terms



chloroform/isoamyl alcohol (CIA)

:

a mixture of two chemicals used in DNA isolation to rid the extract of the contaminating compound phenol




lysis

:

the breaking open of a cell




osmotic shock

:

the lysing of cells by moving them from a liquid environment with a high solute concentration to an environment with a very low solute concentration





phenol


:

a simple chemical used in DNA extraction to precipitate proteins and aid in their removal





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