Role of Sodium in DNA Extraction

Use of Sodium in DNA Extraction

Figure: Structure of DNA

DNA does not float around free inside the nucleus of a cell. It is associated with a variety of different proteins and encased in a cellular membrane. In animal cells, the DNA is also contained within a nuclear membrane. In order to extract DNA from a cell, the associated membranes and proteins must first be removed and then physically separated from the DNA. Sodium can be involved in several of the steps to accomplish this goal.

Sodium as a Detergent

o Sodium is an element. Its chemical symbol is Na for Natrium, the Latin word for sodium. It is a positive ion and often associates with negative ions as part of useful compounds. For example, when sodium ions are bound to chloride ions, they make the compound sodium chloride, which is ordinary table salt.

Several different forms of sodium are used in DNA extraction. Sodium dodecyl sulfate, or SDS. is a sodium-containing detergent. It has the chemical formula of C₁₂H₂₅NaO₄S, where the Na stands for sodium. Detergents are used to break down cell walls and membranes. They work by chemically poking holes in the cell membranes or walls.

Once holes are poked in the membranes, the membranes can be further disrupted mechanically, as with a blender. After that, it is easier to get the contents of the cell out, including the DNA.

Sodium as an Alkaline Agent

o Sodium hydroxide is another compound containing sodium that is used to extract DNA out of a cell. The chemical formula for sodium hydroxide is NaOH. Sodium hydroxide is a base. A solution of sodium hydroxide makes the solution very basic or alkaline. Sodium hydroxide can act by loosening the rigid structure of a cell wall or membrane, thereby releasing the DNA.

Sodium hydroxide is most often used in plasmid DNA extraction. Plasmid DNA in bacteria usually exists in a ring form in the cytoplasm, separate from the chromosomal DNA in the nucleus. While chromosomal DNA programs the bacterial cell's functions and processes, plasmid DNA is often a genetically engineered DNA that codes for a specific gene or genes of interest. Plasmids are highly valuable research tools and their extraction from bacterial cells is a routine laboratory procedure.

To separate the bacterial chromosomal DNA and sheared DNA from plasmid DNA, sodium hydroxide is often used. Chromosomal DNA and sheared DNA are both linear, whereas plasmid DNA is circular. When the solution is basic, for example, when sodium hydroxide is added, double-stranded DNA molecules separate. This is known as denaturation. Their complementary bases are no longer associated with each other. This can be thought of much like the two complementary sides of a zipper. When DNA is double-stranded, the zipper is zipped up. When the DNA is denatured, the zipper is not only unzipped, but the two strands are completely separated from each other, like in a jacket.

On the other hand, plasmid DNA molecules, although they are unzipped, are not separated. The circular strands can easily find their complementary strands and "renature" back into a circular double-stranded plasmid DNA molecule once the solution is no longer alkaline. This is one of the unique properties of plasmids that allow them to be separated from chromosomal DNA. In this way, the plasmid DNA with the desired gene of interest can be removed and separated from the regular bacterial chromosomal DNA.

Sodium Acetate's Role

Sodium can also be in the form of sodium acetate. Like sodium hydroxide, sodium acetate is used to help separate plasmid DNA from chromosomal DNA, but by a much different mechanism and at a different part of the DNA extraction procedure.

Single strands of linear DNA are insoluble in high salt. They will precipitate out, forming a solid. Adding sodium acetate to SDS detergent solutions forms solids of cellular debris as well as denatured chromosomal linear DNA. Circular plasmid DNA is not insoluble in high salt. Plasmid DNA will remain in solution, thus separating the desired plasmid DNA from the rest of the DNA in the cell.

Sodium hydroxide provides the basic solution to denature and unzip the DNA strands, both plasmid and chromosomal. Once the DNA is no longer in the alkaline solution, only the plasmid DNA can zip back up. In order to separate the denatured, unzipped, chromosomal DNA from the renatured, zipped-up plasmid DNA, sodium acetate is used to selectively precipitate the chromosomal DNA and other cellular debris away from the desired double-stranded plasmid DNA.

Role of Sodium in DNA Precipitation

Precipitated chromosomal DNA and cellular debris can be removed from the soluble plasmid DNA still in solution by centrifugation, a high-speed spinning process that causes the solids to be forced into the bottom of a tube as a small pellet, allowing the liquid at the top containing plasmid DNA to be separated out.

This plasmid DNA can then be precipitated out of the solution by adding an alcohol and a salt. It is often desirable to precipitate out the plasmid DNA in order to concentrate its amount in solution and in order to bring it back up into a solution that is stabilizing to its chemical structure. The salt used to precipitate the plasmid DNA can be sodium chloride or sodium acetate, for example, but can also be ammonium acetate or lithium chloride.

Sodium is a positively charged ion. In a solution of sodium chloride, table salt, for example, the sodium chloride molecule separates into sodium ions and chloride ions. DNA, on the other hand, is highly negatively charged. The large negative charge of the DNA molecule is neutralized by the positive sodium ions in solution. This neutralization of the negative charges on DNA allows it to precipitate in alcohol. Without the salt, the DNA remains negatively charged and will stay in the aqueous part of the solution.

If this mixture is centrifuged, the precipitated plasmid DNA will become a pellet at the bottom of the tube. The liquid portion can be removed and the DNA can then be put back into solution, or resuspended, in a different solution at the desired concentration.

Sodium as Part of the Buffer Solution

DNA is usually resuspended in a solution containing Tris and EDTA. This is called a buffer solution. EDTA stands for the chemical ethylenediamine tetracetic acid, and usually exists in the lab as a disodium salt, Na₂C₁₀H₁₆N₂O₈. Buffers are used to prevent drastic pH changes, and in this case, Tris/EDTA keeps the DNA in a solution in a pH range of about 7.0 to 9.0.