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.
