DNA
ISOLATION AND PURIFICATION
Basic to all biotechnology
research is the ability to manipulate DNA. First and foremost for recombinant
DNA work, researchers need a method to isolate DNA from different organisms.
Isolating DNA from bacteria is the easiest procedure because bacterial cells
have little structure beyond the cell wall and cell membrane. Bacteria such as E. coli are the preferred organisms for
manipulating any type of gene because of the ease at which DNA can be isolated.
E. coli maintain both genomic and
plasmid DNA within the cell. Genomic DNA is much larger than plasmid DNA, allowing
the two different forms to be separated by size.
To release the DNA from a
cell, the cell membrane must be destroyed. For bacteria, an enzyme called lysozyme digests the peptidoglycan,
which is the main component of the cell wall. Next, a detergent bursts the cell
membranes by disrupting the lipid bilayer. For other organisms, bursting the
cells depends on their architecture. Tissue samples from animals and plants
have to be ground up to release the intracellular components. Plant cells are
mechanically sheared in a blender to break up the tough cell walls, and then
the wall tissue is digested with enzymes that break the long polymers into
monomers. DNA from the tail tip of a mouse is isolated after enzymes degrade
the connective tissue. Every organism or tissue needs slight variations in the
procedure for releasing intracellular components.
Once released, the
intracellular components are separated from the remains of the outer structures
by either centrifugation or chemical extraction. Centrifugation separates
components according to size, because heavier or larger molecules sediment at a
faster rate than smaller molecules. For example, after the cell wall has been
digested, its fragments are smaller than the large DNA molecules.
Centrifugation causes the DNA to form a pellet, but the soluble cell wall
fragments stay in solution. Chemical extraction uses the properties of phenol
to remove unwanted proteins from the DNA. Phenol is an acid that dissolves 60%
to 70% of all living matter, especially proteins. Phenol is poorly water
soluble, and when it is mixed with an aqueous sample of DNA and protein, the
two phases separate, much like oil and water. The protein dissolves in the
phenol layer and the nucleic acids in the aqueous layer. The two phases are separated
by centrifugation, and the aqueous DNA layer is removed from the phenol.
Once the proteins are
removed, the sample still contains RNA along with the DNA. Because this is also
a nucleic acid, it is not soluble in phenol. Luckily, the enzyme ribonuclease (RNase) digests RNA into ribonucleotides. Ribonuclease treatment
leaves a sample of DNA in a solution
containing short pieces of RNA and ribonucleotides. When an equal volume of
alcohol is added, the extremely large DNA falls out of the aqueous phase and is
isolated by centrifugation. The smaller ribonucleotides stay soluble. The DNA
is ready for use in various experiments.
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