Biomedical Engineering Reference
In-Depth Information
isolation. The organic extraction method uses organic solvents such as a
mixture of phenol, chloroform and isoamyl alcohol to separate DNA that
is soluble in the aqueous phase from other contaminants that are soluble in
the organic phase and to recover DNA from the aqueous phase by alcohol
precipitation (Ausubel
et al.
, 1994; Sambrook and Russell, 2001). Though
inexpensive, this method is fairly time-consuming and produces hazardous
organic waste, and the extract may not be suffi ciently pure for direct use in
subsequent applications, such as PCR (Verhagen
et al.
, 1999).
The salting out method uses a high salt concentration to precipitate pro-
teins and other contaminants and remove the precipitate by centrifugation
(Miller
et al.
, 1988). This method avoids use of organic solvents but still
requires an alcohol precipitation step to remove salts and concentrate the
DNA, even though low quality DNA tends to be obtained.
In the CsCl density gradient method, DNA is isolated from foreign
contaminants through phase separation under a high centrifugal force
(300 000 ×
g
-525 000 ×
g
) (Richards
et al.
, 1994). DNA is often mixed with
ethidium bromide to produce fl uorescence for visualization to enhance
DNA collection. Utilizing this method, DNA can be easily collected with-
out excess contaminants after ultracentrifugation (Carr, 2009). Additional
clean-up procedures are commonly required to remove ethidium bromide.
DNA isolated by this method is of high quality, but the process is highly
time-consuming (Duncan
et al.
, 2003).
Unlike the methods mentioned above that use chemical reagents to
extract DNA, the silica-based and anion-exchange methods utilize solid
substrates to isolate DNA. The silica-based method is based on the selective
adsorption of nucleic acids to silica under high chaotropic salt conditions,
such as guanidine hydrochloride, guanidine isothiocyanate, sodium iodide
and sodium perchlorate (Boom
et al.
, 1990). Both RNA and DNA bind to
silica but a buffering solution with optimized ionic conditions can effectively
prevent the RNA from binding to silica, thus separating DNA from RNA.
This method is simple, fast, reproducible, and most importantly, produces
high-quality DNA. In addition, DNA isolation kits that incorporate the
silica-based membrane technology, such as spin columns, are widely avail-
able from different commercial sources (Promega, 2010; QIAGEN, 2010a;
Invitrogen, 2010). A shortcoming of this technique is that the average size
of DNA isolated by a silica gel membrane is around 20-50 kb, and methods
based on the silica-based membrane technology are not suitable for isola-
tion of high-molecular-weight DNA (>100 kb) (Duncan
et al.
, 2003).
Lastly, the anion-exchange method is based on the mechanism that pos-
itively charged substrates attract negatively charged molecules to separate
DNA from proteins and other contaminants under certain salt conditions
when a sample passes through resin particles. This method also requires fur-
ther alcohol precipitation but can be used to extract high-quality DNA with
