Investigating CSI – Background material Table of Contents I ...

DNA Extraction Techniques
Organic-based Extraction
Phenol extraction is a common technique used to purify a DNA sample.
Generally, samples are extracted by addition of one-half volume of neutralized
(with TE buffer, pH 7.5) phenol to the sample, followed by vigorous mixing for a
few seconds to form an emulsion. Following centrifugation for a few minutes, the
aqueous (top) phase containing the nucleic acid is recovered and transferred to a
clean tube. Residual phenol then is removed by extraction with an equal volume
of water-saturated diethyl ether. Following centrifugation to separate the phases,
the ether (upper) phase is discarded and the DNA is concentrated by ethanol
precipitated.
Chelex
Chelex extraction is used to isolate the DNA away from the rest of the contents
of the cells. The protocol consists of mixing cells with 5% chelex and Proteinase
K and incubating the solution at 56 o C. The proteinase K is an enzyme that
digests proteins (cell membranes for example). The chemical chelex, in the form
of minute beads, binds to most of the contents of the cell except DNA. It does
this by chelating magnesium. A boiling step has been added to further breakup
the cells in order to free the DNA. The chelex beads (with the non-DNA cellular
components) are then removed by centrifugation leaving behind the DNA. This
DNA is single-stranded and ready for the polymerase chain reaction (PCR).
Silica
This extraction method consists of flowing a prepared DNA sample over silica
micro beads that attract and capture the DNA strands onto the beads. Upon
capture of the DNA strands, the DNA is washed and collected. In the presence of
high salt DNA binds to silica particles then the silica with adsorbed DNA washed
to remove salt and impurities from the original sample, and the clean DNA eluted
in water or buffer.
Silica extraction works with a wide size range of DNA and allows efficient
recovery (90%) of product. Using silica you can purify DNA as small as 100bp.
There is no upper size limit on recovery efficiency of DNA. However, precautions
should be taken during purification of longer DNA fragments (20kb) to avoid
shearing. Proteins and RNA do not bind to silica and are eliminated during
washes and for this reason it is also an ideal tool to purify and concentrate DNA
directly from various reaction mixtures. Because silica does not bind
oligonucleotides with high efficiency (