Biology Reference
In-Depth Information
Add 5% Chelex
®
100 solution.
Remove
swab
Incubate 56
C
then 100
°
C
°
Centrifuge
Centrifuge
DNA in solution
Cellular material
Precipitated
protein
(a) (b) (c) (d)
Figure 4.1
The Chelex
100 extraction is quick and easy to perform. (a) The cellular material
is added to 1 ml of TE (1 mm EDTA, 10 mm Tris: pH 8.0) and incubated at room temperature for
10-15 minutes. (b) The tube is centrifuged at high speed to pellet the cellular material and the
supernatant is removed. (c) The pellet of cellular material is resuspended in 5% Chelex
, the tube
is incubated at 56
◦
C for 15-30 minutes and then placed in a boiling water bath for 8 minutes.
The tube is centrifuged at high speed for 2-3 minutes to pellet precipitated protein. (d) The
supernatant contains the DNA and can be used directly in a PCR
has a very high affinity for polyvalent metal ions, such as magnesium (Mg
2
+
); it
chelates the polyvalent metal ions and effectively removes them from solution. The
extraction procedure is very simple, the Chelex
100 resin, which is supplied as
beads, is made into a 5% suspension using distilled water. The cellular material
is incubated with the Chelex
100 suspension at 56
◦
C for up to 30 minutes.
Proteinase K, which digests most cellular protein, is often added at this point. This
incubation is followed by 8 - 10 minutes at 100
◦
C to ensure that all the cells have
ruptured and that the protein has denatured. The tube is then simply centrifuged to
pellet the Chelex
100 resin and the denatured protein at the bottom of the tube,
leaving the aqueous solution containing the DNA to be used in PCR (Figure 4.1).
The Chelex
100 suspension is alkaline, between pH 9.0 and 11.0, and as a result
DNA that is isolated using this procedure is single-stranded.
The major advantages of this method are it is quick, taking around a hour; it is
simple and does not involve the movement of liquid between tubes, thereby reducing
the possibility of accidentally mixing samples; the cost is very low; and it avoids the
use of harmful chemicals. Importantly, it is amenable to a wide range of forensic
samples [9]. The DNA extract produced using this method is relatively crude but
sufficiently clean in most cases to generate a DNA profile.
Silica-based DNA extraction
Within molecular biology generally, the 'salting out' procedure has been widely
used [1]. The first stage of the extraction involves incubating the cellular material
in a lysis buffer that contains a detergent along with proteinase K. The commonly
used detergents are sodium dodecyl sulphate (SDS), Tween 20, Triton X-100 and
