Biology Reference
In-Depth Information
Another crude preparation method involves boiling the insect with subse-
quent dilution. Lysing cells in boiling water is a quick and effective method of
preparing DNA for the PCR, although only a small volume of the extract can be
used because cellular debris may inhibit the PCR.
Cells in complex biological fluids or cells resistant to lysis require additional
processing. Rapid and inexpensive DNA extractions can be achieved using
Chelex
®
, a polyvalent chelating agent in resin form, which reduces degrada-
tion of DNA heated in low-ionic-strength buffers, probably by chelating heavy
metal ions that may serve as catalysts in the breakdown of DNA (
Singer-Sam
et al. 1989
). Adding Chelex during boiling appears to increase the amount of
DNA produced from samples containing small amounts of template. Chelex
is nontoxic, provides rapid results, and can be used to isolate DNA from hun-
dreds of individual insects or mites suitable for either the standard PCR or RAPD-
PCR (
Edwards and Hoy 1994
) (
Table 8.3
). A disadvantage to Chelex is that the
extracted DNA is unstable and must be used within a few days.
A variety of DNA extraction methods have been tested for the PCR
(
Goldenberger et al. 1995, Steiner et al. 1995, Hammond et al. 1996, Shahjahan
et al. 1995, Aljanabi and Martinez 1997
), and many commercial kits are avail-
able. However, these may be expensive if used to extract DNA from large
Table 8.3: A Rapid Method for Extracting DNA from a Single Insect or Mite Using Chelex
®
100
Chelating Resin.
1.
Add a single insect or mite to a microcentrifuge tube. Insects can be alive or frozen at
−
80 °C.
2.
Tap tube sharply to move the insect to the bottom of the tube. If the insect or mite is difficult to detect
visually, add a small amount of buffer and spin in a microfuge tube to ensure the specimen is at the
bottom.
3.
Immerse the bottom of each microfuge in liquid nitrogen. Freeze a plastic pestle in liquid nitrogen.
(Pestles are prepared in advance by melting the ends of 200-
μ
l pipettor tips and molding them to the
bottoms of microcentrifuge tubes.)
4.
Macerate the frozen specimen well within the tube with the frozen pestle.
5.
Add 200
μ
l of a 5% (w/v) Chelex
®
solution (Bio-Rad Laboratories).
6.
Vortex the solution vigorously to thaw it.
7.
Remove the pestle and place tube into a temperature cycler and heat to 56 °C for 15 minutes.
8.
Centrifuge the sample (
>
100 g) for 15 seconds in a nanofuge to allow removal of the DNA solution from
the top of the tube. Avoid removing any Chelex resin from the bottom of the tube. The DNA can be used
for both traditional and RAPD-PCR. The DNA is not suitable for cutting with restriction enzymes, ligation
reactions, or DNA sequencing. The DNA can be stored for a few days only at
−
20 °C.
(Adapted from Edwards and Hoy 1994.)
