Biology Reference
In-Depth Information
numbers of samples. The “best” DNA-extraction method will depend on the
goals of your experiment. Preliminary experiments should be conducted to
determine which DNA-extraction method is appropriate. For example, if the
goal is to process large numbers of insects for ecological studies, then rapid,
nontoxic and inexpensive extraction methods, such as Chelex, may be useful and
sufficient, especially if amplifying multiple-copy genes in mitochondria or ribo-
somes. However, Chelex may yield some false-negative results and the extracted
DNA cannot be stored for long periods of time. If it is important to maintain live
insects for further study, it is possible to extract insect hemolymph from large
insects, extract the DNA from the hemolymph with Chelex, and yield DNA suit-
able for microsatellite analysis (
Gerken et al. 1998
).
Sometimes, it is important to preserve the intact arthropod specimen so that
morphological analyses can be made (
Paquin and Vink 2009, Jeyaprakash and
Hoy 2010
). Several protocols have been developed that would allow nuclear
and mitochondrial DNA to be extracted from whole insects or mites by soak-
ing the specimen in an extraction buffer, yet allow the specimen to be pre-
served (
Table 8.4
). Methods developed to extract DNA and RNA from fossil
bones and teeth have been used to extract DNA from arthropods (
Cox 1968,
Hoss and Paabo 1993, Rohland et al. 2004, Gilbert et al. 2007, Rowley et al. 2007,
Jeyaprakash and Hoy 2010
) without destroying the specimen. DNA and RNA can
be extracted from fossil bones (
Hoss and Paabo 1993
) using a salt (guanidinium
thiocyanate [GuSCN]) or guanidinium hydrochloride (GuHCl) (
Table 8.4
). It was
Table 8.4: Extraction of DNA From a Mite by Soaking in a GuSCN Buffer Followed by Isolation
Using a Silica Matrix Leaves the Body Intact for Morphological Analysis.
1.
Prepare GuSCN buffer with 5 M GuSCN, 50 mM Tris, pH 8.0, 25 mM NaCl, 20 mM ethylene dinitrilotetra-
acetic acid (EDTA) and 1.3% Triton X-100.
2.
Prepare the nucleic-acid-binding silica matrix by mixing 15 g of silicone dioxide with 125 ml of distilled
water and allow it to settle for a week. After removing and discarding the supernatant, add fresh
distilled water (125 ml), mix well, and allow the silica matrix to settle for another week. Resuspend the
settled silica matrix in 25 ml of fresh distilled water plus 25 ml of HCl, then autoclave and store at room
temperature in a dark bottle (Boom et al. 1990).
3.
Place single specimens (fresh or stored in 95% EtOH) in 200 ml of GuSCN buffer at 60 °C for 16 hours.
After soaking, 190 ml of the supernatant containing the DNA can be removed, leaving the specimen in
10 ml. It can be stored in alcohol or slide mounted.
4.
To isolate the genomic DNA, the supernatant is mixed with 10 ml of silica matrix, incubated at 25 °C for
10 minutes and then centrifuged at 12,000 rpm at room temperature. The pellet produced is washed with
70% EtOH, resuspended in 10 ml of sterile water, incubated at 60 °C for 5 minutes, centrifuged again to
remove the supernatant containing DNA from the silica matrix, then stored at
−
20 °C.
(Modified from Jeyaprakash and Hoy 2010.)
