Biology Reference
In-Depth Information
using the protocol outlined in Section 5.3.1, there are no true vouchers (
Rohland
et al. 2004, Rowley et al. 2007
). A less well-known method to extract DNA and
RNA involves using a
chaotropic salt
, a salt that disrupts the structure of or
denatures proteins and nucleic acids. Guanidinium thiocyanate (GuSCN) or gua-
nidinium hydrochloride (GuHCl) are both chaotropic salts that can be used to
denature proteins and therefore can destroy nucleases that can degrade DNA or
adhere to the DNA (
Cox 1968, Bowtell 1987
). The addition of a silica-based puri-
fication method can improve extraction efficiency (
Hoss and Paabo 1993
).
Jeyaprakash and Hoy (2010)
evaluated four different protocols for extracting
DNA from single predatory mites (Phytoseiidae) using a “soaking” method and
evaluated the resultant nuclear and mitochondrial DNA by polymerase chain
reaction (PCR). (For a description of the PCR, see Chapter 8.) The DNA obtained
was suitable for amplification by the
R
andom
A
mpliication of
P
olymorphic DNA
(RAPD)-PCR protocol, as well. One extraction protocol used the GuSCN buffer
alone, the second protocol used a GuSCN-PTB buffer, the third protocol used a
GuSCN buffer followed by isolation using a silica matrix, and the fourth protocol
used a GuSCN-PTB buffer followed by isolation of DNA with the silica matrix.
Sufficient DNA was extracted after soaking intact specimens using the third pro-
tocol (GuSCN buffer and a silica matrix that binds the DNA) to yield DNA for the
PCR, although eggs of the mites had to be pricked to provide sufficient DNA.
The quality of DNA produced was high, and the DNA produced stored well
at
−
20°C. The adult mites soaked in the GuSCN buffer appeared normal after
they were slide mounted and examined under high magnification.
Rowley et al. (2007)
evaluated whether mitochondrial DNA could be extracted
from tenuipalpid mites, a spider (Araneidae), a lady beetle (Coccinellidae), a
dipteran (Ulidiidae), and a eurytomid wasp without damaging the specimens.
Specimens were removed from 80% EtOH, air-dried, and soaked in GuSCN-based
extraction buffer. DNA was precipitated with isopropanol, stored overnight
at
−
20°C, and centrifuged for 20 minutes at 40°C. The DNA was then vacuum-
dried, resuspended in TE buffer, and stored at
−
20°C. Specimens were generally
well preserved and suitable as voucher specimens, and the DNA obtained was
used to amplify mitochondrial DNA by the PCR.
5.3.3 Assessing the Quality of Extracted DNA
The accurate determination of DNA concentration and purity is essential for
many molecular genetic protocols. Knowing the concentration of DNA is essen-
tial in ensuring reproducibility, enhancing amplification of DNA for the PCR, and
cloning or sequencing protocols. Ultraviolet (UV) spectrometry often is used to
determine DNA purity and quantity. The nucleic-acid sample, which may include
