Biology Reference
In-Depth Information
This chapter provides an introduction to the procedures that could be used
to carry out the experiment illustrated in
Figure 5.1
. Simplified protocols of
some procedures are provided for those interested in knowing something about
the steps involved, although the methods described are illustrative rather than
complete laboratory protocols. There are many excellent laboratory manu-
als available that provide detailed techniques (see General References) as well
as websites with up-to-the-minute protocols. As noted, many techniques have
been simplified and are available in kits provided by commercial sources. This
led one molecular biologist to conclude that the availability of kits has led to a
“paradigm shift” in biology (
Gilbert 1991
).
5.3 Extracting DNA from Insects
Most DNA extraction protocols require that the insect or specific tissues be
ground up. However, methods have been developed that allow the specimen
to be preserved (Section 5.3.2). Whatever method is used to extract the DNA
from insects, it must be purified before it can be used in most molecular biology
projects. The degree of purity needed is determined by the goals of the experi-
ment. Any time DNA is to be cut, or ligated, or cloned, it must be
very clean
.
Proteins, lipids, sugars and nucleases that could interfere with these procedures
should be removed. Insects should be killed rapidly so that nucleases (DNases or
RNases) are not allowed to damage the DNA or RNA being extracted. Insects can
be stored in 95% ethanol (EtOH) at room temperature or in a freezer (
−
20°C)
until their DNA is extracted. Alternatively, the living insect can be ground up in
an extraction buffer that stops degradation of the DNA or RNA.
5.3.1 DNA Extraction Resulting in Loss of the Specimens
One of the most common methods formerly used to extract and purify nucleic
acids required phenol (
Table 5.1
). Cells were disrupted mechanically by grinding
to release the genomic DNA; usually the insect sample was ground in a solu-
tion containing protease K (
Jowett 1986, Sambrook and Russell 2001
). Once the
DNA is released, phenol can be used to extract DNA (or RNA) in large- or small-
scale procedures. A plethora of different phenol-extraction methods have been
published, but the primary function of phenol is to remove proteins from an
aqueous solution containing nucleic acids. Proteins need to be removed because
some of them are nucleases that could damage the DNA, whereas others sim-
ply could interfere with later manipulations. Ethylenediaminetetra-acetic acid
(EDTA) is often added; it is a chelating agent that binds magnesium (Mg
2
+
) ions
that are required for nucleases to act on the DNA.
