Biology Reference
In-Depth Information
Selecting primers for allele-specific PCR remains somewhat empirical,
although computer programs have been developed to aid in their design
(
Table 8.2
) (
Lexa et al. 2001
). It is desirable, where possible, to select primer pairs
with a G
+
C content of
≈
50% and a random base distribution (except at the 3
′
end). It is important to avoid complementary 3
′
ends of the primer pairs to avoid
primer-dimer artifacts that will reduce the yield of the desired DNA.
Runs of three or more Cs or Gs at the 3
′
ends of primers may promote
mispriming at G
+
C-rich regions. Primers with T, C, or G as the 3
′
(last) nucle-
otide results in a more specific PCR product than if the primer ends in an A
(
Ayyadevara et al. 2000
). Amplification efficiency is reduced when T and A
occupy the penultimate 3
′
position of the primer (
Ayyadevara et al. 2000
).
Palindromic sequences within primers should be avoided. Sequences that will
yield a significant secondary structure should be avoided. In some cases, primers
with two Gs and/or Cs at the 3
′
end (“G/C clamp”) will ensure the primer anneals
strongly to the template to promote specific priming (
Roux 1995
).
Sometimes, suboptimal primers, perhaps containing high amounts of A and
T, must be used due to the nature of the target sequence. Low concentrations
of tetramethylammonium chloride (TMAC) could reduce mispriming and thus
reduce nonspecific amplification (
Chevet et al. 1995
).
8.3.7 Storing Insects for the PCR
Ideal killing and storage techniques include placing the insects into an ultralow
freezer (
−
80 °C) or into liquid nitrogen, or dry ice. Rapid killing reduces damage
to DNA by endogenous DNases. Storage of insects under inappropriate condi-
tions can have detrimental effects on the quality and quantity of DNA available
for the PCR (
Dick et al. 1993
). However, it is not always possible to kill and store
insects under optimal conditions in remote field sites.
Alternative killing and storage methods include the use of ethanol (EtOH)
at 95 or 100% (
Quicke et al. 1999
). The use of EtOH at
<
95% is undesirable
because the water in insects dilutes the EtOH, which can result in degradation
of DNA. If your insects are large, it may be desirable to kill them in 100% EtOH,
pour it off, and replace it with fresh to reduce the dilution with endogenous
water. Storage in methanol and chloroform, as well as low concentrations of
EtOH, can result in poor preservation of DNA (
Fukatsu 1999
).
Other killing and storage methods may provide useful DNA, although
loss in quality and quantity may occur. Desiccation with silica gel has pre-
served DNA of tiger beetles for several months (
Vogler and Pearson 1996
).