Biology Reference
In-Depth Information
have melting temperatures near 65-70 °C permit the use of higher annealing
temperatures to enhance reaction specificity. Thermal cycling profiles in Long
PCR typically use a hot start at 78-80 °C, initial denaturation at 94°C for 1 min-
ute, 25-40 cycles of denaturation at 94°C for 15 seconds, and annealing and
extension steps at 60-68°C for 30-60 kb of target DNA. Typically, extension times
are increased for each subsequent cycle to facilitate production of long product
molecules.
Other factors that influence the success of the Long PCR include the integrity
of the target DNA, which means that DNA extraction methods must be carefully
considered. Longer targets can be amplified best from DNA with little shearing
damage (
Cheng and Kolmodin 1998
). Several DNA extraction methods produce
large DNA fragments from insects (
Ebert 1996
), although shearing of DNA may
be difficult to avoid when extracting DNA from adult insects because their exo-
skeleton can damage the DNA during grinding. One solution is to extract DNA
from embryos (
Rabinow et al. 1993
).
The Long PCR protocol has been used for another application—amplifying
microbial DNA when mixed with arthropod DNA (
Jeyaprakash and Hoy 2000,
Hoy et al. 2001
). When insect and microbial DNA are mixed, efficient amplifi-
cation of the microbial DNA seems to be inhibited, for unknown reasons. For
example, the ability to detect the endosymbiont
Wolbachia
within the bodies
of various arthropods was greatly enhanced when the Long PCR, rather than
a standard PCR, protocol was used;
Wolbachia
were found in 76% of the 63
arthropods examined in 13 orders. The Long PCR thus can be used to increase
sensitivity or fidelity of the PCR even when shorter DNA targets are amplified.
The Long PCR protocol is
≈
5-7 orders of magnitude more sensitive in ampli-
fying
Wolbachia
DNA than the standard PCR (
Jeyaprakash and Hoy 2000
).
When standard and Long PCR protocols were compared using known amounts
of
Wolbachia
template DNA mixed with known amounts of insect DNA, the
Long PCR could amplify as few as 100 copies of
Wolbachia
DNA consistently. By
contrast, standard PCR was only able to reliably detect
Wolbachia
DNA when
at least 100 million copies of plasmid DNA were present. Similar results were
obtained in the amplification of the bacterium causing citrus greening disease
(Hoy et al. 2001) (
Figure 8.4
).
Long PCR should have many applications whenever long DNA fragments are
useful; for example, Long PCR has been used to develop rapid restriction maps of
DNA fragments of 8-18 kb (
Her and Weinshilboum 1995
). Long PCR can be used
to clone large genes or be a labor-saving alternative for studying larger genome
segments such as entire mitochondria that are 16-20 kb (
Nelson et al. 1996
).
