Biology Reference
In-Depth Information
The PCR has revolutionized the way in which much of our research is conducted.
However, the speed and ease of using the PCR and MDA can also result in erroneous
conclusions (
Box 8.2
).
End users and commercial developers continue to develop sophisticated tech-
niques for integrating the PCR into more applications (
Waters et al. 1998, Smyth
et al. 2010
). For example, it has long been a goal to reduce the time to obtain
results in conventional PCR machines. Thermal cycler heating and cooling rates
have constrained this goal and typical reactions take an hour or more. However,
Wheeler et al. (2011)
found that a new thermocycler could amplify DNA in less
than three minutes using two commercially available enzymes; the ultrafast PCR
machine has a superior heat transfer due to convective flow through a porous
media substrate, allowing very short cycle times.
Box 8.2 A Cautionary Tale of the Insidious Nature of Contamination and the
Sensitivity of the PCR and Whole Genome Amplification
A brief history of analyses of
Wolbachia
in the predatory mite
Metaseiulus occidentalis
will illustrate some of
the issues associated with advances in PCR methods, the high degree of sensitivity of the PCR and whole
genome amplification (WGA), as well as the possibility of contamination leading to false conclusions.
This predatory mite was known to have populations that were partially incompatible with others and to
contain two microbial symbionts, although the bacteria were not identified at that time. Once
Wolbachia
was discovered and 16S PCR primers became available, positive results were obtained using an allele-
specific PCR protocol, leading us to conclude that
M. occidentalis
was infected with
Wolbachia
, although
not all reactions were positive. Furthermore, heat treatment resulted in strains that appeared to lack
Wolbachia
based on negative PCR results and, when “
Wolbachia
-negative” females were mated with males
positive for
Wolbachia
, they exhibited reproductive incompatibility. However, the two-spotted spider mite
Tetranychus urticae
, the prey fed to these predators, also were infected with
Wolbachia
and sequencing of 16S
sequences from both predator and prey indicated they had identical sequences. This led to speculation
that horizontal transfer could have occurred between predator and prey. Subsequent PCR analyses using
wsp
sequences also indicated both predator and prey had nearly identical sequences, supporting this
hypothesis. Furthermore, although we starved the predators for 6 to 8 hours prior to extracting their DNA,
they still retained the
Wolbachia
sequences. However, PCRs of groups of predator eggs gave inconsistent
results; some were positive and some were negative, even using the highly sensitive high-fidelity PCR
protocol. We expected all eggs to be positive if transovarial transmission was involved, as it is with other
Wolbachia
infections of arthropods. When whole genome amplification became available, we used this
technique to amplify
Wolbachia
sequences prior to conducting high-fidelity PCR analyses on single eggs.
The results were again variable; some eggs were positive and some were negative (
Jeyaprakash and Hoy
2004
). During this time,
Wolbachia
was the only symbiont known to cause reproductive incompatibility.
Cardinium
, another bacterium associated with reproductive incompatibility, subsequently was discovered,
so we tested
M. occidentalis
and
T. urticae
and discovered that
M. occidentalis
was infected with
Cardinium
but their
T. urticae
prey was not. During these studies, surface decontamination of the mites to eliminate
contaminating DNA was not done. However, when bleach was used to surface-decontaminate eggs and
adults of
M. occidentalis
, combined with starvation for more than 72 hours, the positive high-fidelity PCR
signal for
Wolbachia
disappeared. This indicated that previous detection of
Wolbachia
in
M. occidentalis
was
