Biology Reference
In-Depth Information
the nonworking primer pairs while reducing the concentration of the effec-
tive primer pairs. Other recommendations for optimizing multiplex PCR can be
found in Sambrook and Russell (2001) . Examples of the use of multiplex PCR
include efforts to detect honey bee viruses (acute bee paralysis virus, Kashmir
bee virus and Deformed wing virus) in bumble bees using reverse-transcriptase
PCR ( Meeus et al. 2010 ) and Nosema apis and Nosema ceranae infections in
honey bees ( Hamiduzzaman et al. 2010 ). Rugman-Jones et al. (2011) developed
a method to identify any life stage of 10 parasitoids of soft scales in California
citrus, using ribosomal RNA genes and three multiplex PCR protocols.
8.4.10 Nested PCR
Nested PCR involves a two-step procedure in which one pair of primers is used
to amplify a fragment. Subsequently, a second pair of primers is used to amplify
a smaller fragment from an aliquot of the product of the first PCR. Nested PCR is
designed to be both sensitive and specific.
Nested PCR of a 16S rRNA gene from the causative agent of granulocytic
ehrlichiae ( Ehrlichia chaffeensis ), a disease of humans, was found to be so
sensitive that as few as two copies of the 16S gene could be detected when a
spiking experiment was conducted ( Massung et al. 1998 ). Spiking experiments
were conducted using known quantities of a plasmid containing the 16S rRNA
gene spiked into background human genomic DNA. The use of serial dilutions
to determine how repeatable and reliable a PCR assay is should be done when-
ever it is important to resolve how often false negatives are likely to occur in an
experiment.
8.4.11 PCR-RFLP
PCR-RFLP eliminates some of the disadvantages to traditional restriction frag-
ment length polymorphism (RFLP) analysis for analyzing population variation
using DNA isolated from individual insects ( Karl and Avise 1993 ). If no prim-
ers are available from the literature, a genomic DNA library is constructed and
clones are isolated. Clones with inserts of 500-2000 bp are chosen and sequences
of the first 100-150 nt from both ends are obtained so that PCR primers can be
designed. Nuclear DNA is amplified by the PCR using these primers and digested
with appropriate restriction enzymes. The cut DNA is visualized after electro-
phoresis by staining with ethidium bromide. The advantage to PCR-RFLP is that
DNA extracted from a single individual is sufficient, after amplification, to pro-
vide electrophoretic bands that can be visualized without having to be hybrid-
ized with radiolabeled probes.
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