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possible to extract sufficient DNA from mite eggs that PCR could be conducted
using the GuSCN buffer followed by silica-matrix purification, which resulted in
DNA suitable for amplification of mitochondrial and single-copy nuclear genes
( Jeyaprakash and Hoy 2010 ).
Some experiments require specialized extraction methods ( Mauel et al.
1999 ). For example, different numbers of false-negative results were obtained
when DNA from ticks infected with a pathogen, granulocytic ehrlichiosis, was
extracted by three different methods. Blood-fed ticks have inhibitors of the PCR
that cannot be extracted easily with standard extraction methods. Inhibition
of the PCR also was observed when amplifying Borrelia burgdorferi DNA from
blood-fed ticks ( Schwartz et al. 1997 ).
Some PCR protocols require higher-quality DNA than others. For example,
A mpliied F ragment L ength P olymorphism PCR (AFLP-PCR) allows insects to be
“finger-printed” (see Section 8.4.1), but requires very pure and high quality DNA
that can be cut completely by restriction endonucleases. When different DNA
extraction methods were compared, two of the complex methods failed to pro-
duce adequate amounts of DNA, one simple method produced only poor quality
DNA, but three treatments (two complex DNA methods involving phenol treat-
ments plus a CTAB-based protocol) produced an adequate quality and quantity
of DNA ( Reineke et al. 1998 ).
The PCR is inhibited by a variety of impurities including: complex polysaccharides,
heme in blood, humic substances in soil, proteases, urea in urine, phenol, and deter-
gents ( Schwartz et al. 1997, Al-Soud and Radstrom 1998 ). One approach to reduce
the amount of impurities is to dilute them ( Table 8.2 ). Upon dilution, however, the
template DNA must remain sufficiently abundant. Other methods for eliminating
inhibitors include the use of dialysis or centrifugation in cesium chloride gradients,
but these methods can result in the loss of large amounts of the template DNA.
Experimental procedures sometimes can introduce inhibitors of the PCR. For
example, Lee and Cooper (1995) found that PCR carried out on DNA cloned into
E. coli failed when the bacterial colonies containing the clones were picked from
plates with wooden toothpicks. The nature of the water-soluble inhibitor in the
wooden toothpicks is unknown, but the toothpicks negatively affected both Taq
and Vent DNA polymerases.
If inhibition is a serious problem, it might be reduced by embedding whole
cells in low-melting point agarose blocks, then immersing the block in a lysis
buffer, which results in intact genomic DNA with minimal shearing damage.
The agarose is then washed and cellular debris and other contaminants dif-
fuse out during the lysis and washing steps, resulting in highly purified genomic
 
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