Biology Reference
In-Depth Information
disease spirochete (
Borrelia burgdorferi
) DNA extracted from the midgut of ticks
(
Ixodes dammini
) stored for 50 years in 70% EtOH could be amplified by the PCR
(
Persing et al. 1990
). Individual tick specimens were removed from the EtOH
with flame-sterilized forceps and air-dried on filter paper disks for 5 minutes.
Then, 200
μ
l of 0.5-mm glass beads were incubated in 1 ml of 1% bovine serum
albumin in distilled water at 37 °C for 30 minutes and then washed twice in 1 ml
of distilled water. Ticks were placed whole into 0.6-ml microcentrifuge tubes
containing slurries (20
μ
l) of the treated glass beads. Specimens were crushed
into the beads with a disposable plastic dowel for 30-45 seconds to liberate the
midgut contents, and 25
μ
l of PCR buffer was added. Samples were boiled for
5 minutes, and then cooled on ice; 5-
μ
l portions of the supernatant fluid were
used for the PCR.
A simpler protocol was used by
Azad et al. (1990)
to determine whether indi-
vidual ticks or fleas were infected with rickettsia. Individual ticks or fleas were
placed in 100
μ
l of
brain heart
infusion broth and boiled for 10 minutes. The PCR
was carried out with 10
μ
l of the suspension. Because the PCR can be applied
to frozen or formalin-fixed tissues, dried museum specimens, and alcohol-
preserved specimens, PCR can reduce potential dangers involved in maintain-
ing and transporting live infectious disease vectors from the field. Detection of
pathogens by the PCR is significantly more sensitive than enzyme-linked immu-
nosorbent assay (ELISA) (
Azad et al. 1990
).
Dried, pinned specimens of the
Anopheles gambiae
mosquito complex, rang-
ing in age from 15-93 years, were tested to determine whether ribosomal DNA
could be amplified by the PCR (
Townson et al. 1999
). Most of the specimens
yielded amplifiable DNA from entire abdomens, but extractions from single hind
legs from these old, dried specimens were unsuccessful. By contrast, single legs
from a fresh specimen produced sufficient DNA to yield a PCR product. Note,
however, that ribosomal genes are present in high copy numbers.
The PCR has been used to amplify DNA from tissues preserved in formalin fol-
lowed by paraffin embedding. Specimens up to 40 years old have yielded DNA
up to 800bp in length (
Wright and Manos 1990
). However,
Watts et al. (2007)
found that air-dried insect legs
>
50 years old had limited usefulness for studies
of nuclear loci. The integrity of the DNA and the duration of fixation affect the
length of the product that can be amplified.
8.5.3 Amplifying RNA
The PCR can be used to amplify messenger RNA sequences using reverse tran-
scriptase (
Kawasaki 1990
). This process allows analysis of gene expression during
