Biology Reference
In-Depth Information
amplification. RAPD-PCR uses short, randomly chosen primers to amplify multi-
ple DNA segments in the genome. The resulting banding patterns (similar to bar
codes) provide information about genetic variation within the entire genome of
insects.
The power of the PCR to amplify DNA is dramatic; theoretically even a single
molecule can be amplified, although efficiency usually is lower. This power cre-
ates formidable problems with contamination and requires careful organization
of PCR experiments and the use of adequate controls. However, because the PCR
is relatively easy, novices in molecular biology can use it to study molecular sys-
tematics, evolution, ecology, behavior, and development.
8.2 Introduction
The
polymerase chain reaction
(
PCR
) is an
in vitro
or cell-free method for syn-
thesizing DNA sequences in nearly any amount required starting with a small
initial quantity. The PCR is one of the most accessible and versatile techniques
available to entomologists interested in both basic and applied problems. The
PCR is powerful because it can be used to isolate specific DNA fragments, end
label DNA, mutagenize specific DNA fragments, clone cDNA and genomic DNA,
sequence DNA, quantitate RNA and DNA, and alter a variety of sequences to
study gene expression.
DNA polymerase, as used in the PCR, was designated the “Molecule of the
Year” by
Science
in 1989 (
Guyer and Koshland 1989
). The PCR became a stan-
dard laboratory method in an extraordinarily short time after it was invented in
1985 (
Mullis 1987, Rabinow 1996
). In 1993, Kary Mullis received the Nobel Prize
for chemistry for his work on the PCR, although some believe other scientists
should have shared credit for the invention (
Rabinow 1996
).
Since 1985, improvements in, and optimization of, the PCR have led to its use
by numerous scientists (
Dieffenbach and Dveksler 2003, Park 2010, Kennedy and
Oswald 2011
). The PCR has become a common procedure in forensics and diag-
nostics, and it is revolutionizing studies of basic biology, ecology, systematics,
and evolution. This relatively simple technique has provided scientists with lim-
ited experience in molecular biology the opportunity to apply molecular tech-
niques to diverse problems (
White et al. 1989, Arnheim et al. 1990, Erlich and
Arnheim 1992, King 2010
).
Although the PCR is conceptually simple, the process is, in fact, not com-
pletely understood. The PCR involves complex kinetic interactions between the
template (or target) DNA, product DNA, oligonucleotide primers (polymers
