Biology Reference
In-Depth Information
AFLP-PCR with North American populations, from which they were suspected to
have derived (
Forneck et al. 2000
). Two distinct populations were found in Europe,
and AFLP-PCR patterns suggest that two different introductions occurred, one from
the northeastern United States and the other from the south central United States.
A simplified version of AFLP was developed to discriminate between European
and African honey bees (
Suazo and Hall 1999
). The protocol involved digest-
ing DNA and ligating the adapters in one reaction rather than two; one restric-
tion enzyme was used rather than two; and amplification was accomplished in
one reaction rather than two. Finally, the PCR products were electrophoresed in
agarose-Synergel instead of polyacrylamide and visualized by ethidium bromide
staining rather than autoradiography of labeled primers. These modifications in
AFLP-PCR may reduce the amount of polymorphism detected.
8.4.2 Anchored PCR
If only one sequence is known that is suitable for a primer (rather than two),
anchored PCR
can be used. The procedure involves synthesis of cDNA with the
known primer from mRNA (
Collasius et al. 1991
). A poly(G) tail is added to the
cDNA. The second primer is developed by synthesizing a primer with a poly(C)
sequence, which allows amplification of a second DNA strand that is comple-
mentary to the cDNA. Subsequent cycles yield amplified DNA from both strands.
8.4.3 Arbitrary Primers
Ecologists, evolutionary biologists, and geneticists often wish to develop genetic
markers for insects for which little genetic information is available.
Arbitrarily
primed PCR
(AP-PCR) can produce a characteristic “fingerprint” pattern for any
genome, which could be useful for developing markers for breeding programs,
genetic mapping, population genetics, or epidemiology (
Welsh and McClelland
1990, Welsh et al. 1992, McClelland and Welsh 1994
).
AP-PCR involves two cycles of low-stringency amplification, followed by cycles
conducted at higher stringency, using a single primer of arbitrary sequence. The
term
stringency
refers to PCR conditions such as the annealing temperature. If
a high annealing temperature is used, then the primers will only anneal to the
template DNA if a high proportion of the sequences match. Lower annealing
temperatures allow some mismatches. Full-length primers (20-34nt long) that
have been used include the Universal M13 sequencing primer, the M13 reverse
sequencing primer, and the T3 sequencing primer.
How does AP-PCR work? At lower temperatures, an arbitrary primer can
anneal to many sequences with some mismatches. By chance, some primers will
