Biology Reference
In-Depth Information
be able to anneal to the target DNA within a few hundred bases of each other
and on opposite strands. Sequences between these positions then will be ampli-
fied. The extent to which sequences amplify depends on the efficiency of prim-
ing and the efficiency of extension. During early cycles, those sequences that
prime most efficiently will predominate. During later cycles, those that amplify
most efficiently will predominate.
Between three and 20 DNA products typically are produced in AP-PCR, which
allows differentiation between closely related strains of some species (
Welsh
et al. 1990
). AP-PCR also has been used to amplify RNA in order to detect and
clone mRNAs that are differentially expressed in different cells (
McClelland
et al. 1995
). Clones of the aphid
Ceratovacuna nekoashi
from a single gall were
shown by AP-PCR to be genetically identical, whereas aphids from different galls
on the same twig were successfully differentiated, indicating that members of
a gall constitute a clonal population, a gall is founded by a single female, and
intergall migration is absent or rare (
Fukatsu and Ishikawa 1994
).
A modification of AP-PCR was developed and called DALP, or
D
irect
A
mpliication of
L
ength
P
olymorphisms (
Desmarais et al. 1998
). DALP uses the
M13 sequencing forward primer as a core sequence for the forward primer and
the M13 reverse primer. These primers produce specific multibanded patterns
that show inter-individual length variations. Each band then can be sequenced
with the universal sequencing primers.
8.4.4 Asymmetric PCR
Single-stranded DNA can be produced by asymmetric PCR. By providing an
excess of primer for one of the two strands, typically in ratios of 50:1 to 100:1,
amplification results in product that is primarily single-stranded. Early in the
reaction, both strands are produced, but as the low-concentration primer is
depleted, the strand primed by the abundant primer accumulates arithmetically.
Such ss DNA is particularly useful for sequencing (see Chapter 7).
8.4.5 Degenerate Primers
If only a limited portion of a protein sequence is known for a target gene,
degenerate primers
may allow detection of new or uncharacterized sequences
in a related family of genes, or may amplify members of a gene family.
Degenerate primers are a mixture of oligonucleotides varying in base sequence,
but with the same number of bases.
Designing degenerate primers for the PCR requires several considerations.
You will recall that the genetic code is degenerate (with more than one codon
