46 Landgraf and Wolfes
Allele-specific oligodeoxynucleotide (ASO) PCR (57) is based on
the fact that only fully complementary primers will initiate the reac-
tion. Therefore, amplification will be prohibited, if the complemen-
tary sequence is deleted or mutated. A primer specific for the mutated
sequence is used as positive control. Point mutations, deletions, and
insertions in the a- 1-antitrypsin gene were monitored (58) with prim-
ers harboring a 3' base mismatch with respect to the unmutated gene.
One 3' mismatch is necessary and sufficient to ensure reliable detec-
tion. Primers bearing a mismatch located in the center of the primer
sequence are not suitable for allele-specific hybridization.
A different technique to find point mutations is competitive
oligodeoxynucleotide priming (COP)(19). When the annealing of a
primer mixture is performed at low stringency, the perfectly matched
primer will be favored with a level of discrimination greater than
100:1. An extension of COP and ASO-PCR involves the use of oligo-
deoxynucleotides conjugated with a fluorescent dye marker; the prod-
ucts are analyzed in a fluorimeter, hence rendering the electrophoretic
analysis obsolete (30,59).
4.3. Application of Specifically Designed Primers
For the reason that a primer needs to be complementary only at its
3' end to the sequence to be analyzed, mismatched primers can be of
advantage in introducing new restriction sites in the amplified prod-
uct. The product can be traced by restriction enzyme analysis even at
high background (55). Also, the fragment produced can be cloned
readily, one more benefit of this method (32).
Sometimes it is difficult to detect a single mutation in longer frag-
ments using denaturation gradient gel electrophoresis (DGGE). This
drawback can be overcome by attachment of a 40-bp GC-rich stretch
to the primers (60). The analysis of the PCR product, melting at a
higher temperature, will enhance the sensitivity of this method.
Mixed oligodeoxynucleotide primers designed on the basis of the amino
acid sequence of urate oxidase were used successfully to clone the gene
for this enzyme (34). If the primers crosshybridize to repetitive sequences,
even an optimal adaptation of the annealing temperature will not elimi-
nate unspecific bands. In this case, the amplification products can be
diluted and reamplified with a second set of primers (61,62). The use of
dc7GTP in a ratio of 3:1 over GTP is recommended to improve the PCR
of sequences with distinct secondary structures (8,63).