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and some fungi, this amplification would create too many fragments. In those cases,
the preselective amplification is performed with additional nucleotides on the end of
each primer (see page 10-7). Each added nucleotide reduces the number of
sequences by a factor of four.
The thermal cycling conditions of the preselective amplification step have been
optimized to generate a constant final mass of fragments. Band intensity in
subsequent reactions can therefore be correlated with relative differences in
representation of the fragments within the genome, and not to the overall amount of
genomic DNA that went into the initial restriction-ligation mix.
It is not necessary to perform this step if:
Selective
Amplification—
Microbial
Fingerprinting
♦
relative peak height information is not desired
♦
methods are available to normalize the final signal
♦
very accurate quantitation of the input DNA is performed routinely
Additional PCR amplifications are run to reduce the complexity of the mixture further
so that the fragments can be resolved on a polyacrylamide gel. These amplifications
use primers chosen from the 18 available AFLP Microbial Fingerprinting Kit Selective
Primers (nine EcoRI fluorescent dye-labeled primers and nine unlabeled MseI
primers). After PCR amplification with these primers, a portion of the samples is
analyzed on a Applied Biosystems DNA Sequencer.
Selective amplification with an EcoRI and an MseI primer amplifies primarily
EcoRI-MseI-ended fragments. The EcoRI-EcoRI fragments do not amplify well. The
MseI-MseI fragments are not visualized because they do not contain fluorescent dye
labels. Only the EcoRI-containing strands are detected (Figure 10-7).
Figure 10-7 Selective amplification with fluorescent dye-labeled primers
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10-6 AFLP Mapping