For AFLP we must do Restriction-Ligation with two Adapter and restriction enzymes.
Maybe the DNA is cut with same restriction enzyme, therefore PCR is performed with one primer. How we can identify this PCR product?
I am not quite sure if I understood correctly the question, but if you cut with only one enzyme (even if it is the rare-cutter EcoRI), use one adapter and then amplify with only one primer you will end with lots of fragments, impossible to resolve with polyacrylamide gel electrophoresis (I'm not even sure PCR would be that efficient to amplify them all).
AFLP involves steps for reducing this high number of fragments, doing a pre-amplification with the primer sequence corresponding to the adapter plus an additional nucleotide (amplifying only those genomic sequences starting with the complementary base). Later, this sub-population of fragments undergoes an amplification using the same primer sequence in the previous step plus two additional nucleotides, so at the end the amplification is restricted to fragments containing specifically those three nucleotides next to the restriction site.
You can play with different combinations of the selective nucleotides to analyze different sub-populations of fragments.
- Badges
- Science method