This approach has not been yet tried in the lab .
We will explain the theoretical experiment performed when filling the
form as shown in the picture:
As it can be seen in the example,
I overhang ends are
type A and B fragments, and in type C and D fragments. Type A and D
fragments and type B and C are identical.
To amplify all fragments
will need adaptors for Eco
end and for the two types of Afl
I adapters A and B).
Adapters required to amplified all EcoRI-AflI and AflI-EcoRI fragments
EcoRI Adapter: 5'-AATTNNNNNNNNNNNNNN-3'
AflI Adapter A: 5'-GACNNNNNNNNNNNNNN-3' (will amplify fragment types A and D)
AflI Adapter B: 5'-GTCNNNNNNNNNNNNNN-3' (will amplify fragment types B and C)
In this experiment, we have selected the checkbox indicated by the
arrow ("For non-palindromic endonuclease, discern complementary"). By
selecting this checkbox we are indicating to the program that
fragments from EcoRI to AflI must be
computed (and AflI to EcoRI) where the recognition
for AflI is GGACC
not the complementary (GGTCC). So
recognition sequence indicated is discerned from its complementary. As
a consequence, the program will
only compute fragment types A and
(and fragment B and
C will not be computed).
The use of non-palindromic endonucleases in AFLP-PCR experiments may be
considered as a strategy to reduce the number of fragments amplified.
of this strategy will be similar to the ue of selective nucleotides
of number of amplified fragments). In this experiment with M genitalium
, from a total of
(fragments types A, B, C and D), only 94 are
(fragments types A and D).
Two non-palindromic endonucleases may be used in the experiment, and
selective nucleotides may also be included in the experiment.