In silico Double Digestion Fingerprinting (DDF)

Simulation of AFLP-PCR experiment when using one non-palindromic endonuclease

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:

In this experiment we will use two endonucleases to perform an AFLP-PCR like experiment (it is an AFLP-PCR experiment, but it is not a classic one, because we are using one non-palindromic endonuclease). The maximum length of fragments has been set up to 3000 bp.

In the example we will use this two endonucleases:

Endonuclease number 1 (RE1) is palindromic
e.g.: EcoRI, with recognition G'AATT_C.
This enzyme has been selected from the list of palindromic endonucleases in the form,
Endonuclease number 2 (RE2) is not palindromic.
p.e: AflI, with recognition sequence G'GWC_C (G'GAC_C or G'GTC_C)
The recognition sequence GGACC has been included in the form. Sequence GGTCC is omitted.
Finally, we have selected the checkbox shown by the arrow ("For non-palindromic endonuclease, discern complementary").

When using the selected endonucleases, the following fragments will be yielded:

EcoRI-AflI fragments
 
Type A : from GAATTC to GGACC
         AATTCNNNNNNNNNNNNNNNNNNNNNNNNNG        
             GNNNNNNNNNNNNNNNNNNNNNNNNNCCTG                          

Type B : from GAATTC to GGTCC
         AATTCNNNNNNNNNNNNNNNNNNNNNNNNNG
             GNNNNNNNNNNNNNNNNNNNNNNNNNCCAG

AflI-EcoRI fragments

Type C: from GGACC to GAATTC
        GACCNNNNNNNNNNNNNNNNNNNNNNNNNG                
           GNNNNNNNNNNNNNNNNNNNNNNNNNCTTAA                        

Type D: from GGTCC to GAATTC
        GTCCNNNNNNNNNNNNNNNNNNNNNNNNNG        
           GNNNNNNNNNNNNNNNNNNNNNNNNNCTTAA

As it can be seen in the example, the AflI overhang ends are different in 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 we will need adaptors for EcoRI end and for the two types of AflI ends (AflI adapters A and B).

Adapters required to amplified all EcoRI-AflI and AflI-EcoRI fragments

EcoRI Adapter: 5'-AATTNNNNNNNNNNNNNN-3'
                   3'-NNNNNNNNNNNNNN-5' 


AflI Adapter A: 5'-GACNNNNNNNNNNNNNN-3'                (will amplify fragment types A and D)
                   3'-NNNNNNNNNNNNNN-5'


AflI Adapter B: 5'-GTCNNNNNNNNNNNNNN-3'                (will amplify fragment types B and C)
                   3'-NNNNNNNNNNNNNN-5'

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 sequence for AflI is GGACC, but not the complementary (GGTCC). So the recognition sequence indicated is discerned from its complementary. As a consequence, the program will only compute fragment types A and D (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. The effect of this strategy will be similar to the ue of selective nucleotides (reduction of number of amplified fragments). In this experiment with M genitalium, from a total of 152 cEoRI-AflI fragments (fragments types A, B, C and D), only 94 are amplified (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.