In silico Double Digestion Fingerprinting (DDF)

Simulation of AFLP-PCR experiment when using a non-palindromic endonuclease with degenerated (N) nucleotide

It is important to understand example 1 before going forward with this example. Example 1 is used as reference in this theoretical experiment. This approach has not yet been tried in the lab.

We will explain the 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 does not use a palindromic endonuclease of totally defined recognition sequence). The maximum length of fragments has been set up to 3000 bp.

We will use these two endonucleases:

Endonuclease number 1 (RE1) is palindromic.
e.g.: EcoRI, with recognition sequence G'AATT_C.
This enzyme has been selected from the list of palindromic endonucleases in the form.
Endonuclease number 2 (RE2) is not palindromic and it has a N within recognition sequence.
e.g.: AspS9I, with recognition sequence G'GNC_C.
In this experiment we will amplify only some fragments yielded by double digestion with EcoRI and AspS9I. The fragments to be amplified will be those from EcoRI to recognition sequence GGACC, but not the ones from EcoRI to recognition sequences GGTCC, GGGCC and GGCCC.
Checkbox shown by the arrow ("For non-palindromic endonuclease, discern complementary") will be selected as in example 1.

This experiment is similar to example 1 (which uses endonucleases EcoRI and AflI -G'GWC_C- ). In this case we have added sequence GGNCC in the "Exclusion sequence" field.

When using endonucleases EcoRI and AspS9I, the following fragments will be yielded:

EcoRI-AspS9I fragments
                                                    Adapters for GGACC end

Type A : from GAATTC to GGACC
         AATTCNNNNNNNNNNNNNNNNNNNNNNNNNG        Adaptor for GGACC: 5'-GACNNNNNNNNNNNNNN-3'
             GNNNNNNNNNNNNNNNNNNNNNNNNNCCTG                              3´-NNNNNNNNNNNNNN-5'

Type B : from GAATTC to GGTCC
         AATTCNNNNNNNNNNNNNNNNNNNNNNNNNG        
             GNNNNNNNNNNNNNNNNNNNNNNNNNCCAG        
                   
Type C : from GAATTC to GGGCC
         AATTCNNNNNNNNNNNNNNNNNNNNNNNNNG
             GNNNNNNNNNNNNNNNNNNNNNNNNNCCCG

Type D : from GAATTC to GGCCC
         AATTCNNNNNNNNNNNNNNNNNNNNNNNNNG        
             GNNNNNNNNNNNNNNNNNNNNNNNNNCCGG        

AspS9I-EcoRI fragments

Type E: from GGACC to GAATTC
        GACCNNNNNNNNNNNNNNNNNNNNNNNNNG                
           GNNNNNNNNNNNNNNNNNNNNNNNNNCTTAA                        

Type F: from GGTCC to GAATTC
        GTCCNNNNNNNNNNNNNNNNNNNNNNNNNG                Adaptor for GGACC: 5'-NNNNNNNNNNNNNN-3'
           GNNNNNNNNNNNNNNNNNNNNNNNNNCTTAA                           3'-NNNNNNNNNNNNNNCAG-5'

Type G: from GGGCC to GAATTC
        GGCCNNNNNNNNNNNNNNNNNNNNNNNNNG                
           GNNNNNNNNNNNNNNNNNNNNNNNNNCTTAA                        

Type H: from GGCCC to GAATTC
        GCCCNNNNNNNNNNNNNNNNNNNNNNNNNG                
           GNNNNNNNNNNNNNNNNNNNNNNNNNCTTAA

The program, in a first step, will select the following kind of fragments (as in example 1):

from EcoRI to GGACC (type A fragments and a few more; see below)
         AATTCNNNNNNNNNNNNNNNNNNNNNNNNNG
             GNNNNNNNNNNNNNNNNNNNNNNNNNCCTG

from GGTCC to EcoRI (type A fragments and a few more; see below)
        GTCCNNNNNNNNNNNNNNNNNNNNNNNNNG
           GNNNNNNNNNNNNNNNNNNNNNNNNNCTTAA

In our example, the total number of fragments for both types are 94.

Within some of these fragments there may be GGGCC and GGCCC internal sequences, but as GGNCC sequence has been introduced in the experiment as an "Exclusion Sequence", in a second step, those fragments with GGNCC internal sequence will be removed from results. Consequently, the number of fragments is reduced to 70.

EcoRI enzyme may be substituted by a second enzyme similar to AspS9I. Selective nucleotides may also be included in the experiment.