In silico Double Digestion
Simulation of AFLP-PCR
experiment when using a non-palindromic endonuclease with degenerated
It is important to understand example 1
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
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
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:
Adapters for GGACC end
Type A : from GAATTC to GGACC
AATTCNNNNNNNNNNNNNNNNNNNNNNNNNG Adaptor for GGACC: 5'-GACNNNNNNNNNNNNNN-3'
Type B : from GAATTC to GGTCC
Type C : from GAATTC to GGGCC
Type D : from GAATTC to GGCCC
Type E: from GGACC to GAATTC
Type F: from GGTCC to GAATTC
GTCCNNNNNNNNNNNNNNNNNNNNNNNNNG Adaptor for GGACC: 5'-NNNNNNNNNNNNNN-3'
Type G: from GGGCC to GAATTC
Type H: from GGCCC to GAATTC
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)
from GGTCC to EcoRI (type A fragments and a few more; see below)
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.
RI enzyme may be
substituted by a second enzyme similar to Asp
S9I. Selective nucleotides may
also be included in the experiment.