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The procedure of this technique is divided into three steps:

1. Digestion of total cellular DNA with one or more restriction enzymes and
ligation of restriction half-site specific adaptors to all restriction fragments.

2. Selective amplification of some of these fragments with two PCR primers


that have corresponding adaptor and restriction site specific sequences.

3. Electrophoretic separation and amplicons on a gel matrix, followed by


visualisation of the band pattern.

The aim of this tool is to perform a theoretical AFLP-PCR experiment by using


the same principles, and to suggest the adaptors and primers needed in the
experiment.

Choosing bacteria species

Select the bacterial genera you want to work with. A form will appear where we
may select the species, and the restriction enzymes and selective nucleotides for
the experiment.

We may also include the plasmids if available. For bacterial species with two
chromosomes, both chromosomes will be used in the experiment. In some
bacterial species, chromosomes are linear, and this fact has also been considered
in the experiment.

Digestion and ligation to adaptors

After selection of genome, the information necessary to perform the experiment


must be selected. The form has been partially reproduced below:

5'-
AG 3000 GT

-3'

Select restriction enzymes from the lists (restriction enzyme 1 from RE1 list,
and restriction enzyme 2 from RE2 list). They will be used to perform a
complete theoretical DNA digestion of the genome. Selective nucleotides may
also be introduced (see selective nucleotides in the form above: "AG" in the 5'
end, and "GT" in the 3' end).

The restriction enzymes available in the form have the following characterictics:

1. They all will cleave DNA within recognition sequence

2. The recognized sequence is not ambiguous (no degenerated nucleotides)

3. They will all yeild overhang ends (no blund ends)

Selective nucleotides introduced in the form must match the upper strand of
DNA (see the grey segments in the picture).

As a result of DNA digestion, three types of DNA fragments will be produced:


1. Fragments cleaved in both ends by the same restriction enzyme (RE1 or
RE2)

2. Fragments cleaved in 5' end by RE1 and by RE2 in 3' end

3. Fragments cleaved in 3' end by RE1 and by RE2 in 5' end

In AFLP-PCR, only type 2 and 3 fragments will be amplified to yeild visible


bands. This is due to ligation of different adaptors in each site of the fragment
which will allow a geometric increase of this kind of bands when PCR
amplification is perform. As a consequence, type 1 fragments have been
eliminated from calculations in the theoretical experiment. Adaptors will be
ligated to DNA fragments as shown in the picture below.

Ligation of adaptors in AFLP-PCR experiments

The sequence of these adaptors are partially defined in the response page. Some
nucleotides of the adaptors are defined by the recognition sequence of the
restriction enzymes (in pale blue and pale green). The sequence of adaptors
which does not match the endonuclease recognition sequence (in magenta and
red) must be designed to avoid recognition of genomic DNA of the species used
in the experiment and to prevent aberrant results.

If the restriction enzymes used to cut the genomic DNA are not heat labile, or
restriction and ligation are performed simultaneously, the adaptor sequence must
not regenerate the original recognition sequence. To avoid this regeneration they
must be used adaptors mustbe used that produce a base change in the recognition
sequence. An example is shown below:

DNA sequence with


//-------GAATTC------//------ EcoRI and MseI
TTAA------// recognition
//-------CTTAAG------//------ sequences
AATT------//
EcoRI MseI
//-------G AATTC------//------T DNA restriction
TAA------//
//-------CTTAA G------//------AAT
T------//

NNNNNNA AATTC------//------T Addition of


TACnnnnnn adaptors.
nnnnnnTTTAA G------//------AAT As nucleotides in red
GNNNNNN are
different to the
original
ones (blue),
NNNNNNAAATTC------//------TTACnnnnnn restriction
nnnnnnTTTAAG------//------AATGNNNNNN sites are not
reconstructed.

In the results page of theoretical AFLP-PCR partial sequence of adaptors allow


the reconstruction of th original recognition sequence, which is a valid option
only when heat labile endonucleases are used, and restriction and ligation are
performed separately.

PCR amplification with adaptor specific primers

In AFLP-PCR experiments, the primers must be designed to allow PCR


amplification of the fragments cleaved by RE1 in 5' end and RE2 in 3' end, so
that they will be complementary to sequence defined by adaptors and sequence
recognised by restriction enzymes. The amplification will be performed as shown
in the picture:
AFLP-PCR with primers matching adaptors and recognised restriction enzymes sequences.

Partial sequence of primers necessary to perform AFLP-PCR are suggested in the


response page.

When experiments are perfformed as shown in this page, several combinations of


restriction enzymes used in AFLP-PCR will yeild a large number of amplicons,
so that interpretation will be very difficult. In fact, AFLP-PCR experiments
performed by this service will not show bands when their number is above 50.

In order to avoid getting a large number of bands, in clasical AFLP-PCR, two


consecutive PCR amplifications are performed. The first one is performed with
primers which match only adaptors and sequences recognized by restriction
enzymes (as in the experiment above). The resulting amplicons are used in a
second PCR with slightly longer primers: those primers will match adaptors,
restriction enzyme recognized sequence and they will contain one or more
nucleotides in the 3' end (selective nucleotides), so that the number of amplicons
yeilded in this second PCR are fewer (as shown in the picture below). This way,
thenumber of bands will be lower and interpretation of band pattern easier.

AFLP-PCR with primers matching adaptors, restriction enzymes recognised sequences and
containning aditional selective bases in the 3' end. Usage of selective primers will reduce
the number of bands amplified.

In silico AFLP-PCR experiment allows the calculation of band pattern yeilded


when selective bases are added to primers. The selective bases must be
introduced in the form as shown above.

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