In silico Double Digestion Fingerprinting (DDF)


The aim of in silico Double Digestion Fingerprinting (DDF) tool is to simulate fingerprinting techniques which use two endonucleases. This tool also allows to try novel aproaches based on double digestion, and even techniques using three endonucleases.

Up to date, three techniques using double digestion have been described:
  • Amplified Fragment Length Polymorphisms or AFLP-PCR (1),
  • Infrecuent Restriction Site PCR or  IRS-PCR(2), which is basically AFLP-PCR, but uses at least one endonuclease cutting DNA infrecuentely, and selective nucleotides are not used
  • Subtracted Restriction Fingerprinting or SRF (3).
For AFLP-PCR and SRF with bacteria specific simulation tools are available, and they both include a suggestion tool (a helpful tool to find apropiate combinations of endonucleases in the selected microorganism).

By using DDF tool, all the above techniques can be simulated. But those ones are not the only options available. In fact, when developing this tool we have consider novel fingerprinting techniques which have not been previously described. Although we have not tried them in the laboratory, we have developed these hypothesis because they were necessary for correct development of a universal double digestion based fingerprinting tool.

You will find below the customizable parameters of this simulation tool:

SELECTION OF GENOME  OR UPLOADING SEQUENCES

Complete bacterial sequences have been obtained from NCBI. All up to date sequences bacterial genome sequences are included in this service.

Users may also upload their sequences to the server. Sequences must be in Fasta format in a text/plain type file. All data non-compatible with a DNA sequence (for example digits) will be remove from file.

Uploaded files will be associated to theIP of users. This association allow us to avoid the ue of password protection systems. Up to 5 sequences may be uploaded by each user, and they will be removed after 1 month.

We will not record data related to uploaded sequences or their use, only for statistics.

SELECTION OF FRAGMENTS

Digestion of DNA with two endonucleases will yield 4 types of fragments:

Type 1: From endonuclease 1 to endonuclease 2   RE1-RE2
Type 2: From endonuclease 2 to endonuclease 1   RE2-RE1
Type 3: From endonuclease 1 to endonuclease 1   RE1-RE1
Type 4: From endonuclease 2 to endonuclease 2   RE2-RE2

Selection of fragments may be manual, or we may select the technique (by selecting the checkbox for AFLP-PCR or SRF techniques, the correct fragments will be selected).
  • AFLP-PCR/IRS-PCR will amplify type 1 and 2 fragments.
  • SRF will only detect type 3 fragments.
MAXIMUM LENGTH OF FRAGMENTS

The allowed length of fragments is between 20 and 10000 bp. 3000 bp is the default value. The selected length includes the recognition sequence of endonucleases.

EXCLUSION SEQUENCE

Selected fragment types will be checked for presence of DNA sequence included in this  textbox, and in case the sequence is present the fragment will be eliminated (it will not be detected). This exclusion sequence may correspond to a third endonuclease used in the experiment. So it is possible to simulate a triple digestion experiment.

SELECTION OF ENDONUCLEASES

The two endonucleases used in the experiment are named RE1 and RE2 (for restriction enzyme 1 and restriction enzyme 2). They may be selected in two ways:
  • From a list of commercially available palindromic restriction enzymes (the list has been obtained from REBASE).
  • By introducing the recognition sequence (Degenerated nucleotides are allowed).
Both palindromic and non-palindromic endonucleases may be used in the experiment. Non-palindromic sequences will recognize two or more different sequences in the positive strand, and consecuently, more than one overhang end types will be yielded. An example is available here.

A checkbox allows to discern between overhang ends yielded by non-palindromic endonucleases (whether we want to select fragments cutting the DNA only with selected sequence in the direction introduced in the form, or whether we do not want to discern between cutting directions).

This simulation tool for Double Digestion Fingerprinting techniques allows to simulated AFLP-PCR and SRF techniques to be simulated, and also novel aproaches. We have included a few examples to show the use of this tool.

  link Simulation of AFLP-PCR experiment
  link Simulation of Subtracted Restriction Fingerprinting (SRF) experiment
  link Simulation of Double Digestion Fingerprinting experiment when using one non-palindromic endonuclease
  link Simulation of Double Digestion Fingerprinting experiment when using a non-palindromic endonuclease with degenerated (N) nucleotides
  link Simulation of Double Digestion Fingerprinting experiment when using only one endonuclease


DNA TEMPLATE

The DNA template will correspond to the sequenced bacterial DNA chosen in the form, and when available, plasmids may be included in the experiment. When the bacterial species has more than one chromosome (e.g. Agrobacterium tumefaciens, circular chromosome), both will be used. In the experiment, bacterial chromosomal DNA will be considered as circular unless is it linear (eg. Agrobacterium tumefaciens, linear chromosome). Plasmids will always be considered circular.

RESULTS

The Rresults page will show the starting position of amplicon in the chromosome or plasmid and the length of each amplicon. Amplicons obtained in each chromosome or plasmid will be shown separately in tables.

Amplicons will also be shown in a figure which includes a 100 bp ladder. On the right of the figre sorted length of all amplicons will be shown.

When clicking the starting position of amplicons in the tables the following information will be provided:
  • Source of DNA, and starting and ending positions of amplicon,
  • DNA sequence of amplicon
  • List of ORF to which the amplicons belongs to.
  • A link to NCBI which shows a map of the chromosome around the amplicon.


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