In Silico Biology 5, 0031 (2005); ©2005, Bioinformation Systems e.V.  

In silico simulation of fingerprinting techniques based on double endonuclease digestion of genomic DNA


Rosario San Millán, Javier Garaizar and Joseba Bikandi*


Department of Immunology, Microbiology and Parasitology, University of the Basque Country
Paseo de la Universidad, 7, 01006 Vitoria-Gasteiz, Spain.

* Corresponding author; Email: oipbibij@lg.ehu.es; fax: +34-945-013014



Edited by H. Michael; received March 14, 2005; revised and accepted April 06, 2005; published April 11, 2005



Abstract

We have developed an online generic tool for simulation of fingerprinting techniques based on the double endonuclease digestion of DNA. This tool allows modelling and modifications of already existing techniques, as well as new theoretical approaches not yet tried in the lab. It allows the use of any combination of recognition patterns and discrimination of end types yielded by restriction with non palindromic recognition sizes. Re-creation of experimental conditions in silico saves time and reduces laboratory costs. This tool allows simulation of Amplified Fragment Length Polymorphism (AFLP-PCR), Subtracted Restriction Fingerprinting (SRF), and additional novel fingerprinting techniques. Simulation may be performed against custom sequences uploaded to the server, or against all sequenced bacterial genomes. Different endonuclease types may be selected from a list, or a recognition sequence may be introduced in the form. After double digestion of DNA, four fragment types are yielded, and the program allows their customised selection. Selective nucleotides may be used in the experiment. Scripts for specific simulation of AFLP-PCR and SRF techniques are available, and both include a suggestion tool for the selection of endonucleases. This is the first program available for the simulation of SRF fingerprinting.

Availability: This free online tool is available at http://www.in-silico.com/DDF/.

Keywords: restriction digest, double digest, AFLP-PCR, Subtracted Restriction Fingerprinting (SRF), data mining, in silico analysis, genotyping, endonuclease, bacterial genomes



Introduction

Fingerprinting of microorganisms for epidemiological purposes is available through many genomic techniques, including those based on the double endonuclease digestion of the genome. The online tool described in this work is suitable for simulation of already described fingerprinting techniques based on double digestion and their modifications, but it also opens a wide research space: allows simulation of experiments with all possible recognition sites (all commercial endonucleases and also user defined recognition patterns), discriminating the ends yielded by restriction with non- palindromic recognition sizes, and showing a wide range of fragment sizes in the results. Additionally, a growing number of bacterial genomes may be used in the experiments (over 200 at this moment), and it also allows using sequences uploaded to the server. Without a doubt, simulating performance of fingerprinting techniques prior to the real experiment allows reduction of costs and wet-lab efforts.

Until now, two basic techniques based on double digestion have been described (Fig. 1): Amplified Fragment Length Polymorphisms PCR or AFLP-PCR [Vos et al., 1995] and Subtracted Restriction Fingerprinting or SRF [Terletski et al., 2003]. These techniques are based on the same principle: a genome is cleaved with two restriction enzymes (RE1 and RE2) to yield four fragment types (RE1-RE1, RE1-RE2, RE2-RE1 and RE2-RE2), but only a subset of them are amplified or detected. A third technique called Infrequent Restriction Site PCR or IRS-PCR [Mazurek et al., 1996] is very similar to AFLP-PCR. As restriction sites are very sensitive to small genomic variations, band patterns may be used for strain characterization in epidemiological studies.



Figure 1: Genotyping techniques based on double digestion of genome. Cleavage with two restriction enzymes (RE1 and RE2) yields four fragment types. As the number of fragments is very high, only some fragments are selected or detected in the following steps.
  • In AFLP-PCR, overhang ends yielded after digestion are ligated to adaptors, and PCR amplification is performed by using specific primers (which are complementary to adaptors, recognition sequence for endonuclease and a small number of nucleotides within fragments, called selective nucleotides). Only a few RE1-RE2 and RE2-RE1 fragments are amplified, separated in a gel, stained and detected.
  • In SRF fingerprinting, after double digestion, a filling reaction is performed, which allows the labelling of overhang ends with digoxigenin (for RE1 end) or biotin (for RE2 end). A subtraction step allows the removal of fragments labelled on one or both ends with biotin, and the remaining fragments (RE1-RE1) are separated in a gel, transferred and detected after staining.


To date, only AFLP-PCR simulation tools had been developed (Table 1), but the potential use of double digestion for genotyping is not limited to techniques already described. We describe here a multipurpose tool to simulate all techniques described in the literature and additional theoretical approaches.


Table 1: Software availability for simulation of AFLP-PCR.
Programs for AFLP simulation Reference Use
AFLPinSilico (http://intra.psb.ugent.be:8080/bioinformatics/AFLPinSilico.html) Rombauts et al., 2003 Free, online
In silico AFLP (http://www.in-silico.com/s_AFLP/) Bikandi et al., 2004 Free, online
ALFIE (http://bioinfosoftware.phls.org.uk/aflp.html) PHLS, UK Free, online
RemComb (http://www.keygene.com) Keygene, NL Commercial



Double digestion fingerprinting simulation


We have developed a program for in silico simulation of several fingerprinting techniques based on the double digestion of a genome, including AFLP-PCR and SRF. Additional approaches are proposed as examples of the potential use of the program. Although we have not tried those approaches in the lab they may become valuable fingerprinting techniques.

The program was developed with open-source software (PHP running on an Apache server with Linux operating system), and it can be used for free. To use this service Mozilla/Firefox is recommended.

This tool may run against preloaded bacterial genomes, or against sequences uploaded to our server by users (Fig. 2). Over 200 completely sequenced bacterial genomes are included at www.in-silico.com [Bikandi et al., 2004], and they are regularly updated. Plasmids may be included in the experiment. Sequences and other related files were obtained from the National Center for Biotechnology Information (NCBI). Additionally, up to 5 sequences may be uploaded to the server by each user (5 million bp/sequence). Fragment types to be detected after double digestion may be manually selected, or selection can be automatic after choosing a technique (AFLP-PCR or a SRF). Commercially available endonucleases from a list or recognition sequence (degenerated nucleotides are allowed) can be used for double digestion. The list of endonucleases was obtained from the Restriction Enzyme Database [Roberts et al., 2003]. The program allows the use of non-palindromic endonucleases (which are avoided in AFLP-PCR and SRF). By double restriction with non-palindromic enzymes, 8 fragment types are obtained (4 fragment types are obtained when using palindromic ones), and a checkbox allows selection of fragments cleaved in a specific strand. Selective nucleotides may be used as in other programs for AFLP-PCR. Users may define an exclusion sequence, which allows the removal of all fragments containing the specified sequence.



Figure 2: Main form for the Double Digestion Fingerprinting program. Selecting the parameters shown in the image will simulate SRF.


On the following page (Fig. 3), the program shows the starting position of each fragment, its length, and a simulation of their electrophoretic mobility. Further information from each fragment may be obtained by following the corresponding links: DNA sequences and list of ORFs included for each fragment.



Figure 3: Response page for SRF fingerprinting experiment. Links allow retrieval of fragment sequence and ORF information.


The Double Digestion Fingerprinting simulation program described in this paper has been integrated with programs at www.in-silico.com. This site already includes an AFLP-PCR simulation tool and an AFLP-PCR suggestion. The suggestion tool provides a list of endonuclease pairs for each genome that will produce a small number of fragments without using selective nucleotides. Similar tools had been developed for SRF fingerprinting.



Specific tools for SRF simulation

A simplified program has also been developed for specific in silico simulation of SRF fingerprinting against preloaded bacterial genomes. The program allows selection of commercially available endonucleases suitable for this technique. A suggestion tool has also been developed. As the biggest problem when using SRF fingerprinting is the selection of suitable endonuclease pairs (necessary to yield a small number of fragments for correct detection of bands), this suggestion tool will be very useful. Its use is described in Fig. 4. This tool is the first reported attempt to simulate SRF fingerprinting.



Figure 4: Use of SRF suggestion tool. After selection of a genome, the minimum and maximum number of bands are selected. The response page will show a list of all restriction enzyme pairs that, theoretically, will yield bands within the selected range. By clicking the number of bands, in silico simulation is shown.




Acknowledgements

This work was supported by project IE019 sub-project DIAMOLFUN from the Basque Government and project 1/UPV00093.125-E-15896/204 from the University of The Basque Country.



Abbreviations

AFLP-PCRAmplified Fragment Length Polymorphism PCR
SRFSubtracted Restriction Fingerprinting
IRS-PCRInfrequent Restriction Site PCR




References