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Nucleic Acids Research Advance Access published online on June 16, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp499
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods online

ORMA: a tool for identification of species-specific variations in 16S rRNA gene and oligonucleotides design

Marco Severgnini1,*, Paola Cremonesi2, Clarissa Consolandi1, Giada Caredda1, Gianluca De Bellis1 and Bianca Castiglioni2

1Institute of Biomedical Technologies, Italian National Research Council, Via Fratelli Cervi 93, Segrate and 2Institute of Agricultural Biology and Biotechnology, Italian National Research Council, Via Bassini 15, Milan, Italy

*To whom correspondence should be addressed. Tel: +39 02 26422705; Fax: +39 02 26422770; Email: marco.severgnini{at}itb.cnr.it

Received February 3, 2009. Revised April 30, 2009. Accepted May 24, 2009.

16S rRNA gene is one of the preferred targets for resolving species phylogenesis issues in microbiological-related contexts. However, the identification of single-nucleotide variations capable of distinguishing a sequence among a set of homologous ones can be problematic. Here we present ORMA (Oligonucleotide Retrieving for Molecular Applications), a set of scripts for discriminating positions search and for performing the selection of high-quality oligonucleotide probes to be used in molecular applications. Two assays based on Ligase Detection Reaction (LDR) are presented. First, a new set of probe pairs on cyanobacteria 16S rRNA sequences of 18 different species was compared to that of a previous study. Then, a set of LDR probe pairs for the discrimination of 13 pathogens contaminating bovine milk was evaluated. The software determined more than 100 candidate probe pairs per dataset, from more than 300 16S rRNA sequences, in less than 5 min. Results demonstrated how ORMA improved the performance of the LDR assay on cyanobacteria, correctly identifying 12 out of 14 samples, and allowed the perfect discrimination among the 13 milk pathogenic-related species. ORMA represents a significant improvement from other contexts where enzyme-based techniques have been employed on already known mutations of a single base or on entire subsequences.


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