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

Severgnini, Marco; Cremonesi, Paola; Consolandi, Clarissa; Caredda, Giada; De Bellis, Gianluca; Castiglioni, Bianca

In hybridization-based techniques, mutations are identified on the basis of the higher thermal stability of the perfectly-matched probes as compared to mismatched probes. Although this has been the most frequently applied technique, it is characterized by many hindrances which make hybridization-based strategy function poorly in high-complexity biological samples. Therefore, for analytical and diagnostic purposes, hybridization is generally combined with some other selection or enrichment procedures. Enzyme-mediated ligation methods, on the other hand, rely on interrogation of a mutation by a couple of oligonucleotides annealing immediately adjacent to each other on a target DNA, with one of the probes having its 3 0 -end complementary to the point mutation. In this case, the search is for a single base that characterizes a species against all the others in a group of interest. The presence of a point mutation is assessed by the ligation of the two adjacent oligonucleotides, which occurs only when both are correctly base-paired (6) . The Ligation Detection Reaction (LDR) (7) , for instance, represents a reliable technique for identifying one or more sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of targetnucleotide sequences. This enzymatic in vitro reaction is based on the design of two oligonucleotide probes for each target sequence: a probe specific for the variation (called 'Discriminating Probe', or DS), which is 5 0 -fluorescently labeled, and a 5 0 -phosphorylated 'Common Probe' (or CP), starting one base 3 0 -downstream of the DS. The previously polymerase chain reaction (PCR)-amplified sample, the oligonucleotide probe pairs and a thermostable DNA ligase are blended to form a mixture: the two probes hybridize consecutively along the template and the DNA ligase joins their ends only in the case of a perfect match. This reaction is cycled to increase product yield. The PCR-LDR approach, usually, is associated to the hybridization onto a Universal Array (UA), where a set of artificial sequences, called Zip-codes are arranged (7) . This entire approach was proven to be rapid, flexible and easily adaptable from one target to another, useful, for example, in environmental monitoring (8, 9) , forensics (10) and the food industry (11, 12) .