For the design of genus- and species-specific probes the ITS regions of the rRNA gene cassette were exploited. These coding regions show a high degree of variation [19] and analysis of the fungal ITS alignments revealed significant differences among the different fungi. However, analysis of the ITS regions of Fusarium species showed that they have similar sequences which could have cross hybridized on the array, making it non-specific. Kane et al. [20] found that in 50mer oligonucleotide arrays, cross-hybridization occurred between fragments of relatively low sequence similarity. The highly repetitive DNA content of plant genomes resulted in cross-hybridization

of DNA fragments to printed-probe DNA #AZD1152 research buy randurls[1|1|,|CHEM1|]# and the overall spot intensity of many probes was increased. Therefore, the EF regions were used for the design of species-specific probes for Fusarium species. For some probes with similar sequences

the chances of cross hybridization were minimized by substituting a single oligonucleotide in the probe sequence using a high affinity DNA analogue known as locked nucleic acid (LNA) at three specific points to increase the specificity and the Tm of a probe. The LNAs were inserted at a single nucleotide polymorphism (SNP) site for improved performance of the probe. Letowski et al. [21] found that probes containing polymorphisms toward the centre of the probe showed a higher discrimination power. If LNAs enough are to be included then they must be inserted in a triplicate series around the centre of the probe. Further, G-T mismatch sites must be avoided and should preferably be inserted at sites Trichostatin A concentration where adenine is the identity of the base [18]. Cross hybridization has also been reported in several microarray-based species detection

studies where single regions were used for identification. Anthony et al. [22] found that in oligonucleotide arrays, cross-hybridization occured between Listeria species and it was necessary to include additional probes to the array. In a similar study done by Volokhov et al [23], E. coli and Salmonella isolates produced indistinguishable hybridization profiles when single probes were used. However, they showed that multiple probes improve the sensitivity of the array when compared with the single diagnostic probes that could be unsuitable for a group of closely related organisms. In this study, the probes spotted onto the array were a mixture of single and multiple probes for each species that were either genus-, species-specific or specific for genes leading to toxin production. When multiple probe sequences were used the discriminatory power of the array increased as a sample hybridized to at least one probe of the multiple probes on the array. In addition, probes for the array construction were designed around a Tm of 56°C so that all probes would hybridize under similar conditions.