Hybridization and washing procedures were carried out as described previously (Tobino et al., 2011). Chemiluminescent detection was performed using an antidigoxigenin antibody conjugated with alkaline phosphatase and CSPD (both Roche) according to the instruction manual (DIG Application Manual for Filter Hybridization, Roche), and the signal was GSK126 mouse recorded by LAS-4000 mini (Fujifilm, Tokyo, Japan) using a 10 min exposure. Signals were background corrected and considered positive when the signal to noise ratio was > 3 in all the replicated
spots. Partial sequences from both ends (60–700 bp) of each probe were read using SP6 and T7 primers as described previously (Tobino et al., 2011). The full probe sequence was defined as the segment that was on and within both end sequences in the genome, found using the blastn tool from the National Center for Biotechnology Information (NCBI). INK 128 concentration The full probe sequences were then searched against the target genome sequences using
blastn in NCBI under the default settings. The match that had the least e-value was selected as the representative similarity pair between the probe and the target genome. To eliminate short alignments and anomalous high signals, caused by the high gene copy number, those pairs that had < 500 bp alignment or significant multiple hits were rejected in the subsequent analysis. Specific responses were observed from probes corresponding to the target genome at all hybridization temperatures tested (Fig. S2). Visible signals were also found from some probes whose origins were different from the target genome, Phosphoprotein phosphatase indicating the occurrence
of cross-hybridization (i.e. false positives). As shown in Table 1, the level of false positive signals was 64.7% (216 of 334) at 55 °C but decreased steadily to 22.5% (75 of 334) at 70 °C and was almost completely absent (1.5%; 5 of 334) at 75 °C. In contrast, very few probes (0.6%; 1 of 167) corresponding to the target genome fell in negative and were only found at hybridization temperatures above 70 °C. These results suggest that randomly generated genomic fragments (~ 2000 bp) can function as specific probes to discriminate species in the genus Pseudomonas under highly stringent conditions. Sequence similarity searches between the fragment probes and target genomes produced a total of 496 similarity pairs (Fig. S3). With the exception of probes that originated from the target genome (resulting in 100% similarity), most of the pairs had < 90% similarity, while only two pairs sharing a partial sequence of rrn operon were found to have > 90% similarity of > 500 bp.