Such stringent conditions compromised signal intensities; however, specific signals remained IDO inhibitor detectable at the highest stringency (at 75 °C hybridization) with negligible false negatives. These results suggest that, without any probe design or selection, genomic

fragments can provide a reasonable specificity for microbial diagnostics or species delineation by DNA–DNA similarities. Microarray-based technology, with its advantage of highly parallel detection, has been applied to both population profiling and to functional studies of complex microbial communities in the environment (Loy et al., 2002; Palmer et al., 2006; He et al., 2007; Iwai et al., 2008). Recent studies have used synthesized oligonucleotides as probes because of their flexibility in design and preparation, FK228 with intensive specificity evaluation applied to the probe design criteria (He et al., 2005). In addition, several studies have reported the

use of PCR-amplified genomic fragment sequences as probes. Such microarrays have been used for the detection of specific bacteria (Kim et al., 2004, 2005), species determination (Cho & Tiedje, 2001), and screening of environmental sequences related to a certain function within a community (Yokoi et al., 2007; Tobino et al., 2011). As the probes in these studies are randomly prepared by shotgun cloning of genomic DNAs, this kind of microarray is independent of sequence information found in public databases and hence offers unique potential for exploring unsequenced

microorganisms. Gemcitabine mw However, the specificity of genomic fragment probes has not been evaluated in detail. In this study, we prepared genomic fragment probes from pure cultures whose whole genome sequences are available and then evaluated hybridization specificity in terms of sequence similarity between probe and target. Genomic fragment probes were prepared from genomic DNAs of three Pseudomonas strains (Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens Pf-5, and Pseudomonas putida KT2440) by shotgun cloning as described previously (Tobino et al., 2011). The probe set consisted of 167 fragment probes (55, 56, and 56 probes from PAO1, Pf-5, and KT2440, respectively) of ~ 2000 bp in length (Supporting Information, Table S1) and four control probes (see the figure legend of Fig. S1 for the preparation of control probes). Each fragment probe was spotted onto nylon membranes (5 ng per spot) in duplicate, and the spotted membranes were alkaline denatured, baked, and stored in a plastic bag until use (see Fig. S1 for probe layout). Genomic DNAs of pure cultures, plus control DNA (yeast gene ACT, included in the probe set as a positive control) were individually labeled with digoxigenin (DIG) by random priming according to the manufacturer’s instructions (DIG High Prime; Roche, Basel, Switzerland). Labeled products were sonicated to an average length of 400 bp.