Using the software package ARB - basic steps ...

For FISH, probes with a length of 15 - 30 nucleotides are most commonly used. Sequence signatures serving as suitable target sites for nucleic acid probing can be convenient and automatically identified using the PROBE_DESIGN tool of the ARB software package (Ludwig et al., 2004).

First, a target group of organisms must be specified, e.g., in a phylogenetic tree within ARB. The PROBE_DESIGN tool searches for all possible signature sequences that are diagnostic for the selected species. The tool excludes potential probe sequences, which contain self-complementary regions with more than three nucleotides. The GC-content of probe sequences influences their melting behavior. By default this parameter is set in PROBE_DESIGN between 50 to 100% to ensure a tight binding but usually it ranges between 50 to 70%.

Sometimes the PROBE_DESIGN tool can not find a suitable probe. However, the program provides options for modifying the search parameters to look for signatures in subsets of the group originally selected, or to allow the signature to be found in a defined number of species outside the target group. By the combinatorial use of probes with overlapping specificity the selected group of organism may be fully targeted.

Another strategy of probe selection is searching for already published probes. Loy and coworkers established an rRNA-targeted oligonucleotide probe database named 'probeBase' with currently more than 1300 entries, mostly developed for FISH (Loy et al., 2003).

www.microbial-ecology.net/probebase

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