3.1.1. Probe sequence
Requirements for oligonucleotide probes used in FISH are partially the same as those for oligonucleotide primers used in conventional PCR, and as such, general primer design software and web-based services can be used as a starting point for designing probes (e.g. Primer 3 (Rozen and Skaletsky, 2000), SILVA(Quast et al., 2013) and probeBase (Loy et al., 2007)). The probeBase database represents a good resource to analyse potential probe sequences (http://www.microbialecology.net/probebase/default.asp). At this web site, one can also find published oligonucleotides that have been shown to work in a given target bacterium.
Probes should be designed using the following guidelines:
- 15-25 bases in length
- 50-70% GC content
- 50-60˚C Tm
- Specificity of a FISH probe is increased by including unique bases to a given target bacterium (or bacterial group) either in the middle part of the primer or evenly distributed over the entire sequence.
- Choose a suitable region of the 16S rRNA molecule to target for hybridization. The brightness of a FISH signal is largely affected by the accessibility of a given region for probe hybridization (Fuchs et al., 1998; Behrens et al., 2003, Yilmaz et al. 2006). This factor was experimentally examined for a few bacterial species. Because secondary structures of rRNAs can slightly differ between bacterial species, regions known to result in good hybridization efficiency in these bacterial species might not be the most suitable ones in another target bacterium.
- Examine the specificity of a designed probe by conducting diagnostic PCR using an unlabelled oligonucleotide with the same sequence as the probe and a universal forward primer for the bacterial 16S rRNA gene such as 16SA1 (5’-AGAGTTTGATCMTGGCTCAG-3’) or 16SA2 (5’-GTGCCAGCAGCCGCGGTAATAC-3’).
- The unlabelled oligonucleotide is also useful for competitive suppression control experiments, which are later described in this chapter. You will need at least 500 µM of the unlabelled oligonucleotide.