mathFISH, a Web Tool That Uses Thermodynamics-Based Mathematical Models for In Silico Evaluation of Oligonucleotide Probes for Fluorescence In Situ Hybridization

Abstract

Mathematical models of RNA-targeted fluorescence in situ hybridization (FISH) for perfectly matched and mismatched probe/target pairs are organized and automated in web-based mathFISH (http://mathfish.cee.wisc .edu). Offering the users up-to-date knowledge of hybridization thermodynamics within a theoretical framework, mathFISH is expected to maximize the probability of success during oligonucleotide probe design.Fluorescence in situ hybridization (FISH) has been used for more than 2 decades in microbial ecology (since the publication of the article by DeLong et al. [5] in 1989) for the detection of whole cells of interest in environmental samples. Microbial ecological applications of FISH generally target rRNA as the phylogenetic marker and rely on the specific hybridization of labeled synthetic DNA probes with this complex molecule inside fixed cells. FISH offers unparalleled capabilities such as the quantification of target groups of organisms and visualization of their spatial distribution.The main challenge in FISH applications is the optimization of sensitivity and specificity during probe design (23). Throughout the years, several experimental techniques have been developed for the optimization of probe performance, such as the use of formamide for mismatch discrimination (13,19), the addition of unlabeled competitor oligonucleotides for improving mismatch discrimination (13), the empirical assessment of target accessibility (2, 7), the use of unlabeled helper oligonucleotides for improving accessibility (6), the amplification of signal using enzyme-labeled probes (catalyzed reporter deposition [CARD]-FISH) (16), and the use of cloned target for the determination of the formamide denaturation profiles (Clone-FISH) (18). Although highly useful, these tools have increased the complexity of probe design. In addition to the need for trial and error, experimental approaches without theoretical guidelines are limited by the physical availability of either organisms or clones belonging to target and nontarget microbial groups, making optimization cumbersome in some cases (24).There is a plethora of literature on the universal physicochemical properties of nucleic acid hybridizations (e.g., see the review by Turner [22]), which can potentially help the prediction of probe performance in FISH. To facilitate the use of this knowledge in FISH applications, we have recently developed thermodynamics-based mathematical models of FISH (24-26). The models simulate FISH in silico using thermodynamically established parameters that define DNA/RNA interactions between the probe and targeted rRNA, DNA/DNA interactions within the probe, and RNA/RNA interactions within the rRNA.Having been tested with model organisms (24-27), the mathematical models are expected to facilitate probe design. The models could potentially reduce the number of cycles of trial and error to reach the right probe and optimal hybridization conditions, provide a basis for evaluating the confidence level in experimental results (24), and serve as a subst...

“Candidatus Finniella” (Rickettsiales, Alphaproteobacteria), Novel Endosymbionts of Viridiraptorid Amoeboflagellates (Cercozoa, Rhizaria)

“…The proposed probes were checked for specificity in silico with the probeCheck online database (24) and with BLAST of NCBI (http: //blast.ncbi.nlm.nih.gov/). Probes with high specificity were subjected to mismatch analyses with the online tool mathFISH to determine parameters (temperature and formamide concentration) for optimal stringency of the probe (25,26). If needed, probes were manually modified according to these results and checked again with probeCheck, BLAST, and math-FISH.…”

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk

“…Optimal hybridization conditions were predicted using the mathFISH program (mathfish.cee.wisc. edu/) (47). Probes were diluted in hybridization buffer [30% formamide (vol/vol), 1 M NaCl, 20 mM Tris (pH 7.4), 0.01% SDS] to a final concentration of 500 nM each and incubated with tissue sections on slides for 2 h at 35°C.…”

Community Shift from Phototrophic to Chemotrophic Sulfide Oxidation following Anoxic Holomixis in a Stratified Seawater Lake

“…The specificity of the probe was evaluated with the ARB PROBE_MATCH tool. The expected half-dissociation temperature was calculated in silico with mathFISH (44) and evaluated by in situ hybridization at 46°C over a formamide range of 25 to 40% with environmental samples from Lake Rogoznica. The optimal formamide concentration for hybridization at 46°C was determined to be 30%.…”