designs polynucleotide probe mixtures for the detection of a single target sequence. These probe mixtures consist of multiple, non-overlapping polynucleotides distributed across the length of the target region. The selected polynucleotides have similar melting properties, and thus, can be used in as a single hybridization probe.
Such polynucleotide probe mixtures are required for gene detection in microbial cells by direct-geneFISH, phageFISH, geneFISH and geneELISA.
In a series of steps, gene-PROBER guides the user through the probe design process, from similarity searches between the target and non-targets, to the selection of polynucleotides with similar melting temperatures and of their primers (when the probes will be synthesized by PCR).
Gene-PROBER can be used whenever single targets need to be detected, as for example:
- lytic or temperate viruses infecting bacterial or archaeal cells
- plasmids or chromosomal regions in bacteria or archaea
Gene-PROBER is not meant for the design of probes to detect multiple targets, e.g. multiple alleles of a single gene. Once the probe mix has been designed for a single target, the coverage across the other alleles can be checked with
software. For more details about designing polynucleotide probes for multiple alleles, check the following publication:
Moraru, C., Moraru, G., Fuchs, B. & Amann, R. Concepts and software for a rational design of polynucleotide probes. Environmental Microbiology Reports 3, 69-78 (2011).
For a detailed description of the gene and phage hybridization methods, please read the following publications:
Moraru, C., Lam, P., Fuchs, B., Kuypers, M. & Amann, R. GeneFISH - an in situ technique for linking gene presence and cell identity in environmental microorganisms. Environmental Microbiology 12, (2010).
Allers, E. et al. Single-cell and population level viral infection dynamics revealed by phageFISH, a method to visualize intracellular and free viruses. Environmental microbiology 15, 2306–18 (2013).
Barrero-Canosa, J., Moraru, C., Zeugner, L., Fuchs, B. & Amann, R. Direct-geneFISH: a simplified protocol for the simultaneous detection and quantification of genes and rRNA in microorganisms. Environmental microbiology, (2016), doi:10.1111/1462-2920.13432.
If you are using gene-PROBER, please cite the following: TO BE UPDATED. The reference will be updated immediately after online publication.
If you have suggestions for gene-PROBER or if you encounter problems during probe design, please email to: liliana.cristina.moraru( at )uni-oldenburg.de and mention gene-PROBER in the subject