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.
How to access the manual:
A comprehensive manual detailing the use of genePROBER can be accessed below. The manual contains also two short tutorials. The sequence datasets used to in the tutorials can be downloaded below as well.
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Some steps (especially 4b, which calculates primer pairs) can require a long time (e.g. days), depending on the input parameters (target length, GC range). In those cases, restricting the input parameters will help reduce the number of polynucleotides/primers being calculated, and thus, reduce the computational time.
How to cite gene-PROBER:
If you are using gene-PROBER, please cite the following: TO BE UPDATED. The reference will be updated immediately after online publication.
How to report problems or make suggestions:
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.