In situ hybridization/Polynucleotide Probe Design software
In situ hybridization of genes and mRNA is most often based on polynucleotide probes. However, the specificity of polynucleotide probes has not been thoroughly investigated and a rational probe design concept was still missing, because the well established concept for oligonucleotide probe design cannot be transferred to polynucleotides.
PolyPro consists of three modules:
1. This paper describes the step by step algorithms used in PolyPro software.
Moraru, C., Moraru, G., Fuchs, B.M., and Amann, R. (2011) Concepts and software for a rational design of polynucleotide probes. Environ. Microbiol. Reports, 3: 69-78.
Fluorescence in situ hybridization (FISH) of genes and mRNA is most often based on polynucleotide probes. However, the well-established concepts for oligonucleotide probe design cannot be transferred to polynucleotides. Due to the high allele diversity of genes, a single probe is not sufficient to detect all alleles of a gene.
The main objective of this study was to develop a concept and software (PolyPro) for rational design of polynucleotide probe mixes to target particular genes.
PolyPro consists of three modules: a GenBank Taxonomy Extractor (GTE), a Polynucleotide Probe Designer (PPD) and a Hybridization Parameters Calculator (HPC). The new concept proposes the construction of defined polynucleotide mixes to target the habitat specific sequence diversity of a particular gene. The concept and the software are intended as a first step towards a more frequent application of polynucleotides for in situ identification of mRNA and genes in environmental microbiology.
2. In this paper a step by step example of probe design of amoA genes is given (see supplemental information)
The knowledge about the metabolic potentials of as yet to be cultured microorganisms has increased with the advance of sequencing technologies and the consequent discoveries of novel genes. On the other hand, it is often difficult to reliably assign a particular gene to a phylogenetic clade, because these sequences are usually found on genomic fragments that carry no direct marker of cell identity, such as rRNA genes.
The aim of the present study was to develop geneFISH – a protocol for linking gene presence with cell identity in environmental samples, the signals of which can be visualized at a single cell level. This protocol combines rRNA-targeted catalysed reporter deposition – fluorescence in situ hybridization and in situ gene detection. To test the geneFISH protocol, it was applied to seawater samples from the Benguela upwelling system.
For gene detection, a polynucleotide probe mix was used, which was designed based on crenarchaeotal amoA clone libraries prepared from each seawater sample. Each probe in the mix was selected to bind to targets with up to 5% mismatches. For probe design, the PolyPro software was used. To determine the hybridization parameters, the Tm of probes, targets and hybrids was estimated based on theoretical calculations and in vitro measurements.
It was shown that at least 30%, but potentially the majority of the Crenarchaeota present in these samples harboured the amoA gene and were therefore likely to be catalyzing the oxidation of ammonia.
Polynucleotide Probe Design software - Main interface
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