Polynucleotide probe design
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Polynucleotide probe design. In situ hybridization parameters
PolyPro
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.


We recently developed a concept and software (PolyPro) for rational design of polynucleotide probe mixes to identify particular genes in defined taxa.

PolyPro consists of three modules:

 

Download

 

Name PolyPro - Polynucleotide probe design
Version 1.8.4
Package size ~ 8.5 MB
Platform Windows
Download time less than 10 seconds
Price freeware
Download Polynucleotide probe design software

 

 

Installation instructions

  1. Download the ZIP file
  2. Extract all files and folders from it  (this is mandatory!)
    The program will not work with UNC (network) paths like '\\ServerName\UserName\PolyPro\'. Please install it on your local drive instead. For example: 'c:\PolyPro\'
  3. Double click the PolyPro.exe application to run it

 

References

 

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.

 

Abstract:

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)
Moraru, C., Lam, P., Fuchs, B.M., Kuypers, M.M.M. and Amann, R. (2010) GeneFISH – an in situ technique for linking gene presence and cell identity in environmental microorganisms. Environ. Microbiol. 12: 3057-3073.

 

Abstract:

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.

 

 

Screen shots

 

Rational design of polynucleotide probe mixes to identify particular genes in defined taxa

Polynucleotide Probe Design software - Main interface

 

GenBank Taxonomy Extractor software module

1. GenBank Taxonomy Extractor software module

 

Hybridization Parameters Calculator software module

2. Hybridization Parameters Calculator software module

 

Polynucleotide Probe Design software module

3. Polynucleotide Probe Design software module

 

 

 

 

 

 

 

 

Genes/mRNA based on polynucleotide probes. Specificity of polynucleotide probes
 
In situ hybridization of genes/mRNA
In situ hybridization software by
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