A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, andbioinformatics data, derived from the simulation of the secondary structure folding and prediction ofhybridisation reaction, was carried out in order to rationalize the selection of ssDNA probes for thedetection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis.Six ssDNA probes (from 11 to 25 bases in length, 2 thiolated and 4 biotinylated) were selected withindifferent regions of B. ostreae and B. exitiosa PCR amplicons (300 and 304 bases, respectively) with the aimto discriminate between these parasite species. ssDNA amplicons and probes were analyzed separatelyusing the “Mfold Web Server” simulating the secondary structure folding behaviour. The hybridisation ofamplicon–probe was predicted by means of “Dinamelt Web Server”. The results were evaluated consideringthe number of hydrogen bonds broken and formed in the simulated folding and hybridisation process,variance in gaps for each sequence and number of available bases. In the experimental part, thermallydenatured PCR products were captured at the sensor interface via sandwich hybridisation with surfacetetheredprobes (thiolated probes) and biotinylated signalling probes. A convergence between analyticalsignals and simulated results was observed, indicating the possibility to use bioinformatic data for ssDNAprobes selection to be incorporated in genosensors.[...]

Electrochemical genosensors for the detection of Bonamia parasite. Selection of single strand-DNA (ssDNA) probes by simulation of the secondary structure folding

MASCINI, Marcello;DEL CARLO, MICHELE;TISCAR, Pietro Giorgio;COMPAGNONE, DARIO
2011-01-01

Abstract

A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, andbioinformatics data, derived from the simulation of the secondary structure folding and prediction ofhybridisation reaction, was carried out in order to rationalize the selection of ssDNA probes for thedetection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis.Six ssDNA probes (from 11 to 25 bases in length, 2 thiolated and 4 biotinylated) were selected withindifferent regions of B. ostreae and B. exitiosa PCR amplicons (300 and 304 bases, respectively) with the aimto discriminate between these parasite species. ssDNA amplicons and probes were analyzed separatelyusing the “Mfold Web Server” simulating the secondary structure folding behaviour. The hybridisation ofamplicon–probe was predicted by means of “Dinamelt Web Server”. The results were evaluated consideringthe number of hydrogen bonds broken and formed in the simulated folding and hybridisation process,variance in gaps for each sequence and number of available bases. In the experimental part, thermallydenatured PCR products were captured at the sensor interface via sandwich hybridisation with surfacetetheredprobes (thiolated probes) and biotinylated signalling probes. A convergence between analyticalsignals and simulated results was observed, indicating the possibility to use bioinformatic data for ssDNAprobes selection to be incorporated in genosensors.[...]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11575/16744
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