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

A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, and bioinformatics data, derived from the simulation of the secondary structure folding and prediction of hybridisation reaction, was carried out in order to rationalize the selection of ssDNA probes for the detection 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 within different regions of B. ostreae and B. exitiosa PCR amplicons (300 and 304 bases, respectively) with the aim to discriminate between these parasite species. ssDNA amplicons and probes were analyzed separately using the "Mfold Web Server" simulating the secondary structure folding behaviour. The hybridisation of amplicon-probe was predicted by means of "Dinamelt Web Server". The results were evaluated considering the 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, thermally denatured PCR products were captured at the sensor interface via sandwich hybridisation with surface-tethered probes (thiolated probes) and biotinylated signalling probes. A convergence between analytical signals and simulated results was observed, indicating the possibility to use bioinformatic data for ssDNA probes selection to be incorporated in genosensors. (C) 2011 Elsevier B.V. All rights reserved.

Carbon fiber microelectrodes for adsorptive stripping analysis of trace nucleic acids

Carbon fiber ultramicroelectrodes are shown to be suitable for adsorptive stripping potentiometric measurements of trace DNA and RNA. The origin of the carbon fiber has a profound effect upon its suitability for trace analysis of nucleic acids, with the 'Aesar' materials performing most favorably. The resulting ultramicroelectrodes offer effective adsorptive accumulation of DNA and RNA from unstirred microliter-volume solutions, and are shown to be useful in adsorptive stripping transfer experiments. The influence of the surface pretreatment and accumulation conditions is described, along with the analytical-performance characteristics. The detection limits are 6, 15 and 40 mu g/l tRNA, ssDNA and dsDNA, respectively (5 min accumulation). (C) 1998 Elsevier B.V. S.A.

Targeted Nucleotide Exchange in Bovine Myostatin Gene

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Adsorptive potentiometric stripping analysis of nucleic acids at mercury electrodes

The application of adsorptive stripping potentiometry to the reductive detection of nucleic acids at mercury electrodes is reported. Compared to analogous voltammetric stripping modes, constant current potentiometric stripping analysis (PSA) effectively addresses the hydrogen discharge background problem, and hence greatly improves the characteristics of the superimposed cytosine/adenine (CA) reduction peak. Compared to earlier schemes for trace measurements of nucleic acids at mercury or carbon electrodes that rely on anodic signals arising from the guanine residue, convenient quantitation can now be carried out in connection with the cytosine and adenine residues. Variables influencing the adsorptive PSA response are explored and optimized. With five minute accumulation, the detection limits for tRNA, ssDNA and dsDNA are 30 mu g l(-1), 60 mu g l(-1) and 2 mg l(-1), respectively. Such different values reflect the strong dependence of the PSA CA signal upon the nucleic-acid structure. This allows the quantitation of ssDNA or tRNA in the presence of dsDNA, and offers new possibilities for electrochemical studies of DNA structure and interactions.

Tethered DNA scaffolds on optical sensor platforms for detection of hipO gene from Campylobacter jejuni

DNA biosensors have gained increased attention over traditional diagnostic methods due to their fast and responsive operation and cost-effective design. The specificity of DNA biosensors relies on single-stranded oligonucleotide probes immobilized to a transduction platform. Here, we report the development of biosensors to detect the hippuricase gene (hipO) from Campylobacter jejuni using direct covalent coupling of thiol- and biotin-labeled single-stranded DNA (ssDNA) on both surface plasmon resonance (SPR) and diffraction optics technology (DOT, dotLab) transduction platforms. This is the first known report of the dotLab to detect targeted DNA. Application of 6-mercapto-1-hexanol as a spacer thiol for SPR gold surface created a self-assembled monolayer that removed unbound ssDNA and minimized non-specific detection. The detection limit of SPR sensors was shown to be 2.5 nM DNA while dotLab sensors demonstrated a slightly decreased detection limit of 5.0 nM (0.005 μM). It was possible to reuse the SPR sensor due to the negligible changes in sensor sensitivity (∼9.7 × 10 -7 ΔRU) and minimal damage to immobilized probes following use, whereas dotLab sensors could not be reused. Results indicated feasibility of optical biosensors for rapid and sensitive detection of the hipO gene of Campylobacter jejuni using specific ssDNA as a probe. © 2011 Elsevier B.V.

RPA-1 from Leishmania amazonensis (LaRPA-1) structurally differs from other eukaryote RPA-1 and interacts with telomeric DNA via its N-terminal OB-fold domain

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq (R))

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)