-
PloS One 2016Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to...
Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.
Topics: Base Pair Mismatch; DNA Primers; Genotyping Techniques; Oligonucleotide Probes; Real-Time Polymerase Chain Reaction; Taq Polymerase
PubMed: 26863543
DOI: 10.1371/journal.pone.0146950 -
Angewandte Chemie (International Ed. in... Mar 2017Super-resolution microscopy allows optical imaging below the classical diffraction limit of light with currently up to 20× higher spatial resolution. However, the...
Super-resolution microscopy allows optical imaging below the classical diffraction limit of light with currently up to 20× higher spatial resolution. However, the detection of multiple targets (multiplexing) is still hard to implement and time-consuming to conduct. Here, we report a straightforward sequential multiplexing approach based on the fast exchange of DNA probes which enables efficient and rapid multiplexed target detection with common super-resolution techniques such as (d)STORM, STED, and SIM. We assay our approach using DNA origami nanostructures to quantitatively assess labeling, imaging, and washing efficiency. We furthermore demonstrate the applicability of our approach by imaging multiple protein targets in fixed cells.
Topics: DNA; DNA Probes; Microscopy, Fluorescence; Nanostructures; Optical Imaging
PubMed: 28256790
DOI: 10.1002/anie.201611729 -
Scientific Reports May 2024The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal...
The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)] on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease.
Topics: Biosensing Techniques; Citrus; Plant Diseases; DNA Probes; Bacterial Outer Membrane Proteins; Electrochemical Techniques; Electrodes; Nucleic Acid Hybridization; Dielectric Spectroscopy; Limit of Detection; Rhizobiaceae; Liberibacter
PubMed: 38806617
DOI: 10.1038/s41598-024-63112-w -
Langmuir : the ACS Journal of Surfaces... Jan 2015Electrochemical DNA-based (E-DNA) sensors are utilized to detect a variety of targets including complementary DNA, small molecules, and proteins. These sensors typically...
Electrochemical DNA-based (E-DNA) sensors are utilized to detect a variety of targets including complementary DNA, small molecules, and proteins. These sensors typically employ surface-bound single-stranded oligonucleotides that are modified with a redox-active molecule on the distal 3' terminus. Target-induced flexibility changes of the DNA probe alter the efficiency of electron transfer between the redox active methylene blue and the electrode surface, allowing for quantitative detection of target concentration. While numerous studies have utilized the specific and sensitive abilities of E-DNA sensors to quantify target concentration, no studies to date have demonstrated the ability of this class of collision-based sensors to elucidate biochemical-binding mechanisms such as cooperativity. In this study, we demonstrate that E-DNA sensors fabricated with various lengths of surface-bound oligodeoxythymidylate [(dT)n] sensing probes are able to quantitatively distinguish between cooperative and noncooperative binding of a single-stranded DNA-binding protein. Specifically, we demonstrate that oligo(dT) E-DNA sensors are able to quantitatively detect nM levels (50 nM-4 μM) of gene 32 protein (g32p). Furthermore, the sensors exhibit signal that is able to distinguish between the cooperative binding of the full-length g32p and the noncooperative binding of the core domain (*III) fragment to single-stranded DNA. Finally, we demonstrate that this binding is both probe-length- and ionic-strength-dependent. This study illustrates a new quantitative property of this powerful class of biosensor and represents a rapid and simple methodology for understanding protein-DNA binding mechanisms.
Topics: Biosensing Techniques; DNA; DNA Probes; DNA-Binding Proteins; Electrochemistry
PubMed: 25517392
DOI: 10.1021/la504083c -
Briefings in Bioinformatics Jan 2024Target enrichment sequencing techniques are gaining widespread use in the field of genomics, prized for their economic efficiency and swift processing times. However,...
Target enrichment sequencing techniques are gaining widespread use in the field of genomics, prized for their economic efficiency and swift processing times. However, their success depends on the performance of probes and the evenness of sequencing depth among each probe. To accurately predict probe coverage depth, a model called Deqformer is proposed in this study. Deqformer utilizes the oligonucleotides sequence of each probe, drawing inspiration from Watson-Crick base pairing and incorporating two BERT encoders to capture the underlying information from the forward and reverse probe strands, respectively. The encoded data are combined with a feed-forward network to make precise predictions of sequencing depth. The performance of Deqformer is evaluated on four different datasets: SNP panel with 38 200 probes, lncRNA panel with 2000 probes, synthetic panel with 5899 probes and HD-Marker panel for Yesso scallop with 11 000 probes. The SNP and synthetic panels achieve impressive factor 3 of accuracy (F3acc) of 96.24% and 99.66% in 5-fold cross-validation. F3acc rates of over 87.33% and 72.56% are obtained when training on the SNP panel and evaluating performance on the lncRNA and HD-Marker datasets, respectively. Our analysis reveals that Deqformer effectively captures hybridization patterns, making it robust for accurate predictions in various scenarios. Deqformer leads to a novel perspective for probe design pipeline, aiming to enhance efficiency and effectiveness in probe design tasks.
Topics: DNA Probes; Deep Learning; RNA, Long Noncoding; Nucleic Acid Hybridization; Genomics
PubMed: 38305453
DOI: 10.1093/bib/bbae007 -
Biosensors Jun 2023Fast, sensitive, and easy-to-use methods for detecting DNA related to food adulteration, health, religious, and commercial purposes are evolving. In this research, a...
Fast, sensitive, and easy-to-use methods for detecting DNA related to food adulteration, health, religious, and commercial purposes are evolving. In this research, a label-free electrochemical DNA biosensor method was developed for the detection of pork in processed meat samples. Gold electrodeposited screen-printed carbon electrodes (SPCEs) were used and characterized using SEM and cyclic voltammetry. A biotinylated probe DNA sequence of the Cyt b gene mtDNA used as a sensing element containing guanine substituted by inosine bases. The detection of probe-target DNA hybridization on the streptavidin-modified gold SPCE surface was carried out by the peak guanine oxidation of the target using differential pulse voltammetry (DPV). The optimum experimental conditions of data processing using the Box-Behnken design were obtained after 90 min of streptavidin incubation time, at the DNA probe concentration of 1.0 µg/mL, and after 5 min of probe-target DNA hybridization. The detection limit was 0.135 µg/mL, with a linearity range of 0.5-1.5 µg/mL. The resulting current response indicated that this detection method was selective against 5% pork DNA in a mixture of meat samples. This electrochemical biosensor method can be developed into a portable point-of-care detection method for the presence of pork or food adulterations.
Topics: Animals; Swine; DNA, Mitochondrial; Streptavidin; Electrochemical Techniques; Food Contamination; DNA Probes; Biosensing Techniques; Gold; Guanine; Sus scrofa; Electrodes
PubMed: 37367022
DOI: 10.3390/bios13060657 -
Biosensors Aug 2022A new electrochemical DNA biosensor based on mercaptopropionic acid (MPA)-capped ZnS quantum dots (MPA-ZnS QDs) immobilization matrix for covalent binding with 20-base...
A new electrochemical DNA biosensor based on mercaptopropionic acid (MPA)-capped ZnS quantum dots (MPA-ZnS QDs) immobilization matrix for covalent binding with 20-base aminated oligonucleotide has been successfully developed. Prior to the modification, screen-printed carbon paste electrode (SPE) was self-assembled with multilayer gold nanoparticles (AuNPs) and cysteamine (Cys). The inclusion of MPA-ZnS QDs semiconducting material in modified electrodes has enhanced the electron transfer between the SPE transducer and DNA leading to improved bioanalytical assay of target biomolecules. Electrochemical studies performed by cyclic voltammetry (CV) and differential pulsed voltammetry (DPV) demonstrated that the MPA-ZnS QDs modified AuNPs electrode was able to produce a lower charge transfer resistance response and hence higher electrical current response. Under optimal conditions, the immobilized synthetic DNA probe exhibited high selectivity towards synthetic target DNA. Based on the DPV response of the reduction of anthraquinone monosulphonic acid (AQMS) redox probe, the MPA-ZnS QDs-based electrochemical DNA biosensor responded to target DNA concentration from 1 × 10 μM to 1 × 10 μM with a sensitivity 1.2884 ± 0.12 µA, linear correlation coefficient (R) of 0.9848 and limit of detection (LOD) of 1 × 10 μM target DNA. The DNA biosensor exhibited satisfactory reproducibility with an average relative standard deviation (RSD) of 7.4%. The proposed electrochemical transducer substrate has been employed to immobilize the aminated Arowana fish () DNA probe. The DNA biosensor showed linearity to target DNA from 1 × 10 to 1 × 10 µM (R = 0.9785) with sensitivity 1.1251 ± 0.243 µA and LOD of 1 × 10 µM. The biosensor has been successfully used to determine the gender of Arowana fish without incorporating toxic raw materials previously employed in the hazardous processing conditions of polypyrrole chemical conducting polymer, whereby the cleaning step becomes difficult with thicker films due to high levels of toxic residues from the decrease in polymerization efficacy as films grew.
Topics: Animals; Biosensing Techniques; DNA; DNA Probes; Electrochemical Techniques; Electrodes; Gold; Metal Nanoparticles; Polymers; Pyrroles; Quantum Dots; Reproducibility of Results; Sulfides; Zinc Compounds
PubMed: 36005045
DOI: 10.3390/bios12080650 -
STAR Protocols Dec 2022Electrophoretic mobility shift assay (EMSA) is a classical and popular approach for DNA/RNA protein-binding affinity detection in vitro. This protocol describes a...
Electrophoretic mobility shift assay (EMSA) is a classical and popular approach for DNA/RNA protein-binding affinity detection in vitro. This protocol describes a competitive EMSA assay using digoxigenin (DIG)-labeled probe, which solves the safety issues and limitations attributed to the short lifespan of the P-radiolabeled DNA probe. We detail steps for DNA probe preparation, protein-DNA mixture coincubation, EMSA, and competitive EMSA process. We optimize the standard DIG-ddUTP-labeling EMSA protocol to high sensitivity with reproducible results. For complete details on the use and execution of this protocol, please refer to Feng et al. (2022).
Topics: Electrophoretic Mobility Shift Assay; DNA-Binding Proteins; DNA; DNA Probes; RNA-Binding Proteins; Nucleotides
PubMed: 36181685
DOI: 10.1016/j.xpro.2022.101730 -
The Journal of Physical Chemistry. B Feb 2021Electrochemical biosensors have extremely robust applications while offering ease of preparation, miniaturization, and tunability. By adjusting the arrangement and...
Electrochemical biosensors have extremely robust applications while offering ease of preparation, miniaturization, and tunability. By adjusting the arrangement and properties of immobilized probes on the sensor surface to optimize target-probe association, one can design highly sensitive and efficient sensors. In electrochemical nucleic acid biosensors, a self-assembled monolayer (SAM) is widely used as a tunable surface with inserted DNA or RNA probes to detect target sequences. The effects of inhomogeneous probe distribution across surfaces are difficult to study experimentally due to inadequate resolution. Regions of high probe density may inhibit hybridization with targets, and the magnitude of the effect may vary depending on the hybridization mechanism on a given surface. Another fundamental question concerns diffusion and hybridization of DNA taking place on surfaces and whether it speeds up or hinders molecular recognition. We used all-atom Brownian dynamics simulations to help answer these questions by simulating the hybridization process of single-stranded DNA (ssDNA) targets with a ssDNA probe on polar, nonpolar, and anionic SAMs at three different probe surface densities. Moreover, we simulated three tightly packed probe clusters by modeling clusters with different interprobe spacing on two different surfaces. Our results indicate that hybridization efficiency depends strongly on finding a balance that allows attractive forces to steer target DNA toward probes without anchoring it to the surface. Furthermore, we found that the hybridization rate becomes severely hindered when interprobe spacing is less than or equal to the target DNA length, proving the need for a careful design to both enhance target-probe association and avoid steric hindrance. We developed a general kinetic model to predict hybridization times and found that it works accurately for typical probe densities. These findings elucidate basic features of nanoscale biosensors, which can aid in rational design efforts and help explain trends in experimental hybridization rates at different probe densities.
Topics: Biosensing Techniques; DNA; DNA Probes; DNA, Single-Stranded; Nucleic Acid Hybridization; Surface Properties
PubMed: 33591751
DOI: 10.1021/acs.jpcb.0c09723 -
Talanta Mar 2024In this work we describe a highly sensitive method based on a biocatalyzed electrochemiluminescence approach. The system combines, for the first time, the use of...
In this work we describe a highly sensitive method based on a biocatalyzed electrochemiluminescence approach. The system combines, for the first time, the use of few-layer bismuthene (FLB) as a platform for the oriented immobilization of tetrahedral DNA nanostructures (TDNs) specifically designed and synthetized to detect a specific SARS-CoV-2 gene sequence. In one of its vertices, these TDNs contain a DNA capture probe of the open reading frame 1 ab (ORF1ab) of the virus, available for the biorecognition of the target DNA/RNA. At the other three vertices, there are thiol groups that enable the stable anchoring/binding to the FLB surface. This novel geometry/approach enables not only the binding of the TDNs to surfaces, but also the orientation of the capture probe in a direction normal to the bismuthine surface so that it is readily accessible for binding/recognition of the specific SARS-CoV-2 sequence. The analytical signal is based on the anodic electrochemiluminescence (ECL) intensity of luminol which, in turn, arises as a result of the reaction with HO, generated by the enzymatic reaction of glucose oxidation, catalyzed by the biocatalytic label avidin-glucose oxidase conjugate (Av-GOx), which acts as co-reactant in the electrochemiluminescent reaction. The method exhibits a limit of detection (LOD) of 4.31 aM and a wide linear range from 14.4 aM to 1.00 μM, and its applicability was confirmed by detecting SARS-CoV-2 in nasopharyngeal samples from COVID-19 patients without the need of any amplification process.
Topics: Humans; Hydrogen Peroxide; Biosensing Techniques; DNA; Nanostructures; Limit of Detection; DNA Probes; Polymerase Chain Reaction; Luminescent Measurements; Electrochemical Techniques
PubMed: 37984235
DOI: 10.1016/j.talanta.2023.125405