Elementary processes of DNA surface hybridization resolved by single-molecule kinetics: implication for macroscopic device performance

Hybridization of a single DNA molecule on a surface was investigated by electrical conductance measurements. The hybridization efficiency increases with increasing the DNA concentration, in contrast to preceding studies with ensemble studies.

A Label-Free Impedimetric Genosensor for the Nucleic Acid Amplification-Free Detection of Extracted RNA of Dengue Virus

Developing rapid and sensitive diagnostic methods for dengue virus (DENV) infection is of prime priority because DENV infection is the most prevalent mosquito-borne viral disease. This work proposes an electrochemical impedance spectroscopy (EIS)-based genosensor for the label-free and nucleic acid amplification-free detection of extracted DENV RNA intended for a sensitive diagnosis of DENV infection. A concentration ratio of 0.04 mM 6-mercaptohexanoic acid (MHA) to 1 mM 6-mercapto-1-hexanol (MCH) was selected to modify thin-film gold electrodes as a link to control the coverage of self-designed probe DNA (pDNA) at a density of 4.5 ± 0.4 × 1011 pDNA/cm2. The pDNA/MHA/MCH-modified genosensors are proven to improve the hybridization efficiency of a synthetic 160-mer target DNA (160mtDNA) with a 140-mer electrode side overhang as compared to other MHA/MCH ratio-modified genosensors. The MHA(0.04 mM)/MCH(1 mM)-modified genosensors also present good hybridization efficiency with the extracted DENV serotype 1 (DENV1) RNA samples, having the same electrode side overhangs with the 160mtDNA, showing a low detection limit of 20 plaque forming units (PFU)/mL, a linear range of 102–105 PFU/mL and good selectivity for DENV1. The pDNA density-controlled method has great promise to construct sensitive genosensors based on the hybridization of extracted DENV nucleic acids.

Hybridization Chain Reactions Targeting the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

In this work, hybridization chain reactions (HCRs) toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS–CoV-2) nucleocapsid phosphoproteins gene loci and human RNase P are proposed to provide an isothermal amplification screening tool. The proposed chain reactions target the complementary DNA (cDNA) of SARS–CoV-2, with loci corresponding to gold-standard polymerase chain reaction (PCR) loci. Four hybridization chain reaction reactions are demonstrated herein, targeting N1/N2/N3 loci and human RNase P. The design of the hybridization chain reaction, herein, is assisted with an algorithm. The algorithm helps to search target sequences with low local secondary structure and high hybridization efficiency. The loop domain of the fuel hairpin molecule H1 and H2, which are the tunable segments in such reactions, are used as an optimization parameter to improve the hybridization efficiency of the chain reaction. The algorithm-derived HCR reactions were validated with gel electrophoresis. All proposed reactions exhibit a hybridization complex with a molecular mass >1.5k base pairs, which is clear evidence of chain reaction. The hybridization efficiency trend revealed by gel electrophoresis corresponds nicely to the simulated data from the algorithm. The HCR reactions and the corresponding algorithm serve as a basis to further SARS–CoV-2 sensing applications and facilitate better screening strategies for the prevention of on-going pandemics.

Quantification of DNA by a Thermal-Durable Biosensor Modified with Conductive Poly(3,4-ethylenedioxythiophene)

The general clinical procedure for viral DNA detection or gene mutation diagnosis following polymerase chain reaction (PCR) often involves gel electrophoresis and DNA sequencing, which is usually time-consuming. In this study, we have proposed a facile strategy to construct a DNA biosensor, in which the platinum electrode was modified with a dual-film of electrochemically synthesized poly(3,4-ethylenedioxythiophene) (PEDOT) resulting in immobilized gold nanoparticles, with the gold nanoparticles easily immobilized in a uniform distribution. The DNA probe labeled with a SH group was then assembled to the fabricated electrode and employed to capture the target DNA based on the complementary sequence. The hybridization efficiency was evaluated with differential pulse voltammetry (DPV) in the presence of daunorubicin hydrochloride. Our results demonstrated that the peak current in DPV exhibited a linear correlation the concentration of target DNA that was complementary to the probe DNA. Moreover, the electrode could be reused by heating denaturation and re-hybridization, which only brought slight signal decay. In addition, the addition of the oxidized form of nicotinamide adenine dinucleotide (NAD+) could dramatically enhance the sensitivity by more than 5.45-fold, and the limit-of-detection reached about 100 pM.

The value of alien representatives of Prunus L. genus for flowering cherries breeding

Aim. The necessity to search for sources and donors of deficit features for east cherry tree breeding (sakura) and to classify the collection of this promising woody ornamental plant for domestic horticulture determined the conduction of our research. Methods. The availability of some representatives of sakura collection of NDP «Sofiyivka» for breeding was studied with conventional methods, namely, flower color, intensity and duration of flowering, fruit attractiveness, form of a tree crown, vigor and other traits, which determine plant attractiveness for gardens, parks and street plantations, were estimated. Results. Among sakura cultivars which present interest for breeding and are characterized by high ornamental nature along with their adaptability to the conditions of most of the regions in Ukraine, such well-known cultivars as ‘Ama-no-gawa’, ‘Kanzan’, ‘Kiku-shidare-zakura’ and ‘Royal Burgundy’ are to be mentioned. Despite the information concerning the feasibility of spontaneous and the success of artificial intercultivar and interspecific hybridization of Prunus serrulata with other cultivars and other Prunus s. l. genera, at present we have not received P. serrulata hybrids. Conclusions. To enhance the breeding productivity of flowering cherry tree, namely hybridization efficiency of P. serrulata with donors and sources of ornamental traits, it is advisable to involve new initial material on a wide genetic basis not only by economic-valuable features, but also considering the cases of S-genes genetic incompatibility. Keywords: Amygdaloideae Arn., Prunus sensu lato, initial material for breeding, gametophytic self-incompatibility, oriental flowering cherries (sakura).

The Implications of Fragmented Genomic DNA Size Range on the Hybridization Efficiency in NanoGene Assay

DNA hybridization-based assays are well known for their ability to detect and quantify specific bacteria. Assays that employ DNA hybridization include a NanoGene assay, fluorescence in situ hybridization, and microarrays. Involved in DNA hybridization, fragmentation of genomic DNA (gDNA) is necessary to increase the accessibility of the probe DNA to the target gDNA. However, there has been no thorough and systematic characterization of different fragmented gDNA sizes and their effects on hybridization efficiency. An optimum fragmented size range of gDNA for the NanoGene assay is hypothesized in this study. Bacterial gDNA is fragmented via sonication into different size ranges prior to the NanoGene assay. The optimum size range of gDNA is determined via the comparison of respective hybridization efficiencies (in the form of quantification capabilities). Different incubation durations are also investigated. Finally, the quantification capability of the fragmented (at optimum size range) and unfragmented gDNA is compared.