Detection Of Single-stranded DNA Research Articles

Characterization of a Rolling-Circle Replication Plasmid pLR1 from Lactobacillus plantarum LR1

A cryptic plasmid from Lactobacillus plantarum LR1, designated pLR1, was sequenced and characterized. It consisted of a 2066-bp circular molecule with a G + C content of 52.7%. The plasmid pLR1 was predicted to contain five putative ORFs, in which ORF1 shared 93% and 92% identity with Rep proteins of pLP1 and pC30il, members of rolling-circle replication (RCR) pC194 family. Detection of single-stranded DNA (ssDNA) intermediates by Southern hybridization and mung bean nuclease treatment confirmed that pLR1 replicated via the RCR mechanism. Accumulation of ssDNA in rifampicin-treated strains implied that the host-coded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA (dsDNA). Furthermore, the copy number of pLR1 was estimated to be 36 in each cell by real-time polymerase chain reaction.

Dipstick-type biosensor for visual detection of DNA with oligonucleotide-decorated colored polystyrene microspheres as reporters

In recent years, there is a continuously growing interest in the development of biosensors for rapid, simple and inexpensive DNA tests suitable for the small laboratory or for on-site testing. Detection is accomplished through electrochemical, optical or gravimetric transduction. We report on the development of disposable dipstick-type DNA biosensors that employ oligonucleotide-decorated colored polystyrene microspheres as reporters and enable visual detection of DNA sequences without the use of instrumentation. The biosensors have been designed to detect DNA molecules that contain both, a biotin moiety and a segment that is complementary to the oligonucleotide attached on the surface of blue or red microspheres. Capture of the hybrids by immobilized streptavidin at the test zone results in the formation of a colored line. The biosensors were applied to: (a) detection of single-stranded DNA, (b) detection of PCR-amplified double-stranded DNA and (c) genotyping of single nucleotide polymorphisms (SNP). The results were compared with sensors based on gold nanoparticle reporters. It is also demonstrated that the microspheres offer the potential for multicolor detection of specific DNA sequences.

Using enzymatic amplification by aldolase for the optical detection of DNA by an artificial signal cascade

A two-step reaction cascade is applied to the sequence-specific detection of single-stranded DNA, including analyte-triggered re-activation of apo-aldolase by its cofactor Zn2+ and catalytic conversion of a chromophore.

Recognition and detection of ssDNA using 2-mercaptobenzothiazole self-assembled monolayer modified gold electrode

A novel method for sensitive detection of single-stranded DNA (ssDNA) using 2-mercaptobenzothiazole (MBT) self-assembled monolayer (SAM) modified gold electrode (Au-MBT) has been developed. Based on the intercalating effect of MBT into double stranded DNA (dsDNA), Au-MBT electrode possessed the ability to distinguish ssDNA and dsDNA. Cyclic voltammetry (CV) was used to preliminarily characterize the MBT self-assembled monolayer on gold electrode. In the mixture of excessive probe ssDNA and target ssDNA, current signal recorded by differential pulse voltammetry (DPV) after DNA hybridization reflected the extent of the hybridization. Definite relations were observed between the reductive peak currents and the concentration of target ssDNA. The quantitative detection of target ssDNA was achieved with a linear range from 1.59 × 10−8 to 2.4 × 10−6 mol/L. The detection limit was 2.38 × 10−9 mol/L (3σ, n = 11). The Au-MBT electrode possessed high reusability and reproducibility, no significant deterioration of peak currents occurring over 10 detection/regeneration cycles. Combined with the advantages of simplicity and low-cost, the developed electrochemical biosensor shows potential in continuous and quantitative detection of ssDNA in biological samples.

Agrobacterium ParA/MinD-like VirC1 spatially coordinates early conjugative DNA transfer reactions

Agrobacterium tumefaciens translocates T-DNA through a polar VirB/D4 type IV secretion (T4S) system. VirC1, a factor required for efficient T-DNA transfer, bears a deviant Walker A and other sequence motifs characteristic of ParA and MinD ATPases. Here, we show that VirC1 promotes conjugative T-DNA transfer by stimulating generation of multiple copies per cell of the T-DNA substrate (T-complex) through pairwise interactions with the processing factors VirD2 relaxase, VirC2, and VirD1. VirC1 also associates with the polar membrane and recruits T-complexes to cell poles, the site of VirB/D4 T4S machine assembly. VirC1 Walker A mutations abrogate T-complex generation and polar recruitment, whereas the native protein recruits T-complexes to cell poles independently of other polar processing factors (VirC2, VirD1) or T4S components (VirD4 substrate receptor, VirB channel subunits). We propose that A. tumefaciens has appropriated a progenitor ParA/MinD-like ATPase to promote conjugative DNA transfer by: (i) nucleating relaxosome assembly at oriT-like T-DNA border sequences and (ii) spatially positioning the transfer intermediate at the cell pole to coordinate substrate-T4S channel docking.

Isolation and characterization of a new plasmid pSpnP1 from a multidrug-resistant clone of Streptococcus pneumoniae

A novel Streptococcus pneumoniae plasmid (pSpnP1; 5413 bp) has been isolated from the multidrug-resistant clone Poland 23F-16, and its complete nucleotide sequence has been determined. Sequence analysis predicted seven co-directional open reading frames and comparative analyses revealed that plasmid pSpnP1 is different to pDP1, the only previously described pneumococcal plasmid, whereas it is highly similar to pSt08, a plasmid from Streptococcus thermophilus. A double-stranded origin for replication similar to the replication origin of the pC194/pUB110 family was located upstream of the putative rep gene ( orf2). It also contained a 144-bp region with over 60% identity to the single-stranded origin type A of the Streptococcus agalactiae plasmid pMV158/pLS1. Detection of single-stranded DNA by Southern blot analysis indicated that pSpnP1 replicates via a rolling circle mechanism. Interestingly, the product of orf1 has a putative Zonular occludens toxin conserved domain present in toxigenic strains of Vibrio cholerae. Real-time PCR assays revealed that this ORF was expressed. Hybridization experiments showed that the pSpnP1 replicon was unusual among other examined antibiotic-resistant pneumococcal clones, although the recombinant plasmids based on pSpnP1 were able to replicate in Bacillus subtilis and Lactococcus lactis.

Mapping yeast origins of replication via single-stranded DNA detection

Studies in th Saccharomyces cerevisiae have provided a framework for understanding how eukaryotic cells replicate their chromosomal DNA to ensure faithful transmission of genetic information to their daughter cells. In particular, S. cerevisiae is the first eukaryote to have its origins of replication mapped on a genomic scale, by three independent groups using three different microarray-based approaches. Here we describe a new technique of origin mapping via detection of single-stranded DNA in yeast. This method not only identified the majority of previously discovered origins, but also detected new ones. We have also shown that this technique can identify origins in Schizosaccharomyces pombe, illustrating the utility of this method for origin mapping in other eukaryotes.

Voltammetric detection of single base-pair mismatches and quantification of label-free target ssDNA using a competitive binding assay

The application of electrochemical techniques for DNA detection is motivated by their potential to detect hybridisation events in a more rapid, simplistic and cost-effective manner compared to conventional optical assays. Here, we present an electrochemical DNA sensor for the specific and quantitative detection of single-stranded DNA (ssDNA). Probe oligonucleotides were immobilised onto thin gold film electrodes by a 5′-thiol-linker. Hybridisation was detected by means of the electroactive redox-marker methylene blue (MB) covalently attached to the 5′-end of the target ssDNA and voltammetric techniques. MB-labeled target ssDNA was recognised down to 30 pmol. By application of a competitive binding assay, non-labeled ssDNA was detected down to 3 pmol. In addition, the DNA-modified electrodes were capable of sensing single base-pair mismatches at different positions within the sequence of the hybridised double-stranded DNA (dsDNA).

Quantitative Real-Time Measurements of DNA Hybridization with Alkylated Nonoxidized Silicon Nanowires in Electrolyte Solution

The quantitative, real-time detection of single-stranded oligonucleotides with silicon nanowires (SiNWs) in physiologically relevant electrolyte solution is demonstrated. Debye screening of the hybridization event is circumvented by utilizing electrostatically adsorbed primary DNA on an amine-terminated NW surface. Two surface functionalization chemistries are compared: an amine-terminated siloxane monolayer on the native SiO2 surface of the SiNW, and an amine-terminated alkyl monolayer grown directly on a hydrogen-terminated SiNW surface. The SiNWs without the native oxide exhibit improved solution-gated field-effect transistor characteristics and a significantly enhanced sensitivity to single-stranded DNA detection, with an accompanying 2 orders of magnitude improvement in the dynamic range of sensing. A model for the detection of analyte by SiNW sensors is developed and utilized to extract DNA-binding kinetic parameters. Those values are directly compared with values obtained by the standard method of surface plasmon resonance (SPR) and demonstrated to be similar. The nanowires, however, are characterized by higher detection sensitivity. The implication is that SiNWs can be utilized to quantitate the solution-phase concentration of biomolecules at low concentrations. This work also demonstrates the importance of surface chemistry for optimizing biomolecular sensing with silicon nanowires.

Expression of Anti-Apoptotic Protein, Bcl-2, in Liver Regeneration After a Partial Hepatectomy

Although Bcl-2 is well known to have anti-apoptotic activities in vitro and in vivo, the role of Bcl-2 relating to liver regeneration remains controversial. The aim of this study was to document the effect of Bcl-2 expression on liver regeneration in rats undergoing a partial hepatectomy. Adult male Wistar rats (n = 4/group) at 72 h before undergoing a 70% partial hepatectomy (PH) were administered 1 x 10(9) plaque-forming units of adenovirus vector encoding either human Bcl-2 (group 1) or LacZ (group 2) intravenously and were sacrificed at 0, 12 h, and at 1, 2, 3, 7, 14, and 21 days postoperatively. In group 3, normal saline was injected instead of adenovirus vector. Liver regeneration was monitored by measuring the restituted liver mass and proliferating cell nuclear antigen (PCNA) immunostaining. The incidence of apoptosis in the liver was analyzed by the immunohistochemical detection of single-stranded DNA at 14 and 21 days postoperatively. The restituted liver mass showed significantly higher values in group 1 (26.1 +/- 7.2%) than in group 2 (14.7 +/- 6.8%) and 3 (13.6 +/- 5.0%) at 1 day after PH (P < 0.05). The PCNA labeling index was significantly higher in group 1 (47.2 +/- 9.9%) than in groups 2 (19.0 +/- 7.8%) and 3 (19.2 +/- 15.2%) at 1 day after a partial hepatectomy (P < 0.05). The hepatocyte growth factor (HGF) mRNA expression was significantly lower in group 1 than in group 2 at 12 h after PH (P < 0.05). The number of single-stranded DNA-positive cells decreased significantly more in group 1 (5.67 +/- 1.53 positive cells/10 fields per tissue) than those in group 2 (18.33 +/- 7.57 positive cells/10 fields per tissue) at 14 days after PH. These results thus indicated that an overexpression of anti-apoptotic protein Bcl-2 does not necessarily have an anti-apoptotic effect on liver regeneration but appears to have a pro-proliferative effect in the early phase of liver regeneration.

Enzymatically Amplified Surface Plasmon Resonance Imaging Detection of DNA by Exonuclease III Digestion of DNA Microarrays

This paper describes a novel approach utilizing the enzyme exonuclease III in conjunction with 3'-terminated DNA microarrays for the amplified detection of single-stranded DNA (ssDNA) with surface plasmon resonance (SPR) imaging. When ExoIII and target DNA are simultaneously introduced to a 3'-terminated ssDNA microarray, hybridization adsorption of the target ssDNA leads to the direction-dependent ExoIII hydrolysis of probe ssDNA strands and the release of the intact target ssDNA back into the solution. Readsorption of the target ssDNA to another probe creates a repeated hydrolysis process that results over time in a significant negative change in SPR imaging signal. Experiments are presented that demonstrate the direction-dependent surface enzyme reaction of ExoIII with double-stranded DNA as well as this new enzymatically amplified SPR imaging process with a 16-mer target ssDNA detection limit of 10-100 pM. This is a 10(2)-10(3) improvement on previously reported measurements of SPR imaging detection of ssDNA based solely on hybridization adsorption without enzymatic amplification.

Optical sequential readout of microcantilever arrays for biological detection

A technique for the readout of the nanomechanical response of multiple microcantilevers is presented. This combines the optical beam deflection technique and the scanning of a laser beam illuminating the cantilevers of an array sequentially. The individual motion of each microcantilever is obtained by correlating the total photocurrent collected in a photodetector array, the position of the laser beam and the coordinates of the laser spot centroid on the photodetector. The technique provides sub-angstrom resolution in the cantilever deflection and it allows the readout of tens of cantilevers per second. This is applied for the quasi-simultaneous detection of single-stranded DNA with an array of five microcantilevers. Moreover, this technique also allows the measurement of the resonant frequency of individual microcantilevers into an array. This can be applied for multiple detection of several pathogen targets.

Investigation of DNA Sensing Using Piezoresistive Microcantilever Probes

Piezoresistive microcantilever-based sensors maybe used in a variety of sensing applications, including chemical sensing and biological sensing. In these applications, a sensing material is functionalized so as to undergo a volumetric or dimensional change upon analyte exposure. A piezoresistive microcantilever in contact with, or embedded within, the sensing material records the dimensional change as a simple resistance change in the cantilever as it is strained by the volumetric shift in the sensing layer. Here, we describe the detection of single-strand DNA by utilizing a sensing layer material consisting of thiolated single-strand DNA attached to a gold film substrate. A piezoresistive microcantilever in direct contact with this layer in solution immediately responds to the presence of the complimentary (25 base) single strand.

Sequencing and Characterization of Plasmid pUIBI-1 from Bacillus thuringiensis Serovar entomocidus LBIT-113

Plasmid pUIBI-1 from Bacillus thuringiensis svr. entomocidus was sequenced and its replication mechanism analyzed. Sequence analysis revealed that pUIBI-1 contains 4671 bp and a 32% GC content. Plasmid pUIBI-1 also includes at least seven putative open reading frames (ORFs) encoding for proteins ranging from 5 to 50 kDa. ORF-1 encodes for a putative 16-kDa Rep protein, which lacks homology with proteins of similar function. ORF2 encodes for a protein of 50 kDa and shows homology with Mob proteins of plasmids pLUB1000 from Lactobacillus hilgardii (32.2%) and pGI2 from B. thuringiensis (33.7%). Detection of single-stranded DNA (ssDNA) intermediates indicated that pUIBI-1 replicates by the rolling-circle replication mechanism, as demonstrated by S1 treatment and Southern hybridization under non-denaturing conditions.

Detection of single-stranded DNA in the hydropic vestibule after the direct injection of antigen into the endolymphatic sac of guinea pigs

Immunohistochemical study for single-stranded DNA (ssDNA) with vestibule of guinea pigs was performed after the injection of keyhole limpet hemocyanin (KLH) into the right endolymphatic sac. Endolymphatic hydrops became evident by expansion of the Reissner's membrane in the cochlea of all animals 1 day after the injection of KLH. Increased ssDNA expression was detected in the sensory epithelium and transitional area, while temporal bones in the control group did not show any ssDNA immunoreactivities. ssDNA is accompanied with the apoptotic change in the vestibule. Our results suggest that apoptotic changes could be involved in the hydropic vestibule and these phenomena lead inner ear disturbance as seen in endolymphatic hydrops.

Characterization and Replication Properties of the Zymomonas mobilis ATCC 10988 Plasmids pZMO1 and pZMO2

The complete nucleotide sequences of two small cryptic Zymomonas mobilis ATCC 10988 plasmids (pZMO1 and pZMO2) were determined. The plasmids showed 67% homology to each other at their nucleotide level. Plasmid pZMO1 was 1651 bp long with 38% G + C content and contained an open reading frame (ORFZMO1) of 1044 nucleotides. ORFZMO1 is predicted to encode a polypeptide of 348 amino acids and shows a high degree of homology with gram-negative replication proteins of rolling circle replicating plasmids, which belong to the pC194/pUB110 family. Plasmid pZMO2 was found to be 1669 bp long, with a 38.5% G + C content, and it contained an ORF of 552 nucleotides (ORFZMO2) encoding a putative polypeptide of 184 amino acids. This polypeptide also shows a high degree of homology with the replication proteins of RCR plasmids of gram-negative bacteria, but only at their N-termini. The region necessary for replication of both plasmids was determined by stability tests under nonselective conditions, following cloning in pBR325 and introduction in Z. mobilis ATCC 10988 by pRK2013 assisted conjugation. Double- and single-strand origin regions were predicted by sequence analysis. Detection of single-stranded DNA in the extract of exponentially growing cells confirmed experimentally the rolling circle replication mode of at least pZMO2.

The covalent attachment of multiple fluorophores to DNA containing phosphorothioate diesters results in highly sensitive detection of single-stranded DNA

DNA fragments containing multiple internucleotidic phosphorothioate diesters, prepared by either chemical or enzymatic syntheses, are amenable to labeling with the fluorophore monobromobimane. With the incorporation of phosphorothioate diesters at each internucleotidic site, multiple fluorophores, ideally one for each nucleotide residue, can be covalently attached to the DNA fragment. The presence of multiple labels can be expected to interfere with analysis techniques, such as polyacrylamide gel electrophoresis. To avoid such problems, the fluorophores are introduced in a "postassay" fashion, that is, while the fragments are still embedded within the gel matrix. The detection limit (to the naked eye) for multiply labeled single-stranded DNA containing hundreds of base residues is in the low femtomole range. DNA containing greater than 1000 base residues can be visualized in some cases in the subfemtomole range without the use of sophisticated electronic instrumentation.

Detection of single-stranded DNA in scrapie-infected brain by electron microscopy

The nucleic acid content of enriched preparations of mitochondria/tubulofilamentous particles from normal and scrapie-infected hamster brains were examined by electron microscopy. After spreading on collodion-coated grids circular molecules of approximately 15.7 kb corresponding in size to mitochondrial DNA (mtDNA) were observed both in normal and scrapie-infected brains. In nucleic acid preparations from scrapie-infected brains multimeric mtDNA and single-stranded DNA strands of about 0.49 × 10 6 daltons were also visualized. These findings demonstrate the presence of a single-stranded DNA in scrapie-infected brains and are consistent with previous data based on enzyme digestion of nucleic acids isolated from scrapie-infected brains.

Adsorptive transfer stripping voltammetry: Determination of nanogram quantities of DNA immobilized at the electrode surface

In adsorptive transfer stripping voltammetry (AdTSV), DNA is first adsorbed at the electrode, the electrode is washed and transferred (with the adsorbed layer) in the medium not containing DNA, and voltammetric analysis is performed in this medium. Adsorption can be performed from a drop of DNA solution, which makes it possible to reduce the volume of the analyzed sample by two orders of magnitude as compared to that of conventional voltammetry. With the hanging mercury drop electrode the limit of detection of single-stranded DNA is below 0.1 μg/ml; thus if the adsorption is performed from a 10-μl drop of DNA solution subnanogram quantities of single-stranded DNA are sufficient for the analysis. In AdTSV the behavior of single- and double-stranded DNAs markedly differ from each other in a manner similar to that in the conventional voltammetric or polarographic analysis; AdTSV can thus be used in DNA structure analysis. In AdTSV the DNA transport and its adsorption at the electrode are separated from the electrode process; due to this fact it is possible (a) to perform the voltammetric analysis of DNA from media not suitable for voltammetric analysis of the conventional type, (b) to study the interaction of immobilized DNA with other substances in solution without the results of the voltammetric analysis being influenced by DNA interactions in the bulk of solution, and (c) to exploit the differences of adsorbability of DNA and other substances in order to separate them on the electrode.

Ultrastructural detection of single-stranded DNA in rat liver nucleus by the electronmicroscopic immuno-peroxidase method

Single-stranded DNA (ssDNA) in nucleus of rat liver cell was detected electron-microscopically by an indirect immuno-peroxidase technique using anti-thymine antibody. Anti-thymine was made by immunizing a rabbit with thymine-bovine serum albumin (BSA) conjugate. Anti-thymine was prepared from anti-thymine-BSA serum by adsorption with insolubilized BSA. Immunochemical analysis of anti-thymine was performed by gel diffusion and confirmed the reactivity of antithymine with ssDNA. The enzymatic reaction products showing single-stranded DMA were detected in the chromatin areas of nuclei of both normal and regenerating rat liver cells. The specificity of the reaction product was checked by control experiments.