ssDNA Concentration Research Articles

One-step conjugation of aminoferrocene to phosphate groups as electroactive probes for electrochemical detection of sequence-specific DNA

A straightforward electrochemical DNA biosensing approach based on exploiting organometallic compound, aminoferrocene (AFC), as electroactive probes was firstly demonstrated, where the probes could be directly labeled to the free phosphate groups of the hybridized PNA/DNA heteroduplexes merely through one-step conjugation in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and imidazole. Briefly, mercapto-terminated peptide nucleic acid (PNA) was firstly immobilized onto gold electrode and used as the capture probes for the specific recognition of target single-stranded DNA (ssDNA). After hybridization, AFC probes were directly labeled to the free 5′-terminal phosphate groups, which were activated by EDC and imidazole, of the hybridized PNA/DNA heteroduplexes, and then they were exploited as the electroactive probes to monitor the hybridization. As the captured ssDNA was labeled with AFC in the stoichiometric ratio of 1:1, thus the electrochemical analysis of the proportionally labeled AFC based on differential pulse voltammetry (DPV) enabled a quantitative determination of sequence-specific DNA. Under optimal conditions, the approach presented a good linear relationship between the current intensities and logarithm of ssDNA concentrations in the range from 0.1nM to 100nM with a detection limit of 93pM, and it rendered satisfactory analytical performance in serum samples. Furthermore, it exhibited excellent specificity toward single-nucleotide polymorphism (SNP) and precluded complicated protocols. More importantly, the simplicity of this approach together with its compatibility with standard micro-fabrication techniques makes it great potential in practical applications, especially in microarray areas where simple procedures are preferred.

Highly sensitive silicon nanowire biosensor with novel liquid gate control for detection of specific single-stranded DNA molecules.

The study demonstrates the development of a liquid-based gate-control silicon nanowire biosensor for detection of specific single-stranded DNA (ssDNA) molecules. The sensor was fabricated using conventional photolithography coupled with an inductively coupled plasma dry etching process. Prior to the application of DNA to the device, its linear response to pH was confirmed by serial dilution from pH 2 to pH 14. Then, the sensor surface was silanized and directly aminated with (3-aminopropyl) triethoxysilane to create a molecular binding chemistry for biofunctionalization. The resulting Si‒O‒Si‒ components were functionalized with receptor ssDNA, which interacted with the targeted ssDNA to create a field across the silicon nanowire and increase the current. The sensor shows selectivity for the target ssDNA in a linear range from target ssDNA concentrations of 100 pM to 25 nM. With its excellent detection capabilities, this sensor platform is promising for detection of specific biomarkers and other targeted proteins.

Fluorescence based method for specific sequence detection of genetically modified tomato

In recent years genetic engineering resulted to the production of genetically modified organism (GMO) included many important crops. Several attempts have been focused on development of methods for detection of genetically modified organisms (GMOs). In this study fluorescence based method for specific sequence recognition of plant CaMV promoter in transgenic tomato was developed which was upon hybridization of specific probe and amplified targets from transgenic tomato plants. Methylene Blue (MB) as a fluorescence marker has been used and showed quenching effect after addition of different concentration of target ssDNA to the solution. After optimizing of environmental conditions the results showed that decreased fluorescence was proportional with the concentration of 3 × 10 −9 to 1× 10 −7 M of target ssDNA with the detection limited of 2 × 10 -9 M.

PNA-based DNA assay with attomolar detection limit based on polygalacturonic acid mediated in-situ deposition of metallic silver on a gold electrode

An electrochemical method is described for the ultrasensitive determination of sequence-specific DNA by using polysaccharide-mediated in-situ deposition of metallic silver on a gold electrode. Specifically, a thiolated peptide nucleic acid (PNA) is immobilized on the gold electrode via formation of a self-assembled monolayer. Following hybridization between PNA and target single-stranded DNA (ssDNA), polygalacturonic acid (PGUA) is introduced to the PNA/DNA heteroduplexes via phosphate-zirconium-carboxylate coordination interaction. Next, the vicinal hydroxy groups of the polysaccharide backbone are cleaved and oxidized into aldehyde groups. These act as reductants and convert added silver ions into metallic silver which in-situ deposits on the gold electrode, and then is stripped off electrochemically into a solution of KCl where it is accurately determined by differential pulse voltammetry. Under optimal conditions, this assay exhibits a wide linear response range in that the stripping current is related to the logarithm of the concentration of target ssDNA in the 0.1 fM to 10 pM range, with a detection limit as low as 2.5 aM. The method displays excellent specificity in clearly differentiating mismatched oligonucleotide fragments. We therefore believe that this method has a large potential in terms of genotyping of single-nucleotide polymorphism. Moreover, this kind of signal amplification possesses a very large capability with respect to ultrasensitive quantitation of low-abundant biomarkers.

Ultrasensitive electrochemical DNA biosensor by exploiting hematin as efficient biomimetic catalyst toward in situ metallization

In this work, we presented a novel signal amplification approach to construct an electrochemical DNA biosensor for the ultrasensitive determination of sequence-specific DNA by exploiting hematin as biomimetic catalyst toward in situ metallization. Briefly, thiolated peptide nucleic acid (PNA) probes were firstly immobilized onto gold electrode through the formation of self-assembled monolayer (SAM) and then hybridization was accomplished in the ensuing step. After that, hematin molecules were introduced to the hybridized PNA/DNA heteroduplexes by employing phosphate–zirconium–carboxylate coordination chemistry. Next, the attached hematin molecules acted as catalyst in accelerating the reduction of silver ions in the presence of catechol, leading to the in situ deposition of silver particles onto the electrode. Finally, the deposited silver particles were electrochemically stripped into KCl solution and measured by square wave voltammetry (SWV). Under optimal conditions, the hematin-based electrochemical DNA biosensor presented a good linear relationship between the stripping peak currents and logarithm of single-stranded DNA (ssDNA) concentrations in the range from 0.1fM to 0.1nM with a low detection limit of 62.41aM, and it rendered satisfactory analytical performance for the determination of ssDNA in serum samples. Furthermore, it exhibited good reproducibility and stability, meanwhile, it also showed excellent specificity toward single-nucleotide polymorphism (SNP). Therefore, the hematin-based signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level.

DNA methylation detection by a novel fluorimetric nanobiosensor for early cancer diagnosis

A very sensitive and convenient fluorescence nanobiosensor for rapid detection of DNA methylation based on Fe3O4/Au core/shell nanoparticles has been developed. Specific site of CpG islands of adenomatous polyposis coli (APC), a well studied tumor suppressor gene, was used as the detection target DNA sequence. The characteristics of nanoparticles were determined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), UV–visible spectroscopy and X-ray diffraction (XRD) spectroscopy. Fe@Au nanoparticles functionalized by bounding of single stranded DNA (ssDNA) probe through sulfhydryl group at the 5′ phosphate end. Then unmethylated and methylated complementary target ssDNA were hybridized with the immobilized ssDNA probe. Dipyridamole, a pharmaceutical agent used for the first time as a fluorescence probe which significantly interacted with hybridized unmethylated and methylated DNA. Upon the addition of the target unmethylated and methylated ssDNA, the fluorescence intensity increased in linear range by concentration of unmethylated ssDNA from 1.6×10−15 to 6.6×10−13M with detection limit of 1.2×10−16M and on the other hand, fluorescence intensity declined linearly with concentration of 3.2×10−15–8.0×10−13M methylated DNA and detection limit was 3.1×10−16M. We have also shown that nanobiosensor could distinguish ratio of methylation in series of partially methylated DNA targets with identical sequences. A density functional theory (DFT) calculation was also performed to investigate the interaction between Dipyridamole with unmethylated and methylated cytosine. Finally real sample analysis suggested that nanobiosensor could have practical application for methylation detection in human plasma sample.

Binding mode and thermodynamic studies on the interaction of the anticancer drug dacarbazine and dacarbazine–Cu(II) complex with single and double stranded DNA

The binding mode and thermodynamic characteristics of the anticancer drug dacarbazine (Dac) with double and single stranded DNA were investigated in the absence and presence of Cu(II) using cyclic voltammetry, square wave voltammetry and fluorescence spectroscopy. The interaction of Dac and Dac–Cu(II) complex with dsDNA indicated their intercalation into the base stacking domain of dsDNA double helix and the strength of interaction is independent on the ionic strength. The interaction of Dac with dsDNA in the presence of Cu(II) leads to a much stronger intercalation. The interaction mode of Dac molecules with ssDNA is electrostatic attraction via negative phosphate on the exterior of the ssDNA with Dac. The binding constants, stoichiometric coefficients and thermodynamic parameters of Dac and Dac–Cu(II) complex with dsDNA and ssDNA were evaluated. Comparison of the mode interaction of Dac with dsDNA and ssDNA was discussed. The decrease of peak current of Dac was proportional to DNA concentration, which was applied for determination of dsDNA and ssDNA concentration.

Single Electrode Genosensor for Simultaneous Determination of Sequences Encoding Hemagglutinin and Neuraminidase of Avian Influenza Virus Type H5N1

The duo-genosensor consisting of two different oligonucleotide probes immobilized covalently on the surface of one gold electrode via Au-S bond formation was used for simultaneous determination of two different oligonucleotide targets. One of the probes, decorated on its 5'-end with ferrocene (SH-ssDNA-Fc), is complementary to the cDNA representing a sequence encoding part of H5 hemagglutinin from H5N1 virus. The second probe, decorated on its 5'-end with methylene blue (SH-ssDNA-MB), is complementary to cDNA representing the fragment of N1 neuraminidase from the same virus. The presence of both probes on the surface of gold electrodes was confirmed with Osteryoung square-wave voltammetry (OSWV). The changes in redox activity of both redox active complexes before and after the hybridization process were used as analytical signal. The peak at +400 ± 2 mV was observed in the presence of 40 nM ssDNA used as a target for SH-ssDNA-Fc probe. This peak increased with the increase of concentration of target ssDNA. It indicates the "signal on" mode of analytical signal generation. The peak at -250 ± 4 mV, characteristic for SH-ssDNA-MB probe, was decreasing with the increase of the concentration of the complementary ssDNA target starting from 8 to 100 nM. This indicates the generation of electrochemical signal according to the "signal off" mode. The proposed duo-genosensor is capable of simultaneous, specific, and good sensitivity probing for the sequences derived from genes encoding two main markers of the influenza virus, hemagglutinin and neuraminidase.

Influence of ssDNA Immobilization on the Conductance of Solution Gated Graphene Transistors

Carboxyl-modified graphene materials in both oxide and reduced state were explored in parallel for the preparation of field-effect transistors (FET). They were solution gated by phosphate buffer solution (PBS) (pH 7.2). Their conductance were examined and compared with unmodified graphene transistors, firstly. Then, after single strand DNA molecules were immobilized on reduced and oxide graphene transistors, their conductance and compared. Here ssDNA molecules were amino-tagged at the terminal five. It was found that ambipolar characteristic was exhibited by reduced graphene transistors, even they were undergone carboxyl modification. And it was also discovered that there were opposite conductance variation with the increasing of ssDNA concentrations and bigger changes were obtained by reduced carboxyl-modified graphene transistors.

Trashing of Single-Stranded DNA Generated during Processing of Arrested Replication Fork in E. coli

We analyzed formation of single-stranded DNA (ssDNA) related to SOS induction in nalidixilate (Nal)-treated Escherichia coli, using immunofluorescence microscopy accompanied by computer analysis. We found enhancement of both ssDNA concentrations and cells having ssDNA foci that often localized around cellpoles. Analyzing several mutants deficient in DNA repair or replication, we found, after Nal treatment, that recN, recA, uvrD and dnaB failed to increase ssDNA concentration and that recG and particularly ruvA only partially enhanced it. In Nal-treated recB mutant, despite its failure in SOS induction, ssDNA foci positive cells increased with a slight enhancement of its concentration. These observations suggest the existence of a cellular process that sequesters genotoxic ssDNA as inert form, offering a new concept for SOS suppressor genes action.

A novel sensing strategy for the detection of Staphylococcus aureus DNA by using a graphene oxide-based fluorescent probe

A novel sensing strategy for sequence-specific recognition of Staphylococcus aureus (S. aureus) DNA was designed based on the DNA hybridization between dye-labeled single-stranded DNA (ssDNA) and the complementary target DNA. Graphene oxide (GO) can adsorb FITC-labeled probes and quench the fluorescence efficiently via Förster resonance energy transfer (FRET). However, the formation of double-stranded DNA (dsDNA) will alter the conformation of ssDNA and disturb the interaction between GO and ssDNA. Thus the dsDNA-GO mixture exhibits a stronger fluorescence emission than that of the ssDNA-GO mixture, and the relative fluorescence intensity ΔI/I0 (ΔI = I(a) - I(q)) is related to the concentration of ssDNA (S. aureus DNA). Here we illustrate a "post-mixing" strategy in which the fluorescent dye-labeled DNA was allowed to hybridize with S. aureus DNA prior to the addition of GO. In this experiment, the competition between ssDNA-GO adsorption and the dsDNA formation was avoided and the incubation time was shortened to 20 min. Under the optimum conditions, the relative fluorescence intensity ΔI/I0 was proportional to the concentration of S. aureus DNA in the range 0.0125-3.125 nmol L(-1), with a detection limit of 0.00625 nmol L(-1) and good sequence selectivity.

Label-free biosensing with single-molecule force spectroscopy

We demonstrate here a novel single-molecule, label-free bioanalytical system capable of sensing the presence of specific ssDNA oligomer sequences and proteins with high selectivity and sensitivity. An ssDNA concentration of 1 nM and a Lyz concentration of 0.65 nM could be detected.

BioSpotlight

BioTechniquesVol. 53, No. 5 BioSpotlightOpen AccessBioSpotlightPatrick Lo & Kristie NyboPatrick LoSearch for more papers by this author & Kristie NyboSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000113945AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Titer countsPhage display enables researchers to identify interactions of protein sequences fused to a phage coat protein expressed on the viral surface. Displayed sequences can be selected based on their desired binding properties, following which the associated virus is propagated in a bacterial host for isolation and purification. It is common to repeat the selection steps three or more times for enrichment, with the phage concentration determined after each propagation step. Phage concentration is commonly calculated using the plating method, where Escherichia coli are infected with phage, mixed in soft agar, plated, and the resulting plaques or colonies counted. Although this is an effective means of quantification, the procedure is lengthy, requiring an overnight incubation period. UV photometry can be used as a quicker approach to assess phage concentrations, but this method requires complete removal of impurities that absorb UV light and adversely affect readings. In this issue of BioTechniques, Lehmusvuori et al. from the University of Turku, Finland describe a new approach for quantifying bacteriophage using lanthanide fluorescence probes, a technology previously developed by the authors for other applications, and demonstrate its effectiveness using the M13 phage display system. The method requires two non-fluorescent oligonucleotide probes, one carrying a lanthanide ion and the other an antenna probe. When the probes hybridize to adjacent positions on the chloramphenicol acetyl-transferase gene, a selectable marker on the M13 phagemid single stranded DNA, the lanthanide ion and antenna probe form a highly fluorescent lanthanide chelate complex that can be measured as an indication of the concentration of ssDNA in solution. After only four hours in culture, phage could be detected in supernatant, demonstrating a significant time savings for this important step in phage display technology compared to the plating method. Measurements were highly reliable, and even more accurate than phage counts obtained using absorbance measurements. Although demonstrated with the M13 phage display system, this methodology should be applicable to quantification of other ssDNA phage species.Diagram of the complementation probe-based bacteriophage quantification assay detailed in Lehmusvuori, et al.See “Homogenous M13 bacteriophage quantification assay using switchable lanthanide fluorescence probes”.Having a BLAST (on your own)The first step in identifying any novel DNA or protein sequence is to perform a sequence similarity search against databases of known sequences to establish identity or find sequence homologs. The principal algorithm used for such similarity searches is the Basic Local Alignment Search Tool (BLAST), which is conveniently done online using the National Center for Biotechnology Information (NCBI) server, in conjunction with various publicly available databases such as GenBank and UniProt. Under certain circumstances, however, researchers may prefer to carry out offline BLAST searches on a local computer against their own databases of proprietary or unpublished sequences. While NCBI provides stand-alone, command line versions of BLAST that can be freely downloaded and installed on a personal computer, these are not particularly user-friendly, especially for biological researchers lacking computer expertise. There are two graphical front ends with extensive features and options for the BLAST command line software currently available, but these are restricted to Unix-based systems. Windows users unfortunately are restricted to a pair of graphical user interfaces (GUIs) that suffer from several major limitations. In this issue, P. Santiago-Sotelo and J.H. Ramirez-Prado at the Centro de Investigación Científica de Yucatán (Mexico) describe prfectBLAST, an operating system-independent GUI for the NCBI stand-alone suite of the new BLAST+ command line executables. prfectBLAST is free, open-source, completely self-contained, and coded in Java version 6, allowing it to be executed on multiple platforms (including Windows, Mac OS X, and Linux). In addition, the software does not require installation or any dependencies, so it can be run from an external flash or hard drive if desired. Its graphical layout and choice of options was designed to mirror the NCBI web interface for BLAST+ searches, and all BLAST+ parameters can be easily changed through the GUI. Searches using any of the BLAST+ programs are carried out against locally stored, user-customized databases and can be run in the computer background; several searches can be carried out concurrently. prfectBLAST is ideally suited for novice and casual users, yet flexible enough to allow experts to fully utilize all the advanced options available for BLAST+.See “prfectBLAST: a platform independent portable front end for the command terminal BLAST+ stand-alone suite”.FiguresReferencesRelatedDetails Vol. 53, No. 5 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print November 2012 Information© 2012 Author(s)PDF download

Abstract 5559: Detection of IDH1 mutation in the plasma of glioma patients

Abstract Background IDH1 mutation is both a diagnostic and a strong prognostic factor in gliomas. In this work, we investigated the feasibility of detecting the IDH1 mutation in plasma of glioma patients. Patients and Methods The study was conducted in 31 controls and 80 glioma patients with known IDH1 status, and confronted with MRI data. Small Size DNA (ssDNA) was extracted from 4mL plasma and the IDH1R132H mutation was detected by a combination of COLD (co-amplification at lower denaturation temperature) and digital PCR. Results ssDNA concentration (150pb to 200pb) ranged from 0.1 to 6.6 ng/mL (median 1.2 ng/mL) in controls and 0.1 to 50.3 ng/mL (median 1.2 ng/mL) in patients. ssDNA concentration did not correlate with tumor volume in low grade glioma. In contrast, ssDNA concentration in high grade gliomas patients was significantly higher than in healthy controls and correlated with the enhancing volume V (V=0: 0.9 ng/ml [0.1-3.5]; V< 10 cm3: 1.6 ng/ml [0.4-24.9]; V>10 cm3: 14.0 ng/ml [0.6-50.3]). We then analyzed 39 patients (25 with R132H IDH1 mutated glioma, 14 with IDH1 wild type glioma) for the detection of IDH1 mutation on the ssDNA extracted from plasma. The mutation was detected in plasma DNA of 15 out of 25 patients (60%) with IDH1 mutated tumor, and in none of the 14 patients with IDH1 wild type tumor. The detection rate of IDH1 mutation in plasma was higher in high grade (12/17 patients (70.6%) and was correlated with the enhancing tumor volume. Interestingly all the mutant tumors with an enhancing tumor volume higher than 3.5 cm3 (9/9) were detected by digital PCR in plasma. Conclusions Our study shows that (i) tumor DNA is released in glioma patients; (ii) ssDNA concentration is correlated with enhancing tumor volume, and (iii) IDH1 mutation is detectable in plasma DNA. This represents the first step for developing a quantitative plasma biomarker that could be used to monitor glioma status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5559. doi:1538-7445.AM2012-5559

Amino-ethoxilated fluorinated amphiphile: Synthesis, self-assembling properties and interactions with ssDNA

Fluorinated compounds are not only more hydrophobic than their hydrogenated counterparts but also oleophobic. This makes them interesting for biomedical applications. They form a large variety of supramolecular architectures and, if appropriately designed, they spontaneously form vesicles. Herein we report the synthesis of novel monocatenar fluorinated surfactant, named F9NH(EO)2NH2, its use as ssDNA vector with potential applications in gene delivery or RNA interference. The molecular structure of the amphiphilic compound was designed in three parts: (i) a fluorinated hydrophobic tag responsible of self-assembling, (ii) a flexible and hydrophilic ethylenoxide moiety as polar head and (iii) a protonated at physiological pH primary amine responsible of binding. Surface tension, dynamic light scattering (DLS) measurements, and TEM studies showed that the monocatenar surfactant F9NH(EO)2NH2 spontaneously self-assembles into vesicles. This represents another example in addition to the few existing in the literature and it highlights the unique properties of the fluorinated tail. Interactions between ssDNA and cationic monocharged F9NH(EO)2NH2 were probed by static light scattering (SLS), fluorescence and circular dichroism (CD). F9NH(EO)2NH2 was found to exclude ethidium bromide (EB) from ssDNA/EB complex, and this process was dependent on the ssDNA concentration and on the charge ratio surfactant to ssDNA. CD spectra showed that by increasing the surfactant concentration, ssDNA undergoes a continuous conformational transition from native B-form to a more ordered phase (Z+/Z−≤3) and then to a less ordered phase (Z+/Z−≥4), which confirmed the results obtained by fluorimetry and SLS measurements. Cell viability studies reveled that monocatenar fluorinated surfactant F9NH(EO)2NH2 is not toxic for human cells (MRC-5) below 40μM.

Rapid Microarray Detection of DNA and Proteins in Microliter Volumes with Surface Plasmon Resonance Imaging Measurements

A four-chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The 12-element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm(2) area; 45 nm thickness), each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA, respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (ssDNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements are also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss is proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18 000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 s in the microliter volume format.

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

TraI, a bifunctional enzyme containing relaxase and helicase activities, initiates and drives the conjugative transfer of the Escherichia coli F plasmid. Here, we examined the structure and function of the TraI helicase. We show that TraI binds to single-stranded DNA (ssDNA) with a site size of ∼25 nucleotides, which is significantly longer than the site size of other known superfamily I helicases. Low cooperativity was observed with the binding of TraI to ssDNA, and a double-stranded DNA-binding site was identified within the N-terminal region of TraI 1-858, outside the core helicase motifs of TraI. We have revealed that the affinity of TraI for DNA is negatively correlated with the ionic strength of the solution. The binding of AMPPNP or ADP results in a 3-fold increase in the affinity of TraI for ssDNA. Moreover, TraI prefers to bind ssDNA oligomers containing a single type of base. Finally, we elucidated the solution structure of TraI using small angle x-ray scattering. TraI exhibits an ellipsoidal shape in solution with four domains aligning along one axis. Taken together, these data result in the assembly of a model for the multidomain helicase activity of TraI.

Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe

A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/ F 0 = 2.73 C (μM) + 1.14 ( R = 0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N = 3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.

Ultratrace voltammetric method for the detection of DNA sequence related to human immunodeficiency virus type 1

An ultra-trace voltammetric method was developed for the determination of single strand DNA (ss-DNA) related to the human immunodeficiency virus type 1 (HIV-1). It is based on the signal amplification of carbon nanotubes loaded with silver nanoparticles and placed on a gold microelectrode. The capture ss-DNA (a 21-mer) possessing a thiol group at the 3′ end was self-assembled onto the surface of the gold microelectrode. It was then hybridized with target HIV-1 ss-DNA (a 42-mer) and further hybridized with the electrochemical probe (a 18-mer ss-DNA) tagged with multiwall carbon nanotubes and loaded with silver nanoparticles. The resulting formation of a DNA sandwich conjugate led to a strong electrochemical oxidation signal that was linearly proportional to the concentration of HIV-1 ss-DNA in the range from 1.0 to 100 pM. The detection limit was 0.5 pM (at an S/N of 3). This was equivalent to 0.05 fmol of HIV-1 ss-DNA in a volume of 20 μL. The relative standard deviation was 4.0% at 1.0 pM (n = 11). Non-complementary ss-DNA of HIV-1 ss-DNA was effectively discriminated. This work demonstrates that the employment of the microelectrode and a sandwich hybridization model is promising in terms of sensitive and selective electrochemical detection of DNA.

Microbial Inactivation of Pseudomonas putida and Pichia pastoris Using Gene Silencing

Antisense deoxyoligonucleotide (ASO) gene silencing was investigated as a potential disinfection tool for industrial and drinking water treatment application. ASOs bind with their reverse complementary mRNA transcripts thereby blocking protein translation. While ASO silencing has mainly been studied in medicine, it may be useful for modulating gene expression and inactivating microorganisms in environmental applications. In this proof of concept work, gene targets were sh ble (zeocin resistance) and todE (catechol-2,3-dioxygenase) in Pichia pastoris and npt (kanamycin resistance) in Pseudomonas putida. A maximum 0.5-fold decrease in P. pastoris cell numbers was obtained following a 120 min incubation with single-stranded DNA (ssDNA) concentrations ranging from 0.2 to 200 nM as compared to the no ssDNA control. In P. putida, a maximum 5.2-fold decrease was obtained after 90 min with 400 nM ssDNA. While the silencing efficiencies varied for the 25 targets tested, these results suggest that protein activity as well as microbial growth can be altered using ASO gene silencing-based tools. If successful, this technology has the potential to eliminate some of the environmental and health issues associated with the use of strong chemical biocides. However, prior to its dissemination, more research is needed to increase silencing efficiency and develop effective delivery methods.