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We have analyzed and compared the available experimental data (PDB) on the backbone geometry of the DNA in solution (NMR), in crystals (X-rays), and in complexes with proteins (X-rays and cryo-electron microscopy). The deoxyribose (pseudorotational angle τ0) and e/ζ (BI-BII transition in phosphates) flexibilities are practically the same in the four samples. The α/γ mobility is minimal in crystalline DNA: on the histograms, there is one canonical and one noncanonical t/t peak. The α/γ mobility increases in DNA solutions (three more noncanonical peaks) and is maximal in DNA-protein complexes (another additional peak). On a large amount of data, we have confirmed that the three main degrees of freedom of the sugar-phosphate backbone are "orthogonal": changes in any of the angles τ0, (ζ-e), and (γ-α) occur, as a rule, at a constant (usually canonical) value of any other. In the DNA-protein complexes, none of the geometrical parameters commonly used to distinguish the A and B forms of DNA, except for Zp and its simpler analog Zp', show an unambiguous correlation with τ0. Proteins, binding to DNA, in 59% of cases change the local shape of the helix up to the characteristic of the A-form without switching the deoxyribose conformation from south to north. However, we have found simple local characteristics of one nucleotide that correlate with the angles τ0 and (ζ-e). These are the angles C3'C1'N* and C4'C3'P(2), respectively. They are orthogonal in DNA-protein complexes exactly as the pair τ0 and (ζ-e). Most characteristics of DNA in complexes with proteins are the same in X-ray and in cryo-EM data, except for the histogram for the angle τ0. We offer a possible explanation for this difference. We also discuss the artifacts on the e/ζ histogram for DNA in solutions caused by the currently used NMR refinement protocols.

We study buffered aqueous solutions of deoxyribonucleic acid isolated from bacteriophage lambda (λ-DNA) at shear rates up to 105 s−1. The shear rates are accessed with a narrow-gap rheometer at gap widths down to 20 μm. At lower shear rates, our data merge with the literature values. At high shear rates, the viscosity levels off into an infinite-shear viscosity plateau. Hence, the viscosity functions of buffered aqueous DNA solutions are now available for the entire shear-rate range from the first Newtonian plateau to that of infinite-shear viscosity. The latter hardly differs from the solvent viscosity. For the normal-stress differences, we observe a power-law dependence on the shear rate close to previous findings up to shear rates of about 104 s−1. Beyond this shear-rate range, we observe a stepwise change with the shear rate. By means of agarose gel electrophoresis, we confirm that the λ-DNA is not fragmented during our rheometric study at high shear rates. Yet, at the highest shear rates studied, shear-induced changes in the DNA to structures not being able to travel through the gel appear.

The DNA interaction with cis-isomers of photosensitive azobenzene-containing surfactants was studied by both experimental methods and computer simulation. It was shown that before the organization of micelles, such surfactants in the cis-conformation form associates of only a single type with a disordered orientation of molecules. In contrast, for trans-isomers, there exist two types of associates with head-to-head or head-to-tail orientations of molecules in dependence on salt concentration in a solution. The comparison of cis- and trans-isomer binding to DNA and the influence of salt concentration on the formation of their complexes with DNA were studied. It was shown that cis-isomers interact with phosphate groups of DNA and that their molecules were also located along the minor groove of DNA.

Hydration plays a fundamental role in DNA structure and functioning. However, the hydration shell has been studied only up to the scale of 10–20 water molecules per nucleotide. In the current work, hydration shells of DNA were studied in a solution by terahertz time-domain spectroscopy. The THz spectra of three DNA solutions (in water, 40 mm MgCl2 and 150 mM KCl) were transformed using an effective medium model to obtain dielectric permittivities of the water phase of solutions. Then, the parameters of two relaxation bands related to bound and free water molecules, as well as to intermolecular oscillations, were calculated. The hydration shells of DNA differ from undisturbed water by the presence of strongly bound water molecules, a higher number of free molecules and an increased number of hydrogen bonds. The presence of 40 mM MgCl2 in the solution almost does not alter the hydration shell parameters. At the same time, 150 mM KCl significantly attenuates all the found effects of hydration. Different effects of salts on hydration cannot be explained by the difference in ionic strength of solutions, they should be attributed to the specific action of Mg2+ and K+ ions. The obtained results significantly expand the existing knowledge about DNA hydration and demonstrate a high potential for using the THz time-domain spectroscopy method.

The extensional rheology of polymeric liquids has been extensively examined through experiments and theoretical predictions. However, a systematic study of the extensional rheology of polymer solutions in the semidilute regime, in terms of examining the effects of concentration and molecular weight, has not been carried out so far. Prior studies of the shear rheology of semidilute polymer solutions have demonstrated that their behaviour is distinctively different from that observed in the dilute and concentrated regimes. This difference in behaviour is anticipated to be even more pronounced in extensional flows. In this work, the extensional rheology of linear, double-stranded DNA molecules, spanning an order of magnitude of molecular weights (25 to 289 kilobasepairs) and concentrations (0.03 to 0.3 mg/ml), has been investigated. DNA solutions are now used routinely as model polymeric systems due to their near-perfect monodispersity. Measurements have been carried out with a filament stretching rheometer since it is the most reliable method for obtaining an estimate of the elongational stress growth of a polymer solution. Transient and steady-state uniaxial extensional viscosities of DNA dissolved in a solvent under excess salt conditions, with a high concentration of sucrose in order to achieve a sufficiently high solvent viscosity, have been determined in the semidilute regime at room temperature. The dependence of the steady state uniaxial extensional viscosity on molecular weight, concentration and extension rate is measured with a view to determining if data collapse can be observed with an appropriate choice of variables. Steady state shear viscosity measurements suggest that sucrose-DNA interactions might play a role in determining the observed rheological behaviour of semidilute DNA solutions with sucrose as a component in the solvent., 30 pages, 9 figures, submitted to Korea-Australia Rheology Journal

High accuracy, reliability, and reproducibility of genetic analyses in various applications require optimized and validated protocols and standards. Optimal procedures for storing the genetic material extracted from biological samples are equally important. In this study, we investigated the stability of dilute (4000 cp/μL, nominal concentration, equivalent to 0.02 ng/mL) DNA solutions stored at 4, -20, and -80 °C in the presence or absence of nucleic acid carriers. As representative examples, we used different formulations of a linearized plasmid DNA solution considered for characterization as reference materials (RMs) for specific applications. Employing droplet digital PCR, a highly accurate and precise method for quantification of nucleic acid not requiring a calibrant, we demonstrated that inclusion of a carrier nucleic acid in the formulation (at 50 ng/μL) improved the plasmid stability at -20 and -80 °C. For the case of a DNA standard used in real-time PCR assays for human erythropoietin gene, cDNA or transcript, we found that inclusion of yeast RNA in the formulation was preferred over salmon testes DNA as it had no effect on PCR amplification and provided the lowest relative expanded uncertainty for the characterized RM. RNA background may also be preferred as it is applicable to a broader range of DNA RMs. Our findings are important in production of reliable, stable DNA standards, including DNA RMs. These results can be used when selecting protocols for stable storage of DNA either extracted from biological samples or synthesized in a laboratory.

Graphene field-effect transistors (FETs) provide an efficient platform for enabling the label-free detection of DNA molecules. In this study, we used an interfacial polymer brush layer, which is inserted between graphene and SiO2, to enhance the electrical properties of the DNA sensors based on graphene FETs. When a polymer brush with no net dipole moment was used as a surface modification layer of SiO2, high field-effect mobility and stability were obtained in graphene FETs. In addition, it was confirmed that the graphene FETs exhibited stable operation in aqueous environments. To examine the response of DNA sensors based on graphene FETs, four types of DNA oligomers with homogeneous nucleotides (i.e. 12mer of adenine, thymine, cytosine, and guanine) were consecutively dropped onto the graphene surface and changes of electrical properties in the graphene FETs were monitored after complete drying of DNA solutions. These DNA oligomers n-doped the graphene due to the electron-rich characteristics of the nucleobases. In addition, electron and hole mobilities decreased gradually upon the addition of DNA solution because DNA molecules served as charged impurities. Graphene FETs with polymer brush provide a platform for detecting DNA molecules with low concentration.

This paper proposes a cost-effective real-time multiplexed polymerase chain reaction (PCR) chip system for point-of-care (POC) testing. In the proposed system, nucleic acid amplification is performed in a reaction chamber built on a printed-circuit-board (PCB) substrate with a PCB pattern heater and a thermistor. Fluorescence can be detected through the transparent plastic on the other side of the substrate. Open platform cameras were used for miniaturization and cost-effectiveness. We also used simple and cost-effective oblique lighting to stimulate fluorescence. Response performance was investigated by observing the change in the average brightness of the chamber images with various reference dye concentrations. In addition, we investigated the interference properties between different colors by measuring the fluorescence response for each dye concentration mixed with the maximum concentration of the different dyes. Quantitative performance was validated using standard DNA solutions. Experimental results show that the proposed system is suitable for POC real-time multi-PCR systems.

Fabrication of surfactant-modified DNA thin films with high uniformity, specifically DNA–CTMA, has been well considered via drop-casting and spin-coating techniques. However, the fabrication of thin films with pure DNA has not been sufficiently studied. We characterize the uniformity of thin films from aqueous salmon DNA solutions mixed with ethanol, methanol, isopropanol, and acetone. Measurements of thickness and macroscopic uniformity are made via a focused-beam ellipsometer. We discuss important parameters for optimum uniformity and note what the effects of solvent modifications are. We find that methanol- and ethanol-added solutions provide optimal fabrication methods, which more consistently produce high degrees of uniformity with film thickness ranging from 20 to 200 nm adjusted by DNA concentration and the physical parameters of spin-coating methods.

The dielectric properties of calf thymus DNA solutions in water and ethanol were investigated by means of terahertz time-domain spectroscopy in the range of 0.1–1 THz. Complex dielectric permittivity of solutions was fitted by twocomponent relaxation Debye model with an overdamped oscillator and their parameters were estimated. The difference between the properties of the distilled water and all aqueous solutions is the most pronounced, while the ethanol-water mixture with and without DNA shows minimal changes of about 2%, which is comparable with the experimental accuracy.

The photosensitive azobenzene-containing surfactant C-4-Azo-OC(6)TMAB is a promising agent for reversible DNA packaging in a solution. The simulation of the trans-isomer surfactant organization into associates in a solution with and without salt as well as its binding to DNA at different NaCl concentrations was carried out by molecular dynamics. Experimental data obtained by spectral and hydrodynamic methods were used to verify the results of simulation. It was shown that head-to-tail aggregates with close to antiparallel orientation of surfactant molecules were formed at certain NaCl and surfactant concentrations (below critical micelle concentration). Such aggregates have two positively charged ends, and therefore, they can be attracted to negatively charged DNA phosphates far located along the chain, as well as those that belong to different molecules. This contributes to the formation of intermolecular DNA-DNA contacts, and this way, the experimentally observed precipitation of DNA can be explained.

We use video particle tracking microrheology (VPTMR) in order to investigate the viscoelasticity of salmon DNA and correlate it to its steady-flow shear-thinning viscosity. Aqueous solutions of DNA are tested in a wide concentration range from the dilute to the semidilute unentangled concentration regime. The observed mean squared displacement shows power-law scaling with lag-time which is equivalent to power-law behavior of the complex modulus as a function of frequency that is, |G* (ω)| = S ∙ ω α . The relaxation exponent α changes abruptly with concentration in the semidilute regime from about 1 to about 0.5 which is the exponent predicted by the Rouse model. The quasi-property S follows the scaling of viscosity for uncharged polymers near θ-conditions in the semidilute regime that is, η∼c1/3νeff-1 with νeff = 0.50 - 0.51. The shear-thinning exponent observed by viscometry increases gradually towards the value of 0.5 which has been predicted for Rouse chains under flow. Our findings are in agreement with recent studies of DNA solutions where DNA is treated as a model polymer and addresses the low-molar mass regime of DNA viscoelasticity. This work demonstrates that the combination of passive particle tracking with viscometry can provide a complete picture on the viscoelasticity of DNA-based biopolymer materials.

Edge instabilities are believed to be one of the possible causes of shear banding in entangled polymeric fluids. Here, we investigate the effect of edge disturbance on the shear-induced dynamics of well-entangled DNA solutions. Using a custom high-aspect-ratio planar-Couette cell, we systematically measure the velocity profiles of sheared DNA samples at different distances away from the edge of the shear cell. Under a weak oscillatory shear with the corresponding Weissenberg number (Wi) smaller than 1, where DNA solutions exhibit linear velocity profiles with strong wall slip, the penetration depth of the edge disturbance is on the order of the gap thickness of the shear cell, consistent with the behavior of Newtonian fluids. However, under a strong oscillatory shear with Wi > 1 that produces shear-banding flows, the penetration depth is an order of magnitude larger than the gap thickness and becomes spatially anisotropic. Moreover, we find that the shear-banding flows persist deep inside the sheared sample, where the effect of edge disturbance diminishes. Hence, our experiments demonstrate an abnormally long penetration depth of edge disturbance and illustrate the bulk nature of shear-banding flows of entangled polymeric fluids under time-dependent oscillatory shear., 8 pages, 6 figures (accepted by J. Rheol.)

The nonlinear rheological behaviors of concentrated double-stranded (ds) and single-stranded (ss) DNA solutions were studied under standard oscillatory and large amplitude oscillatory shear (LAOS) deformations. To decompose the total stress into the elastic and viscous components, the raw nonlinear waveforms were analyzed using the MITlaos software package. We found that smooth and rigorous intracycle strain stiffening and shear thinning dominate the elastic and viscous decomposition, respectively, of the ds DNA sample. In contrast, the ss DNA sample exhibits initial intracycle strain stiffening, followed by a transition region and a terminal flow behavior. Insights into the nonlinear response of the DNA solutions were achieved by performing particle image velocimetry (PIV) at a wide range of imposed strain amplitudes and two angular frequencies. The rheo-PIV results of the ds DNA sample show a substantially modified flow field with distinct strain-driven shear bands at both angular frequencies examined. The bands formed by the ss DNA sample were weak even at the maximum strain amplitude. The results from this work are insightful from a traditional bulk rheological perspective, as well as for further single-molecular studies.

DNA dynamics and flow properties are of great importance for understanding its functions. DNA is a semiflexible polymer chain characterized by having a large persistence length of around 50 nm and high charge density; DNA chains are interacting efficiently at high concentrations, in dependence of the ionic concentration. In relation with DNA molecular characteristics, it is also known that DNA solutions are able to form liquid crystalline phases over a critical polymer concentration. In this work, the supramolecular organization in calf-thymus DNA solution, with low degree of entanglement, appearing under flow was studied in a wide DNA concentration range from 2 to 10 mg/mL, at a pH of 7.3 and 20 °C. The rheological behavior of the system was studied using steady state flow and oscillatory measurements. Transient regimes were also tested by imposing controlled shear rates on a short time up to steady state. Furthermore, a combination of visual observations and flow birefringence measurements was proposed ...

A study of caffeine effects on the complexation of the DNA molecule with Cu2+ and Mg2+ ions in solution is presented. DNA binding with cations and caffeine is traced from changes in DNA circular dichroism and UV absorption spectra. The complexation of caffeine with M2+ ions is studied by IR spectroscopy. Caffeine is found to form complexes of different structure with Mg2+ and Cu2+ ions in solution. In the presence of caffeine, DNA solutions with MCl2 show signs of formation of intricate complexes involving nitrogenous bases. In DNA solutions with CuCl2, the presence of caffeine causes additional destabilization of the secondary DNA structure. In DNA solutions with MgCl2, the presence of caffeine leads to the formation of new complexes with the participation of Mg2+, caffeine, and bases without disrupting the secondary DNA structure.

Existing digital microfluidic (DMF) chips exploit the electrowetting on dielectric (EWOD) force to perform droplet splitting. However, the current splitting methods are not flexible and the volume of the droplets suffers from a large variation. Herein, we propose a DMF chip featuring a 3D microblade structure to enhance the droplet-splitting performance. By exploiting the EWOD force for shaping and manipulating the mother droplet, we obtain an average dividing error of

Phenylketonuria (PKU), which is a deficiency of an important metabolic enzyme, is caused by several identified point mutations in populations around the world. Several methods have been developed for the detection of DNA mutations so far, including biosensors, especially electrochemical biosensors. In this research, we developed a novel biosensor using a special electrochemical label, hematoxylin. A screen-printed gold electrode (SPGE) modified with thiolated detection probes was used to detect the point mutation IVS10nt-11g→a of phenylalanine hydroxylase (PAH) enzyme. The methodology and its efficiency have been proved using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) studies in [Fe(CN)6]3−/4− solution. Under optimized conditions, the biosensor was able to detect the target DNA ranging from 20 pM to 150 nM with a detection limit of 8.5 pM. In addition, the selectivity of the biosensor was tested by the application of single-base mismatched and three-base mismatched oligonucleotides in comparison to the target DNA. Finally, the interference studies were performed in order to confirm the functionality of the biosensor in real sample applications, which proved good functionality for the biosensor in simulated extracted DNA solutions.

In a PNAS paper, Smith et al. (1) demonstrate spontaneous self-assembly of a DNA analog from a solution of mononucleotide triphosphates, exhibiting concurrently a high degree of Watson–Crick base pair selectivity with complementarity-dependent partitioning of nucleotides, a stacking order corresponding very closely to B-DNA geometry and a mesophase ordering similar to the one observed for much longer segments of DNA. This biopolymer “liquid crystal autocatalysis,” enabled by the three levels of soft, hierarchical assembly and exhibiting key DNA structural elements, can eventually lead to molecular selection, as well as catalytically guide and facilitate the final ligation of prebiotic DNA analogs into structurally stiff DNA. Aqueous solutions of long- as well as short-fragment DNA exhibit a sequence of ordered liquid-crystalline (LC) mesophases at DNA concentrations consistent with in vitro conditions (2). This spontaneous DNA ordering, relevant from the fundamental biophysical point of view (3, 4), is also indispensible for DNA packing within bacteriophage capsids at high osmotic pressures (4), in eukaryotic sperm cells where DNA is packaged by a variety of basic (i.e., positively charged) polypeptide chains (5), as well as in the spatial organization of chromatin inside the eukaryotic nucleus (6). Starting from the high-density end, the sequence of ordered phases in DNA solutions starts with an orthorhombic (OR) crystalline phase (7) with DNA in the A-form conformation, corresponding to its pronounced dehydration (8). Shorter 50-nm nucleosomal DNA fragments are in the OR phase at somewhat higher density but do not show the A-form fingerprint (9). At lower densities, long DNA enters the line hexatic (LH) LC phase (10), characterized by long-range orientational and bond orientational orders but with short-range positional order (11). The OR→LH transition is characterized by a discontinuous DNA density change and the emergence of a sixfold symmetric scattering intensity profile observed for oriented samples (7 … [↵][1]1Email: podgornikrudolf{at}ucas.ac.cn. [1]: #xref-corresp-1-1

Two 600-bp DNA solutions (DNA600-G and DNA600-T) were developed as certified reference material, NMIJ CRM 6205-a, for the validation of DNA quantification methods. Both DNA600-G and DNA600-T are ideal as "spike-in control" because these materials have artificial nucleic acid sequences. The certified values were determined as the mass concentration of total DNA (whole DNA materials in sample solution regardless of sequence) at 25 °C by formic acid hydrolysis/liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) and inductively coupled plasma-mass spectrometry (ICP-MS) based on the amount of phosphorus. DNAs were synthesized, and plasmids including the synthesized DNAs were cloned into Escherichia coli DH5α. The amplified plasmids were digested with a restriction enzyme and highly purified. Then, the purified DNAs were diluted with water to approximately 1 ng/μL. By using the CRM-validated methods in fields where DNA quantification is required, the reliability of DNA quantification could be improved. Graphical abstract.

The surface tension and dilational surface visco-elasticity of the individual solutions of the biopolymer DNA and the azobenzene-containing cationic surfactant AzoTAB, as well as their mixtures were measured using the drop profile analysis tensiometry. The negatively charged DNA molecules form complexes with the cationic surfactant AzoTAB. Mixed DNA + AzoTAB solutions exhibit high surface activity and surface layer elasticity. Extremes in the dependence of these characteristics on the AzoTAB concentration exist within the concentration range of 3 × 10−6–5 × 10−5 M. The surface tension of the mixture shows a minimum with a subsequent maximum. In the same concentration range the elasticity shows first a maximum and then a subsequent minimum. A recently developed thermodynamic model was modified to account for the dependence of the adsorption equilibrium constant of the adsorbed complex on the cationic surfactant concentration. This modified theory shows good agreement with the experimental data both for the surface tension and the elasticity values over the entire range of studied AzoTAB concentrations.

Liquid crystal phase is an emergent phase of biological systems. Experimental studies have shown that DNA oligomers exhibit lyotropic liquid crystal phases. Simple Landau free energy function is presented to describe the isotropic-nematic-columnar lyotropic phase sequence of DNA solutions and transitions between them. The order parameters necessary to describe these lyotropic liquid crystalline phase transitions are identified. General arguments are presented for the topology of the phase diagram in the vicinity of the isotropic-nematic-columnar triple point. Within this model the isotropic-nematic, isotropic-columnar and nematic-columnar lyotropic phase transitions are found to be always first order. The concentration and oligomers length dependent transition temperatures are calculated. Calculations based on this model agree qualitatively with experiments.

Electrohydrodynamic spraying (or electrospaying, ES) of DNA solutions is an attractive technique for applications in mass spectrometry, in microarray fabrication, and in generation of DNA nanoaerosols. Here we report how ES affects DNA structure and evaluate possible ways to reduce DNA damage upon ES. It is shown that under any ES conditions, linear λ-phage DNA is subjected to intensive rupture producing a mixture of fragments. In addition to such fragmentation, notable reversible changes in the DNA structure were revealed by a slight increase in DNA electrophoretic mobility. The degree of fragmentation was shown to decrease with decreased DNA length and with increased flow rate through the ES capillary. Fragments shorter than 5 kbp did not show any notable damage upon ES. Both experimental data and theoretical estimations of the forces acting on DNA during ES indicate that DNA is damaged by mechanical forces, and the damage takes place in the vicinity of the Taylor cone tip, presumably due to the high shear stress or/and viscous drag forces operating there. Condensation of λ-DNA with hexamminecobalt(III) ions completely protected it from any damage upon ES.

The aim of the study was to establish a DNA isolation protocol Nectandra megapotamica (Spreng.) Mez., able to obtain samples of high yield and quality for use in genomic analysis. A commercial kit and four classical methods of DNA extraction were tested, including three cetyltrimethylammonium bromide (CTAB)-based and one sodium dodecyl sulfate (SDS)-based methods. Three drying methods for leaves samples were also evaluated including drying at room temperature (RT), in an oven at 40ºC (S40), and in a microwave oven (FMO). The DNA solutions obtained from different types of leaves samples using the five protocols were assessed in terms of cost, execution time, and quality and yield of extracted DNA. The commercial kit did not extract DNA with sufficient quantity or quality for successful PCR reactions. Among the classic methods, only the protocols of Dellaporta and of Khanuja yielded DNA extractions for all three types of foliar samples that resulted in successful PCR reactions and subsequent enzyme restriction assays. Based on the evaluated variables, the most appropriate DNA extraction method for Nectandra megapotamica (Spreng.) Mez. was that of Dellaporta, regardless of the method used to dry the samples. The selected method has a relatively low cost and total execution time. Moreover, the quality and quantity of DNA extracted using this method was sufficient for DNA sequence amplification using PCR reactions and to get restriction fragments.

Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

With a miniaturized, reflective cavity-coupled fluorometer, a new fluorescence detection technique is presented with a quantitative analysis of its limits and sensitivity in detection. This paper is on a comparative study of fluorescence detection, on the same modularized fluorometer platform, to various combinations of light sources, optical filter lenses, reflectivity of the cavity wall, and fluorophore models. The quantitative comparison results allows for scrutinizing the mechanisms of noises in each measurement, including Raman scattering, and determining the limit of detection (LOD). This fluorometer enables detection of fluorescein at 0.4 nM and of SYBR Green (SG)-stained DNA at 0.05 ng μl-1 when equipped with a 450 nm laser diode, 550 nm cut-on longpass optical filter, and a polished reflective cavity. The detection of DNA can be further improved to a lower LOD by addressing the uncertainty in preparing the SG/DNA solutions with a consistent dye-base ratio throughout all the sample solutions.

Every cell in a living organism is constantly exposed to physical and chemical factors which damage the molecular structure of proteins, lipids, and nucleic acids. Cellular DNA lesions are the most dangerous because the genetic information, critical for the identity and function of each eukaryotic cell, is stored in the DNA. In this review, we describe spectroscopic markers of DNA damage, which can be detected by infrared, Raman, surface-enhanced Raman, and tip-enhanced Raman spectroscopies, using data acquired from DNA solutions and mammalian cells. Various physical and chemical DNA damaging factors are taken into consideration, including ionizing and non-ionizing radiation, chemicals, and chemotherapeutic compounds. All major spectral markers of DNA damage are presented in several tables, to give the reader a possibility of fast identification of the spectral signature related to a particular type of DNA damage.

A label-free biosensor based on a reflective microfiber probe for in-situ real-time DNA hybridization detection is proposed and experimentally demonstrated. The microfiber probe is simply fabricated by snapping a non-adiabatic biconical microfiber through closing the oxyhydrogen flame during fiber stretching. Assisted with the Fresnel reflection at the end of microfiber, a reflective microfiber modal interferometer is realized. The in-situ DNA hybridization relies on the surface functionalization of a monolayer of Poly-L-lysine (PLL) and synthetic DNA sequences that bind to a given target with high specificity. The detection processes of DNA hybridization in various concentration of target DNA solutions are monitored in real-time and the experimental results present a minimum detectable concentration of 10pM with good repeatability. Additionally, the detection specificity is also investigated by immersing the microfiber probe into the non-complementary ssDNA solutions and observing the spectral variation. The proposed biosensor has advantages of high sensitivity, compact size, ease of use and simple fabrication, which makes it has great potential to be applied in a lot of fields such as disease diagnosis, medicine, and environmental science.

The chemical and physical properties of glass do not allow it to be considered as an inert material. Therefore, it is necessary to discuss the ability of laboratory glassware to influence the state and properties of aqueous solutions, including DNA solutions. As a demonstration of this ability, it is shown here how contact with glass affects the state of salts in aqueous solutions. In terms of the topic under discussion, it is especially important that sodium salts are very sensitive to these contacts. Thus, it is shown here that contact with glass can affect the formation of fibers from sodium salts of DNA, which were mainly used in X-ray studies aimed at determining the molecular structure and parameters of DNA molecules. Special attention is paid to the fact that laboratory glassware is also sensitive to contact with aqueous solutions, including DNA solutions. That such sensitivity may be important to biologists is also shown here. In particular, it was shown how ignoring the sensitivity of silica glass, from which the photometric cells are made, to electrization gave rise to misconceptions about the spectral properties of aqueous solutions, including DNA solutions. The effect of air dissolved in aqueous DNA solutions on the spectral properties of both these solutions and the photometric cells containing them is also shown. In particular, it was shown that UV absorption of aqueous DNA solutions, which does not contain air, is completely insensitive to heating, and UV absorption of aqueous DNA solutions, which are saturated with air, is supersensitive to heating. In other words, it is shown here that our understanding of the thermal denaturation of DNA, which is reflected in the UV absorption of its aqueous solutions, was formed due to these two types of sensitivity. In the end, a conclusion is proposed here about the significant contribution of glass and air to the creation of traditional ideas about the structure and properties of DNA.

Systematic investigations using neutron and X-ray small angle scattering in near-physiological salt solutions were made to reveal the effect of polymer concentration, pH, and calcium ion concentration on the structure of semi-dilute solutions of four model biopolymers [polyaspartic acid, DNA, chondroitin sulfate, and hyaluronic acid (HA)] representing typical backbone structures. In the low q range (

This protocol describes a method to purify yeast artificial chromosome (YAC) DNA for microinjection. YAC DNA solutions cannot be concentrated by standard DNA precipitation methods and resuspended into smaller volumes. Attempts to precipitate YAC DNA solutions will, unavoidably, break and destroy the DNA molecules. Therefore YAC DNA must always be kept in a solution that maintains the integrity of the YAC DNA molecules. In the method described here, YAC DNA is extracted from pulsed-field gel electrophoresis (PFGE) agarose plugs by using two gel electrophoresis steps. The first gel electrophoresis step is the PFGE itself. The region of agarose containing the YAC DNA of interest is excised and run on a standard electrophoresis gel setup at a 90° angle to the PFGE run. The YAC DNA migrates out of the agarose slice and enters into a thicker low-melting agarose gel, thereby promoting compaction of YAC DNA molecules. The aim of this method is to convert a slice of agarose into a cube of agarose of smaller volume. The volume of this cube will determine the final concentration of the YAC DNA precipitation.

5-methylcytosine (5mC) is an epigenetic mark which has a profound effect on various fundamental processes in cells. In present study, at first Hydrophilic Interaction Liquid Chromatography (HILIC) was compared with Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) based on their selectivity (α), retention factor (k), and resolution (R) for cytosine (C) and 5mC nucleobases. We tried to justify the separation mechanism on the basis of mobile phase and solute polarity, structural characterization of solute and stationary phases, log Do/w, and pka under both modes. Then, these two modes were compared in order to select the best column for measurement of methylation level in two real samples with less analytical complexity (i.e. animal and bacteria) and a highly complex sample (i.e. plant), after chemical hydrolysis of DNA. In this favor, diol and cyano (CN) columns in HILIC mode as well as C8 and C18 in RP-HPLC were investigated. Optimum separation and the best validation parameters were obtained for CN column with Limit of Detection (LOD) of 1.4 pmol and Limit of Quantification (LOQ) of 4.8 pmol for 5mC. When the CN column was used in HILIC-UV procedure, separation of 5mC and C bases was achieved in all types of hydrolyzed DNA solutions.

Complexes of polycations and DNA, also known as polyplexes, have been extensively studied in the past decade, as potential gene delivery systems. Their stability depends strongly on the characteristics of the polycations, as well as the nature of the added salt. We present here a study of the DNA ionene complexation in which we used fluorescence, UV, and CD spectroscopy, combined with molecular dynamics computer simuations, to systematically examine the influence of the polycation charge density, as well as the influence of the nature of the counterion, on the stability of these systems. Ionenes as polycations, depending on their structural characteristics, have previously been found to possess low cytotoxicity, and are therefore particularly interesting as potential gene delivery agents. The results show that the DNA solutions in the presence of the polycation are more stable in the case of very large or very small ionene charge density, suggesting different mechanism of complexation. The computer simulations show that the ionenes with high charge density bind to the minor groove of the DNA molecules, while the ionenes with lower charge density bind to the major groove of the DNA. The nature of the counterions play only a minor role: precipitation of the DNA molecules occurs at slightly lower ionene concentration when fluoride counterion are present, compared to the bromide counterions.

The current work presents the using of microfluidic hydrodynamic flow focusing for enabling an accurate control of self-assembling nanoparticles. A mixture of surfactant and DNA dispersed in 35% ethanol is focused between two streams of pure water in a microfluidic channel. As a result, a rapid change of solvent quality takes place in the central stream, and the surfactant-bound DNA molecules undergo a fast coil-globule transition. Using this method nanoparticles having a hydrodynamic diameter of 70nm with a polydispersity index below 0.2 were achieved. A second method relied on the controlled diffusive mixing of surfactant and DNA solutions through a water stream of tunable width. Using this method the smallest nanoparticles achieved were about 30 nm in hydrodynamic diameter, meaning that most of them contained a single DNA molecule.

Ultraviolet (UV) radiation has a strong influence in the damage of deoxyribonucleic acid (DNA). In this work, the possibility of a DNA UV radiation dosimeter is evaluated. For that, calf thymus DNA samples, thin films and aqueous solutions, were irradiated with 254 nm wavelength light during different periods of time, being the damage caused by the irradiation analysed by both UV-visible and infrared spectroscopies. As the DNA is a polyelectrolyte, the pH of the DNA samples was also considered as a variable. Results demonstrated that damage in DNA takes place in both thin films and solutions when irradiated at 254 nm, as revealed by a consistent decay in measured absorbance values. However, DNA solutions were seen to give more reliable as the induced damage is easily measured. For this case, the absorbance at 260 nm was seen to exponentially decrease with the irradiation time as a result of radiation damage with the kinetics damage strongly dependent of pH. Consequently, the lifetime of such dosimeter device can be chosen by changing the pH of aqueous solutions.

Determining mechanical properties of single-stranded DNA film grafted on gold surface is critical for analysis and design of DNA-microcantilever biosensors. However, it remains an open issue to quantify the relations among the disordered packing patterns of DNA chains, the mechanical properties of DNA film and the resultant biodetection signals. In this paper, first, the bending experiment of microcantilever is carried out to provide the basic data for a refined multi-scale model of microcantilever deflection induced by ssDNA immobilization. In the model, the complicated interactions in DNA film (consisting of DNA, water molecules and salt ions) are simplified as effective interactions among coarse-grained soft cylinders, which can reveal the varieties of DNA structure in the circumstances of different lengths and salt concentrations; Ohshima's distribution of net charge density is employed to incorporate compositional variations of salt ions along the thickness direction into the Strey's mesoscopic empirical potential on molecular interactions in DNA solutions, and the related model parameters for ssDNA film on substrate are obtained from the curve fitting with our microcantilever bending experiment. Second, the effect of nanoscopic distribution of DNA chains on elastic modulus of ssDNA film is studied by a thought experiment of uniaxial compression, and the disordered patterns of DNA chains are generated by Monte Carlo method. Simulation results point out that nanoscale ssDNA film shows size effect, gradient and diversity in elastic modulus and can achieve maximum stiffness by preferring a disordered and energetically favorable packing pattern collectively induced by electrostatic force, hydration force and configurational entropy.

Investigation of morphology of deoxyribonucleotide triphosphate (DNA) dried on different surfaces by atomic force microscopy (AFM) is important in DNA research that is focused on subjects of condensation for gene therapy, sizing, DNA mapping, and cancer examination. The solvent, the surface type, and the method of drying effect the morphology of DNA on solid surfaces. Ethanol and water were used as solvents, flat mica, silica, and alumina surfaces were used as the substrates in the present study. Different methods such as ambient air-drying, N2-forced flow regime drying, and freeze-drying have been applied to droplets of DNA solutions in water or ethanol on the substrates. Forced flow drying regime causes nonlinear DNA attachment on the surface and self-assembly. DNA vertical distance on mica surface was found to be 6 and 1.4 nm for DNA dried in ambient air from ethanol and water solutions, respectively. It was 1.6 nm for N2 flow drying of aqueous DNA solution on mica surface. It was 4.6, 4.6, and 1.99 nm for ambient, N2 flow, and freeze-dried aqueous DNA on alumina surfaces, respectively. Aqueous solution of DNA dried under N2 flow on silica surface had 0.8 nm vertical distance. The smallest standard deviation of 0.05 nm was observed for DNA dried under N2 flow on alumina surface.

Our recent research results showed that DNA chains undergo a compactisation when DNA solutions are doped with different chromophores. In this paper we will present our attempt to model this behaviour in order to predict the DNA solutions characterisation.

We presume that the primary effect of Near Infrared (NIR) radiation on aqueous solutions of biological molecules concerns modification of hydrogen bonded structures mainly the global and the hydration shell water molecules. Since water has a significant influence on the DNA structure, we expect that the thermal stability of DNA could be modified by NIR radiation. The herring sperm DNA was exposed to NIR radiation (700-1100nm) for 5, 10, and 20min periods. The temperature dependent infrared measurements were done for the thin films formed on the diamond ATR crystal from evaporated DNA solutions exposed and unexposed to NIR radiation. For the NIR-treated samples (at room temperature) the B form was better conserved than in the control sample independently of the irradiation period. Above 50°C a considerable increase in the A form was only observed for 10min NIR exposed samples. The hydrodynamic radius, (Rh), studied by the dynamic light scattering, showed drastic decrease with the increasing irradiation time. Principal components analysis (PCA) allowed to detect the spectral features correlated with the NIR effect and thermal stability of the DNA films. Obtained results strongly support the idea that the photoionization of water by NIR radiation in presence of DNA molecules is the main factor influencing on its physicochemical properties.

Using high-resolution confocal rheometry, we study the shear profiles of well-entangled DNA solutions under large amplitude oscillatory shear (LAOS) in a rectilinear planar shear cell. With increasing Weissenberg number (Wi), we observe successive transitions from normal Newtonian linear shear profiles to wall-slip dominant shear profiles and finally to shear-banding profiles at high Wi. To investigate the microscopic origin of the observed shear banding, we study the dynamics of micron-sized tracers embedded in DNA solutions. Surprisingly, tracer particles in the shear frame exhibit transient super-diffusivity and strong dynamic heterogeneity. The probability distribution functions of particle displacements follow a power-law scaling at large displacements, indicating a Levy-walk-type motion, reminiscent of tracer dynamics in entangled wormlike micelle solutions and sheared colloidal glasses. We further characterize the length and time scales associated with the abnormal dynamics of tracer particles. We hypothesize that the unusual particle dynamics arise from localized shear-induced chain disentanglement., 9 pages, 8 figures, accepted in Phys. Rev. E

The viscosity of dilute and semidilute unentangled DNA solutions, in steady simple shear flow, has been measured across a range of temperatures and concentrations. For polystyrene solutions, measurements of viscosity have been carried out in the semidilute unentangled regime, while results of prior experimental measurements in the dilute regime have been used for the purpose of data analysis, and for comparison with the behaviour of DNA solutions. Interpretation of the shear rate dependence of viscosity in terms of suitably defined non-dimensional variables, is shown to lead to master plots, independent of temperature and concentration, in each of the two concentration regimes. In the case of semidilute unentangled solutions, defining the Weissenberg number in terms of a concentration dependent large scale relaxation time is found not to lead to data collapse across different concentrations. On the other hand, the use of an alternative relaxation time, with the concentration dependence of a single correlation blob, suggests the existence of universal shear thinning behaviour at large shear rates., Comment: 24 pages, 13 figures, supplementary material (see ancillary directory), to appear in Journal of Rheology

The influence of water soluble cationic meso-tetra(4N-hydroxyethyl)pyridyl porphyrin ( H 2 THOEtPyP 4) and it's metal complexes with Ni ( II ), Cu ( II ), Co ( II ) and Zn ( II ) on hydrodynamic and spectral behavior of DNA solutions has been studied by viscometry and UV-vis absorption methods. It was shown that the presence of planar porphyrins, such as H 2 THOEtPyP 4, NiTHOEtPyP 4 and CuTHOEtPyP 4 leads to an increase in viscosity at relatively small concentrations, and then decreases to stable values. Such behavior corresponds to intercalation of these porphyrins in DNA structure, which results in a decrease of helical twist and lengthening of the DNA molecule. In the case of porphyrins with axial ligands, such as ZnTHOEtPyP 4 and CoTHOEtPyP 4, the relative viscosity decreases, which is explained by self-stacking of these porphyrins on DNA surface. Calculation and interpretation of binding parameters (Kb and n) demonstrated good agreement of viscometric and spectrophotometric measurements.

Microparticulate deposits of scorpiand-like azamacrocyclic receptors (L1–L4) attached to graphite electrodes provide distinctive voltammetric features in contact with aqueous DNA solutions at biological pH, denoting the formation of DNA surface complexes. This voltammetry allows for screening dsDNA, ssDNA and G-Quadruplex DNA using L4-modified electrodes. Scanning electrochemical microscopy and scanning tunneling microscopy examination of dsDNA fibers attached to the substrate electrode in contact with DMSO solutions containing ferrocene and receptor indicate that a synergic effect is exerted between electrochemically generated ferrocenium ion and the receptors so that they increase mutually their binding ability to dsDNA. Experiments in air-saturated DMSO suggest that there are specific binding sites in dsDNA able to react with electrochemically generated superoxide ion and that such binding sites can be blocked by coordinating receptors.

Aromatic species of the genus Satureja are rich in secondary metabolites that interfere with DNA isolation procedures. Four protocols based on the standard CTDNA extraction protocol of Doyle and Doyle (1987) were tested in six savory taxa. The polyphenol adsorbents activated charcoal and/or polyvinylpyrrolidone 10 were employed in three procedures (B, C and D); for the elimination of polysaccharides, 4M NaCl was applied in the latter two. The highest DNA yield was obtained with Protocol D and averaged 1420.7±398.3 μg DNA/g of dry leaf tissue. Optimal values of the absorbance ratio 260/280 of all DNA solutions revealed the absence or only negligible contamination by proteins. Contamination by polysaccharides inferred from the absorbance ratio 260/230 showed that Protocol C provided the least contaminated material (average of 1.7±0.4). Enzymatic reactions of DNA solutions obtained by Protocol D showed amplification of both loci in all individuals. In conclusion, Protocol D is suitable for the isolation of high quantities of pure DNA from Satureja spp. [Projekat Ministarstva nauke Republike Srbije, br. 173029 i br. 173005]

Experimental studies have shown that both long and short complementary B-form DNA oligomers exhibit liquid crystals phases. We study the isotropic to cholesteric (I–Ch) phase transition of DNA solutions using Landau theory. We define relevant order parameters for this transition. It is shown that the I–Ch transition temperature is depressed and a two phase region is formed due to the presence of the DNA concentration. The concentration and oligomers length dependent transition temperature is calculated and compared with experimental results. The Kerr effect and Cotton–Mouton coefficient are calculated in the isotropic phase above the I–Ch transition.

UVA-activated psoralens are used to treat hyperproliferative skin conditions due to their ability to form DNA photoadducts, which impair cellular processes and may lead to cell death. Although UVA (320-400 nm) is more commonly used clinically, studies have shown that UVB (280-320 nm) activation of psoralen can also be effective. However, there has been no characterization of UVB-induced adduct formation in DNA alone. As psoralen derivatives have a greater extinction coefficient in the UVB region (11 800 cm(-1) M(-1) at 300 nm) compared with the UVA region (2016 cm(-1) M(-1) at 365 nm), a greater extent of adduct formation is expected. SELDI-TOF, a proteomic technique that combines chromatography with mass spectrometry, was used to detect photoadduct formation in an alternating A-T oligonucleotide. 8-Methoxypsoralen (8-MOP) and DNA solutions were irradiated with either UVA or UVB. An adduct peak was obtained with SELDI-TOF. For UVB-activated 8-MOP, the extent of adducts was three times greater than for UVA. HPLC ESI-MS analysis showed that UVB irradiation yielded high levels of 3,4-monoadducts (78% of total adducts). UVA was more effective than UVB at conversion of 4',5'-monoadducts to crosslinks (17% vs 4%, respectively). This report presents a method for comparing DNA binding efficiencies of interstrand crosslink inducing agents.

A three-dimensional (3-D) microfluidic-channel-based electrochemical DNA biosensor was constructed using anodic aluminum oxide (AAO) filtration membranes as the designing templates by taking advantage of their well-defined cylindrical fluidic channels. By electroless gold plating and through thio chemistry, single stranded DNA sequences with 19 base pairs specified to Bacteroides thetaiotaomicron were immobilized into the AAO fluidic channels to form a probe DNA electrode for electrochemical sensing. Cyclic voltammetry (CV) was used to measure the electrochemical response of the sensor using two different redox indicators, [Fe(CN)6]4− and/or [Ru(NH3)6]3− before and after hybridization with the complementary target DNA sequences. The microfluidic-channel-based DNA biosensor showed detection sensitivity amplified to 10−19 M, which is one order of magnitude higher than attomolar (10−18 M) range. Such enhanced detection sensitivity is attributed to the well-defined fluidic channel structure that gives high surface area for probe DNA immobilization and enables target DNA solutions to pass through its fluidic channels for rapid and efficient hybridization.

High level of humus existing in oil-spilled marsh soil could co-extract with DNA to inhibit PCR amplification. We established a stable DNA extracting protocol by modifying direct lysis and activated sludge DNA extracting procedures: with 0.1g/ml PVPP in extracting buffers to pretreat soils and three cycles of freeze-thawing treatment added, by employing lysozyme and 15% SDS, through reducing duration of water bath and digesting RNA with Rnase A before phenol/chloroform extraction. With high concentration of DNA, this protocol provided lower OD230/OD260 value and appropriate OD260/OD280 ratio, which both were significantly different from 0.1g/ml PVPP & 1% CTAB method, indicating pure DNA solutions. Additionally, DNA purity was further checked by amplifying V3-V5 regions of 16S rRNA on bacterial DNA with primer pair 341F/929R.PCR products confirmed that crude oil-spilled marsh soil pretreated by PVPP protocol and extracted by modified extracting procedure could clear humus better and obtain good quality of DNA.

Summary Systematic investigations using small angle neutron scattering (SANS) were made to reveal the effect of calcium ions on the structure of DNA gels and solutions in near-physiological salt solutions. To describe the neutron scattering response two characteristic distances are required. The shorter length scale, R (≈10 A), was found to be governed by the geometry of the DNA chain and is close to the cross-sectional radius of the double helix. The longer length scale L corresponds to the mesh size of the network of overlapping polymer chains. L decreases with increasing DNA concentration as L ∝ cDNA−0.73. With increasing CaCl2 concentration both L and the scattering intensity increase. The increase in the scattering intensity reflects the reduction of the osmotic modulus as the calcium ion concentration increases. The values of the osmotic modulus derived from osmotic swelling pressure measurements are in reasonable agreement with those obtained from SANS.