Orientation Of DNA Molecules Research Articles

Dielectric-Modulated Field Effect Transistors for DNA Detection: Impact of DNA Orientation

In this letter, we present a study that highlights the importance of electrostatic effects on DNA sequence orientation, and the subsequent influence on the functioning principles of nanogap embedded dielectric-modulated field effect transistor (DMFET) biosensors. We find that the orientation of DNA molecules is responsible for governing the sensitivity and dominance of charge or dielectric constant effect in nanogap embedded FET devices. Using 2-D TCAD simulations, the study not only provides a correct explanation for the observed trend of threshold voltage shifts for DNA detection using n-channel DMFET, which has been loosely attributed to the dominance of the charge effect, but also gives insight into the reason of higher sensitivity of p-channel over n-channel DMFET biosensors.

Polarization-Sensitive Two-Photon Microscopy Study of the Organization of Liquid-Crystalline DNA

Highly concentrated DNA solutions exhibit self-ordering properties such as the generation of liquid-crystalline phases. Such organized domains may play an important role in the global chromatin topology but can also be used as a simple model for the study of more complex 3D DNA structures. In this work, using polarized two-photon fluorescence microscopy, we report on the orientation of DNA molecules in liquid-crystalline phases. For this purpose, we analyze the signal emitted by fluorophores that are noncovalently bound to DNA strands. In nonlinear processes, excitation occurs exclusively in the focal volume, which offers advantages such as the reduction of photobleaching of out-of-focus molecules and intrinsic 3D sectioning capability. Propidium iodide and Hoechst, two fluorophores with different DNA binding modes, have been considered. Polarimetric measurements show that the dyes follow the alignment with respect to the DNA strands and allow the determination of the angles between the emission dipoles and the longitudinal axis of the DNA double strand. These results provide a useful starting point toward the application of two-photon polarimetry techniques to determine the local orientation of condensed DNA in physiological conditions.

Direct observation of long-strand DNA conformational changing in microchannel flow and microfluidic hybridization assay

Conformational control of macromolecules is useful for efficient chemical and biochemical reactions. This article reports a direct observation method for macromolecules, such as long-strand DNA, in microchannel flow as well as a simple method for stretching DNA strands by microfluidics. Stretching and orientation of DNA molecules by control of flow within a microchannel was observed by optical microscopy. This DNA stretching is explained by coil–stretch transition of polymer molecules. This technique is useful for creating chemical reactions with macromolecules. It offers high selectivity and efficiency that are impossible to achieve in bulk solution. We also demonstrate that our microfluidic stretching method can accomplish efficient hybridization of long-strand DNA. This method will be useful for direct hybridization assay of long-strand DNA.

Dielectrophoretic manipulation of surface-bound DNA

Dielectrophoretic manipulation enables the positioning and orientation of DNA molecules for nanometer-scale applications. However, the dependence of the dielectrophoretic force and torque on the electric field magnitude and frequency has to be well characterised to realise fully the potential of this technique. DNA in solution is attracted to the strongest electric field gradient (i.e. the electrode edge) as a result of the dielectrophoretic force, while the dielectrophoretic torque aligns the DNA with its longest axis parallel to the electric field. In this work, the authors attached -DNA fragments (48 and 25 kilobases) to an array of gold microelectrodes via a terminal thiol bond and characterised the orientation and elongation as a function of electric field magnitude (0.1-0.8 MVm) and frequency (0.08-1.1 MHz). Maximum elongation was observed between 200 and 500 kHz for the attached DNA. Dielectrophoresis is limited by thermal randomisation at electric fields below 0.1 MVm and by electrothermal effects above 0.7 MVm. The authors conclude that dielectrophoresis can be used to manipulate surface-immobilised DNA reproducibly.

Anomalous radial migration of single DNA molecules in capillary electrophoresis.

We report the unexpected radial migration of DNA molecules in capillary electrophoresis (CE) with applied Poiseuille flow. Such movement can contribute to anomalous migration times, peak dispersion, and size and shape selectivity in CE. When Poiseuille flow is applied from the cathode to the anode, DNA molecules move toward the center of the capillary, forming a narrow, highly concentrated zone. Conversely, when the flow is applied from the anode to the cathode, DNA molecules move toward the walls, leaving a DNA-depleted zone around the axis. We showed that the deformation and orientation of DNA molecules under Poiseuille flow was responsible for the radial migration. By analyzing the forces acting on the deformed and oriented DNA molecules, we derived an expression for the radial lift force, which explained our results very well under different conditions with Poiseuille flow only, electrophoresis only, and the combination of Poiseuille flow and electrophoresis. Factors governing the direction and velocity of radial migration were elucidated. Potential applications of this phenomenon include an alternative to sheath flow in flow cytometry, improving precision and reliability of single-molecule detection, reduction of wall adsorption, and size separation with a mechanism akin to field-flow fractionation. On the negative side, nonuniform electroosmotic flow along the capillary or microfluidic channel is common in CE, and radial migration of certain analytes cannot be neglected.

Patterns of DNA orientation in Insect sperm: evolutionary convergence

SUMMARYForty nine insect species, belonging to nine orders, and six non-insect species were studied by means of polarized fluorescence microscopy in order to estimate the diversity of the DNA package fashions. It is possible to distinguish five main groups of DNA orientation patterns in relation to the sperm head axis: nearly isotropic, low perpendicular, low parallel, moderate parallel, high parallel. One and the same order includes, as a rule, two or more groups of sperm DNA orientation. All the groups, except for the last, are heterogeneous. The last type of DNA package (group V), with the orientation of DNA molecules nearly parallel to the sperm head axis, represents so far unexplainable evolutionary convergence. It occurs in Aeshnidae (Odonata), Acrididae and Gryllidae (Orthoptera), Aphrophoridae (Hemiptera) and Tenthredinidae (Hymenoptera) insects, whereas their close relatives have quite different DNA orientation patterns. In general, approximately from the level of a family and higher, it is diffi...

A birefringence, electron microscopy, and histochemical survey of chromosomal structure in the sperm nuclei of an echinothurid sea urchin, Araeosoma owstoni

The mature sperm head of Araeosoma owstoni, an echinothurid sea urchin, showed positive birefringence reflecting that the overall orientation of DNA molecules was semiperpendicular toward the nuclear axis of the sperm head. Transmission electron microscopical observation of sperm in this species revealed a highly electron-dense cylindrical coil with an empty central core extending along the major axis of the sperm head. This coil had seven to eight turns along its entire length of 3.5 μm. The maximum width was 0.35 μm near the distal end of the nucleus, and the minimum width was 0.17 μm near the apical end. Lamellar substructures were also present in the sperm nucleus, appearing at the periphery of the electron-dense cylinder in a radial manner. Staining with Feulgen's reaction and acid-orcein indicated that the coil was probably composed of sperm chromosomes.

DNA orientation using specific avidin-ferritin biotin end labelling.

The orientation of DNA molecules has been determined by labelling one of the molecule end with a Biotin-labelled analog of dTTP (Bio-dUTP) and then by complexing the Bio-dUTP with Avidin-Ferritin. DNAs of phi X174, pBR322 and SV40 were end labelled with Bio-dUTP and imaged by Electron Microscopy (EM). This is a rapid, general method to unambiguously determine the orientation of DNA molecules for precise mapping and quantification of DNA secondary structures or protein-DNA interaction sites using EM.

Orientation of DNA molecules in agarose gels by pulsed electric fields.

The electric birefringence of DNA restriction fragments of three different sizes, 622, 1426, and 2936 base pairs, imbedded in agarose gels of different concentrations, was measured. The birefringence relaxation times observed in the gels are equal to the values observed in free solution, if the median pore diameter of the gel is larger than the effective hydrodynamic length of the DNA molecule in solution. However, if the median pore diameter is smaller than the apparent hydrodynamic length, the birefringence relaxation times increase markedly, becoming equal to the values expected for the birefringence relaxation of fully stretched DNA molecules. This apparent elongation indicates that end-on migration, or reptation is a likely mechanism for the electrophoresis of large DNA molecules in agarose gels. The relaxation times of the stretched DNA molecules scale with molecular weight (or contour length) as N2.8, in reasonable agreement with reptation theories.

Dielectric relaxation and orientation of DNA molecules.

AbstractA conductivity dispersion has been measured at very low frequencies (VLF) on several concentrated DNA solutions. By measuring simultaneously their electric birefringence decay, it is shown that the dielectric relaxation (which is related to the conductivity dispersion) is due to the molecular orientation. Different polarization mechanisms are discussed. It is concluded that the DNA polarizability measured in the VLF range can only be explained by the orientation of a permanent ionic dipole. It is suggested that such permanent dipoles could be caused by small differences in the ionic composition between the two molecular “ends;” the difference could either be stable (asymmetrical localization of protein impurities for instance) or transient (fluctuating dipoles explained by the Kirkwood‐Schumaker theory).