DNA sequencing with hydrophilic and hydrophobic polymers at elevated column temperatures

Read length in DNA sequencing by capillary electrophoresis at elevated temperatures is shown to be greatly affected by the extent of hydrophobicity of the polymer separation matrix. At column temperatures of up to 80 degrees C, hydrophilic linear polyacrylamide (LPA) provides superior read length and separation speed compared to poly(N,N-dimethylacrylamide) (PDMA) and a 70:30 copolymer of N,N-dimethylacrylamide and N,N-diethylacrylamide (PDEA30). DNA-polymer and polymer intramolecular interactions are presumed to be a major cause of band broadening and the subsequent loss of separation efficiency with the more hydrophobic polymers at higher column temperatures. With LPA, these interactions were reduced, and a read length of 1000 bases at an optimum temperature of 70 degrees -75 degrees C was achieved in less than 59 min. By comparison, PDMA produced a read length of roughly 800 bases at 50 degrees C, which was close to the read length attained in LPA at the same temperature; however, the migration time was approximately 20% longer, mainly because of the higher polymer concentration required. At 60 degrees C, the maximum read length was 850 bases for PDMA, while at higher temperatures, read lengths for this polymer were substantially lower. With the copolymer DEA30, read length was 650 bases at the optimum temperature of 50 degrees C. Molecular masses of these polymers were determined by tandem gel permeation chromatography-multiangle laser light scattering method (GPC-MALLS). The results indicate that for long read, rapid DNA sequencing and analysis, hydrophilic polymers such as LPA provide the best overall performance.     

Improvement of base-calling in multilane automated DNA sequencing by use of electrophoretic calibration standards, data linearization, and trace alignment

We present a new method for the linearization and alignment of data traces generated by multilane automated DNA sequencing instruments. Application of this method to data generated with the Visible Genetics Open Gene DNA sequencing system (using MicroCel 700 gel cassettes, with a 25 cm separation distance) allows read lengths of > 1,000 nucleotides to be routinely obtained with high confidence and > 97% accuracy. This represents an increase of 10-15% in average read length, relative to data from this system that have not been processed in the fashion described herein. Most importantly, the linearization and alignment method allows usable sequence to be obtained from a fraction of 10-15% of data sets which, because of original trace misalignment problems, would otherwise have to be discarded. Our method involves adding electrophoretic calibration standards to the DNA sequencing fragments. The calibration standards are labeled with a dye that differs spectrally from the dye attached to the sequencing fragments. The calibration standards are identical in all the lanes. Analysis of the mobilities of the calibration standards allows correction for both systematic and random variation of electrophoretic properties between gel lanes. We have successfully used this method with two-dye and three-dye DNA sequencing instruments.     

Stimuli-responsive 2D polyelectrolyte photonic crystals for optically encoded pH sensing

A versatile photonic crystal sensing motif based on a two-dimensional (2D) inverse opal monolayer of stimuli-responsive polyelectrolyte gel with tunable optical properties is reported. The photonic membrane shows prompt response to pH and can be readily read out from either its optical spectra or interference colours.     

Enzymatic Detection of Urinary Steroid-17β-Glucuronides after Gel Filtration

Abstract

An enzymatic detection of urinary steroid-17β-glucuronides is described. The principle of the method is as follows; after gel filtration with Sephadex G-25 β-glucuronidase is added to each effluent fraction and incubated for 20 h at 37 $C. After hydrolysis, 3β, 17β-hydroxysteroid dehydrogenase is added and incubated for 20 min at 37 $C. An absorbance at 500 nm is read aginst sample of first fraction effluent.     

An Electrically Driven and Readable Molecular Monolayer Switch Based on a Solid Electrolyte

The potential application of molecular switches as active elements in information storage has been demonstrated through numerous works. Importantly, such switching capabilities have also been reported for self‐assembled monolayers (SAMs). SAMs of electroactive molecules have recently been exploited as electrochemical switches. Typically, the state of these switches could be read out through their optical and/or magnetic response. These output reading processes are difficult to integrate into devices, and furthermore, there is a need to use liquid environments for switching the redox‐active molecular systems. In this work, both of these challenges were overcome by using an ionic gel as the electrolyte medium, which led to an unprecedented solid‐state device based on a single molecular layer. Moreover, electrochemical impedance has been successfully exploited as the output of the system.     

Imaging as a tool for improving length and accuracy of sequence analysis in automated fluorescence-based DNA sequencing

A new method of signal analysis for automated fluorescence-based DNA sequencing is presented. Signal resolution is a limiting factor in obtaining accurate sequence information beyond 400-450 nucleotides per gel lane. We have developed a computer program for the imaging of DNA bands in sequencing gels. The image analysis shows that distortions in the shapes of the bands decrease resolution of peaks observed served in the standard data plots. Reconstruction of the undistorted band shape prior to signal analysis substantially improves the resolution of peaks and may improve the accuracy and length of the contiguous sequence read. Image analysis identified other factors limiting the accuracy and length of automated DNA sequence analysis and provided a tool for evaluating various remedies. Our techniques should also be applicable in other systems, for example, in gel electrophoresis of proteins and DNA restriction fragments, and in scranning densitometry.     

Adaptive weighted least squares method for the estimation of DNA fragment lengths from agarose gels

The size of DNA fragments is most frequently estimated from their electrophoretic mobilities. Agarose gels are used to estimate the size of DNA fragments ranging from a few hundred nucleotides to more than 20 kbp. The common practice when estimating the unknown fragment sizes is to plot the log of the size of molecular weight standards against their mobility and read the values of unknowns from this graph. However, due to perturbations in the gel, such plots often show pronounced curvature which may introduce significant subjectivity into the interpolation process. We present a new method "adaptive weighted least squares (AWLS)" based on the significance test to choose the order of polynomial. We compare this with the method introduced by Schaffer based on the modification of the Southern method. The results obtained by AWLS are significantly better than the method introduced by Schaffer. Different lanes are tested for consistency.     

K and mixed K+O adlayers on Rh(110)

The evolution of the structure of the adlayers and the substrate during adsorption of K and coadsorption of K and O on Rh(110) is studied by scanning tunneling microscopy and low-energy electron diffraction. The K adsorption at temperature above 450 K leads to consecutive (1x4), (1x3), and (1x2) missing-row reconstructions for coverage up to 0.12 ML, which revert back to (1x3) and (1x4) with increasing coverage up to 0.21 ML. The coadsorption of different oxygen amount at T>450 K and eventually following reduction-reoxidation cycles led to a wealth of coadsorbate structures, all involving substrate missing-row-type reconstructions, some including segmentation of Rh rows along the [110] direction. The presence of K stabilizes the (1x2) missing-row reconstruction, which facilitates the formation of a great variety of very open (10x2)-type reconstructions at high oxygen coverage, not observed in the single adsorbate systems.     

A computational study of the reconstruction of amorphous mesoporous materials from gas adsorption isotherms and structure factors via evolutionary optimization

A general method for the three-dimensional reconstruction of mesoporous materials by evolutionary optimization against target data is developed. The method is applied specifically in reconstruction of amorphous material models using gas adsorption data, structure factor data, or a combination of both. A recently introduced lattice-gas approach is used to model adsorption in these calculations, and a high-pass limited Fourier representation is used to facilitate evolution of large-scale structures during the optimization. Reconstructions are made of several material models which mimic real materials obtained either by phase separation and etching or by sol-gel processing. Analysis of the reconstructions provides considerable insight into the type and quantity of structural information probed by gas adsorption and small-angle scattering experiments. We find that reconstructions based only on structure factors tend to underestimate the mean pore size. We also find that in many cases excellent reconstructions can be obtained using only adsorption-branch data, and that in all cases reconstructions based jointly on both types of data are superior to those based only on one, suggesting that these measures contain "complementary" information. It is also found that in most cases the use of desorption data is not warranted, and that the use of adsorption data taken at many temperatures will not improve reconstructions. The reproducibility of the method is shown to be satisfactory. The method can be computationally expensive if gas adsorption data are used, but it is easily parallelized, and therefore results can still be obtained in reasonable time. Finally, the possible application of this approach to real systems, including templated porous materials, is discussed.     

Limits of the plane wave approximation in the measurement of molecular properties

Rescattering electrons offer great potential as probes of molecular properties on ultrafast timescales. The most famous example is molecular tomography, in which high harmonic spectra of oriented molecules are mapped to "tomographic images" of the relevant molecular orbitals. The accuracy of such reconstructions can be greatly affected by the distortion of scattering wave functions from their asymptotic forms due to interactions with the parent ion. We investigate the validity of the commonly used plane wave approximation in molecular tomography, showing how such distortions affect the resulting orbital reconstructions.     

A versatile microfabricated platform for electrophoresis of double- and single-stranded DNA

We demonstrate a versatile microfabricated electrophoresis platform, incorporating arrays of integrated on-chip electrodes, heaters, and temperature sensors. This design allows a range of different sieving gels to be used within the same device to perform separations involving both single- and double-stranded DNA over distances on the order of 1 cm. We use this device to compare linear and cross-linked polyacrylamide, agarose, and thermo-reversible Pluronic-F127 gels on the basis of gel casting ease, reusability, and overall separation performance using a 100 base pair double-stranded DNA ladder as a standard sample. While cross-linked polyacrylamide matrices provide consistently high-quality separations in our system over a wide range of DNA fragment sizes, Pluronic gels also offer compelling advantages in terms of the ability to remove and reload the gel. Agarose gels offer good separation performance, however, additional care must be exercised to ensure consistent gel properties as a consequence of the need for elevated gel loading temperatures. We also demonstrate the use of denaturing cross-linked polyacrylamide gels at concentrations up to 19% to separate single-stranded DNA fragments ranging in size from 18 to 400 bases in length. Primers differing by 4 bases at a read length of 30 bases can be separated with a resolution of 0.9-1.0 in under 20 min. This level of performance is sufficient to conduct a variety of genotyping assays including the rapid detection of single nucleotide polymorphisms (SNPs) in a microfabricated platform. The ability to use a single microelectrophoresis system to satisfy a wide range of separation applications offers molecular biologists an unprecedented level of flexibility in a portable and inexpensive format.     

A theoretical study of the possible use of electroosmotic flow to extend the read length of DNA sequencing by end-labeled free solution electrophoresis

End-labeled free solution electrophoresis (ELFSE) provides a means of separating DNA with free-solution CE, eliminating the need for gels and polymer solutions which increase the run time and can be difficult to load into a capillary. In free-solution electrophoresis, DNA is normally free-draining and all fragments reach the detector at the same time, whereas ELFSE uses an uncharged label molecule attached to each DNA fragment in order to render the electrophoretic mobility size-dependent. With ELFSE, however, the larger molecules are not separated enough (limiting the read length in the case of ssDNA sequencing) while the smaller ones are overseparated; the larger ones are too fast while the shorter ones are too slow, which is the opposite of traditional gel-based methods. In this article, we show how an EOF could be used to overcome these problems and extend the DNA sequencing read length of ELFSE. This counterflow would allow the larger, previously unresolved molecules more time to separate and thereby increase the read length. Through our theoretical investigation, we predict that an EOF mobility of approximately the same magnitude as that of unlabeled DNA would provide the best results for the regime where all molecules move in the same direction. Even better resolution would be possible for smaller values of EOF which allow different directions of migration; however, the migration times then would become too large. The flow would need to be well controlled since the gain in read length decreases as the magnitude of the counterflow increases; an EOF mobility double that of unlabeled DNA would no longer increase the read length, although ELFSE would still benefit from a reduction in migration time.     

Optical and MRI investigations of an optimized acrylamide-based polymer gel dosimeter

The first purpose of this research was improvement of sensitivity of the normoxic acrylamide-based polymer gel dosimeter. Another aim of this study was investigation of the absorbance of the irradiated gels as well as their relaxation rate variations. In addition, a new optical parameter, area under the absorbance spectrum (AUS), was investigated. Sensitivity improvement was performed by adding glucose and urea to the previously reported acrylamide-based polymer gel formulation and new formulation was named PAGATUG. The formulation which gives the nearest tissue elemental composition has been determined to be 3 % bis, 3 % AA, 5 % gelatine, 5 mM THPC, 0.01 mM HQ, 8.5 % glucose, and 3 % urea. The differences in electron density, number of electrons per gram and effective atomic number of PAGATUG gel were no more than 1, 0.5, and 0.8 % of the corresponding values for the soft tissue respectively. PAGATUG gels were irradiated by ⁶⁰Co radiotherapy unit photon beams with different doses and imaged using a 1.5T Siemens Avanto MRI scanner for different post irradiation times. In addition, the absorbance of the irradiated gels were evaluated using a double beam spectrophotometer. We found that the R ₂-sensitivity of polymer gel was improved by a factor of more than 2.6 in respect of the previously reported PAGAT polymer gel. Dose–absorbance sensitivity was obtained as 0.89 Au Gy^?1 and the results showed more stable response in respect of R ₂ investigation. An AUS-sensitivity of 107.7 Au nm Gy^?1 indicated to steep response variation. This read out parameter showed an acceptable linearity and dynamic dose range.     

Optimal hybrid sequencing and assembly: Feasibility conditions for accurate genome reconstruction and cost minimization strategy

Recent advances in high-throughput genome sequencing technologies have enabled the systematic study of various genomes by making whole genome sequencing affordable. Modern sequencers generate a huge number of small sequence fragments called reads, where the read length and the per-base sequencing cost depend on the technology used. To date, many hybrid genome assembly algorithms have been developed that can take reads from multiple read sources to reconstruct the original genome. However, rigorous investigation of the feasibility conditions for complete genome reconstruction and the optimal sequencing strategy for minimizing the sequencing cost has been conspicuously missing. An important aspect of hybrid sequencing and assembly is that the feasibility conditions for genome reconstruction can be satisfied by different combinations of the available read sources, opening up the possibility of optimally combining the sources to minimize the sequencing cost while ensuring accurate genome reconstruction. In this paper, we derive the conditions for whole genome reconstruction from multiple read sources at a given confidence level and also introduce the optimal strategy for combining reads from different sources to minimize the overall sequencing cost. We show that the optimal read set, which simultaneously satisfies the feasibility conditions for genome reconstruction and minimizes the sequencing cost, can be effectively predicted through constrained discrete optimization. Through extensive evaluations based on several genomes and different read sets, we verify the derived feasibility conditions and demonstrate the performance of the proposed optimal hybrid sequencing and assembly strategy.     

Nonvolatile polymer memory device based on bistable electrical switching in a thin film of poly(N-vinylcarbazole) with covalently bonded C60

A functional polymer (PVK-C60), containing carbazole moieties (electron donors) and fullerene moieties (electron-acceptors) in a molar ratio of about 100:1, was synthesized via covalent tethering of C60 to poly(N-vinylcarbazole) (PVK). The molecular structure and composition of PVK-C60 were characterized by FTIR, Raman, and UV-vis absorption spectroscopy, gel permeation chromatography (GPC), X-ray photoelectron spectroscopy (XPS), and cyclic voltammetry (CyV). The C60-modified PVK exhibited an enhanced glass-transition temperature (Tg = 226 degrees C) and good solubility in organic solvents such as toluene, tetrahydrofuran, chloroform, and N,N-dimethylformamide (DMF). It could be cast into transparent films from solutions. For a thin film of PVK-C60 sandwiched between an indium tin oxide (ITO) electrode and an Al electrode (ITO/PVK-C60/Al), the device behaved as nonvolatile flash (rewritable) memory with accessible electronic states that could be written, read, and erased. The polymer memory exhibited an ON/OFF current ratio of more than 105 and write/erase voltages around -2.8 V/+3.0 V. Both the ON and OFF states were stable under a constant voltage stress of -1 V for 12 h and survived up to 108 read cycles at -1 V under ambient conditions.     

GPC,CD and sedimentation analysis of poly-Lys and branched chain poly-Lys-poly-DL-Ala polypeptides

Three different poly-L-lysine (pLys) samples and a branched polypeptide poly-[L-Lys-(DL-Ala)_m] (poly-L-Lys-poly-DL-Ala, AK) were synthetized. Relative molar mass distribution, its average and the degree of polymerisation were determined by sedimentation analysis and gel permeation chromatography (GPC). The conformation of the polymers in solution was studied by circular dichroism (CD) spectroscopy at various pH values and ionic strengths. The data obtained by different methods were compared.A preliminary account of this work was read at the 4^th Conference on Colloid Chemistry, Eger, 1983.     

Bistable electrical switching and memory effects in a thin film of copolymer containing electron donor-acceptor moieties and europium complexes

A nonconjugated methacrylate copolymer (PCzOxEu) containing carbazole moieties (electron donors), 1,3,4-oxadiazole moieties (electron acceptors), and europium complexes in the pendant groups was synthesized via free radical copolymerization of methacrylate monomers containing the respective functional groups. The molecular structure and composition of PCzOxEu was characterized by elemental analysis, FT-IR, 1H NMR, 13C NMR, UV-vis absorption and fluorescence spectroscopies, gel permeation chromatography (GPC), X-ray photoelectron spectroscopy (XPS), and cyclic voltammetry (CyV). The resulting copolymer exhibited a relatively high glass transition temperature (Tg approximately 125 degrees C) and good solubility in common organic solvents. It could be cast into transparent films from solutions. For a thin film of PCzOxEu sandwiched between an indium-tin oxide (ITO) electrode and an Al electrode (ITO/PCzOxEu/Al), the structure behaved as a nonvolatile flash (rewritable) memory with accessible electronic states that could be written, read, and erased. The polymer memory exhibited an ON/OFF current ratio up to 10(5), switching response time of approximately 1.5 micros, more than 10(6) read cycles, retention time of more than 8 h, and write/erase voltages of about 4.4 V/-2.8 V under ambient conditions. The roles of oxadiazole moieties in improving the response time and retention time of the memory device were elucidated from the molecular simulation results.     

Synthesis and dynamic random access memory behavior of a functional polyimide

The device under testing was a plastic dynamic random access memory based on a donor-functionalized polyimide (TP6F-PI), which exhibited the ability to write, read, erase, and refresh the electrical states. The device had an ON/OFF current ratio up to 105, promising minimal misreading error. Both the on and off states were stable under a constant voltage stress of 1 V and survived up to 108 read cycles at 1 V.     

An improvement on the two-dimensional convolution method of image reconstruction and its application to SPECT

In single-photon emission computed tomography (SPECT) and X-ray CT one-dimensional (1-D) convolution method is used for their image reconstruction from projections. The method makes a 1-D convolution filtering on projection data with a 1-D filter in the space domain, and back projects the filtered data for reconstruction. Images can also be reconstructed by first forming the 2-D backprojection images from projections and then convoluting them with a 2-D space-domain filter. This is the reconstruction by the 2-D convolution method, and it has the opposite reconstruction process to the 1-D convolution method. Since the 2-D convolution method is inferior to the 1-D convolution method in speed in reconstruction, it has no practical use. In the actual reconstruction by the 2-D convolution method, convolution is made on a finite plane which is called convolution window. A convolution window of size N X N needs a 2-D discrete filter of the same size. If better reconstructions are achieved with small convolution windows, the reconstruction time for the 2-D convolution method can be reduced. For this purpose, 2-D filters of a simple function form are proposed which can give good reconstructions with small convolution windows. They are here defined on a finite plane, depending on the window size used, although a filter function is usually defined on the infinite plane. They are however set so that they better approximate the property of a 2-D filter function defined on the infinite plane. Filters of size N X N are thus determined. Their value varies with window size. The filters are applied to image reconstructions of SPECT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Near-infrared time-resolved fluorescence lifetime determinations in poly(methylmethacrylate) microchip electrophoresis devices

High aspect-ratio microstructures were hot-embossed in polymer substrates with a molding tool fabricated using lithography/electroplating/forming (LIGA). The resulting devices were used for the electrophoretic separation of oligonucleotides labeled with near-infrared (near-IR) dyes. Near-IR time-resolved fluorescence was used as an identification method for the labeling dyes. The detection apparatus consisted of a pulsed laser diode operating at 680 nm, a single-photon avalanche diode, an integrated microscope, and a PC-board incorporating time-correlated single photon counting electronics. Investigation of the optical quality and amount of autofluorescence generated from different polymer substrates was carried out in the near-IR region for determining compatibility with time-resolved fluorescence. Our results revealed that of several poly(methylmethacrylate)(PMMA) substrates, brand Plexiglas offered minimal replication errors in the embossed features using appropriate embossing conditions with low background fluorescence contributions to the observed decay. Near-IR dye-labeled oligonucleotides were separated to determine the applicability of fluorescence lifetime discrimination between Cy5.5 (tauf = 930 ps) and IRD700 (tauf = 851 ps) labeling dyes during the microchip separation. These dyes were used to label T-fragments (thymine) of an M13mp18 ssDNA template. The DNA ladders were electrophoresed at 130 V/cm in a 4% linear polyacrylamide gel (LPA) gel matrix in a 9.5 cm long serpentine channel heated to 50 degrees C. The electropherogram revealed that the lifetimes could be accurately read well beyond 450 bases, although single-base pair resolution in the electropherogram was difficult to achieve due to potential solute-wall interactions in the polymer microdevice or the electroosmotic flow (EOF) properties of the device. The relative standard deviations secured for individual bands in the electropherogram were similar to those obtained using capillary gel electrophoresis, in spite of the lower load volume.