High-efficiency DNA separation by capillary electrophoresis in a polymer solution with ultralow viscosity

The viscosities of some polymer solutions for DNA separation in capillary electrophoresis are generally very high, which makes them hard to pump into the capillaries. We have developed a novel sieving buffer, based on low-molecular-weight hydroxypropylmethylcellulose, to separate DNA fragments. The viscosity of this sieving matrix was at least 1 order of magnitude lower than that of traditional buffers with similar sieving effect. The influence of additives such as urea and mannitol was investigated. It was found that the double-stranded DNA (ds DNA) fragments began to denature in 3.5 M urea, and 7 M urea can denature the ds DNA completely. The presence of mannitol will decrease the overlap threshold of the polymer solution (the concentration at which the polymer molecules begin to entangle with each other), which makes it possible to separate DNA fragments in a polymer solution of relatively low concentration. The influence of the electrical field was also investigated, and it was found that the mobility of DNA fragments up to 2000 bp in length did not change greatly with different electric fields. This phenomenon implies that the DNA fragments at this range do not change their conformation with the increase of electric field as was previously believed. The possible mechanism for the separation of DNA fragments is also discussed.     

Capillary electrophoresis of supercoiled DNA molecules: parameters governing the resolution of topoisomers and their separation from open forms

We describe the separation of covalently closed and open circular DNA forms with capillary electrophoresis. This technique is expected to be applied in the research of novel anticancer molecules targeting the activity of topoisomerase I. The separation of a plasmid mixture containing fully supercoiled molecules, single topoisomers, and their relaxed and open circular forms was tested in an electric field of 200 V/cm using Tris/borate buffer with the addition of magnesium ions at low concentrations and various sieving polymers. The resulting separation is quite simple to achieve and is clearly comparable to that obtained in agarose gels run at low voltage, but with an improved resolution, a higher quantitativity, and a higher speed of analysis. We identified three main parameters that influence the separation: (I) Low concentrations of MgCl2 in the separation buffer are required for a good resolution of topoisomers. (II) Cellulose derivatives can be used as sieving polymers; in our hands, HPMC and HEC worked best. (III) High molecular mass forms of sieving polymers allow the best separations.     

DNA capillary electrophoresis in entangled dynamic polymers of surfactant molecules

Aqueous solutions of monomeric nonionic surfactants, n-alkyl polyoxyethylene ethers (C16E6, C16E8, C14E6), can be used as sieving matrixes for the separation of DNA fragments by capillary electrophoresis. Unlike ordinary polymer solutions, these surfactant solutions behave as dynamic polymers. By combining the "reversible gel" theory of DNA electrophoresis and the static and dynamic properties of wormlike surfactant micelles, a model is developed for describing the migration behavior of DNA molecules in these solutions. According to the model, the separation limit can be extended at low surfactant concentrations. Surfactant solutions as a separation medium provide many advantages over ordinary polymers, such as ease of preparation, solution homogeneity, stable structure, low viscosity, and self-coating property for reducing electroosmotic flow. More importantly, the properties of wormlike micelles (micelle size, entanglement concentration) can be adjusted by simply changing the monomer concentration, denaturant, and temperature to allow the separation of different size ranges of DNA fragments. Fast separation is achieved for DNA fragments ranging from 10 bp to 5 kb by using bare fused-silica columns. DNA sequencing fragments of BigDye G-labeled M13 up to 600 bases were separated within 60 min.     

Evaluation of the Film-Coating Properties of a Hydroxyethyl Cellulose/Hydroxypropyl Methylcellulose Polymer System

ABSTRACT

The effect of different grades of hydroxyethyl cellulose (HEC) and hydroxypropyl methylcellulose (HPMC) on the film-formation and taste-masking ability for ibuprofen granules was evaluated. Three batches of coated ibuprofen granules were prepared using a roto-granulator, each with a different coating composition. Two grades of HEC [MW 300,000 (H) and MW 90,000 (L)] were combined with three different grades of HPMC [MW 11,000 (L), MW 25,000 (M) and MW 35,000 (H)] to prepare the coating solutions. Mechanical strength and physical properties of the polymer films were evaluated. Films made from HPMC (L)/HEC (H), HPMC (M)/HEC (H), and HPMC (H)/HEC (H) were stronger and more flexible than the HPMC (L)/HEC (L) films. The assay, dissolution, particle size distribution, and environmental scanning electron microscopy (ESEM) data of the three batches of the coated ibuprofen granules were similar. These data indicated that the two grades of HEC had equivalent film-coating properties. However, the HPMC (L)/HEC (L) film-coated granules showed better taste-masking characteristics (no burning after-taste) than the HPMC (L)/HEC (H) and HPMC (M)/HEC (H) film-coated granules. The ESEM data of the polymer films indicated that both HPMC (L)/HEC (H) and HPMC (M)/HEC (H) films exhibited more roughness and contained larger particles than the HPMC (L)/HEC (L) films. A hydration/dehydration study of the films revealed that HPMC (L)/HEC (H) and HPMC (M)/HEC (H) films were more susceptible to moisture effects, which subsequently led to a faster hydration rate of the polymer films. These data suggest that the molecular weight of the HEC affects the taste-masking ability of the resultant polymer film. The HEC (L) mixed well with the HPMC (L) to yield a uniform film that was more resistant to moisture effects. Hence, for optimum coating applications, particular attention should be paid to the molecular weight of the coating polymers to ensure that they are comparable to each other.     

Evaluation of the film-coating properties of a hydroxyethyl cellulose/hydroxypropyl methylcellulose polymer system

The effect of different grades of hydroxyethyl cellulose (HEC) and hydroxypropyl methylcellulose (HPMC) on the film-formation and taste-masking ability for ibuprofen granules was evaluated. Three batches of coated ibuprofen granules were prepared using a roto-granulator, each with a different coating composition. Two grades of HEC [MW 300,000 (H) and MW 90,000 (L)] were combined with three different grades of HPMC [MW 11,000 (L), MW 25,000 (M) and MW 35,000 (H)] to prepare the coating solutions. Mechanical strength and physical properties of the polymer films were evaluated. Films made from HPMC (L)/HEC (H), HPMC (M)/HEC (H), and HPMC (H)/HEC (H) were stronger and more flexible than the HPMC (L)/HEC (L) films. The assay, dissolution, particle size distribution, and environmental scanning electron microscopy (ESEM) data of the three batches of the coated ibuprofen granules were similar. These data indicated that the two grades of HEC had equivalent film-coating properties. However, the HPMC (L)/HEC (L) film-coated granules showed better taste-masking characteristics (no burning after-taste) than the HPMC (L)/HEC (H) and HPMC (M)/HEC (H) film-coated granules. The ESEM data of the polymer films indicated that both HPMC (L)/HEC (H) and HPMC (M)/HEC (H) films exhibited more roughness and contained larger particles than the HPMC (L)/HEC (L) films. A hydration/dehydration study of the films revealed that HPMC (L)/HEC (H) and HPMC (M)/HEC (H) films were more susceptible to moisture effects, which subsequently led to a faster hydration rate of the polymer films. These data suggest that the molecular weight of the HEC affects the taste-masking ability of the resultant polymer film. The HEC (L) mixed well with the HPMC (L) to yield a uniform film that was more resistant to moisture effects. Hence, for optimum coating applications, particular attention should be paid to the molecular weight of the coating polymers to ensure that they are comparable to each other.     

Separation of double- and single-stranded DNA restriction fragments: capillary electrophoresis with polymer solutions under alkaline conditions.

Capillary electrophoresis in buffers containing hydroxyethyl cellulose (HEC) was used to separate double- and single-stranded DNA restriction fragments under neutral and alkaline conditions in epoxy-coated capillaries. It was found that better resolution was achieved using highly entangled HEC solutions for a narrow range of DNA fragment sizes, while lower resolution was obtained over a wide separation range using diluted HEC solutions. Optimal resolution of these DNA fragments was obtained using buffers containing 0.5% HEC at pH 11 with plate numbers exceeding 3 x 10(6) plates/m. It was also found that the diffusion coefficients and electrophoretic mobilities of DNA fragments decreased with increasing pH. This may indicate a more extended DNA conformation and, therefore, enhancement of transient entanglement coupling between DNA and HEC polymers under alkaline condition. At pH 12, ss-DNA were well separated in entangled HEC solutions; however, the resolution of ss-DNA was significantly decreased in diluted polymer solution.     

Effective capillary electrophoresis-based heteroduplex analysis through optimization of surface coating and polymer networks

The efficacy of capillary electrophoresis for detecting DNA mutations via heteroduplex analysis (HDA) is dependent upon both the effective passivition of the capillary surface and the choice of the correct polymer network for sieving. Using HDA with laser-induced fluorescence detection of fluorescently labeled DNA fragments, an effective coating and optimal polymer matrix were sought. Optimized separation conditions were determined through the methodological evaluation of a number of different silanizing reagents, polymeric coatings, and polymer networks for resolving the PCR-amplified DNA fragments associated with five mutations (185delAG, 1294del40, 4446C > G, 5382insC, 5677insA) in the breast cancer susceptibility gene (BRCA1). For capillary coating, allyldimethylchlorosilane, 4-chlorobutyldimethylchlorosilane, (gamma-methacryloxypropyl)trimethoxysilane, chlorodimethyloctylsilane (OCT), and 7-octenyltrimethoxysilane were evaluated as silanizing reagents in combination with poly(vinylprrolidone) (PVP) and polyacrylamide (PA) as the polymeric coat. The HDA results were compared with those obtained using a commercial (FC) coated capillary. Of these, the OCT-PVP combination was found to be most effective. Using this modified capillary, HDA with polymer networks that included hydroxyethylcellulose (HEC), linear polyacrylamide, and PVP showed that a PVP-, PA-, or FC-coated capillary, in combination with HEC as the sieving polymer, could be used effectively to discriminate the mutations in less than 10 min. However, optimal performance was observed with the OCT-PVP-coated capillary and HEC as the polymer network.     

黏度法研究非离子型高分子对盐水溶液中油酸钾聚集行为的影响

测定了在不同浓度聚丙二醇(PPG)、聚乙二醇(PEG)和聚乙烯基吡咯烷酮(PVP)存在条件下油酸钾盐水溶液的黏度,研究了非离子型高分子对盐水溶液中油酸钾表面活性剂聚集行为的影响,结果表明,随着非离子型高分子的加入,油酸钾盐水溶液的黏度迅速下降,但是程度显著不同,不仅如此,加入更加疏水的聚丙二醇以后,油酸钾盐水溶液黏度下降的程度最低。由此可见,非离子型高分子疏水程度不是导致表面活性剂盐水溶液黏度下降的唯一因素,高分子与表面活性剂疏水和亲水基团之间的相互作用,以及由此形成的高分子/表面活性剂聚集体的形态,是决定表面活性剂盐水溶液黏度下降最主要的原因。     

In-situ sampling and separation of RNA from individual mammalian cells

In this investigation RNA was directly sampled and separated at the single-cell level (without extraction) by capillary electrophoresis (CE). Laser-induced fluorescence (LIF) was employed to detect ethidium bromide-labeled RNA molecules under native conditions. Hydroxypropylmethylcellulose was used as a matrix for molecular sieving. Additives to the polymer solution included poly(vinylpyrrolidone) to eliminate the electroosmotic flow and mannitol to enhance the separation. Peak identities were confirmed as RNA by enzymatic treatment with RNase I. The individual Chinese Hamster Ovary (CHO-K1) cells were injected into a capillary and the cells were lysed online with sodium dodecyl sulfate (SDS) solutions before running electrophoresis. Low molecular mass (LMM) RNAs as well as larger fragments (tentatively identified as 18S and 28S ribosomal RNA by comparison with the literature) were detected with this system, which corresponds to a detected amount of approximately equals 10-20 pg of RNA/cell. A Proteinase K study showed that proteins incorporated with RNA molecules were eliminated by SDS treatment and thus did not influence the migration of RNA. Experiments were also performed with this technique to detect nucleic acid damage. Changes in the peak pattern were detected in the cells treated with hydrogen peroxide, which meant that strand breaks occurred in DNA and RNA. It was found that 60 mM caused the most severe damage to the nucleic acids.     

Sample stacking during membrane-mediated loading in automated DNA sequencing.

Microporous membrane-mediated loading is a novel and efficient sample injection technique for ultrathin slab gel electrophoresis-based automated DNA sequence analysis. The sequencing reaction mixture is spotted directly onto the tabs of the membrane loader, which is then inserted to close proximity of the straight edge of the separation gel. The use of a higher viscosity (> 60 cSt), low ionic strength (conductivity < 320 microS) well solution with a pH of more than one unit lower that of the separation and gel buffer system makes possible efficient stacking of the DNA sequencing fragments at the interface of the gel matrix. When the injection/separation process is initiated by the application of the electric field, a high local electric potential drop is formed across the low ionic strength well solution zone. This results in very fast migration of the DNA sequencing fragments toward the interface of the separation gel, where they stack up against the higher conductivity sieving matrix. During this stacking process, primarily the local pH mediates the actual mobility of the buffer co-ions (borate), forming the leading and terminating zones in the well solution and separation gel, respectively.     

Stochastic single-molecule videomicroscopy methods to measure electrophoretic DNA migration modalities in polymer solutions above and below entanglement

We have studied the effects of polymer molar mass and concentration on the electrophoretic migration modalities of individual molecules of DNA in LPA, HEC, and PEO solutions via epifluorescent videomicroscopy. While both transient entanglement coupling (TEC) and reptation have been studied in the past, the transition between them has not. Understanding this transition will allow for polymer network properties to be optimized to enhance the speed and resolution of DNA separations in microfluidic devices. Near the overlap threshold concentration, C*, TEC is the dominant observed mode of DNA migration, and the observation frequency of TEC increases with increasing polymer molar mass. As polymer concentration is increased, observed TEC events reduce to zero while DNA reptation events become the only detected mechanism. Individual DNA molecules undergoing both migration mechanisms were counted in solutions of varying polymer molar masses and concentrations and were plotted against a dimensionless polymer concentration, C/C*. The data for LPA reduce to form universal curves with a sharp increase in DNA reptation at approximately 6.5C*. Analogous transition concentrations for PEO and HEC were observed at 5C* and 3.5C*, respectively, reflecting the different physical properties of these polymers. This transition correlates closely with the polymer network entanglement concentration, Ce, as measured by rheological techniques. The electrophoretic mobility of lambda-DNA in LPA polymer solutions was also measured and shows how a balance can be struck between DNA resolution and separation speed by choosing the desired prevalence of DNA reptation.     

Viscosity properties of sodium borophosphate glasses

The viscosity behavior of (1 − x)NaPO₃−xNa₂B₄O₇ glasses (x = 0.05-0.20) have been measured as a function of temperature using beam-bending and parallel-plate viscometry. The viscosity was found to shift to higher temperatures with increasing sodium borate content. The kinetic fragility parameter, m, estimated from the viscosity curve, decreases from 52 to 33 when x increases from 0.05 to 0.20 indicating that the glass network transforms from fragile to strong with the addition of Na₂B₄O₇. The decrease in fragility with increasing x is due to the progressive depolymerization of the phosphate network by the preferred four-coordinated boron atoms present in the low alkali borate glasses. As confirmed by Raman spectroscopy increasing alkali borate leads to enhanced B-O-P linkages realized with the accompanying transition from solely four-coordinated boron (in BO₄ units) to mixed BO₄/BO₃ structures. The glass viscosity characteristics of the investigated glasses were compared to those of P-SF67 and N-FK5 commercial glasses from SCHOTT. We showed that the dependence of the viscosity of P-SF67 was similar to the investigated glasses due to similar phosphate network organization confirmed by Raman spectroscopy, whereas N-FK5 exhibited a very different viscosity curve and fragility parameter due to its highly coordinated silicate network.     

Study of nickel passivation in a borate medium

The adsorption of borate ions at the nickel and/or nickel oxide-electrolyte electrochemical interface was studied at various concentrations and pH values in lithium and borate solutions. First, the passivation range of nickel was estimated using cyclic voltammetry. The nickel passive layer formation kinetics (transfer resistance, capacitance of passive film formed, adsorption capacitance), as well as the semiconducting properties of this oxide layer, were studied using electrochemical impedance spectroscopy (E.I.S.). These electrochemical techniques were used in conjunction with adsorption measurements performed with an electrochemical quartz crystal microbalance (E.Q.C.M.) and with surface analyses (Auger spectroscopy). The nickel oxide showed type p semiconducting properties and was depleted at, corrosion potential. Moreover, very little borate adsorption was observed during the different tests. This may have been the result of the negative surface charge, in the pH and potential conditions applied.     

Joint processing of the parallel and crossed polarized Raman spectra and uniqueness in blind nonnegative source separation

Polarization analysis in Raman spectroscopy provides a powerful tool in chemical physics to identify the symmetry of the vibration modes of molecules, macromolecules and crystals, according to their crystallographic structure and local spatial orientation. In this paper we propose two new approaches in polarized Raman spectroscopy for data with rotational and spatial diversity, respectively. We show that the joint use of parallel and crossed polarization data yields more accurate source separation results and improves the uniqueness properties of the solution in blind nonnegative source separation algorithms. The proposed approaches are validated on two real polarized Raman data sets.     

Characteristics of di- and tri-block copolymers: polymer disperse liquid crystal display

In the reported work the block copolymers are used in the polymer disperse liquid crystal (PDLC) films. The present work has been performed to investigate the effect of block copolymer addition and block ratios on the PDLC characteristics. From our experimental finding, addition of block copolymer in PDLC shows variation in droplet size, electro-optical properties, extent of phase separation, and phase transition temperature. These finding indicate the alteration in solubility parameters of solutions with the addition of block copolymers. Moreover, the tri-block copolymer shows enlarge droplet size, enhancement in the degree of phase separation, and predict improvement in electro-optical properties, as compared to di-block copolymer. Similarly upon such comparison, the study suggests the tri-block copolymer have a relative lower molecular interaction with the liquid crystal molecules.     

Redox polymer and probe DNA tethered to gold electrodes for enzyme-amplified amperometric detection of DNA hybridization

The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+/2+), and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 x 10(-9) to 2 x 10(-6) M. The detection limit is approximately 1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach.     

温敏性聚合物P(AM-Macromer)及其与表面活性剂的复合性能

以含氧乙基结构的烯类单体(Macromer)和丙烯酰胺(AM)为原料,制备了具有温度敏感性的疏水缔合水溶性共聚物P(AM-Macromer).研究了P(AM-Macromer)溶液与不同表面活性剂的相互作用.结果表明,P(AM-Macromer)在水溶液中与表面活性剂存在明显相互作用,复合效应与聚合物浓度、表面活性剂结构及浓度有关.在较高浓度聚合物溶液中,少量表面活性剂的加入能显著提高溶液的黏度;在低浓度聚合物溶液中,表面活性剂的加入使溶液表观黏度下降.表面活性剂对共聚物P(AM-Macromer)溶液黏度的影响强弱顺序为:SDBSCTABOP-10.     

Synthesis of polymeric titanium and zirconium precursors and preparation of carbide fibres and films

Polymeric precursors for carbothermal reactions were prepared from the chelate derivatives of titanium and zirconium alkoxides L2M(OR)2 (L is an acetylacetonato or ethyl acetoacetato group) in alcohols by reaction with organic compounds having two or more reactive OH groups, such as ethylene glycol, saccharose, tartaric acid or dihydroxybenzenes. These organic groups act as bridging ligands in transesterification and condensation polymerization yielding either spinnable viscous solutions or elastic gels. The rheological properties of the concentrated solutions allowed for the preparation of polymer fibres and films. At temperatures up to 1600 °C, bulk precursors as well as fibres and films were thermally converted into carbide powders, films or coatings. The structural transformations of the polymeric materials into the carbides were investigated using thermogravimetric–differential thermal analyses (TGA–DTA), X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infra-red (FTIR) analysis and Raman spectroscopy.     

Foaming binary solution mixtures of low molecular surfactant and polyelectrolyte

The lifetime of water solution foams of sodium dodecylsulfate (DDS, low molecular weight surfactant) and sodium carboxymethylcellulose (SCMC, polyelectrolyte) and their binary mixtures was experimentally investigated. The effects of ionic strength and acidity on the foam life were also determined. In binary solutions, a synergic effect of DDS and SCMC on the surface tension reduction, most likely resulting from the interaction of the surfactant with polymer, was found. The addition of NaCl into solution or increasing the ionic strength was found to decrease the surface tension and reduce interfacial mobility, hence increased foam lifetime. The relatively low lifetime of binary solution foams in acidic medium was attributed to the reaction between SCMC and acid, which resulted in relatively small reductions in the viscosity and consequently lowered the solution viscosity.     

基于NIPAM和HEMA的温敏性共聚物水溶液性能研究

以N-异丙基丙烯酰胺(NIPAM)、甲基丙烯酸羟乙酯(HEMA)、丙烯酰胺(AM)、丙烯酸(AA)为原料,采用水溶液自由基聚合法制备了N-异丙基丙烯酰胺基温敏水溶性共聚物P(NIPAM-HEMA-AM)和P(NIPAM-HEMA-AM-NaAA)。研究了无机盐及聚合物结构对共聚物低临界溶解温度(LCST)的影响,考察了共聚物浓度、盐浓度、表面活性剂浓度对共聚物溶液流变性能特别是粘温特性的影响。结果显示,随着盐浓度的增加,共聚物溶液的LCST呈下降趋势。共聚物浓度较高时,在LCST附近表现出显著的升温增稠性。在P(NIPAM-HEMA-AM-NaAA)溶液中加入NaCl后溶液黏度增加,升温增稠效应明显。