Mannitol influence on the separation of DNA fragments by capillary electrophoresis in entangled polymer solutions

A new kind of sieving matrix is presented in this paper to allow satisfactory separation of DNA fragments in a relatively low viscous solution. When a certain amount of mannitol was added to cellulose solution not concentrated enough to separate PGEM-3Zf(+)/HaeIII standards well, a polymer solution with low viscosity but with very good separation effects was obtained. The separation result of this sieving buffer was comparable with those using highly concentrated cellulose solutions. The sieving ability of solutions with different cellulose concentrations and different amounts of mannitol has been investigated. It was proved that 0.5% was the minimum hydroxypropylmethylcellulose (HPMC) concentration that could be used to separate DNA fragments satisfactorily. HPMC solutions with a concentration of less than 0.5% could not separate the standard DNA fragments even in the presence of mannitol. It was found that 6% was the optimized mannitol concentration because either more or less mannitol will lead a decrease of resolution. The principle of the positive influence of mannitol has also been discussed.     

基于微芯片电泳的脱氧核糖核酸片段的浓缩和分离

采用超负荷电动供给(electrokinetic supercharging, EKS)预浓缩技术,在微芯片电泳(MCE)上对脱氧核糖核酸（DNA）片段进行浓缩和分离。EKS是集合样品电动进样(EKI)和过渡等速电泳(tITP)的一种在线浓缩方法。研究表明：采用该方法后,在40.5 mm长的单通道芯片上能够实现对低浓度样品的大量进样、浓缩和基线分离。在普通的紫外检测条件(检测波长为260 nm)下,对DNA片段的平均检出限(S/N=3)约为0.07 mg/L,仅为十字芯片上的微芯片电泳检出限的1/40。本文还对浓缩过程中的一些关键因素和定性分析进行了探讨。     

Microelectrophoresis for the separation of DNA fragments.

A methodology has been developed which significantly reduces the linear dimension necessary for the electrophoretic separation of DNA fragments and oligonucleotides. DNA fragments are rapidly separated into compact, resolvable microscopic banding patterns which can be detected using a high-resolution electronic imaging system. Separations can be carried out in either capillary tube or thin-layer (slab) microgel formats of one centimeter or less in length. The complete separation of all eleven fragments (1353 to 72 base pairs) of the pi X174 DNA/HaeIII restriction ladder was achieved in a total running distance of less than 2 mm and in less than 2 min. The observed band widths for the larger fragments (1353-603 bp) ranged from 18 to 25 microns, with the intermediate and smaller fragments (310 to 72 bp) ranging from 30 microns to 60 microns. The ethidium bromide-stained microgels were analyzed using an epifluorescent microscope combined with an intensified charged coupled device imaging system. In other experiments, single-base resolution of fluoresceinated oligonucleotides in the 20-30 nucleotide range was demonstrated. DNA sequencing may be possible with further optimization. This new methodology departs from the conventional gel formulations and electrophoretic procedures used for the separation DNA fragments. High voltage gradients and the use of highly concentrated and crosslinked homogeneous polyacrylamide gels effects the rapid separation of DNA fragments in very short distances. Analysis of the microgels with proteins of known size (Stokes radius) indicates that separations are occurring in gels with pore sizes close to the diameter of double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inorganic monoliths in separation science: A review

The practical application of rigid, macro-porous organic polymer and silica based monolithic stationary phases as separation media has been described in the literature since 1992 and 1996, respectively. Today these materials are extensively used in chromatography and electrochromatography and several detailed reviews appear annually describing these materials, their synthesis and application. To compliment these publications, this review focuses upon the less commonly utilised materials for monolith synthesis, both those that have already been applied within separation science, and those that have found applications elsewhere, such as catalysis and water filtration, but have the clear potential to be explored as novel stationary phases in the near future. For the purpose of the review monoliths formed from these various alternative materials will be termed ‘Exotic Monoliths’, as these new substrates in many cases have only just begun to be explored for chromatographic separations, and in many instances have unusual and highly selective surface chemistries, which are attractive in terms of broadening the choice of monolithic materials for separation science. An extensive range of monolithic materials based on the following elements and their compounds (mostly oxides) are covered: Zr, Ti, Al, Hf, C, Au, Ag, Ce, Ge and hydroxyapatite, together with their relevant properties, methods of synthesis, and current and potential applications in separation science.     

Micropreparative separation of peptides derived from sodium dodecyl sulphate-solubilized proteins

A systematic investigation of the influence of the detergent sodium dodecyl sulphate (SDS) on micropreparative peptide separations on microbore reversed-phase high-performance liquid chromatographic columns is reported. A tryptic digest of bovine serum albumin and a mixture of synthetic peptides were used to monitor the separation behaviour of a 1.6 mm I.D. Nucleosil C18 column in the presence of various amounts of SDS. The data demonstrate that even traces of SDS in the sample reduce the separation efficiency and peptide recovery. An extraction method is presented which reduces the SDS content in peptide mixtures below the critical concentration without affecting significantly the recovery of individual peptides. After acidification of the sample, the detergent is extracted into heptane-isoamyl alcohol (4:1, v/v). In combination with chemical or enzymatic fragmentation techniques, this extraction method facilitates the sequence analysis of minute amounts of SDS-solubilized hydrophobic proteins. The applicability of the method is demonstrated on the example of the integral membrane protein bacteriorhodopsin.     

Micropreparative separation of transfer ribonucleic acids by high-performance liquid chromatography

A method was developed for the micropreparative separation of individual species of tRNA using reversed-phase high-performance liquid chromatography on large pore spherical silica bonded with C3 alkyl chains. Columns were eluted with linear gradients of decreasing sodium chloride and increasing methanol concentrations. The decreasing salt gradient gradually abolished hydrophobic interactions and a significantly higher selectivity was thus obtained when compared with increasing gradients of salts usually employed in reversed-phase separations of tRNA. The acceptance of tRNA fractions was tested by charging them with fifteen different amino acids. Significantly different separations were obtained with tRNA from Escherichia coli and from rat liver. tRNAGlu and tRNATyr from E. coli were obtained in a pure form, all other tRNAs were more or less contaminated by adjoining tRNAs for other amino acids. Rechromatography under suitable isocratic conditions was required to obtain pure tRNA species from rat liver. Isoaccepting tRNAs for several amino acids were separated from rat liver. The method described seems suitable for preliminary fractionations of complex mixtures of tRNA and for a simple purification of isoaccepting species if the presence of tRNAs for other amino acids is not an hindrance.     

Effect of temperature on the separation of long DNA fragments in polymer solution

Electrophoresis of long DNA fragments in polymer solutions is still attractive when performed in short capillaries. Then the separations can be accomplished in minutes rather than hours as is usual in various slab electrophoresis techniques. In this paper we focused on the behavior of large DNA fragments in pulsed field capillary electrophoresis under various temperature conditions. The mobility dependence of fragments of lambda-DNA single-cut mixture on various frequencies at three different temperatures showed that the antiresonance mobility minima are shifted to higher frequencies at higher temperatures. This interesting result is explained in terms of the geometration model of DNA motion.     

聚合物整体柱在生物大分子分离中的应用

近年来,高效液相色谱有机聚合物整体柱以其独有的优势在分离大分子物质中得到了广泛的应用和发展。本文结合本实验室的研究工作,并参考有一定影响力的相关文献,综述了近几年来有机聚合物整体柱的特征及其在生物大分子分离应用中的新进展。     

Novel hydroxyethylcellulose-graft-poly acrylamide copolymer for separation of double-stranded DNA fragments by CE

A novel separation medium, hydroxyethylcellulose-graft-polyacrylamide (HEC-g-PAM) synthesized by atom transfer radical polymerization (ATRP), used for dsDNA separation by CE is presented. The separation performance of HEC-g-PAM, which has the same graft density and different graft length, has been investigated in Tris-boric acid-EDTA (TBE) buffer solvent mixtures. The temperature-dependent rheological behavior of HEC-g-PAM was also studied by steady-shear rheometry. The results showed that dsDNA fragments between 72 and 1353 bp was achieved with a 30 cm effective capillary length at 150 V/cm using this type of graft copolymer as a separation medium in bare fused-silica capillaries, and separation improvement is obtained in HEC-g-PAM compared with HEC and poly(dimethylacrylamide (PDMA).     

CEC separation of aromatic compounds and proteins on hexylamine-functionalized N-acryloxysuccinimide monoliths

Reactive organic polymer monoliths were prepared in fused-silica capillaries by UV-initiated free radical polymerization of N-acryloxysuccinimide (NAS) as reactive monomer, ethylene dimethacrylate as crosslinker, azobisisobutyronitrile as initiator, and toluene as porogen. In a second synthetic step, chemical derivatization of the activated-ester moieties was performed in situ through alkylation reaction with alkylamines to afford monolithic stationary phases with potential reversed-phase properties. A correlation between the synthesis conditions--composition of the reactive solution--chemical characteristics of the reactive polymer monoliths--nitrogen/NAS content--and the reversed-phase separation properties of the functionalized monolithic columns--selectivity towards homologous series of akylbenzenes--was clearly established. This finding offers the possibility of adjusting the experimental conditions with respect to the target applications. The monolithic stationary phases with optimized chemical and porous structures were used for the CEC separation of alkylbenzenes, phenols, anilines, organic acids, amino acids, and proteins. The data indicate that depending on the nature of the analytes (charge, hydrophobic/hydrophilic balance, size) reversed-phase or mixed modes may account for the observed separation.     

Preparation of epoxy monoliths via chemically induced phase separation

Epoxy porous monoliths were prepared from a commercial epoxy resin, D.E.R. 331, that cured with a tertiary amine, 2,4,6-tris-(dimethylaminomethyl) phenol, in the presence of a solvent, diisobutyl ketone (DIBK). During the curing process, polymers were formed and a decrease in its solubility in DIBK; the solution thus phase-separated, usually referred to as chemically induced phase separation. The phase separation formed interconnected polymer-poor phase that then became interconnected pores after the removal of DIBK. By varying the content of DIBK from 32 to 40 vol.%, epoxy monoliths with interconnected pores were prepared, with surface pore size ranging from 0.20 to 2.33 μm, overall porosity from 0.41 to 0.60, and ethanol permeability from 10 to 4,717 L/(m²?h^?1?bar^?1). The glass transition temperatures of the epoxy monoliths, measured with differential scanning calorimetry, were all higher than 100 °C, and temperatures of 5 % weight loss, analyzed by thermal gravimetry, were higher than 350 °C, evidencing the monoliths’ high thermal stability. Also, the monolith morphology was found to be strongly related to the reaction mechanism of polymerization. The results indicate that the mechanism of chain initiation and propagation associated with the tertiary amine can effectively form monoliths with interconnected pores, which cannot be easily prepared with a stepwise polymerization mechanism associated with using primary amine as the curing agent.     

Thick silica gel coatings on methylsilsesquioxane monoliths using anisotropic phase separation

Silica gel coatings on methyltrimethoxysilane (MTMS)-derived monoliths have been studied using wetting transition. Wetting transition is observed in a small confined space, where a coating solution phase-separates into a well-coarsened dimension, making all the phase-separating polymerizing silica phase dynamically flow onto the existing surface of a mold. Bulk coating experiments have shown reductions of both macropore volume and diameter due to the coated layer. Comparing HPLC efficiencies of the coated monolith with those of the non-coated MTMS monolith revealed that the retention factors drastically increased in both normal- and reversed-phase modes. This is attributed to the existence of considerable amounts of accessible micropores left inside the coated layer, where analyte molecules travel and adsorb for a considerable period of time.     

Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.     

Portable capillary electrophoresis system with potential gradient detection for separation of DNA fragments

A portable capillary electrophoresis (CE) system with a novel potential gradient detection (PGD) was utilized to separate DNA fragments. For the first time it was demonstrated that separation of DNA fragments in polymer solution could be detected by a portable CE system integrated with PGD, with a limit of detection (LOD) comparable to that of the CE-ultraviolet (UV) method. Effects of buffer solution, sieving medium, and applied voltage were also investigated. The portable CE-PGD system shows several potential advantages, such as simplicity, cost effectiveness, and miniaturization.     

An automated capillary electrophoresis system for high-speed separation of DNA fragments based on a short capillary

A high-speed DNA fragment separation system was developed based on a short capillary and a slotted-vial array automated sample introduction system. The injection process of DNA sample in a short capillary was investigated systematically with three injection techniques including constant-field-strength, low-field-strength and translational spontaneous injections. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ca. 20-μm plug length) were obtained, which ensure the high-speed and high-efficiency separation for DNA fragments with a short effective separation length. Other separation conditions including the sieving matrix concentration, separation field strength and effective separation length were also optimized. The present system was applied in the separation of ΦX174-Hae III digest DNA marker. With an effective separation length of 2.5 cm, the separation could be achieved in <100 s with plate heights ranging from 0.21 to 0.74 μm (corresponding to plate numbers from 4.86 × 10(6) to 1.36 × 10(6)/m). The repeatabilities for the migration time of the eleven fragments were between 0.4 and 1.1% RSD (n=8). By using the automated continuous injection method, the separation for four different DNA samples could be achieved within 250 s. The present system was further applied in the fast sizing of real DNA samples of PCR products.     

Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies

Monoliths are considered as the fourth-generation chromatography material. Their use for preparative separation of biomolecules has been evolved over the past decade. Monolithic columns up to 8L in size are already commercially available for separation of large biomolecules such as proteins, protein aggregates, plasmid DNA, and viruses. These applications leverage monoliths' inherent properties, such as fast operation and high capacity for large biomolecules. The height equivalent to a theoretical plate (HETP) and dynamic binding capacity do not change with velocity. This is explained by the convective transport through the channels with a diameter of above 1000 nm and has been experimentally verified and also supported by theoretical analyses. Despite low absolute surface area, these large channels provide enough area for adsorption of these large biomolecules, which cannot penetrate into conventional chromatography media designed for protein separation. Monoliths for preparative separations are mainly cast as polymethacrylate or polyacrylamide blocks and have been functionalized as ion exchangers or hydrophobic interaction chromatography media. So-called cryogels have channels more than 30 microm wide, enabling efficient processing of suspensions or even cell-chromatography. This review discusses the pressure drop characteristics, mass transfer properties, scale-up, and applications of monoliths in the context of conventional chromatography media.     

Affinity cryogel monoliths for screening for optimal separation conditions and chromatographic separation of cells

Suitable conditions for separating cells using a chromatographic procedure were evaluated in parallel chromatography on minicolumns. A 96-hole minicolumn plate filled with cryogel monoliths (18.8 mm x 7.1 mm ?) with immobilized concanavalin A was used. Chromatographic columns (113 mm x 7.1 mm ?) were used for chromatographic resolution of a mixture of Saccharomyces cerevisiae and Escherichia coli cells. Separation of a cell mixture containing equal amounts of cells of both types performed in a column format under the determined optimal conditions, resulted in a quantitative capture of applied S. cerevisiae cells, while E. coli passed through the column. Bound S. cerevisiae cells were released by flow-induced detachment and by compression of the adsorbent in the presence of 0.3 M methyl alpha-D-manno-pyranoside. The flowthrough and the eluted fractions were analyzed by plate counting and by registering metabolic activity of S. cerevisiae cells in the eluted fractions after capturing on ConA-cryogel monoliths in a 96-minicolumn plate format. The flowthrough fraction contained E. coli cells with nearly 100% purity, whereas the fraction eluted by compression of the adsorbent contained viable S. cerevisiae cells with 95% purity. Thus, an efficient chromatographic separation of cells was achieved using affinity cryogel column.     

Hierarchically organized silica monoliths: influence of different acids on macro- and mesoporous formation

The influence of different acids, such as hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid and acetic acid on the polymerization-induced phase separation process in the formation of hierarchically organized silica monoliths was investigated in detail. Special emphasis is given to systems synthesized from tetrakis(2-hydroxyethoxy)silane (EGMS) or tetramethoxysilane (TMOS) as the silica source in the presence of Pluronic^® P123 serving as structure-directing agent. The obtained silica monoliths exhibited a co-continuous and cellular macroporous structure comprising 2D hexagonally arranged mesopores with high specific surface areas ranging from 320–787 m² g^?1 independent of the applied silane precursor and regardless whether hydrochloric acid or sulfuric acid was used. A drastic change in macropore morphology to closed pores or particulate structures was observed for nitric, bromic as well as acetic acid. For sulfuric and nitric acid, the influence on the mesostructure was not as pronounced and 2D hexagonally arranged mesopores were obtained. With bromic and acetic acid a loss in mesopore ordering has been observed. Best developed hierarchically organized networks with respect to a co-continuous, cellular macroporous network, specific surface area and 2D hexagonally arranged mesopores were obtained for EGMS as well as for TMOS with P123 in sulfuric acid.