Nonconventional synthesis and characterization of ultrahigh-molar-mass polyacrylamides

In spite of the significant progresses in the field of replaceable sieving matrices for separating DNA in capillary electrophoresis (CE), an intense research activity is still going on to improve the separation of large size DNA sequencing fragments. There are evidences, both from experimental and theoretical sides that the resolution of these fragments, at the single base, requires the use of sieving matrices comprised of long chain linear polymers. In the separation of DNA fragments by CE are of upmost importance: (i) the complete solubility of the polymer, (ii) the linearity of the chain, (iii) the achievement of ultrahigh viscosity in dilute solutions. The aim of this work is the synthesis of ultrahigh-molecular-weight polymers which possess the three requirements mentioned above by employing a nonconventional method. We demonstrate that the sieving performance of polyacrylamide is directly correlated to its intrinsic viscosity.     

无胶筛分毛细管电泳分析几百个碱基对核酸的条件优化

通过正交设计实验综合分析了内充羟丙基甲基纤维素（ＨＰＭＣ）无胶筛分毛细管电泳中的分离场强、ＨＰＭＣ浓度、柱长度和柱内径对核酸分离的影响。结果表明,柱长度越长、柱内径越小、分离场强越小,分离效果越好。考虑实际情况,为能在短时间内使几百个碱基对的核酸得到有效分离,一般选择３７ｃｍ×７５μｍｉ．ｄ．的涂壁毛细管、柱内质量浓度为８ｇ／Ｌ的ＨＰＭＣ、场强为３２４Ｖ／ｃｍ的条件,并在此种条件下分析了ＡｐｏＢ１００基因的低浓度聚合酶链式反应（ＰＣＲ）扩增产物（７１０ｂｐ）。     

Reversible phospholipid nanogels for deoxyribonucleic acid fragment size determinations up to 1500 base pairs and integrated sample stacking

Phospholipid additives are a cost-effective medium to separate deoxyribonucleic acid (DNA) fragments and possess a thermally-responsive viscosity. This provides a mechanism to easily create and replace a highly viscous nanogel in a narrow bore capillary with only a 10 °C change in temperature. Preparations composed of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble, forming structures such as nanodisks and wormlike micelles. Factors that influence the morphology of a particular DMPC–DHPC preparation include the concentration of lipid in solution, the temperature, and the ratio of DMPC and DHPC. It has previously been established that an aqueous solution containing 10% phospholipid with a ratio of [DMPC]/[DHPC] = 2.5 separates DNA fragments with nearly single base resolution for DNA fragments up to 500 base pairs in length, but beyond this size the resolution decreases dramatically. A new DMPC–DHPC medium is developed to effectively separate and size DNA fragments up to 1500 base pairs by decreasing the total lipid concentration to 2.5%. A 2.5% phospholipid nanogel generates a resolution of 1% of the DNA fragment size up to 1500 base pairs. This increase in the upper size limit is accomplished using commercially available phospholipids at an even lower material cost than is achieved with the 10% preparation. The separation additive is used to evaluate size markers ranging between 200 and 1500 base pairs in order to distinguish invasive strains of Streptococcus pyogenes and Aspergillus species by harnessing differences in gene sequences of collagen-like proteins in these organisms. For the first time, a reversible stacking gel is integrated in a capillary sieving separation by utilizing the thermally-responsive viscosity of these self-assembled phospholipid preparations. A discontinuous matrix is created that is composed of a cartridge of highly viscous phospholipid assimilated into a separation matrix of low viscosity. DNA sample stacking is facilitated with longer injection times without sacrificing separation efficiency.     

Size exclusive capillary electrophoresis separation of DNA oligonucleotides in small size linear polyacrylamide polymer solution

The mobility of analytes in capillary electrophoresis using polymer gels and solutions is usually described as having an inverse relationship with the molecular size (mobility decreases as molecular size increases). The most commonly used models for predicting such mobility are the Ogston model and the Reptation model. However, in this study a new separation phenomenon was observed in which the mobility of DNA oligonucleotides increased with molecular size in a capillary electrophoresis phase (CEP) coated capillary column. The polymer system used was a 11% linear polyacrylamide (Mr = 1500) solution. The log-transformed number of base pairs (log N) of three double stranded oligonucleotides had an inverse linear relationship (r2 > 0.9981) with their migration time in the capillary column. Such a relationship is similar to that observed with size exclusive chromatography.     

Mobility, diffusion and dispersion of single-stranded DNA in sequencing gels

Electrophoresis of single-stranded DNA in denaturing polyacrylamide gels is presently a standard procedure for the sequencing of DNA fragments. A thorough understanding of the factors that determine the resolution of DNA fractionated in polyacrylamide gels is necessary to optimize the performance of DNA sequencers. Significant research on the mobility of double-stranded (ds)DNA molecules in agarose and polyacrylamide gels has been performed, and the phenomenon of band broadening of single-stranded (ss)DNA fragments in DNA sequencing gels has received attention only recently. In this paper, we present a detailed study of mobility, diffusion and dispersion of ssDNA in sequencing gels as a function of molecular size, gel concentration and electric field strength. DNA mobility is shown to be essentially independent of electric field in the range of 0-60 V/cm. The band broadening is greatly enhanced in the presence of an electric field and the dispersion coefficient (DE) can be an order of magnitude higher than the field-free diffusion coefficient. The measured migration parameters approximately follow the predictions of the biased reptation including fluctuations (BRF) theory. However, deviations due to nonidealities of the separation conditions are observed. The measured migration parameters can be used to optimize the performance of separation systems.     

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)     

Separation of double-stranded DNA fragments by capillary electrophoresis: Impacts of poly(ethylene oxide), gold nanoparticles, ethidium bromide, and pH

The separation of DNA by capillary electrophoresis using poly(ethylene oxide) (PEO) containing gold nanoparticles (GNPs) is presented. The impacts of PEO, GNPs, ethidium bromide (EtBr), and pH on the separation of double-stranded DNA have been carefully explored. Using a capillary dynamically coated with 5.0% poly(vinylpyrrolidone) and filled with 0.2% PEO containing 0.3 x GNPs (the viscosity less than 15 cP), we have demonstrated the separation of DNA markers V and VI within 5 min at pH 8.0 and 9.0. In terms of resolution and reproducibility, GNPs have a greater impact on the separation of DNA at pH 9.0. Resolution improvements for large DNA fragments (> 300 base pairs, bp) are greater than those for small ones in the presence of GNPs. It is important to point out that reproducibility is excellent (relative standard deviations for the migration times less than 0.5%) and thus no further dynamic coating is required in at least 20 consecutive runs in the presence of GNPs. Using 0.2% PEO (pH 9.0) containing 0.3 x GNPs, the separation of DNA fragments ranging in size from 21 to 23,130 bp was accomplished in 7 min. The results presented in this study show the advantage of PEO containing GNPs for DNA separation, including rapidity, high resolving power, excellent reproducibility, and ease of filling capillaries.     

A new quasi-interpenetrating network formed by poly(N-acryloyl-tris-(hydroxymethyl)aminomethane and polyvinylpyrrolidone: separation matrix for double-stranded DNA and single-stranded DNA fragments by capillary electrophoresis with UV detection

The preparation of a new separation matrix, quasi-interpenetrating networks (quasi-IPNs) formed by poly(N-acryloyl-Tris) (poly(tris-A)) and PVP, and its application for dsDNA and ssDNA fragments separation by CE with UV detection, are presented. This new quasi-IPN exhibited high sieving performance, good dynamic coating ability, and low viscosity. Single-base resolutions of dsDNA fragments (Rs = 0.92 for 123/124 bp) and ssDNA fragments (Rs = 0.65 for 123/124 base, Rs = 0.48 for 309/310 base) were achieved by using the quasi-IPN of poly(tris-A)/PVP (2% + 2%) solution in a 31 cm effective length linear polyacrylamide (LPA)-coated column. Single-base separation of dsDNA fragments (Rs = 0.92 for 123/124 bp) was also obtained within 28 min in a 46.7 cm effective length bare column at higher 160 V/cm electric field strength by using the same quasi-IPN solution. The RSD of the migration time measured for each DNA fragments was less than 1.5% in the bare column for nine continuous runs. The effects of temperature and electric field strength on the DNA separation were also investigated.     

Effect of the matrix on DNA electrophoretic mobility

DNA electrophoretic mobilities are highly dependent on the nature of the matrix in which the separation takes place. This review describes the effect of the matrix on DNA separations in agarose gels, polyacrylamide gels and solutions containing entangled linear polymers, correlating the electrophoretic mobilities with information obtained from other types of studies. DNA mobilities in various sieving media are determined by the interplay of three factors: the relative size of the DNA molecule with respect to the effective pore size of the matrix, the effect of the electric field on the matrix, and specific interactions of DNA with the matrix during electrophoresis.     

Separation of single-stranded DNA fragments at a 10-nucleotide resolution by stretching in microfluidic channels

We report a novel DNA separation method by tethering DNA chains to a solid surface and then stretching the DNA chains with an electric field. The anchor is such designed that the critical force to detach a DNA chain is independent of its size. Because the stretching force is proportional to the DNA net charge, a gradual increase of the electric field leads to size-based removal of the DNA strands from the surface and thus DNA separation. Here we show that this method, originally proposed for separation of long double-stranded DNA chains (>10,000 base pairs), is also applicable to single-stranded (ss) DNA fragments with less than 100 nucleotides (nt). Theoretical analysis indicates that the separation resolution is limited by the fluctuation forces on tethered DNA chains. By employing a microfluidic platform with narrow channels filled with a buffer of low ionic conductivity, we are able to apply a strong electric field to the DNA fragments with negligible Joule heating. Upon stepwise increments of the electric field, we demonstrate efficient separation of short ssDNA fragments at a 10-nt resolution.     

Capillary and microelectrophoretic separations of ligase detection reaction products produced from low-abundant point mutations in genomic DNA

Capillary gel electrophoresis (CGE) and polymer-based microelectrophoretic platforms were investigated to analyze low-abundant point mutations in certain gene fragments with high diagnostic value for colorectal cancers. The electrophoretic separations were carried out on single-stranded DNA (ssDNA) products generated from an allele-specific ligation assay (ligase detection reaction, LDR), which was used to screen for a single base mutation at codon 12 in the K-ras oncogene. The presence of the mutation generated a ssDNA fragment that was >40 base pairs (bp) in length, while the primers used for the ligation assay were <30 bp in length. Various separation matrices were investigated, with the success of the matrix assessed by its ability to resolve the ligation product from the large molar excess of unligated primers when the mutant allele was lower in copy number compared to the wild-type allele. Using CGE, LDR product models (44 and 51 bp) could be analyzed in a cross-linked polyacrylamide gel with a 1000-fold molar excess of LDR primers (25 bp) in approximately 45 min. However, when using linear polyacrylamide gels, these same fragments could not be detected due to significant electrokinetic biasing during injection. A poly(methylmethacrylate) (PMMA) microchip of 3.5 cm effective column length was used with a 4% linear polyacrylamide gel to analyze the products generated from an LDR. When the reaction contained a 100-fold molar excess of wild-type DNA compared to a G12.2D mutant allele, the 44 bp ligation product could be effectively resolved from unligated primers in under 120 s, nearly 17 times faster than the CGE format. In addition, sample cleanup was simplified using the microchip format by not requiring desalting of the LDR prior to loading.     

Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions

Separations of phiX-174/HaeIII DNA restriction fragments have been performed in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose. During the separation, polymer solutions entered the capillary by EOF. When using LPA solutions, bulk EOF is small due to adsorption on the capillary wall. On the other hand, separation is faster and better for the large DNA fragments (> 872 base pairs, bp) using derivative celluloses and PEO solutions. Several approaches to optimum resolution and speed by controlling EOF and/or altering electrophoretic mobility of DNA have been developed, including (i) stepwise changes of ethidium bromide (0.5-5 microg/mL), (ii) voltage programming (125-375 V/cm), (iii) use of mixed polymer solutions, and (iv) use of high concentrations of Tris-borate (TB) buffers. The DNA fragments ranging from 434 to 653 bp that were not separated using 2% PEO (8,000,000) under isocratic conditions have been completely resolved by either stepwise changes of ethidium bromide or voltage programming. Compared to PEO solutions, mixed polymer solutions prepared from PEO and HEC provide higher resolving power. Using a capillary filled with 600 mM TB buffers, pH 10.0, high-speed (< 15 min) separation of DNA (pBR 322/HaeIII digest, pBR 328/ Bg/l digest and pBR 328/Hinfl digest) has been achieved in 1.5% PEO.     

Separation of long DNA fragments by inversion field capillary electrophoresis

This study reports improved pulsed field capillary electrophoresis (PFCE) for separation of large DNA ladders. Important analytical conditions, including gel polymer concentration, ratio of forward to backward pulse duration, and separation potential, were investigated for their effects on the separation performance of DNA ranging in size from 0.1 to 10.0 kilo base pairs (kbp). Results show that DNA fragments from 0.1 to 8.0 kbp can be resolved with high resolution, simultaneously, in a short time. The ratio of forward to backward pulse duration affects the separation performance for DNA fragments greater than 1.5 kbp, and 3 or 4 is the optimum value of the ratio for separation of DNA up to 10 kbp. Furthermore, the separations that were obtained with 74–19,329 bp λ-DNA restriction fragments clearly demonstrate a dramatic improvement in the separation time and resolution over the conventionally used square-wave PFCE. The inversion field capillary electrophoresis reported here may help enable future DNA analysis studies to be performed quickly and effectively.     

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.     

An integrated system for enzymatic cleavage and electrostretching of freely-suspended single DNA molecules

A novel polyacrylamide gel-based femtolitre microchamber system for performing single-molecule restriction enzyme assay on freely-suspended DNA molecules and subsequent DNA electrostretching by applying an alternating electric field has been developed. We attempted the integration by firstly initiating restriction enzyme reaction on a fluorescent-stained lambdaDNA molecule, encapsulated in a microchamber, using magnesium as an external trigger. Upon complete digestion, the cleaved DNA fragments were electrostretched to analyze the DNA lengths optically. The critical parameters for electrostretching of encapsulated DNA were investigated and optimum stretching was achieved by using 1.5 kHz pulses with electric field strength in the order of 10(3) V cm(-1) in 7% linear polyacrylamide (LPA) solution. LPA was adopted to minimize the adverse effects of ionic thermal agitation on molecular dielectrophoretic elongation in the microchamber. In our experiments, as the fragments were not immobilized throughout the entire protocol, it was found from repeated tests that digestion always occurred, producing the expected number of cleaved fragments. This versatile microchamber approach realized direct observation of these biological reactions on real-time basis at a single-molecule level. Furthermore, with the employment of porous polyacrylamide gel, the effective manipulation of DNA assays and the ability to combine conventionally independent bioanalytical processes have been demonstrated.     

Ultrafast, efficient separations of large-sized dsDNA in a blended polymer matrix by microfluidic chip electrophoresis: a design of experiments approach

Double-stranded (ds) DNA fragments over a wide size range were successfully separated in blended polymer matrices by microfluidic chip electrophoresis. Novel blended polymer matrices composed of two types of polymers with three different molar masses were developed to provide improved separations of large dsDNA without negatively impacting the separation of small dsDNA. Hydroxyethyl celluloses with average molar masses of ～27 kDa and ～1 MDa were blended with a second class of polymer, high-molar mass (～7 MDa) linear polyacrylamide. Fast and highly efficient separations of commercially available DNA ladders were achieved on a borosilicate glass microchip. A distinct separation of a 1-kb DNA extension ladder (200-40,000 bp) was completed in 2 min. An orthogonal design of experiments was used to optimize experimental parameters for DNA separations over a wide size range. We find that the two dominant factors are the applied electric field strength and the inclusion of a high concentration of low-molar mass polymer in the matrix solution. These two factors exerted different effects on the separations of small dsDNA fragments below 1 kbp, medium dsDNA fragments between 1 and 10 kbp, and large dsDNA fragments above 10 kbp.     

线性聚丙烯酰胺凝胶毛细管电泳的迁移特性

使用线性聚丙烯酰胺作为筛分介质,对片段长度为80～584bp的标准DNA样品进行毛细管电泳,利用激光诱导荧光方法检测信号,荧光染料为溴化乙啶。改变电场强度100～375V/cm,得到的迁移率曲线与电场强度和DNA片段长度成复杂的函数关系,已有的经典理论模型:Ogston模型、Reptation无拉伸模型和Reptation拉伸模型都不能正确地描述实验观察到的迁移率随电场强度和DNA片段长度的变化情况。因此,提出一种修正的Ogston筛分理论,假定迁移的DNA分子在电场强度方向延展拉伸,如同小分子穿过凝胶筛孔。在该修正模型中,DNA的迁移率仅依赖于电场强度、筛分介质浓度和片段长度,很好地解释了实验现象。     

Divergent dispersion behavior of ssDNA fragments during microchip electrophoresis in pDMA and LPA entangled polymer networks

Resolution of DNA fragments separated by electrophoresis in polymer solutions ("matrices") is determined by both the spacing between peaks and the width of the peaks. Prior research on the development of high-performance separation matrices has been focused primarily on optimizing DNA mobility and matrix selectivity, and gave less attention to peak broadening. Quantitative data are rare for peak broadening in systems in which high electric field strengths are used (>150 V/cm), which is surprising since capillary and microchip-based systems commonly run at these field strengths. Here, we report results for a study of band broadening behavior for ssDNA fragments on a glass microfluidic chip, for electric field strengths up to 320 V/cm. We compare dispersion coefficients obtained in a poly(N,N-dimethylacrylamide) (pDMA) separation matrix that was developed for chip-based DNA sequencing with a commercially available linear polyacrylamide (LPA) matrix commonly used in capillaries. Much larger DNA dispersion coefficients were measured in the LPA matrix as compared to the pDMA matrix, and the dependence of dispersion coefficient on DNA size and electric field strength were found to differ quite starkly in the two matrices. These observations lead us to propose that DNA migration mechanisms differ substantially in our custom pDMA matrix compared to the commercially available LPA matrix. We discuss the implications of these results in terms of developing optimal matrices for specific separation (microchip or capillary) platforms.     

DNA separation by microchip electrophoresis using low-viscosity hydroxypropylmethylcellulose-50 solutions enhanced by polyhydroxy compounds

Low-viscosity polymer solutions have potential for double-stranded (ds) DNA separations in micrototal analysis systems (micro-TAS). In this paper, we report dilute, low-viscosity hydroxypropylmethylcellulose-50 (HPMC-50, 11.5 kDa) solutions containing polyhydroxy additives as separation media. Predominant operational variables, such as applied electric field strength, fluorescent intercalator (YOPro-1) concentration, polymer concentration, and additive concentration, are thoroughly investigated. Fast (within 170 s) and excellent separation of DNA restriction fragments ranging in size from 72 to 1353 base pairs (bp) is achieved in a 30 mm length channel of polymethylmethacrylate (PMMA) microchips at an electric field strength of 300 V/cm, by introducing 8% mannitol, 8% glucose or 10% glycerol additives into a 2% HPMC-50/1 x Tris-borate-EDTA (TBE) solution. The low-viscosity (40 cP) matrix formulation provides both coating of the microchannels and separation of DNA in one step. The performance in the solution surpasses that in highly concentrated HPMC-50 solution. In addition, separation using 1xTris-EDTA buffer in the 2% HPMC-50 matrix containing polyhydroxy additives also exhibits a notably increased performance. This is presumably due to formation of hydrogen-bonding interactions of polyhydroxy additives with HPMC-50 matrix and DNA so as to increase the coupling interactions between matrix and DNA molecules during electrophoresis. The result reflects that boric acid is not a prerequisite in polyhydroxy-enhanced HPMC-50 solution for separation.