Simultaneous detection of 19 K‐ras mutations by free‐solution conjugate electrophoresis of ligase detection reaction products on glass microchips

We demonstrate here the power and flexibility of free‐solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild‐type DNA. Here, four large drag‐tags are used to achieve free‐solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K‐ras oncogene. LDR‐FSCE enabled electrophoretic resolution of these 19 LDR‐FSCE products by CE in 13.5 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free‐solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR‐FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K‐ras mutations on integrated “sample‐in/answer‐out” devices with amplification, LDR, and detection all on one platform.     

End-labeled free-solution electrophoresis of DNA

DNA is a free-draining polymer. This subtle but "unfortunate" property of highly charged polyelectrolytes makes it impossible to separate nucleic acids by free-flow electrophoresis. This is why one must typically use a sieving matrix, such as a gel or an entangled polymer solution, in order to obtain some electrophoretic size separation. An alternative approach consists of breaking the charge to friction balance of free-draining DNA molecules. This can be achieved by labeling the DNA with a large, uncharged molecule (essentially a hydrodynamic parachute, which we also call a drag-tag) prior to electrophoresis; the resulting methodology is called end-labeled free-solution electrophoresis (ELFSE). In this article, we review the development of ELFSE over the last decade. In particular, we examine the theoretical concepts used to predict the ultimate performance of ELFSE for single-stranded (ssDNA) sequencing, the experimental results showing that ELFSE can indeed overcome the free-draining issue raised above, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies. Finally, we also review the reverse process, called free-solution conjugate electrophoresis (FSCE), wherein uncharged polymers of different sizes can be analyzed using a short DNA molecule as an electrophoretic engine.     

Poly-N-hydroxyethylacrylamide (polyDuramide): a novel,hydrophilic, self-coating polymer matrix for DNA sequencing by capillary electrophoresis

A replaceable polymer matrix, based on the novel monomer N-hydroxyethylacrylamide (HEA), has been synthesized for application in DNA separation by microchannel electrophoresis. The monomer was found by micellar electrokinetic chromatography analysis of monomer partitioning between water and 1-octanol to be more hydrophilic than acrylamide and N,N-dimethylacrylamide. Polymers were synthesized by free radical polymerization in aqueous solution. The weight-average molar mass of purified polymer was characterized by tandem gel permeation chromatography-multiangle laser light scattering. The steady-shear rheological behavior of the novel DNA sequencing matrix was also characterized, and it was found that the viscosity of the novel matrix decreases by more than 2 orders of magnitude as the shear rate is increased from 0.1 to 1000 s(-1). Moreover, in the shear-thinning region, the rate of change of matrix viscosity with shear rate increases with increasing polymer concentration. Poly-N-hydroxyethylacrylamide (PHEA) exhibits good capillary-coating ability, via adsorption from aqueous solution, efficiently suppressing electroosmotic flow (EOF) in a manner comparable to that of poly-N,N-dimethylacrylamide. Under DNA sequencing conditions, adsorptive PHEA coatings proved to be stable and to maintain negligible EOF for over 600 h of electrophoresis. Resolution of DNA sequencing fragments, particularly fragments > 500 bases, in PHEA matrices generally improves with increasing polymer concentration and decreasing electric field strength. When PHEA is used both as a separation matrix and as a dynamic coating in bare silica capillaries, the matrix can resolve over 620 bases of contiguous DNA sequence within 3 h. These results demonstrate the good potential of PHEA matrices for high-throughput DNA analysis by microchannel electrophoresis.     

Free-solution electrophoretic separations of DNA-drag-tag conjugates on glass microchips with no polymer network and no loss of resolution at increased electric field strength

Here, we demonstrate the potential for high-resolution electrophoretic separations of ssDNA-protein conjugates in borosilicate glass microfluidic chips, with no sieving media and excellent repeatability. Using polynucleotides of two different lengths conjugated to moderately cationic protein polymer drag-tags, we measured separation efficiency as a function of applied electric field. In excellent agreement with prior theoretical predictions of Slater et al., resolution is found to remain constant as applied field is increased up to 700 V/cm, the highest field we were able to apply. This remarkable result illustrates the fundamentally different physical limitations of free-solution conjugate electrophoresis (FSCE)-based DNA separations relative to matrix-based DNA electrophoresis. ssDNA separations in "gels" have always shown rapidly declining resolution as the field strength is increased; this is especially true for ssDNA > 400 bases in length. FSCE's ability to decouple DNA peak resolution from applied electric field suggests the future possibility of ultra-rapid FSCE sequencing on chips. We investigated sources of peak broadening for FSCE separations on borosilicate glass microchips, using six different protein polymer drag-tags. For drag-tags with four or more positive charges, electrostatic and adsorptive interactions with poly(N-hydroxyethylacrylamide)-coated microchannel walls led to appreciable band-broadening, while much sharper peaks were seen for bioconjugates with nearly charge-neutral protein drag-tags.     

Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis

We demonstrate the feasibility of end-labeled free-solution electrophoresis (ELFSE) separation of DNA using genetically engineered protein polymers as drag-tags. Protein polymers are promising candidates for ELFSE drag-tags because their sequences and lengths are controllable not only to generate monodisperse polymers with high frictional drag, but also to meet other drag-tag requirements for high-resolution separations by microchannel electrophoresis. A series of repetitive polypeptides was designed, expressed in Escherichia coli, and purified. By performing an end-on conjugation of the protein polymers to a fluorescently labeled DNA oligomer (22 bases) and analyzing the electrophoretic mobilities of the conjugate molecules by free-solution capillary electrophoresis (CE), effects of the size and charge of the protein polymer drag-tags were investigated. In addition, the electrophoretic behavior of bioconjugates comprising relatively long DNA fragments (108 and 208 bases) and attached to uncharged drag-tags was observed, by conjugating fluorescently labeled polymerase chain reaction (PCR) products to charge-neutral protein polymers, and analyzing via CE. We calculated the amount of friction generated by the various drag-tags, and estimated the potential read-lengths that could be obtained if these drag-tags were used for DNA sequencing in our current system. The results of these studies indicate that larger and uncharged drag-tags will have the best DNA-resolving capability for ELFSE separations, and that theoretically, up to 233 DNA bases could be sequenced using one of the protein polymer drag-tags we produced, which is electrostatically neutral with a chain length of 337 amino acids. We also show that denatured (unfolded) polypeptide chains impose much greater frictional drag per unit molecular weight than folded proteins, such as streptavidin, which has been used as a drag-tag before.     

Separation of single‐base sequential isomers of single‐stranded DNA by capillary electrophoresis and its application in the discrimination of single‐base DNA mutations

Separation of single‐base substitution sequential DNA isomers remains one of the most challenging tasks in DNA separation by capillary electrophoresis. We developed a simple, versatile capillary electrophoresis technique for the separation of single‐base sequential isomers of DNA having the same chain length. This technique is based on charge differences resulting from the different protonation (acid dissociation) properties of the four DNA bases. A mixture of 13 single‐base sequential isomers of 12‐mer single‐stranded DNA was separated by using an electrophoretic buffer solution containing 20 mM phosphoric acid (pH 2.0) and 8 M urea. We demonstrated that our method could separate all possible mutation patterns under identical experimental conditions. In addition, application of our method to the separation of the polymerase chain reaction product of a 68‐mer gene fragment and its single‐base isomers indicates that in combination with the appropriate genomic DNA extraction techniques, the method can detect single‐base gene mutations.     

用于毛细管电泳DNA分离的合成聚合物*

毛细管电泳的无胶筛分方法在DNA片段分离、DNA 测序方面取得了显著的成绩并已成功应用于人类基因组计划.该法是在毛细管柱中充入一定浓度和组成的线性高分子溶液,利用其对样品组分电泳迁移时的阻滞作用,按分子量大小对DNA等生物大分子进行筛分分离分析.因此,聚合物筛分介质的类型、组成和性质会显著影响分离效果.近年来,由于受到基因组计划的影响,出现了许多用于DNA片段分离和DNA测序的水溶性高分子聚合物,并取得很大进展.本文按照均聚物和共聚物的分类,综述了作为筛分介质的各种合成聚合物及其应用效果,并简要介绍了有关的筛分理论和分离的评价指标.     

Sparsely cross-linked "nanogels" for microchannel DNA sequencing

We have developed sparsely cross-linked "nanogels", sub-colloidal polymer structures composed of covalently linked, linear polyacrylamide chains, as novel DNA sequencing matrices for capillary electrophoresis. The presence of covalent cross-links affords nanogel matrices with enhanced network stability relative to standard, linear polyacrylamide (LPA), improving the separation of large DNA fragments. Nanogels were synthesized via inverse emulsion (water-in-oil) copolymerization of acrylamide and N,N-methylenebisacrylamide (Bis). In order to retain the fluidity necessary in a replaceable polymer matrix for capillary array electrophoresis (CAE), a low percentage of the Bis cross-linker (< 10(-4) mol%) was used. Nanogels were characterized by multiangle laser light scattering and rheometry, and were tested for DNA sequencing by CAE with four-color laser-induced fluorescence (LIF) detection. The properties and performance of nanogel matrices were compared to those of a commercially available LPA network, which was matched for both weight-average molar mass (Mw) and extent of interchain entanglements (c/c*). Nanogels presented in this work have an average radius of gyration of 226 nm and a weight-average molar mass of 8.8 x 10(6) g/mol. At concentrations above the overlap threshold, nanogels form a clear, viscous solution, similar to the LPA matrix (Mw approximately 8.9 x 10(6) g/mol). The two matrices have similar flow and viscosity characteristics. However, because of the physical network stability provided by the internally cross-linked structure of the nanogels, a substantially longer read length ( approximately 63 bases, a 10.4% improvement) is obtained with the nanogel matrix at 98.5% accuracy of base-calling. The nanogel network provides higher-selectivity separation of ssDNA sequencing fragments longer than 375 bases. Moreover, nanogel matrices require 30% less polymer per unit volume than LPA. This is the first report of a sequencing matrix that provides better performance than LPA, in a side-by-side comparison of polymer matrices matched for Mw and extent of interchain entanglements.     

Electrophoretic behavior of DNA‐methyl‐CpG‐binding domain protein complexes revealed by capillary electrophoreses laser‐induced fluorescence

The free solution electrophoretic behavior of DNA‐protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA‐protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA‐methyl‐CpG‐binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA‐protein complexes. This method is based on the effective separation of free DNA and DNA‐protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide‐coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA‐MBD2b complexes, we found that the values of (μ₀/μ)‐1 were directly proportional to the charge‐to‐mass ratios of formed complexes, where the μ₀ and μ are the mobility of free DNA probe and DNA‐protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge‐to‐mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA‐binding properties of the purified MBD2b protein and help to analyze other DNA‐protein complexes. Additionally, this study enhances the understanding of the influence of the charge‐to‐mass ratios of formed DNA‐protein complexes on their separation and electrophoretic behaviors.     

Further Study on Separation of DNA Fragments by Capillary Electrophoresis by Quasi-interpenetrating Network of Polyacryamide and Polyvinylpyrrolidone with UV Detection

Quasi-interpenetrating network of polyacrylamide （PAA） and polyvinylpyrrolidone （PVP） had been successfully used for single-base resolution of double-stranded DNA （0.76 for 123 bp/124 bp） and single-stranded DNA fragments （0.97 for 123 b/124 b） with UV detection. This quasi-IPN （interpenetrating network） sieving matrix showed low viscosity （23.5 mPa·s at 25 ℃） and decreased with increasing temperature. This polymer also exhibited dynamically coating capacity and could be used in the uncoated capillary. The effects of temperature and electric field strength on the DNA separation of quasi-IPN matrix were also investigated and found that the temperature and electric field strength could markedly affected the mobility behavior of DNA fragments. This polymer matrix has also applied to separate the bigger DNA fragments by capillary electrophoresis with UV detection. Under the denaturing conditions, this matrix separated the samples with last fragment of 1353 base in 40 rain, in which the doublet of 309/310 base was partial separated and the resolution was 0.88.     

Star-shaped polymers for DNA sequencing by capillary electrophoresis

The separation and sequencing of DNA are the main objectives of the Human Genome Project, and this project has also been very useful for gene analysis and disease diagnosis. Capillary electrophoresis (CE) is one of the most common techniques for the separation and analysis of DNA. DNA separations are usually achieved using capillary gel electrophoresis (CGE) mode, in which polymer gel is packed into the capillary. Compared with a traditional CGE matrix, a hydrophilic polymer matrix, which can be adsorb by the capillary wall has numerous advantages, including stability, reproducibility and ease of automation. Various water-soluble additives, such as linear poly(acrylamide) (PAA) and poly(N,N-dimethylacrylamide) (PDMA), have been employed as media. In this study, different star-shaped PDMA polymers were designed and synthesized to achieve lower polymer solution viscosity. DNA separations with these polymers avoid the disadvantages of high viscosity and long separation time while maintaining high resolution (10 bp between 271 bp and 281 bp). The influences of the polymer concentration and structure on DNA separation were also determined in this study; higher polymer concentration yielded better separation performance, and star-like polymers were superior to linear polymers. This work indicates that modification of the polymer structure is a potential strategy for optimizing DNA separation.     

Structure‐Independent Conductance of Thiophene‐Based Single‐Stacking Junctions

The experimental investigation of intermolecular charge transport in π‐conjugated materials is challenging. Herein, we describe the investigation of charge transport through intermolecular and intramolecular paths in single‐molecule and single‐stacking thiophene junctions by the mechanically controllable break junction (MCBJ) technique. We found that the ability for intermolecular charge transport through different single‐stacking junctions was approximately independent of the molecular structure, which contrasts with the strong length dependence of conductance in single‐molecule junctions with the same building blocks, and the dominant charge‐transport path of molecules with two anchors transited from an intramolecular to an intermolecular path when the degree of conjugation increased. An increase in conjugation further led to higher binding probability owing to the variation in binding energies, as supported by DFT calculations.     

Sieving properties of end group‐halogenated Pluronic polymer matrix in DNA separation under nondenaturing CE analysis

CE‐SSCP analysis is a well‐established DNA separation method that is based on variations in mobility caused by sequence‐induced differences in the conformation of single‐stranded DNA. The resolution of CE‐SSCP analysis was improved by using a Pluronic polymer matrix, and it has been successfully applied in various genetic analyses. Because the Pluronic polymer forms a micellar cubic structure in the capillary, it provides a stable internal structure for high‐resolution CE‐SSCP analysis. We hypothesized that formation of micellar cubic structure is influenced by the end hydroxyl group of the Pluronic polymer, which affords structural stability through hydrogen bonding. To test this hypothesis, the hydroxyl group was halogenated to eliminate the hydrogen bonding without disturbing the polarity of polymer matrix. CE‐SSCP resolution of two DNA fragments with a single base difference was significantly worse in the halogenated polymer matrices due to band broadening. The viscoelastic properties of control (which has hydroxyl group), chlorinated, and brominated F108 solution upon heating were also investigated by rheological experiments, and we found that gelation was significantly associated with resolution. In this series of experiments, the effect of the hydroxyl group in Pluronic polymer matrix on separation resolution of CE‐SSCP analysis was demonstrated.     

Non‐hybridization single nucleotide polymorphism detection and genotyping assay through direct discrimination of single base mutation by capillary electrophoretic separation of single‐stranded DNA

The significant demands for single nucleotide polymorphism detection and genotyping assays have grown. Most common assays are based on the recognition of the target sequence by the hybridization with its specific probe having the complementary sequence of the target. Herein, a simple, label‐free, and economical non‐hybridization assay was developed for single nucleotide polymorphism detection and genotyping, based on the direct discrimination of single base mutation by simple capillary electrophoresis separation for single‐stranded DNA in an acidic electrophoretic buffer solution containing urea. Capillary electrophoresis separation of single‐base sequential isomers of DNA was achieved due to charge differences resulting from the different protonation properties of the DNA bases. Single nucleotide polymorphism detection and genotyping were achieved by discriminating the electropherogram pattern change, that is, peak number in the electropherogram, obtained by the proposed method. The successful practical application of the proposed method was demonstrated through single nucleotide polymorphism detection and genotyping on a known gene region of 84‐mer, in which guanine to adenine single‐base mutation is commonly observed, using a human hair sample in combination with genomic DNA extraction, polymerase chain reaction amplification, DNA purification from polymerase chain reaction products, and capillary electrophoresis separation.     

Efficient π‐conjugated interrupted host polymer by metal‐free polymerization for blue/green phosphorescent light‐emitting diodes

A new aromatic host polymer poly{[1,4‐bis(9‐decylcarbazole‐3‐yl)‐2,3,5,6‐tetrafluorobenzene‐3,3′‐diyl]‐alt‐[N‐methylisatin‐2‐one‐3,3‐diyl]} (PICzFB) containing carbazole–tetrafluorinebeneze–carbazole moiety in the π‐conjugated interrupted polymer backbone was synthesized by superacid‐catalyzed metal‐free polyhydroxyalkylation. The resulted copolymer PICzFB showed a comparatively wide band gap up to 3.32 eV and high triplet energy (E_T) of 2.73 eV due to confined conjugation by the δ? C bond interrupted polymer backbone. Blue and green light‐emitting devices with PICzFB as host, FIrpic and Ir(mppy)₃ as phosphorescent dopants showed the maximum luminous efficiencies of 5.0 and 27.6 cd/A, respectively. The results suggested that the strategy of incorporating bipolar unit into the π‐conjugated interrupted polymer backbone can be a promising approach to obtain host polymer with high triplet level for solution‐processed blue and green phosphorescent polymer light‐emitting diodes. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 1037–1046     

Reducing the Exciton Binding Energy of Donor–Acceptor‐Based Conjugated Polymers to Promote Charge‐Induced Reactions

Exciton binding energy has been regarded as a crucial parameter for mediating charge separation in polymeric photocatalysts. Minimizing the exciton binding energy of the polymers can increase the yield of charge‐carrier generation and thus improve the photocatalytic activities, but the realization of this approach remains a great challenge. Herein, a series of linear donor–acceptor conjugated polymers has been developed to minimize the exciton binding energy by modulating the charge‐transfer pathway. The results reveal that the reduced energy loss of the charge‐transfer state can facilitate the electron transfer from donor to acceptor, and thus, more electrons are ready for subsequent reduction reactions. The optimized polymer, FSO‐FS, exhibits a remarkable photochemical performance under visible light irradiation.     

Evaluation of different nucleic acid stains for sensitive double-stranded DNA analysis with capillary electrophoretic separation

This paper outlines the first use of SYTOX Orange, SYTO 82 and SYTO 25 nucleic acid stains for on-column staining of double-stranded DNA (dsDNA) fragments separated by capillary electrophoresis (CE). Low-viscosity, replaceable poly(vinylpyrrolidone) (PVP) polymer solution was used as the sieving matrix on an uncoated fused-silica capillary. The effects of PVP concentration, electric field strength, and incorporated nucleic acid stain concentrations on separation efficiency were examined for a wide range of DNA fragment sizes. Our study was focused on using nucleic acid stains efficiently excitable at a wavelength of 532 nm. Among the five tested nucleic acid stains, SYTOX Orange stain was shown to have the best sensitivity for dsDNA detection by CE. About a 500-fold lower detection limit was obtained compared to commonly used ethidium bromide and propidium iodide. SYTOX Orange stain also provided a wide linear dynamic range for direct DNA quantitation with on-line CE detection. Use of SYTOX Orange stain can greatly improve the measurement of DNA fragments by CE, which will enable an expanded set of applications in genomics and diagnostics.     

Novel π‐Electron Extension System via Chromophores Self‐Polymerization to Enhance the NLO Efficiency

A new strategy for the self‐polymerization of chromophores is investigated to develop a 2,7‐carbazole‐based nonlinear optical (NLO) conjugated polymer with an increasing conjugation length of chromophores. Elongation of the conjugation‐path length in chromophores has established engineering guidelines to enhance optical nonlinearity. Compared with the traditional synthesis of an NLO polymer, the chromophores should be well‐designed at a limited conjugation spacer, and then incorporated into a polymer matrix. In this research, the π‐conjugation spacer of chromophores extended perpendicularly to the dipole of chromophores during the polymerization process. Furthermore, this study marks the first research of integrating the π‐electrons of chromophores and conjugated polymers. These conjugated backbones promote a bulk‐polarization response, leading to large NLO coefficients.

     

A viscosity-tunable polymer for DNA separation by microchip electrophoresis

A thermo-responsive separation matrix, consisting of Pluronic F127 tri-block copolymers of poly(ethylene oxide) and poly(propylene oxide), was used to separate DNA fragments by microchip electrophoresis. At low temperature, the polymer matrix was low in viscosity and allowed rapid loading into a microchannel under low pressure. With increasing temperatures above 25°C, the Pluronic F127 solution forms a liquid crystalline phase consisting of spherical micelles with diameters of 17–19 nm. The solution can be used to separate DNA fragments from 100 bp to 1500 bp on poly(methyl methacrylate) (PMMA) chips. This temperature-sensitive and viscosity-tunable polymer provided excellent resolution over a wide range of DNA sizes. Separation is based on a different mechanism compared with conventional matrices such as methylcellulose. To illustrate the separation mechanism of DNA in a Pluronic F127 solution, DNA molecular imaging was performed by fluorescence microscopy with F127 polymer as the separation matrix in microchip electrophoresis. Figure Temperature dependence of the viscosity of 20% w/w Pluronic F127 solution in 1xTBE buffer. Dotted approximates resultant curve.