Electrophoresis (ELECTROPHORESIS)

Publications in this journal

• Article: Dielectrophoretic manipulation of fluorescing single-walled carbon nanotubes.

  Natacha Mureau, Ernest Mendoza, S Ravi P Silva
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  ABSTRACT: We investigate the behavior of fluorescing single-walled carbon nanotubes (SWCNTs) under dielectrophoretic conditions and demonstrate their collection with fluorescence microscopy. SWCNTs are dispersed in water with the aid of a nonionic surfactant, Triton X-100, and labeled through noncovalent binding with the dye 3,3'-dihexyloxacarbocyanine iodide (diOC(6)). The chromophore's affinity to the SWCNTs is due to pi-stacking interactions. Carbon nanotube (CNT) localization is clearly identified on the fluorescence images, showing that the nanotubes concentrate between the electrodes and align along the electric field lines.
  Electrophoresis 02/2013; 28(10):1495-8.
• Article: Metabolic viability of Escherichia coli trapped by dielectrophoresis in microfluidics

  S S Donato, V Chu, D M F Prazeres, J P Conde
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  ABSTRACT: The spatial and temporal control of biological species is essential in complex microfluidic biosystems. In addition, if the biological species is a cell, microfluidic handling must ensure that the cell's metabolic viability is maintained. The use of DEP for cell manipulation in microfluidics has many advantages because it is remote and fast, and the voltages required for cell trapping scale well with miniaturization. In this paper, the conditions for bacterial cell (Escherichia coli) trapping using a quadrupole electrode configuration in a PDMS microfluidic channel were developed both for stagnant and for in-flow fluidic situations. The effect of the electrical conductivity of the fluid, the applied electric field and frequency, and the fluid-flow velocity were studied. A dynamic exchange between captured and free-flowing cells during DEP trapping was demonstrated. The metabolic activity of trapped cells was confirmed by using E. coli cells genetically engineered to express green fluorescent protein under the control of an inducible promoter. Noninduced cells trapped by negative DEP and positive DEP were able to express green fluorescent protein minutes after the inducer was inserted in the microchannel system immediately after DEP trapping. Longer times of trapping prior to exposure to the inducer indicated first a degradation of the cell metabolic activity and finally cell death.
  Electrophoresis 01/2013; 34(4):575-582.
• Article: Electrokinetic injection across supported liquid membranes – new sample pretreatment technique for on-line coupling to capillary electrophoresis. Direct analysis of perchlorate in biological samples.

  Pavel Kubáň, Isaac K. Kiplagat, Petr Boček
  Electrophoresis 04/2012;
• Article: Errata: Study of interaction between drug enantiomers and human serum albumin by flow injection-capillary electrophoresis frontal analysis.

  Xiumei Liu, Xingguo Chen, Yuanyuan Yue, Jingshu Zhang, Yuqin Song
  Electrophoresis 02/2009; 29(24):5067.
• Source

  Available from: cas.cz

  Article: Errata: Detection and analysis of protein-protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes.

  Frank Krause
  Electrophoresis 02/2009; 29(24):5067.
• Article: Errata: Boronate affinity saccharide electrophoresis: A novel carbohydrate analysis tool.

  Thomas R Jackson, Jeremy S Springall, Damien Rogalle, Naoko Masumoto, Hung Ching Li, François D'Hooge, Semali P Perera, Toby A Jenkins, Tony D James, John S Fossey, Jean M H van den Elsen
  Electrophoresis 02/2009; 29(24):5067.
• Article: Ligase detection reaction for the analysis of point mutations using free-solution conjugate electrophoresis in a polymer microfluidic device.

  Rondedrick Sinville, Jennifer Coyne, Robert J Meagher, Yu-Wei Cheng, Francis Barany, Annelise Barron, Steven A Soper
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  ABSTRACT: We have developed a new method for the analysis of low abundant point mutations in genomic DNA using a combination of an allele-specific ligase detection reaction (LDR) with free-solution conjugate electrophoresis (FSCE) to generate and analyze the genetic products. FSCE eliminates the need for a polymer sieving matrix by conjugating chemically synthesized polyamide "drag-tags" onto the LDR primers. The additional drag of the charge-neutral drag-tag breaks the linear scaling of the charge-to-friction ratio of DNA and enables size-based separations of DNA in free solution using electrophoresis with no sieving matrix. We successfully demonstrate the conjugation of polyamide drag-tags onto a set of four LDR primers designed to probe the K-ras oncogene for mutations highly associated with colorectal cancer, the simultaneous generation of fluorescently labeled LDR/drag-tag conjugate (LDR-dt) products in a multiplexed, single-tube format with mutant:WT ratios as low as 1:100, respectively, and the single-base, high-resolution separation of all four LDR-dt products. Separations were conducted in free solution with no polymer network using both a commercial capillary array electrophoresis (CAE) system and a PMMA microchip replicated via hot-embossing with only a Tris-based running buffer containing additives to suppress the EOF. Typical analysis times for LDR-dt were 11 min using the CAE system and as low as 85 s for the PMMA microchips. With resolution comparable to traditional gel-based CAE, FSCE along with microchip electrophoresis decreased the separation time by more than a factor of 40.
  Electrophoresis 01/2009; 29(23):4751-60.
• Article: Investigating DNA migration in pulsed fields using a miniaturized FIGE system.

  Xiaojia Chen, Victor M Ugaz
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  ABSTRACT: PFGE is a well-established technique for fractionation of DNA fragments ranging from kilobases to megabases in length. But many of these separations require an undesirable combination of long experiment times (often approaching tens of hours) and application of high voltages (often approaching tens of kV). Here, we present a simple miniaturized FIGE apparatus capable of separating DNA fragments up to 32.5 kb in length within 3 h using a modest applied potential of 20 V. The device is small enough to be imaged under a fluorescence microscope, permitting the migrating DNA bands to be observed during the course of the separation run. We use this capability to investigate how separation performance is affected by parameters including the ratio of forward and backward voltage, pulse time, and temperature. We also characterize the dependence of DNA mobility on fragment size N, and observe a scaling in the vicinity of N(-0.5) over the size range investigated. The high speed, low power consumption, and simple design of this system may help enable future studies of DNA migration in PFGE to be performed quickly and inexpensively.
  Electrophoresis 01/2009; 29(23):4761-7.
• Article: DNA sequencing by microchip electrophoresis using mixtures of high- and low-molar mass poly(N,N-dimethylacrylamide) matrices.

  Daniel G Hert, Christopher P Fredlake, Annelise E Barron
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  ABSTRACT: Previous studies have reported that mixed molar mass polymer matrices show enhanced DNA sequencing fragment separation compared with matrices formulated from a single average molar mass. Here, we describe a systematic study to investigate the effects of varying the amounts of two different average molar mass polymers on the DNA sequencing ability of poly(N,N-dimethylacrylamide) (pDMA) sequencing matrices in microfluidic chips. Two polydisperse samples of pDMA, with weight-average molar masses of 3.5 MDa and 770 kDa, were mixed at various fractional concentrations while maintaining the overall polymer concentration at 5% w/v. We show that although the separation of short DNA fragments depends strongly on the overall solution concentration of the polymer, inclusion of the high-molar mass polymer is essential to achieve read lengths of interest (>400 bases) for many sequencing applications. Our results also show that one of the blended matrices, comprised of 3% 3.5 MDa pDMA and 2% 770 kDa pDMA, yields similar sequencing read lengths (>520 bases on average) to the high-molar mass matrix alone, while also providing a fivefold reduction in zero-shear viscosity. These results indicate that the long read lengths achieved in a viscous, high-molar mass polymer matrix are also possible to achieve in a tuned, blended matrix of high- and low-molar mass polymers with a much lower overall solution viscosity.
  Electrophoresis 01/2009; 29(23):4663-8.
• Article: Difference gel electrophoresis

  J S Minden, S R Dowd, H E Meyer, K Stühler
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  ABSTRACT: Difference gel electrophoresis (DIGE) was invented to circumvent the inherent variability of 2-DE. This variability is a natural consequence of separating thousands of proteins over a large space, such as a 15 x 20 cm slab of polyacrylamide gel. The originators of 2-DE envisioned being able to compare cancerous cells and normal cells to understand what makes these cells different. Gel-to-gel variability made this an extremely difficult task. We reasoned that if both samples could be run on the same gel, then the inherent variability would be obviated. Thus, we created matched sets of fluorescent dyes that allows one to compare two or three protein samples on a single gel. In the 12 years since the description of DIGE first appeared in Electrophoresis, this founding paper has been cited over 660 times. This review highlights some of the improvements and applications of DIGE. We hope these examples are illustrative of what has been done and where the field is headed.
  Electrophoresis 01/2009; 30 Suppl 1:S156-S161.
• Article: Strategies for enhancing the speed and integration of microchip genetic amplification.

  Viet N Hoang, Govind V Kaigala, Alexey Atrazhev, Linda M Pilarski, Christopher J Backhouse
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  ABSTRACT: In this work, we explore the use of methods that allow a significant acceleration of genetic analysis within microchips fabricated from low thermal conductivity materials such as glass or polymers. Although these materials are highly suitable for integrating a number of genetic analysis techniques onto lab-on-a-chip devices, their low thermal conductivity limits the rate at which heat can be transferred and hence lowers the speed of thermal cycling. However, short thermal cycling times are the key to bringing PCR to clinical point-of-care applications. Although shrinking the PCR reaction chamber volume can increase the speed of thermal cycling, this strategy is not always suitable, particularly when dealing with clinical samples with low analyte concentrations. In the present work, we combine two alternate strategies for decreasing the time required to perform PCR: implementing a heat sink and optimizing the PCR protocol. First, the heat sink substantially reduces the thermal resistance opposing heat dissipation into the ambient environment, and eliminates the parasitic thermal capacitance of the regions in the microchip that do not require heating. The low thermal conductivity of glass is used to our advantage to design the heat-sink placement to achieve fast thermal transitions while maintaining low power consumption. Second, we explore the application of two-stage PCR to provide a further reduction in the time required to perform genetic amplification by merging the annealing and extension stages of the commonly used three-stage PCR approach. In combination, we reduce the time required to perform thermal cycling by roughly a factor of 3 while improving the temperature control.
  Electrophoresis 01/2009; 29(23):4684-94.
• Article: New applications and developments in the use of multiplex ligation-dependent probe amplification.

  Piotr Kozlowski, Anna J Jasinska, David J Kwiatkowski
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  ABSTRACT: Multiplex ligation-dependent probe amplification (MLPA) is a commonly used technique for determining relative DNA sequence dosage (or copy number) in a complex DNA sample. Originally MLPA was designed as a copy number analysis tool for detecting disease-causing genomic mutations and has been successfully applied in the testing and identification of hundreds of genomic mutations in numerous genes including DMD, BRCA1, NF1, and TSC2. More recently, several modifications of the original technique have been implemented. Arguably the most important enhancement of MLPA has been probe generation by chemical synthesis, enabling the facile creation of novel probe sets for any desired application. Other newer applications of MLPA include methylation status determination, copy number analysis in segmentally duplicated regions, expression profiling, and transgene genotyping. MLPA has a potential major role in the analysis of common copy number variation in genome-wide association analyses, which may be enhanced by future improvements to increase throughput and lower costs, such as array-MLPA.
  Electrophoresis 01/2009; 29(23):4627-36.
• Article: Identification of mouse inbred strains through mitochondrial DNA single-nucleotide extension.

  Ana Goios, Leonor Gusmão, Ana Mafalda Rocha, Amélia Fonseca, Luísa Pereira, Molly Bogue, António Amorim
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  ABSTRACT: Inbred mouse strains are used as model organisms for biomedical research in laboratories throughout the world. The most widely used of these strains had their genome sequenced recently, and phylogenetic studies have been performed, namely, based on mitochondrial DNA (mtDNA). This has allowed determining that few polymorphisms distinguish the mtDNAs of the common inbred strains, but a high number of differences are observed among the wild-derived strains. Taking advantage of these observations, we here present a single base extension typing strategy that, with only a pair of multiplex reactions, allows the distinction between common inbred and wild-derived mice strains, and provides the identification of ten different common inbred and six wild-derived mice mtDNA haplotypes. Given that all the animals inside a strain present the same mtDNA, this strategy allows a rapid identification of the strains without the need for probability calculations. We further test this approach in an island population of wild mice, which provides both an indication on its applicability in wild mice, and a comparison of evolutionary processes on inbred and wild mice that are restricted to a limited space. Rapid genotyping methods that allow the distinction of the different strains are important for both the distinction of materials such as tissue and cell collections and to identify the origin of new strains. Moreover, it may also prove valuable in forensic identification of materials collected in laboratory accidents, as well as in cases of scientific fraud.
  Electrophoresis 01/2009; 29(23):4795-802.
• Article: High-throughput analysis of DNA fragments using a miniaturized CE system combined with a slotted-vial array sample introduction system.

  Xiao-Feng Fan, Qi Li, Shi-Li Wang, Zhang-Run Xu, Wen-Bin Du, Qun Fang, Zhao-Lun Fang
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  ABSTRACT: An automated nanoliter sample introduction system was combined to a liquid-core waveguide (LCW)-based microfluidic CE system for high-throughput analysis of DNA fragments. The main component of the sample introduction system was a motor-driven plate, on which a circular array of bottom-slotted vials containing sample/buffer solutions was placed. A 7 cm-long LCW capillary served as both the sample probe and separation channel. The inlet terminal of the capillary could pass through the slots of the vials for electrokinetic sample introduction, and the capillary outlet was immersed in the solution of a reservoir, behind which a PMT facing directly to the outlet was positioned. A diode laser was used as excitation source for LCW LIF detection. Performance of the system was demonstrated through the separation of DNA fragments. Baseline separation was achieved for all 11 fragments of PhiX174-HaeIII digest DNA with a throughput of 33/h. Theoretical plate number for 603 bp fragment was 7.3x10(6)/m, corresponding to a plate height 0.14 microm. The detection limitation for 603 bp fragment was 0.4 ng/microL with a precision of 2.2% RSD for the peak height. Automated sample changing and introduction were achieved with only 0.3 nL gross sample consumption for each cycle.
  Electrophoresis 01/2009; 29(23):4733-8.
• Source

  Available from: stanford.edu

  Article: Thermoresponsive N-alkoxyalkylacrylamide polymers as a sieving matrix for high-resolution DNA separations on a microfluidic chip.

  Brian E Root, Mallory L Hammock, Annelise E Barron
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  ABSTRACT: In recent years, there has been an increasing demand for a wide range of DNA separations that require the development of materials to meet the needs of high resolution and high throughput. Here, we demonstrate the use of thermoresponsive N-alkoxyalkylacrylamide polymers as a sieving matrix for DNA separations on a microfluidic chip. The viscosities of the N-alkoxyalkylacrylamide polymers are more than an order of magnitude lower than that of a linear polyacrylamide (LPA) of corresponding molecular weight, allowing rapid loading of the microchip. At 25 degrees C, N-alkoxyalkylacrylamide polymers can provide improved DNA separations compared with LPA in terms of reduced separation time and increased separation efficiency, particularly for the larger DNA fragments. The improved separation efficiency in N-alkoxyalkylacrylamide polymers is attributed to the peak widths increasing only slightly with DNA fragment size, while the peak widths increase appreciably above 150 bp using an LPA matrix. Upon elevating the temperature to 50 degrees C, the increase in viscosity of the N-alkoxyalkylacrylamide solutions is dependent upon their overall degree of hydrophobicity. The most hydrophobic polymers exhibit a lower critical solution temperature below 50 degrees C, undergoing a coil-to-globule transition followed by chain aggregation. DNA separation efficiency at 50 degrees C therefore decreases significantly with increasing hydrophobic character of the polymers, and no separations were possible with solutions with a lower critical solution temperature below 50 degrees C. The work reported here demonstrates the potential for this class of polymers to be used for applications such as PCR product and RFLP sizing, and provides insight into the effect of polymer hydrophobicity on DNA separations.
  Electrophoresis 01/2009; 29(23):4677-83.
• Source

  Available from: martytornil.com

  Article: Hydrophobically modified polyacrylamide block copolymers for fast, high-resolution DNA sequencing in microfluidic chips.

  Ryan E Forster, Thomas N Chiesl, Christopher P Fredlake, Corin V White, Annelise E Barron
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  ABSTRACT: By using a microfluidic electrophoresis platform to perform DNA sequencing, genomic information can be obtained more quickly and affordably than the currently employed capillary array electrophoresis instruments. Previous research in our group has shown that physically cross-linked, hydrophobically modified polyacrylamide matrices separate dsDNA more effectively than linear polyacrylamide (LPA) solutions. Expanding upon this work, we have synthesized a series of LPA-co-dihexylacrylamide block copolymers specifically designed to electrophoretically sequence ssDNA quickly and efficiently on a microfluidic device. By incorporating very small amounts of N,N-dihexylacrylamide, a hydrophobic monomer, these copolymer solutions achieved up to approximately 10% increases in average DNA sequencing read length over LPA homopolymer solutions of matched molar mass. Additionally, the inclusion of the small amount of hydrophobe does not significantly increase the polymer solution viscosities, relative to LPA solutions, so that channel loading times between the copolymers and the homopolymers are similar. The resulting polymer solutions are capable of providing enhanced sequencing separations in a short period of time without compromising the ability to rapidly load and unload the matrix from a microfluidic device.
  Electrophoresis 01/2009; 29(23):4669-76.
• Article: The next generation of DNA profiling--STR typing by multiplexed PCR--ion-pair RP LC-ESI time-of-flight MS.

  Florian Pitterl, Harald Niederstätter, Gabriela Huber, Bettina Zimmermann, Herbert Oberacher, Walther Parson
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  ABSTRACT: For the first time a multiplexed PCR approach suitable for mass spectrometric STR allele identification is presented. Thirteen forensically important STR markers (vWA, D21S11, D3S1358, D16S539, D8S1179, D7S820, D13S317, D5S818, TPOX, CSF1PO, D2S441, D10S1248, and D22S1045) and the gender typing locus amelogenin were simultaneously amplified. Ion-pair reversed-phase high-performance liquid chromatography electrospray-ionization time-of-flight mass spectrometry (ICEMS) was applied for genotyping, and allowed for highly efficient characterization of multiple PCR amplicons. Compared with electrophoretic sizing ICEMS enabled for the simultaneous detection of length and nucleotide variations. Thus, the obtained amount of biological information present within STR profiles was significantly increased even though the compatibility of typing results with electrophoretically generated data(bases) was maintained. Other advantages of the ICEMS platform included the abandonment of internal size standards, allelic ladders, and any kind of spectral calibration. The 14-plex PCR was tailor-made for ICEMS analysis by designing primer pairs that bind close to the repeat region, by using a proof reading polymerase for amplification, and by implementing molecular mass modifiers for prevention of molecular mass overlaps. In a series of experiments, the performance of the multiplexed PCR-ICEMS assay was evaluated. The ICEMS-based DNA profiling assay was found to be competitive regarding detection sensitivity and analyzability of degraded and casework samples with commercially available electrophoretic typing approaches, which suggests that multiplexed PCR-ICEMS assays could represent a valuable tool for (forensic) genetics.
  Electrophoresis 01/2009; 29(23):4739-50.
• Source

  Available from: stanford.edu

  Article: Advantages and limitations of next-generation sequencing technologies: a comparison of electrophoresis and non-electrophoresis methods.

  Daniel G Hert, Christopher P Fredlake, Annelise E Barron
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  ABSTRACT: The reference human genome provides an adequate basis for biological researchers to study the relationship between genotype and the associated phenotypes, but a large push is underway to sequence many more genomes to determine the role of various specificities among different individuals that control these relationships and to enable the use of human genome data for personalized and preventative healthcare. The current electrophoretic methodology for sequencing an entire mammalian genome, which includes standard molecular biology techniques for genomic sample preparation and the separation of DNA fragments using capillary array electrophoresis, remains far too expensive ($5 million) to make genome sequencing ubiquitous. The National Human Genome Research Institute has put forth goals to reduce the cost of human genome sequencing to $100,000 in the short term and $1000 in the long term to spur the innovative development of technologies that will permit the routine sequencing of human genomes for use as a diagnostic tool for disease. Since the announcement of these goals, several companies have developed and released new, non-electrophoresis-based sequencing instruments that enable massive throughput in the gathering of genomic information. In this review, we discuss the advantages and limitations of these new, massively parallel sequencers and compare them with the currently developing next generation of electrophoresis-based genetic analysis platforms, specifically microchip electrophoresis devices, in the context of three distinct types of genetic analysis.
  Electrophoresis 01/2009; 29(23):4618-26.
• Source

  Available from: stanford.edu

  Article: Polymer systems designed specifically for DNA sequencing by microchip electrophoresis: a comparison with commercially available materials.

  Christopher P Fredlake, Daniel G Hert, Brian E Root, Annelise E Barron
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  ABSTRACT: Electrophoresis-based DNA sequencing is the only proven technology for the de novo sequencing of large and complex genomes. Miniaturization of capillary array electrophoresis (CAE) instruments can increase sequencing throughput and decrease cost while maintaining the high quality and long read lengths that has made CAE so successful for de novo sequencing. The limited availability of high-performance polymer matrices and wall coatings designed specifically for microchip-sequencing platforms continues to be a major barrier to the successful development of a commercial microchip-sequencing instrument. It has been generally assumed that the matrices and wall coatings that have been developed for use in commercial CAE instruments will be able to be implemented directly into microchip devices with little to no change in sequencing performance. Here, we show that sequencing matrices developed specifically for microchip electrophoresis systems can deliver read lengths that are 150-300 bases longer on chip than some of the most widely used polymer-sequencing matrices available commercially. Additionally, we show that the coating ability of commercial matrices is much less effective in the borosilicate chips used in this study. These results lead to the conclusion that new materials must be developed to make high-performance microfabricated DNA-sequencing instruments a reality.
  Electrophoresis 01/2009; 29(23):4652-62.