Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.     

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.     

Ultrathin-layer gel electrophoresis of biopolymers

Emerging need for large-scale, high-resolution analysis of biopolymers, such as DNA sequencing polymerase chain reaction, (PCR) product sizing, single nucleotide polymorphism (SNP) hunting and analysis of protein molecules necessitated the development of automated and high-throughput gel electrophoresis based methods enabling rapid, high-performance separations in a wide molecular weight range. Scaling down electric field mediated separation processes supports higher throughput due to the applicability of higher voltages, thus speeding up analysis time. Indeed, efforts in miniaturization resulted in faster, easier, less costly and more convenient analyses, fulfilling the needs of the emerging biotechnology industry for microscale and massively parallel assays. The two primary approaches in miniaturizing electrophoresis dimensions are the capillary and microslab formats. This latter one evolved towards ultrathin-layer gel electrophoresis which is, except from the thickness of the separation platform, slightly in the upper side of the scale, resulting in considerably easier handling. Ultrathin-layer gel electrophoresis combines the advantages of conventional slab-gel electrophoresis (multilane format) and capillary gel electrophoresis (rapid, high-efficiency separations). It is readily automated, automatic versions of it have been extensively used for large-scale DNA sequencing in the Human Genome Project and more recently became popular in high throughput DNA fragment analysis. Ultrathin-layer techniques are the first step towards the wider use of electrophoresis microchips in perfecting a user-friendly interface between the user and the microdevice.     

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.     

Miniaturization of Slab Gel Electrophoresis Using Thermal Lens Microscope and its Application to Genetic Diagnostics

Slab gel electrophoresis is the most widely used separation method for DNA fragments, proteins and carbohydrates, and miniaturization of this process is expected to provide fast, inexpensive and convenient analyses. However, two problems concerning the miniaturization of gel electrophoresis have to be solved:the separation performance and spatial resolution of the detector. We demonstrated that the separation performance was improved by using a discontinuous gel in which a concentrating gel was used to stack the sample to a sharp band, and using thermal lens microscope (TLM), which is highly sensitive and has a spatial resolution of micron level even in light scattering matrices as a gel, such sharpened separated bands were successfully detected. In this paper, we developed a miniaturized slab gel electrophoresis apparatus, demonstrated high speed separation of DNA fragments, and applied it to genetic diagnosis of coronary heart disease.     

Membrane-mediated ultrafast restriction digestion and subsequent rapid gel microchip electrophoresis of DNA

Ultrafast, membrane-mediated restriction digestion of DNA molecules followed by rapid gel microchip electrophoresis of the resulting fragments is described. Combination of restriction endonuclease digestion on small pore-size microfibrous membranes with sample loading and electrophoresis analysis in a multilane (up to 96) format resulted in very fast restriction digest based microscale DNA analysis. Complete digestion of several nanogram target DNA was accomplished on the microporous membrane at room temperature just in a few minutes with a single or a combination of various restriction enzymes, using only submicroliter quantities of samples and reagents. The reaction mixture containing membrane also served as sample loading device for the subsequent gel microchip electrophoresis based analysis. This work establishes methods for high-speed, high-throughput DNA analysis, featuring extremely low sample and reagent consumption, and fast restriction digestion in combination with sample loading and rapid gel microchip analysis of the resulting fragments. The entire restriction digestion, sample loading and electrophoresis analysis process required less than 20 min.     

A simple capillary gel electrophoresis approach for efficient and reproducible DNA separations. Analysis of genetically modified soy and maize

It is generally assumed that in order to achieve suitable separations of DNA fragments, capillary gel electrophoresis (CGE)-coated capillaries should be used. In this work, a new method is presented that allows to obtain reproducible CGE separations of DNA fragments using bare fused-silica capillaries without any previous coating step. The proposed method only requires: (i) a capillary washing with 0.1 M hydrochloric acid between injections and (ii) a running buffer composed of Tris-phosphate-ethylenediamine tetraacetic acid (EDTA) and 4.5% of 2-hydroxyethyl cellulose (HEC) as sieving polymer. The use of this new CGE procedure gives highly resolved and reproducible separations of DNA fragments ranging from 50 to 750 bp. The separation of these DNA fragments is accomplished in less than 30 min with efficiencies up to 1.7 x 10(6) plates/m. Reproducibility values of migration times (given as %RSD) for the analyzed DNA fragments are better than 1.0% (n = 4) for the same day, 2.2% (n = 16) for four different days, and 2.3% (n = 16) for four different capillaries. The usefulness of this separation method is demonstrated by detecting genetically modified maize and genetically modified soy after DNA amplification by PCR. This new CGE procedure together with LIF as detector provides sensitive analysis of 0.9% of Bt11 maize, Mon810 maize, and Roundup Ready soy in flours with S/ N up to 542. These results demonstrate the usefulness of this procedure to fulfill the European regulation on detection of genetically modified organisms in foods.     

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)     

Cross-linked polyacrylamide gel electrophoresis of single-stranded DNA for microfabricated genomic analysis systems

Microfabricated devices are poised to offer inexpensive self-contained alternatives to conventional benchtop-scale laboratory equipment for performing a variety of important DNA analysis assays. In order to realize the dramatic cost savings possible through photolithographic fabrication techniques, these devices must occupy an extremely compact footprint on the silicon wafer. This requirement implies that electrophoretic separations must be performed over ultrashort distances. Employing cross-linked polyacrylamide gels in place of conventional uncross-linked sieving media offers a convenient strategy to achieve this goal. In this paper, we show how the increased resolving power offered by cross-linked polyacrylamide gels, along with improved sample injection techniques, can be exploited to enhance separation performance in microscale systems. We use these techniques to perform high-resolution gel electrophoresis of single-stranded DNA fragments in microfabricated devices over separation distances of 1.5 cm or less. The results presented here are in agreement with theoretical predictions and suggest that it is possible to perform DNA sequencing on compact microchips. More importantly, the separation performance demonstrated in this work is already more than adequate to perform a number of important genomic assays imposing less stringent resolution requirements than sequencing. Successfully adapting even a few of these assays to the microdevice format has the potential to provide a new generation of inexpensive and portable devices suitable for direct end-user applications.     

Multiplexed DNA sizing by capillary electrophoresis using entangled polymer solutions and diode array detection

We developed a method for the analysis of multiplexed double-stranded DNA (dsDNA) samples complexed to various intercalating dyes using entangled polymer solution. A commercial single-column capillary electrophoresis (CE) instrument with diode array detection was used for multiplexed detection of DNA samples by addition of intercalating fluorescent molecules. A Phi X174HinfI and a pGEM DNA ladder (1 mg/mL) were used for the electrophoretic separation of dsDNA fragments ranging in size from 24 to 726 and 36 to 2645 bp, respectively. The results suggested that simultaneous electrophoretic separation of different DNA ladders multiplexed with different dyes could be performed in the same capillary yielding fast DNA sizing separations. CE analysis, which is often overpowered by slab gel in sample throughput, could now overcome this disadvantage by allowing multiplexed sample analysis in a fraction of the time needed for slab gel analysis. The separation efficiency of stained DNA molecules with both dyes were dramatically improved with buffers containing a large cation such as tetrapentylammonium ion (Npe(4) (+)) as the only cation in the buffer.     

Separating DNA sequencing fragments without a sieving matrix.

The possibility of separating appropriately labeled DNA fragments using free-flow capillary electrophoresis was predicted a few years ago based on simple theoretical arguments. Free-flow separation of double-stranded DNA (dsDNA) fragments in the 100-1000 base range was later demonstrated using a streptavidin label. In this article, we now report that end-labeled free-flow electrophoresis (ELFSE) can also be used to sequence single-stranded DNA (ssDNA). The first 100 bases of a DNA sequencing reaction were read without any sieving matrix when fractionated streptavidin was added to the 5'-end of the ssDNA fragments. These separations required only 18 min and did not require coated capillaries. An analysis of the results indicates that sample injection, analyte-wall interactions and thermal diffusion are the limiting factors at this time. Extrapolating from our data, we predict that several hundred bases could be sequenced in less than 30 min with the proper conditions. ELFSE thus offers an attractive potential alternative to polymer solutions for DNA sequencing in capillaries and microchips.     

Fast parallel detection of feline panleukopenia virus DNA by multi‐channel microchip electrophoresis with programmed step electric field strength

A multi‐channel microchip electrophoresis using a programmed step electric field strength (PSEFS) method was investigated for fast parallel detection of feline panleukopenia virus (FPV) DNA. An expanded laser beam, a 10× objective lens, and a charge‐coupled device camera were used to simultaneously detect the separations in three parallel channels using laser‐induced fluorescence detection. The parallel separations of a 100‐bp DNA ladder were demonstrated on the system using a sieving gel matrix of 0.5% poly(ethylene oxide) (M_r = 8 000 000) in the individual channels. In addition, the PSEFS method was also applied for faster DNA separation without loss of resolving power. A DNA size marker, FPV DNA sample, and a negative control were simultaneously analyzed with single‐run and one‐step detection. The FPV DNA was clearly distinguished within 30 s, which was more than 100 times faster than with conventional slab gel electrophoresis. The proposed multi‐channel microchip electrophoresis with PSEFS was demonstrated to be a simple and powerful diagnostic method to analyze multiple disease‐related DNA fragments in parallel with high speed, throughput, and accuracy.     

Denaturing gradient-based two-dimensional gene mutation scanning in a polymer microfluidic network

An integrated two-dimensional (2-D) DNA separation platform, combining standard gel electrophoresis with temperature gradient gel electrophoresis (TGGE) on a polymer microfluidic chip, is reported. Rather than sequentially sampling DNA fragments eluted from standard gel electrophoresis, size-resolved fragments are simultaneously electrokinetically transferred into an array of orthogonal microchannels and screened for the presence of sequence heterogeneity by TGGE in a parallel and high throughput format. A bulk heater assembly is designed and employed to externally generate a temporal temperature gradient along an array of TGGE channels. Extensive finite element modeling is performed to determine the optimal geometries of the microfluidic network for minimizing analyte band dispersion caused by interconnected channels in the network. A pH-mediated on-chip analyte stacking strategy is employed prior to the parallel TGGE separations to further reduce additional band broadening acquired during the electrokinetic transfer of DNA fragments between the first and second separation dimensions. A comprehensive 2-D DNA separation is completed in less than 5 min for positive detection of single-nucleotide polymorphisms in multiplex PCR products that vary in size and sequence.     

DNA fragment analysis by an affordable multiple-channel capillary electrophoresis system

We are demonstrating a cost-effective multichannel capillary electrophoresis system for a high-efficiency double-stranded DNA (dsDNA) fragments analysis. This bench-type high-performance DNA analysis (HDA) system uses fluorescence-type detection with inexpensive solid-state light sources and nonmoving integrated emission collection micro-optics. DNA samples are analyzed simultaneously by using a multiple usage and disposable multicapillary cartridge, which contains integrated capillary channels, optical fibers and an integrated sieving gel reservoir. Using commercially available dsDNA size markers as indicators, the HDA system provides high resolving power in 7 min separations. The system can hold a total of 192 samples in two 96-well polymerase chain reaction (PCR) plates, which can be automatically analyzed within 2.5 h. This affordable system can be used in laboratories to replace slab gel electrophoresis for routine and high-throughput dsDNA analysis.     

Rapid fabrication of a poly(dimethylsiloxane) microfluidic capillary gel electrophoresis system utilizing high precision machining

In this work, we demonstrate a rapid protocol to address one of the major barriers that exists in the fabrication of chip devices, creating the micron-sized structures in the substrate material. This approach makes it possible to design, produce, and fabricate a microfluidic system with channel features >10 microm in poly(dimethylsiloxane)(PDMS) in under 8 hours utilizing instrumentation common to most machine shops. The procedure involves the creation of a master template with negative features, using high precision machining. This master is then employed to create an acrylic mold that is used in the final fabrication step to cast channel structures into the PDMS substrate. The performance of the microfluidic system prepared using this fabrication procedure is evaluated by constructing a miniaturized capillary gel electrophoresis (micro-CGE) system for the analysis of DNA fragments. Agarose is utilized as the sieving medium in the micro-CGE device and is shown to give reproducible (RSD (n= 34) approximately 5.0%) results for about 34 individual separations without replenishing the gel. To demonstrate the functionality of the micro-CGE device, a DNA restriction ladder (spanning 26-700 base pairs) and DNA fragments generated by PCR are separated and detected with laser-induced fluorescence (LIF). The microchip is shown to achieve a separation efficiency of 2.53 x 10(5) plates m(-1).     

DNA fingerprinting of Vibrio cholerae and Aeromonas species by pulsed-field minigel electrophoresis

DNA molecules of Vibrio cholerae and Aeromonas species were prepared by incubating immobilized cells for 4 and 2 h, respectively, with a nonenzymatic solution that contains chemical reagents only (NDSUPlus). This method gave results as reproducible as the enzymatic one that uses proteinase K, and rendered DNA molecules suitable for fingerprinting by mini-CHEF electrophoresis. As rapid DNA separations at high electric field are achieved in mini-CHEF chamber with low heat evolution, DNA restriction fragments were separated in 5 h at 10 V/cm in a single resolution window. Then, fragment separations in three resolution windows were done in 15 h. This time is shorter than the one needed by the large CHEF chamber for resolving fragments in a single resolution window. Three windows permitted to include larger numbers of restriction fragments in the calculation of isolate similarities. Both sample preparation and mini-CHEF electrophoresis may represent an alternative for performing massive epidemiological studies of V. cholerae and Aeromonas species.     

Simultaneously Sizing and Quantitating Zeptomole‐Level DNA at High Throughput in Free Solution

Determining the sizes and measuring the quantities of DNA molecules are fundamental tasks in molecular biology. DNA sizes are usually evaluated by gel electrophoresis, but this method cannot simultaneously size and quantitate a DNA at low zeptomole (zmol) levels of concentration. We have recently developed a new technique, called bare‐narrow‐capillary/hydrodynamic‐chromatography or BaNC‐HDC, for resolving DNA based on their sizes without using any sieving matrices. In this report, we utilize BaNC‐HDC for measuring the sizes and quantities of DNA fragments at zmol to several‐molecule levels of concentration. DNA ranging from a few base pairs to dozens of kilo base pairs are accurately sized and quantitated at a throughput of 15 samples per hour, and each sample contains dozens of DNA strands of different lengths. BaNC‐HDC can be a cost‐effective means and an excellent tool for high‐throughput DNA sizing and quantitation at extremely low quantity level.     

The Use of Polystyrene Gels in Preparative Recycle-HPLC for the Separation of Structurally Related Molecules

Abstract

This paper deals with the use of polystyrene gels in high performance liquid chromatography for preparative separations of small molecules with very similar structures. The preparative chromatograph, equipped with a recycle device and made in our laboratory, is described. The column set, consisted of three columns packed with low porosity polystyrene gel. The mobile phase was diisopropylether. Three particle sizes (10μ, 20μ, 50μ) were studied for improving the preparative performance. We checked the very high efficiency of our recycling system versus sample load. High sample loadings (up to 100 grams) can be injected when the separation does not require a large number of plates but typical sample loads are 10–20 grams for high performance separations. Some examples of separation of diastereoisomers, configurational isomers or related structure molecules are given. The advantages of our preparative system are discussed.     

Gel immobilization of acrylamide-modified single-stranded DNA template for pyrosequencing

A novel two-step process was developed to prepare ssDNA templates for pyrosequencing. First, PCR-amplified DNA templates modified with an acrylamide group and acrylamide monomers were copolymerized in 0.1 M NaOH solution to form polyacrylamide gel spots. Second, ssDNA templates for pyrosequencing were prepared by removing electrophoretically unbound complementary strands, unmodified PCR primers, inorganic pyrophosphate (PPi), and excess deoxyribonucleotides under alkali conditions. The results show that the 3-D polyacrylamide gel network has a high immobilization capacity and the modified PCR fragments are efficiently captured. After electrophoresis, gel spots copolymerized from 10 microL of the crude PCR products and the acrylamide monomers contain template molecules on the order of pmol, which generate enough light to be detected by a regular photomultiplier tube. The porous structure of gel spots facilitated the fast transportation of the enzyme, dNTPs and other reagents, and the solution-mimicking microenvironment guaranteed polymerase efficiency for pyrosequencing. Successful genotyping from the crude PCR products was demonstrated. This method can be applied in any laboratory; it is cheap, fast, simple, and has the potential to be incorporated into a DNA-chip format for high-throughput pyrosequencing analysis.     

Effects of sequence and structure in the separation of nucleic acids using ion pair reverse phase liquid chromatography

Ion pair reverse phase high performance liquid chromatography on non-porous alkylated poly(styrene-divinylbenzene) particles enables the high resolution separation of double stranded DNA fragments. To further understand the separation mechanisms involved in ion pair reverse phase liquid chromatography we have analysed the effects of curved or "bent" DNA fragments with respect to their separation using both gel electrophoresis and ion pair reverse phase liquid chromatography. Size dependent separations of curved DNA fragments that migrate anomalously during gel electrophoresis were observed using ion pair reverse phase liquid chromatography. To further study the sequence effect and resulting changes in hydrophobicity of the duplex DNA, PCR fragments were generated that contain uracil in place of thymine. The resulting fragments were shown to elute with shorter retention times, demonstrating that sequence-specific effects can alter the retention of duplex DNA. The study was extended to the investigation of non-canonical B-DNA structures (Holliday junctions) under various chromatographic conditions, demonstrating that the coaxial stacking of the helices in such structures, in the presence of magnesium causes a change in retention.