DNA Capillary Electrophoresis Research Articles

Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip for forensic human identification

A fully integrated microdevice and process for forensic short tandem repeat (STR) analysis has been developed that includes sequence-specific DNA template purification, polymerase chain reaction (PCR), post-PCR cleanup and inline injection, and capillary electrophoresis (CE). Fragmented genomic DNA is hybridized with biotin-labeled capture oligos and pumped through a fluidized bed of magnetically immobilized streptavidin-coated beads in microchannels where the target DNA is bound to the beads. The bead-DNA conjugates are then transferred into a 250 nL PCR reactor for autosomal STR amplification using one biotin and one fluorescence-labeled primer. The resulting biotin-labeled PCR products are electrophoretically injected through a streptavidin-modified capture gel where they are captured to form a concentrated and purified injection plug. The thermally released sample plug is injected into a 14 cm long CE column for fragment separation and detection. The DNA template capture efficiency provided by the on-chip sequence-specific template purification is determined to be 5.4% using K562 standard DNA. This system can produce full 9-plex STR profiles from 2.5 ng input standard DNA and obtain STR profiles from oral swabs in about 3 hours. This fully integrated microsystem with sample-in-answer-out capability is a significant advance in the development of rapid, sensitive, and reliable micro-total analysis systems for on-site human identification.

A simple method of fabricating mask-free microfluidic devices for biological analysis.

We report a simple, low-cost, rapid, and mask-free method to fabricate two-dimensional (2D) and three-dimensional (3D) microfluidic chip for biological analysis researches. In this fabrication process, a laser system is used to cut through paper to form intricate patterns and differently configured channels for specific purposes. Bonded with cyanoacrylate-based resin, the prepared paper sheet is sandwiched between glass slides (hydrophilic) or polymer-based plates (hydrophobic) to obtain a multilayer structure. In order to examine the chip's biocompatibility and applicability, protein concentration was measured while DNA capillary electrophoresis was carried out, and both of them show positive results. With the utilization of direct laser cutting and one-step gas-sacrificing techniques, the whole fabrication processes for complicated 2D and 3D microfluidic devices are shorten into several minutes which make it a good alternative of poly(dimethylsiloxane) microfluidic chips used in biological analysis researches.

A compact LED-based module for DNA capillary electrophoresis

A setup consisting of a bifurcated optical fiber made from high-transmission fused-silica cores with relatively high numerical apertures (NA=0.22), high-power cyan light-emitting diodes (LEDs) and Peltier cooling elements controlled by a proportional–integrative–derivative (PID) module is introduced to replace bulky, power- consuming lasers conventionally used in laser induced fluorescence (LIF) microchip capillary electrophoresis (μCE). The output fiber beam size, divergence, power distribution and power stability over time are documented. A modified epifluorescence microscope arrangement is used in conjunction with a compact fixed spectrometer aligned with a cooled charge-coupled device (CCD) camera for added sensitivity. Fluorescent dyes such as fluorescein, 6-carboxyfluorescein (6-FAM) and rhodamine B can be detected in cyclic olefin copolymer (COC) and glass microchannels at submicromolar levels. A single-stranded DNA oligonucleotide (10-mer) labeled with 6-FAM is also detected with reasonable signal-to-noise ratio when electrophoretically migrated at 100 V/cm. The compact LED excitation system presented herein will allow using capillary electrophoresis for DNA detection in compact mobile devices.

Spatially adaptive stationary wavelet thresholding for the denoising of DNA capillary electrophoresis signal

The spatially adaptive thresholding based on the stationary wavelet transform was applied to the noise removal in the signal of DNA separated and determined by capillary electrophoresis. The threshold is derived in a Bayesian framework, and previously used on the wavelet coefficients is the generalized Gaussian distribution. The threshold is simple and closed form, which is adaptive to each subband because it depends on data-driven estimates of the parameters. Using this strategy, the noise in the signal of the DNA-separated analysis by capillary electrophoresis could be removed adequately. Experimental results show that the proposed denoising method is effective, and the spatially adaptive thresholding yields a lower root-mean-square error than the universal thresholding.

Identification of PCR Products Using PNA Amphiphiles in Micellar Electrokinetic Chromatography

We present a method to identify single-stranded PCR products of varying lengths by hybridization of n-alkylated peptide nucleic acids (PNA amphiphiles) to the products, followed by separation with micellar electrokinetic chromatography (MEKC). These end-attached PNA amphiphiles (PNAA) partition to nonionic micelles in the running buffer (Triton X-100), linking the tagged DNA to the micellar drag-tag. This linkage shifts the electrophoretic mobility of a tagged component away from both untagged DNA and tagged DNA of different lengths. The mobility of the tagged DNA is established by its extent of partitioning to the micelle phase as well as its size relative to the attached micelle. A model is presented that can be used to determine the length of an unknown oligomer given an experimentally obtained mobility. We find that the collective action of micelles that transiently attach to the tagged DNA impart about the same hydrodynamic drag as covalently bound "drag-tags" of a similar size. With the use of the PNAA-MEKC method, PCR products of 88, 134, 216, and 447 bases are clearly resolved in less than 5 min. To our knowledge, this work represents the first use of surfactant micelles as drag-tags to separate DNA in capillary electrophoresis. Furthermore, the PNAA tag only attaches to DNA containing a target sequence, helping ensure that only the desired PCR products are analyzed.

PH effect on dynamic coating for capillary electrophoresis of DNA

A buffer consisting of tris(hydroxymethyl)aminomethane, 2-(N-moropholino)ethanesulfonic acid (Mes) and EDTA with constant ion strength was used to investigate the effect of buffer pH on the dynamic coating behavior of poly(N-isopropylacrylamide) (PNIPAM) for DNA separation. The atomic force microscopy (AFM) image illustrated that PNIPAM in lower-pH buffer was much more efficient in covering a silica wafer than that in higher-pH buffer. The coating performance of PNIPAM was also quantitatively analyzed by Fourier transform IR attenuated total reflectance spectroscopy and by measuring the electroosmotic flow (EOF). These results indicated that the stability of the dynamic coating was dependent on the pH of the sieving matrix and was improved by reducing the pH to the weak-acid range. The lower pH of the sieving buffer may induce the polymer more efficiently to adsorb on the capillary wall to suppress EOF and DNA-capillary wall interaction for DNA separation. The enhanced dynamic coating capacity of PNIPAM in lower-pH buffer may be attributed to the hydrogen bonds between the hydroxyl groups of the silica surface and the oxygen atom of the carbonyl groups of PNIPAM.

Designing polymer matrix for microchip-based double-stranded DNA capillary electrophoresis

Polyacrylamide (PAM) was used as a model polymer to build up an empirical model that relates polymer molecular weight, polymer concentration and solution viscosity. The desired random copolymers of acrylamide (AM) and N, N-dimethylacrylamide (DMA) used as DNA separation media for different specifications were synthesized under the guidance of the empirical model. The separation performances of rationally designed copolymers were tested in a 1.2 cm long separation channel, simulating microchip-based capillary electrophoresis. pBR322/ HaeIII digest was successfully separated with good separation resolution and fast speed. Validation of the sieving ability of our polymers was performed in the Agilent 2100 Bioanalyzer. The results of the 10 bp (base pair) DNA ladder separation demonstrate the potential of our approach for the sieving matrix in microchip-based electrophoresis.

Affinity capillary electrophoresis of DNA for detection of single-nucleotide polymorphisms and point mutations: Comprehensive study for optimization of the weak affinity

A single-stranded DNA and its point mutant can be separated with affinity capillary electrophoresis (ACE) in which an appropriate ligand DNA is used as a pseudo-stationary affinity phase. In this paper, we systematically examine the effects of ligand sequence, capillary temperature, and cation concentration on the ACE separation quality, which is quantitatively evaluated in terms of resolution and peak height. For fine tuning of the affinity, control of MgCl 2 concentration and insertion of a spacer sequence into the ligand DNA are more effective than control of the capillary temperature. For design of the ligand DNA, a simple strategy is proposed, based on the prediction of melting temperature. This strategy was tested with eleven different sample sequences. All of them were separated under the same conditions (250 μM MgCl 2 and 25 °C), and nine cases of them had satisfactory separation qualities.

Manipulation of Polymer Chain Entanglement and Self‐Assembly for DNA Capillary Electrophoresis

AbstractA conceptual model has been developed and tested experimentally on five different aspects, relating to the architecture and topology of the polymers making up the transient network that constitutes the separation matrix. The concept can be applied for DNA sequencing analysis and separations of short fragments double‐stranded DNA as well as oligonucleotides. Examples are provided to illustrate the success of the concept.

Association Behavior of PDMA-g-PMMA in Mixed Solvents and Its Application as a DNA Separation Medium

PDMA-g-PMMA (poly(N,N-dimethylacrylamide)-graft-poly(methyl methacrylate)) graft copolymers were synthesized by radical copolymerization of DMA (N,N-dimethylacrylamide) and vinyl-PMMA macromonomers with AIBN (2,2‘-azobis(isobutyronitrile)) as an initiator. The association behavior of high molecular weight graft copolymers of PDMA-g-PMMA with low graft density in methanol−TBE (tris(hydroxymethyl)aminomethane−boric acid−EDTA) buffer solvent mixtures has been investigated by laser light scattering. With changing solvent quality, the PMMA side chains could be associated. The association points acted as cross-linking points which effectively increased the molecular weight of the associated graft copolymers and also prevented the entangled backbone chains from sliding away against one another. This association behavior could be used to improve the quasi-network formation of such copolymers as better separation media in DNA capillary electrophoresis. pBR322/Hae III digest was successfully separated within 10 min in a 10 cm long capillary with one base pair (bp) resolution (123/124 bp). The methanol component in the solvent mixture affected not only the polymer conformation in solution but also the DNA conformation. The average hydrodynamic radius of DNA decreased from 76.2 nm in 1×TBE buffer to 43.7 nm in 30% methanol + 70% 1×TBE (volume fraction) mixed solvent, resulting in a corresponding change in electrophoretic mobility.

Matrix conditioning for lengthened capillary DNA sequencing

Capillary electrophoresis (CE) is currently the preferred format for both DNA sequencing and small DNA fragment analysis. The present study provides a simple revision of the procedure used for CE of DNA with a commercial DNA sequencing apparatus from Applied Biosystems. The revision is electrophoretic conditioning of the sieving matrix (typically POP-6) before sample injection. The effects of this preconditioning are revealed during subsequent analyses performed without replenishing the sieving matrix. The primary effect of preconditioning is to increase peak separations during a subsequent CE. The preconditioning has the following characteristics: (i) The effect on peak separation progressively increases as the preconditioning time increases to at least 6 h. (ii) The effect on peak separation scales approximately as the product of the preconditioning time and the magnitude of the electrical field (162 - 320 V/cm) during preconditioning. (iii) The preconditioning persists for more than 72 h at zero field. Preconditioning of the matrix substantially improves resolution of fragment analysis in the range of 700-2000 nucleotides. For DNA sequencing, the primary impact of preconditioning is, thus far, extension of the range of low-quality base calls at the end of sequence reading. Matrix preconditioning is a new factor to consider when interpreting data obtained by CE in polymer solutions. The mechanism of preconditioning is not yet known.

Capillary Electrophoresis of DNA

Capillary electrophoresis (CE) is an alternative to conventional slab gel electrophoresis for the separation of DNA fragments. CE offers a number of advantages over slab gel separations in terms of speed, resolution, sensitivity, and data handling. Separation times are generally only a few minutes and the DNA is detected either by UV absorption or by fluorescent labeling. The quantity of DNA required for separation is in the nanogram range. Single-base resolution can be obtained on fragments up to several hundred base pairs. In the presence of appropriate standards, fragments can be accurately sized based on relative electrophoretic mobility. A protocol for the analysis of synthetic oligonucleotides in a flowable matrix is described in this unit.

Polymer solutions and entropic-based systems for double-stranded DNA capillary electrophoresis and microchip electrophoresis.

We give an overview of recent development of low-viscosity polymer solutions and entropic trapping networks for double-stranded DNA (dsDNA) separations by conventional capillary electrophoresis and microchip electrophoresis. Theoretical models for describing separation mechanisms, commonly used noncross-linked polymer solutions, thermoresponsive (viscosity-adjustable) polymer solutions, and novel entropic trapping networks are included. The thermoresponsive polymer solutions can be loaded at one temperature into microchannels at lower viscosities, and used in separation at another temperature at entanglement threshold concentrations and higher viscosities. The entropic-based separations use only arrays of regular obstacles acting as size-separations and do not need viscous polymer solutions. These progresses have potential in integration to automated capillary and microfluidic chip systems, enabling better reusability of separation microchannels, much shorter DNA separation times, and higher reproducibility due to less matrix degradation.

Application of wavelet transforms and an approximate deconvolution method for the resolution of noisy overlapped peaks in DNA capillary electrophoresis.

A new procedure for resolving noisy overlapped peaks in DNA separations by capillary electrophoresis (CE) is developed. The procedure combines both a wavelet-based denoising method that effectively denoises the signal and a novel approximate deconvolution technique that resolves the fragment peaks and improves the ability to separate highly overlapped peaks early in the electrophoresis process. Different kinds of overlapped peaks with and without noise simulated by computer as well as some DNA experimental electropherograms were submitted to the new procedure. A second order differential operator with variable coefficients is applied to the entire electrophoresis signal at any given time and approximate deconvolutions of the individual Gaussian peaks are performed. The operator incorporates the effect of the superposition and gives exact annihilation in the neighborhood of each peak. Overlapped peaks with a resolution higher than 0.46 can be resolved directly. Also, the method can determine the peak components of signals with a signal to noise ratio higher than 1.4

Using DNA-binding proteins as an analytical tool.

We propose that DNA-binding proteins can be used as highly efficient and versatile tools in analyses of DNA, RNA, and proteins. This work reports assays applying specific affinity probes: hybridization probes for analyses of DNA and RNA, and aptamer probes for analyses of proteins. Both types of probes are single-stranded DNA. In affinity analyses, in general, the probe (P) binds to a target molecule (T), and the amounts of the probe-target complex (P.T) and unbound P are determined. Distinguishing between P and P.T can be achieved by electrophoretic separation. If the electrophoretic mobilities of P and P.T are close in gel-free media, which is always the case for hybridization analyses, separation typically requires the use of a sieving matrix. Here we utilized a single-stranded DNA binding protein (SSB) to facilitate highly efficient gel-free separation of P and P.T in capillary electrophoresis (CE) for three types of targets: DNA, RNA, and proteins. When present in the CE run buffer, SSB binds differently to P and P.T. Due to this selective binding, SSB induces difference in electrophoretic mobilities of P and P.T in an SSB concentration-dependent fashion. The difference in the electrophoretic mobilities allows for affinity analyses of DNA, RNA, and proteins in gel-free CE. The large number of well-characterized DNA- and RNA-binding proteins and the diversity of their properties will allow researchers to design a comprehensive tool set for quantitative analyses of DNA, RNA, and proteins. Such analyses will facilitate identification of genomic DNA in ultra-small samples without error-prone and time-consuming PCR. They can also be used for monitoring gene expression at both mRNA and protein levels.

DNA capillary electrophoresis using poly(vinyl alcohol). II. Separation media.

We represent the first extensive study on DNA capillary electrophoresis using various poly(vinyl alcohol) (PVAL)-related polymers as separation media. As a separation medium, PVAL homopolymer has shown poor resolutions probably due to its very strong hydrogen-bonding characteristics, resulting in extensive self-aggregation. On the other hand, poly(vinyl alcohol-co-vinyl acetate) and poly(vinyl alcohol-co-1-vinyl-2-pyrrolidone) (PVAL-VP), both with a degree of polymerization of approximately 3.0x10(3), have been found to give excellent electropherograms with good resolutions for the analysis of double-stranded (ds)DNA. PVAL-VP, with hydrolytic stability in high and low pH regions, has also yielded fair electropherograms for single-stranded (ss)DNA under neutral and alkaline conditions, although further investigation is essential in order to increase the resolutions necessary for DNA sequencing analysis. The separations obtained under alkaline conditions have shown significantly shorter retention times, one-third of that for the current commercial separation media, due to the higher ionization of phosphate groups in the DNA chains.

DNA capillary electrophoresis using poly(vinyl alcohol). I. Inner capillary coating.

Two new methods of inner capillary coating with poly(vinyl alcohol) (PVAL) have been investigated and evaluated by performing DNA capillary electrophoresis (CE) using PVAL as a separation medium and by measuring the electroosmotic flow (EOF) mobility. The treatment of capillaries with a silanol-group modified PVAL (PVAL-Si) has been found to give good coating effects for improving the resolution of DNA CE and for reducing the EOF. This coating must be effectively achieved by combining the adsorptive property of PVAL chains onto silica with the reaction between the silanol groups of PVAL-Si and the silica surface. The adsorption of PVAL onto silica has been observed by using atomic force microscopy (AFM) for PVAL-Si as well as for a nonmodified PVAL as a control. The coating with PVAL that links to the capillary wall surface with more hydrolytically stable bonding, -Si-C-, has been formed by performing the Grignard reaction, followed by in-capillary polymerization of vinyl acetate (VAc) and hydrolysis. This coating has been found to be effective for improving the resolution of DNA CE and for reducing the EOF.

Affinity Capillary Electrophoresis in a Poly(dimethylsiloxane)-glass Hybrid Microchip

Abstract Affinity capillary electrophoresis of DNA was conducted in a poly(dimethylsiloxane)-glass hybrid microchip with a 100 μm (width) × 130 μm (height) microchannel containing poly(N,N-dimethylacrylamide)-DNA conjugate. By this method, sample 12-mer oligonucleotide composed of c-K-ras codon 10-13 was separated from its single-base substituted mutant within 2 min.

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.

Site-specific fluorescent labeling of DNA using Staudinger ligation.

We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.