Integrated electroosmotically-driven on-line sample purification system for nanoliter DNA sequencing by capillary electrophoresis

An integrated on-line system is developed for DNA sequencing at the nanoliter scale. The technique involves the use of a nanoreactor for small-volume cycle-sequencing reaction, capillary zone electrophoresis (CZE) for purification of the sequencing fragments, and capillary gel electrophoresis (CGE) for separation of the purified DNA fragments. The nanoreactor and CZE are integrated into one capillary, where a 100-nl dye-labeled terminator cycle-sequencing reaction is carried out followed by CZE to separate excess dye-labeled terminators from the sequencing fragments. On-line electrokinetic injection of the purified DNA fragments into the CGE system is accomplished at a small-volume tee connector by which the CZE capillary is interfaced to the CGE system. The utility of the system is demonstrated in sequencing nanoliter volumes of single-stranded DNA (M13mp18) and double-stranded DNA (pGEM). The use of voltage to drive both CZE and CGE makes it feasible for automation and future adaptation of the whole system to a microchip.     

DNA sequencing by capillary electrophoresis using copolymers of acrylamide and N,N-dimethylacrylamide

Copolymers of acrylamide (AM) and N,N-dimethylacrylamide (DMA) with AM to DMA molar ratios of 3:1, 2:1 and 1:1 and molecular weights of about 2.2 MDa were synthesized. The polymers were tested as separation media in DNA sequencing analysis by capillary electrophoresis (CE). The dynamic coating ability of polydimethylacrylamide (PDMA) and the hydrophilicity of polyacrylamide (PAM) have been successfully combined in these random copolymers. A separation efficiency of over 10 million theoretical plates per meter has been reached by using the bare capillaries without the additional polymer coating step. Under optimized separation conditions for longer read length DNA sequencing, the separation ability of the copolymers decreased with decreasing AM to DMA molar ratio from 3:1, 2:1 and 1:1. In comparison with PAM, the copolymer with a 3:1 AM:DMA ratio showed a higher separation efficiency. By using a 2.5% w/v copolymer with 3:1 AM:DMA ratio, one base resolution of 0.55 up to 699 bases and 0.30 up to 963 bases have been achieved in about 80 min at ambient temperatures.     

Continuous flow infrared matrix-assisted laser desorption/ionization with a solvent matrix

Continuous flow matrix-assisted laser desorption/ionization (MALDI) was demonstrated with infrared laser desorption and an ethanol matrix. A capillary was used to deliver an analyte solution dissolved in ethanol to a metal frit embedded in a sample stage. Typical flow rates were 1.7&mgr;L/min. An optical parametric oscillator tuned to 2.8&mgr;m was used for desorption and ionization, and mass analysis was achieved with a 1 m linear time-of-flight mass spectrometer. Flow injection studies were performed with low picomolar quantities of insulin and myoglobin in solutions containing 0.1 to 1.0% glycerol in ethanol. Copyright 2000 John Wiley & Sons, Ltd.     

A new concept for sequencing DNA by capillary electrophoresis.

It is proposed that the scaling symmetry of constant charge density with increasing molecular weight, which prevents the separation by electrophoresis of DNA molecules in solution (with respect to molecular weight) be broken by the attachment of a perturbing entity (protein, virus or charged sphere) to one end of the molecule. An application of this idea to a concept for sequencing DNA by capillary electrophoresis is discussed, and the possibility of using the reattachment of the RecA protein to separate large segments of DNA in solution by electrophoresis following sequence-specific cleavage is mentioned.     

DNA sequencing by capillary electrophoresis using mixtures of polyacrylamide and poly(N,N-dimethylacrylamide)

The possibility of using polymer mixtures with different chemical compositions as a DNA sequencing matrix by capillary electrophoresis (CE) has been exploited. Polyacrylamide (PAM, 2.5%, w/v) having a molecular mass of 2.2 x 10(6) has been mixed with poly(N,N-dimethylacrylamide) (PDMA) having molecular masses of 8000, 470000 and 2.1 x 10(6) at concentrations of 0.2, 0.5 and 1% (w/v). Unlike polymer mixtures of the same polymer with different molecular masses, the use of polymer mixtures with different chemical compositions encounters an incompatibility problem. It was found that the incompatibility increased with increasing PDMA molecular mass and PDMA concentration, which resulted in decreased efficiency in DNA sequencing. Also, the incompatibility had a more pronounced effect on the efficiency as the base number was increased. However, by choosing a low-molecular-mass PDMA of 8000 and a low concentration of 0.2% (w/v), the incompatibility of PAM and PDMA has been alleviated. At the same time, the advantage of using polymer mixtures revealed a higher efficiency for such a polymer mixture when compared with PAM. The mixture also endowed the separation medium with a dynamic coating ability. An efficiency of over 10 x 10(6) theoretical plates per meter has been achieved by using the bare capillaries without the additional chemical coating step.     

Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis.

A low cost, 0.75-mW helium neon laser, operating in the green region at 534.5 nm, is used to excite fluorescence from tetramethylrhodamine isothiocyanate-labelled DNA fragments that have been separated by capillary gel electrophoresis. The detection limit (3 sigma) for the dye is 500 ymol [1 yoctomole (1 ymol) = 10(-24) mol] or 300 analyte molecules in capillary zone electrophoresis; the detection limit for labeled primer separated by capillary gel electrophoresis is 2 zmol [1 zeptomole (1 zmol) = 10(-21) mol]. The Richardson-Tabor peak-height encoded sequencing technique is used to prepare DNA sequencing samples. In 6% T, 5% C acrylamide, 7 M urea gels, sequencing rates of 300 bases/hour are produced at an electric field strength of 200 V/cm; unfortunately, the data are plagued by compressions. These compressions are eliminated with addition of 20% formamide to the sequencing gel; the gel runs slowly and sequencing data are generated at a rate of about 70 bases/hour.     

A highly efficient and versatile microchip capillary electrophoresis method for DNA separation using gold nanoparticle as a tag

A highly efficient and versatile method for DNA separation using Au nanoparticles (Au NPs) as a tag based on microchip capillary electrophoresis (MCE) was developed. The thiol-modified DNA-binding Au NPs were utilized as a tag. Target DNA was sandwiched between Au NPs and probe DNA labeled with horseradish peroxidase (HRP). In electrophoresis separation, the difference in electrophoretic mobility between free probe and probe-target complex was magnified by Au NPs, which enabled the resulting mixture to be separated with high efficiency by microchip capillary electrophoresis. Horseradish peroxidase was used as a catalytic label to achieve sensitive electrochemical DNA detection via fast catalytic reactions. With this protocol, 27-mer DNA fragments with different sequences were separated with high speed and high resolution. The proposed method was critical to achieve improved DNA separations in hybridization analyses.     

Immunoassays using capillary electrophoresis laser induced fluorescence detection for DNA adducts

Human DNA is exposed to a variety of endogenous and environmental agents that may induce a wide range of damage. The critical role of DNA damage in cancer development makes it essential to develop highly sensitive and specific assays for DNA lesions. We describe here ultrasensitive assays for DNA damage, which incorporate immuno-affinity with capillary electrophoresis (CE) separation and laser induced fluorescence (LIF) detection. Both competitive and non-competitive assays using CE/LIF were developed for the determination of DNA adducts of benzo[a]pyrene diol epoxide (BPDE). A fluorescently labeled oligonucleotide containing a single BPDE adduct was synthesized and used as a fluorescent probe for competitive assay. Binding between this synthetic oligonucleotide and a monoclonal antibody (MAb) showed both 1:1 and 1:2 complexes between the MAb and the oligonucleotide. The 1:1 and 1:2 complexes were separated by CE and detected with LIF, revealing binding stoichiometry information consistent with the bidentate nature of the immunoglobulin G antibody. For non-competitive assay, a fluorescently labeled secondary antibody fragment F(ab′)₂ was used as an affinity probe to recognize a primary antibody that was specific for the BPDE-DNA adducts. The ternary complex of BPDE-DNA adducts with the bound antibodies was separated from the unbound antibodies using CE and detected with LIF for quantitation of the DNA adducts. The assay was used for the determination of trace levels of BPDE-DNA adducts in human cells. Analysis of cellular DNA from A549 human lung carcinoma cells that were incubated with low doses of BPDE (32 nM–1 μM) showed a clear dose–response relationship. BPDE is a potent environmental carcinogen, and the ultrasensitive assays for BPDE-DNA adducts are potentially useful for monitoring human exposure to this carcinogen and for studying cellular repair of DNA damage.     

Formamide modified polyacrylamide gels for DNA sequencing by capillary gel electrophoresis.

Compressions are occasionally found during the separation of DNA sequencing fragments, particularly in G/C-rich regions and in gels operated at room temperature. Addition of at least 10% formamide to urea/polyacrylamide sequencing gels improves the denaturing capacity of the gel, minimizing compressions. Addition of 20% or more formamide decreases the separation rate, theoretical plate count, and resolution for normally migrating fragments. An optimum concentration of 10% formamide improves resolution of compressed regions without degrading the other characteristics of the gel. Operation of gels at room temperature simplifies the engineering associated with automated sequencers based on capillary gel electrophoresis.     

Matrix-assisted laser desorption/ionization time-of-flight analysis of Bacillus spores using a 2.94 microm infrared laser

The performance of infrared (2.94 microm) and ultraviolet (337 nm) lasers were compared for analysis of purified spores of B. subtilis, B. cereus and B. globigii on a four-inch end-cap reflectron time-of-flight instrument. Infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectra of these microorganisms displayed a larger number of biomarker peaks above m/z 4000, compared with UV-MALDI. Biomarker peaks were observed at higher m/z values with the IR laser.     

Fast DNA sequencing up to 1,000 bases by capillary electrophoresis using poly(N,N-dimethylacrylamide) as a separation medium

Poly(N,N-dimethylacrylamide) (PDMA) with a molecular mass of 5.2 x 10(6) g/mol has been synthesized and used in DNA sequencing analysis by capillary electrophoresis (CE). A systematic investigation is presented on the effects of different separation conditions, such as injection amount, capillary inner diameter, polymer concentration, effective separation length, electric field and temperature, on the resolution. DNA sequencing up to 800 bases with a resolution (R) limit of 0.5 (and 1,000 bases with a resolution limit of 0.3) and a migration time of 96 min was achieved by using 2.5% w/v polymer, 150 V/cm separation electric field, and 60 cm effective separation length at room temperature on a DNA sample prepared with FAM-labeled--21M13 forward primer on pGEM3Zf(+) and terminated with ddCTP. Ultrafast and fast DNA sequencing up to 420 and 590 bases (R > or = 0.5) were also achieved by using 3% w/v polymer and 40 cm effective separation length with a separation electric field of 525 and 300 V/cm, and a migration time of 12.5 and 31.5 min, respectively. PDMA has low viscosity, long shelf life and dynamic coating ability to the glass surface. The unique properties of PDMA make it a very good candidate as a separation medium for large-scale DNA sequencing by capillary array electrophoresis (CAE).     

Side-entry excitation and detection of square capillary array electrophoresis for DNA sequencing.

In high throughput DNA sequencing based on capillary electrophoresis, efficient coupling of the laser to each capillary is a challenge. Our group previously reported two multiple point irradiation schemes. The present work describes a more efficient excitation and detection method in which the laser light propagates through the capillary array without undergoing a serious reduction in power. An array of square capillaries (340 microns O.D. x 75 microns I.D.) was sandwiched between two fused-silica plates with an index-matching solution in between. The light was directed into the channel across the capillary array from the side. DNA sequences of PGEM/U from 24 capillaries were obtained even with a relatively low-power laser. The excitation scheme can be scaled up to hundreds of capillaries to achieve high-speed, high-throughput DNA sequencing, genetic typing and drug screening.     

In vivo monitoring of amine neurotransmitters using microdialysis with on-line capillary electrophoresis.

Microdialysis sampling was coupled via a flow-gated interface on-line to capillary electrophoresis with laser-induced fluorescence (LIF) detection for in vivo monitoring of neuroactive amino acids and amines. In the instrument, analytes are derivatized precolumn with o-phthaldehyde and beta-mercaptoethanol to form fluorescent isoindole products. The instrument was improved over previous designs by incorporating a sheath-flow cuvette for reduced background in LIF detection which improved sensitivity by 15-fold. The methodology was improved by utilizing a voltage ramped injection which allowed generation of 500000 theoretical plates with 20 s separations. Resolution of the isoindole derivatives was further improved by addition of hydroxypropyl-modified beta-cyclodextrin to the electrophoresis buffer. The new instrumentation and methods allow resolution and detection of glutamate, gamma-aminobutyric acid, glycine, aspartate, serine, taurine, glutamine and dopamine (if levels are elevated) collected from in vivo sampling probes every 20 s. The technique is suited to continuous monitoring for dynamic measurements of these compounds in vivo.     

Metal-complex/DNA conjugates: a versatile building block for DNA nanoarrays

The use of DNA networks as templates for forming nanoarrays of metallic centres shows an exciting potential to generate addressable nanostructures. Inorganic units can be photoactive, electroactive and/or can possess magnetic and catalytic properties and can adopt different spatial arrangements due to their varied coordination nature. All these properties influence both the structure and function of passive DNA scaffolds and provide DNA nanostructures as a new platform for new materials in emerging technologies, such as nanotechnology, biosensing or biocomputing.     

Improvements in ion signal reproducibility obtained using a homogeneous laser beam for on-line laser desorption/ionization of single particles

A major factor limiting on-line single particle mass spectrometry techniques from becoming more quantitative is the large shot-to-shot variability in ion intensities observed in the laser desorption/ionization (LDI) mass spectra.1,2 In previous work, lab-generated particles showed fluctuations of up to 152% in the absolute ion intensities in averaged spectra of 200-300 'identical' particles.2 Most of these fluctuations were attributed to inhomogeneities in the laser beam profile, leading to significant differences in the power each particle encountered depending on the position in the LDI laser beam where it underwent analysis. The goal of the work presented herein is to determine whether a fiber optic actually reduces the observed variability in single particle LDI mass spectral data. Initial results are presented for individual single component organic particles composed of 2,4-dihydroxybenzoic acid (2,4-DHB) analyzed using a low-power flat-top laser beam profile created by sending an ultraviolet (266 nm) DI laser through a fiber optic. Relative standard deviations of the total ion intensities for peaks in individual spectra are reduced to 31%. Single particle spectra, compared with and without laser homogenization at the same nominal laser fluence, show a marked enhancement. Specifically, the ion signal patterns of the 2,4-DHB particle spectra obtained using a homogenous LDI beam look identical to one another (i.e. only one particle type was produced with a commonly used neural network grouping algorithm), whereas without beam homogenization 25 different particle types (based on ion intensity patterns) were obtained. Future publications will explore more particle types and matrices but the initial results described herein are quite encouraging.     

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.     

Rapid high-resolution electrophoresis of multimeric von Willebrand Factor using a thermopiloted gel apparatus

Rapid and highly reproducible nonreducing agarose gel electrophoresis (NRAGE) of von Willebrand Factor (vWF) multimers was performed using a thermostated minigel apparatus that monitors and precisely controls internal gel temperature. The substitution of lithium dodecyl sulfate (LiDS) for sodium dodecyl sulfate (SDS) allowed electrophoresis to be performed below the 16 degrees C Krafft point of SDS and facilitated NRAGE of vWF over the entire range of 0-35 degrees C. Internal gel temperature was regulated by a thermocouple probe inserted directly into the gel during electrophoresis which interfaced with a thermopilot that continually measures and adjusts temperature to within +/- 0.5 degrees C. At 10 degrees C operative temperature, NRAGE at 1.5% agarose concentration was completed in 20 min at 250 V. Electrophoresis could be performed in only 10 min at 500 V, but at such high voltages, localized temperature fluctuations as much as 6 degrees C resulted in perturbation of banding patterns in those vicinities. In the optimized method, both high molecular weight multimers and proteolytic fragments of vWF were separable suggesting clinical applicability of this system for the diagnosis of von Willebrand Disease and thrombotic thrombocytopenic purpura.     

Compensation for mobility inequalities between lanes computed from band signals in on-line fluorescence DNA sequencing.

Among on-line fluorescence DNA sequencing systems, the four-lane method exhibits the potential for reporting an erroneous sequence due to nonuniform mobility of the DNA fragments migrating among the four lanes. This error is manifest in phenomenon commonly called smiling. This paper presents a computational algorithm which compensates for the mobility inequalities between lanes using signal data obtained from the shorter DNA fragments forming the faster migrating bands. The program mainly consists of two routines: (i) calculation of calibration coefficients (mobility ratios between lanes), and (ii) examination of the coefficients by applying them to a later domain of the same signals. Both routines are connected with several feed-back branches for recalculation. Homology analysis of final sequences has shown that the accuracy rate is maximized with this algorithm and any ambiguous result can be assigned to the residual error inherent in the band identification method used.