High speed DNA sequencing by capillary electrophoresis.

A major challenge of the Human Genome Initiative is the development of a rapid, accurate, and efficient DNA sequencing technology. A major limitation of current technology is the relatively long time required to perform the gel electrophoretic separations of DNA fragments produced in the sequencing reactions. We demonstrate here that it is possible to increase the speed of sequence analysis by over an order of magnitude by performing the electrophoresis and detection in ultra thin capillary gels. An instrument which utilizes these high speed separations to simultaneously analyze many samples will constitute a second generation automated DNA sequencer suitable for large-scale sequence analysis.     

DNA sequencing by capillary electrophoresis (review)

DNA sequencing by capillary electrophoresis has been reviewed with an emphasis on progress during the last four years. The effects of sample purification, composition of sieving matrices, electric field strength, temperature, wall coating and DNA labeling on the DNA sequencing performance are discussed. Multicapillary array instrumentation is compared with one-capillary systems. Integrated systems that perform the whole DNA sequencing operation online starting from the DNA amplification through base calling and data processing are discussed.     

DNA sequencing using 96-capillary array electrophoresis

Practical DNA sequencing in a rugged capillary array electrophoresis system coupled directly to 96-well microtiter plates is demonstrated. A CCD detector was used to monitor all capillaries simultaneously with laser-induced fluorescence at 1.75 frames per second. The reconstructed electropherograms show good signal-to-noise ratios and resolution for the entire capillary array. The system used standard dye labeling and image splitting to obtain fluorescence intensities in two wavelength regions to allow calling up to 410 bases for the DNA sequence. The use of a replaceable poly(ethylene oxide) matrix and a protective poly(vinylpyrrolidone) coating allows high separation speed and short turnaround time for high throughput DNA sequencing. Critical evaluation of the system performance over repeated runs with base calling is presented.     

A chemically synthesized peptoid-based drag-tag enhances free-solution DNA sequencing by capillary electrophoresis

We report a capillary-based DNA sequencing read length of 100 bases in 16 min using end-labeled free-solution conjugate electrophoresis (FSCE) with a monodisperse poly-N-substituted glycine (polypeptoid) as a synthetic drag-tag. FSCE enabled rapid separation of single-stranded (ss) DNA sequencing fragments with single-base resolution without the need for a viscous DNA separation matrix. Protein-based drag-tags previously used for FSCE sequencing, for example, streptavidin, are heterogeneous in molar mass (polydisperse); the resultant band-broadening can make it difficult to obtain the single-base resolution necessary for DNA sequencing. In this study, we synthesized and HPLC-purified a 70mer poly-N-(methoxyethyl)glycine (NMEG) drag-tag with a molar mass of - 11 kDa. The NMEG monomers that comprise this peptoid drag-tag are interesting for bioanalytical applications, because the methoxyethyl side chain's chemical structure is reminiscent of the basic monomer unit of polyethylene glycol, a highly biocompatible commercially available polymer, which, however, is not available in monodisperse preparation at an - 11 kDa molar mass. This is the first report of ssDNA separation and of four-color, base-by-base DNA sequencing by FSCE through the use of a chemically synthesized drag-tag. These results show that high-molar mass, chemically synthesized drag-tags based on the polyNMEG structure, if obtained in monodisperse preparation, would serve as ideal drag-tags and could help FSCE reach the commercially relevant read lengths of 100 bases or more.     

Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis.

Multiwavelength detection of laser induced fluorescence for dideoxynucleotide DNA sequencing with four different fluorophores and separation by capillary gel electrophoresis is described. A cryogenically cooled, low readout noise, 2-dimensional charge-coupled device is used as a detector for the on-line, on-column recording of emission spectra. The detection system has no moving parts and provides wavelength selectivity on a single detector device. The detection limit of fluorescently labeled oligonucleotides meets the high sensitivity requirements for capillary DNA sequencing largely due to the efficient operation of the CCD detector with a 94% duty cycle. Using the condition number as a selectivity criterion, multiwavelength detection provides better analytical selectivity than detection with four bandpass filters. Monte Carlo studies and analytical estimates show that base assignment errors are reduced with peak identification based on entire emission spectra. High-speed separation of sequencing samples and the treatment of the 2-dimensional electropherogram data is presented. Comparing the DNA sequence of a sample separated by slab gel electrophoresis with sequence from capillary gel electrophoresis and multiwavelength detection we find no significant difference in the amount of error attributable to the instrumentation.     

Two-label peak-height encoded DNA sequencing by capillary gel electrophoresis: three examples.

We report a modification to the peak-height encoded DNA sequencing technique of Tabor and Richardson. As in the original protocol, the sequencing reaction uses modified T7 polymerase with manganese rather than magnesium to produce very uniform incorporation of each dideoxynucleoside. To improve sequencing accuracy, two fluorescently labeled primers are employed in separate sequencing reactions. As an example, one sequencing reaction uses a FAM-labeled primer with dideoxyadenosine triphosphate and dideoxycytosine triphosphate; the concentrations of ddATP and ddCTP are adjusted to produce a 2:1 variation in the relative intensity of fragments. The second sequencing reaction uses a TAMRA labeled primer with dideoxythymidine triphosphate and dideoxyguanidine triphosphate; the concentrations of ddTTP and ddGTP are adjusted to produce a 2:1 variation in relative intensity of fragments. The pooled reaction products are separated by capillary gel electrophoresis and identified by one of three different detector systems. Use of a 2:1 peak height ratio typically produces a sequencing accuracy of 97.5% for the first 350 bases; a 3:1 peak height ratio improves accuracy to 99.5% for the first 400 bases. For these experiments, capillary electrophoresis is performed at an electric field of 200 V/cm; two to three hours are required to separate sequencing fragments up to 400 nucleotides in length.     

Internal fluorescence labeling with fluorescent deoxynucleotides in two-label peak-height encoded DNA sequencing by capillary electrophoresis.

Fluorescently labeled deoxynucleotides were used for internal labeling of DNA sequencing fragments generated in a two-color peak-height encoded protocol. Sequenase and a manganese-containing buffer were used to generate uniform peak heights. Tetramethyl rhodamine - dATP was used in a labeling step, followed by termination with ddATP and ddCTP in a 3:1 ratio. Fluorescein - dATP was used in a second reaction, followed by termination with ddGTP and ddTTP in a 3:1 ratio. The fragments were pooled and separated by capillary gel electrophoresis. The results were compared with peak-height encoded sequencing based on fluorescently labeled primers. The dye-labeled primers produced higher resolution separations for shorter fragments. However, dye-labeled primer fragments suffered from an earlier onset of biased reptation and produced shorter sequencing reads. Fragments from 50 to at least 500 bases in length could be sequenced with the internal labels.     

Structure of amplified DNA, analyzed by pulsed field gradient gel electrophoresis

Pulsed field gradient electrophoresis allows the separation of large DNA molecules up to 2,000 kilobases (kb) in length and has the potential to close the resolution gap between standard electrophoresis of DNA molecules (smaller than 50 kb) and standard cytogenetics (larger than 2,000 kb). We have analysed the amplified DNA in four cell lines containing double minute chromosomes (DMs) and two lines containing homogeneously staining regions. The cells were immobilized in agarose blocks, lysed, deproteinized, and the liberated DNA was digested in situ with various restriction endonucleases. Following electrophoretic separation by pulsed field gel electrophoresis, the DNA in the gel was analysed by Southern blotting with appropriate probes for the amplified DNA. We find that the DNA in intact DMs is larger than 1,500 kb. Our results are also compatible with the notion that the DNA in DMs is circular, but this remains to be proven. The amplified segment of wild-type DNA covers more than 550 kb in all lines and possibly up to 2,500 kb in some. We confirm that the repeat unit is heterogeneous in some of the amplicons. In two cell lines, however, with low degrees of gene amplification, we find no evidence for heterogeneity of the repeats up to 750 (Y1-DM) and 800 kb (3T6-R50), respectively. We propose that amplicons start out long and homogeneous and that the heterogeneity in the repeat arises through truncation during further amplification events in which cells with shorter repeats have a selective advantage. Even if the repeats are heterogeneous, however, pulsed field gradient gels can be useful to establish linkage of genes over relatively short chromosomal distances (up to 1,000 kb). We discuss some of the promises and pitfalls of pulsed field gel electrophoresis in the analysis of amplified DNA.     

Separation of large DNA molecules by modified pulsed field gradient gel electrophoresis

Resolution of DNA fragments by pulsed field gradient gel electrophoresis is a function of the pulse time, geometry, and strength of the orthogonal electric fields. The first field geometry described had a number of disadvantages. We show that these disadvantages can be largely overcome by a modified electric field geometry together with an altered switch pattern. These changes are shown to have critical consequences for the technique. Resolution is more uniform across the gel, which permits more samples to be analyzed on the same gel. In addition, DNA molecules follow a migration path that is approximately straight down the gel. This aspect also increases the number of usable wells. One important property of the system described here provides some insight into the mechanism whereby DNA molecules are resolved by this method.     

Magnetic bead purification of labeled DNA fragments for high-throughput capillary electrophoresis sequencing

We have developed an automated purification method for dye-terminator-based DNA sequencing products using a magnetic bead approach. This 384-well protocol generates sequence fragments that are essentially free of template DNA, salt, and excess dye-terminator products. In comparison with traditional ethanol precipitation protocols, this method uses no centrifugation, is rapid, completely automated, and increases the phred-20 read length by an average of 40 bases. To date, we have processed over 4 million samples with 94% averaging 641 phred-20 bases on the MegaBACE 1000 and 4000 and the ABI PRISM 3700 capillary instruments.     

Automated one-step DNA sequencing based on nanoliter reaction volumes and capillary electrophoresis

An integrated system with a nano-reactor for cycle-sequencing reaction coupled to on-line purification and capillary gel electrophoresis has been demonstrated. Fifty nanoliters of reagent solution, which includes dye-labeled terminators, polymerase, BSA and template, was aspirated and mixed with the template inside the nano-reactor followed by cycle-sequencing reaction. The reaction products were then purified by a size-exclusion chromatographic column operated at 50°C followed by room temperature on-line injection of the DNA fragments into a capillary for gel electrophoresis. Over 450 bases of DNA can be separated and identified. As little as 25 nl reagent solution can be used for the cycle-sequencing reaction with a slightly shorter read length. Significant savings on reagent cost is achieved because the remaining stock solution can be reused without contamination. The steps of cycle sequencing, on-line purification, injection, DNA separation, capillary regeneration, gel-filling and fluidic manipulation were performed with complete automation. This system can be readily multiplexed for high-throughput DNA sequencing or PCR analysis directly from templates or even biological materials.     

DNA sequencing with direct blotting electrophoresis.

A method for transferring the DNA molecules of sequencing reaction mixtures onto an immobilizing matrix during electrophoresis has been developed. A blotting membrane moves with constant speed across the end of a very short, denaturing gel and collects the molecules according to size. A constant distance between bands for molecules differing in length by one nucleotide is obtained over a large range (approximately 600 nucleotides with a 5% gel), simplifying the determination of DNA sequences considerably. Reliable sequences of 500 nucleotides can be read and sequence features up to greater than 1000 nucleotides are revealed in a single experiment. The sequencing of a potential Z-DNA-forming fragment from Escherichia coli DNA is given as an example and possible further developments are discussed.     

Plasma creatinine and creatine quantification by capillary electrophoresis diode array detector

Traditional clinical assays for nonprotein nitrogen compounds, such as creatine and creatinine, have focused on the use of enzymes or chemical reactions that allow measurement of each analyte separately. Most of these assays are mainly directed to urine quantification, so that their applicability on plasma samples is frequently hard to perform. This work describes a simple free zone capillary electrophoresis method for the simultaneous measurement of creatinine and creatine in human plasma. The effect of analytical parameters such as concentration and pH of Tris-phosphate running buffer and cartridge temperature on resolution, migration times, peak areas, and efficiency was investigated. Good separation was achieved using a 60.2-cm x 75-microm uncoated silica capillary, 75 mmol/L Tris-phosphate buffer, pH 2.25, at 15 degrees C, in less than 8 min. We compared the present method to a validated capillary electrophoresis assay, by measuring plasma creatinine in 120 normal subjects. The obtained data were compared by the Passing-Bablok regression and the Bland-Altman test. Moreover the performance of the developed method was assessed by measuring creatine and creatinine in 16 volunteers prior to and after a moderate physical exercise.     

The theory of DNA separation by capillary electrophoresis

The Human Genome has been sequenced in large part owing to the invention of capillary electrophoresis. Although this technology has matured enough to allow such amazing achievements, the physical mechanisms at play during separation have yet to be completely understood and optimized. Recently, new separation regimes and new physical mechanisms have been investigated. The use of free-flow electrophoresis and new modes of pulsed-field electrophoresis have been suggested, while we have observed a shift towards single nucleotide polymorphism analysis and microchip technologies. A strong theoretical basis remains essential for the efficient development of new methods.     

DNA analysis by microfabricated capillary electrophoresis device.

The LIGA (Lithographie Galvanoformung Abformung) process using synchrotron radiation lithography is applied to the microfabrication of capillary array electrophoresis (CAE) device. Laser-induced fluorescence detection system for the CAE device has been constructed by the modification of laser confocal fluorescence microscopy. DNA molecules were detected during migrating in the microchannels filled with polymer separation matrices under electric field to optimize the separation conditions for DNA analysis. Based on this observation, we demonstrated that microfabricated CAE device is realized the fast separation of DNA.     

DNA annealing and DNA-protein interactions by capillary electrophoresis

This work deals with annealing of single-stranded DNA and the binding of a serum respond factor to a DNA probe containing specific binding site. Capillary electrophoresis (CE) method is explored and compared with the mobility-shift gel electrophoresis (GE) procedure. The results indicate the CE method offers direct and rapid annealing of the DNA strands. It requires no prior incubation with additives (polynucleotides, proteins) to reduce nonspecific DNA-protein interactions. Unwanted nonspecific interactions are not observed in the CE method. The presence of a fluorescein tag to the DNA probe yields identical results to those with the radioactive label. A fluorescein tag in the CE work can be used without any adverse effects. The dissociation constant (Kd) of this protein-DNA complex by the CE method was similar to those determined by the GE method (approximately 10(-6) M). The proposed method is extremely powerful, highly sensitive, quantitative, and fast. It can determine even very small conformational differences of the DNA probe.     

Energy-transfer cassette labeling for capillary array electrophoresis short tandem repeat DNA fragment sizing.

Energy-transfer (ET) dye-labeled primers significantly improve fluorescent DNA detection because they permit excitation at a single common wavelength and they produce well separated and intense acceptor dye emission. Recently, a new ET cassette technology was developed [Berti, L. et al. (2001) Anal. Biochem. 292, 188-197] that can be used to label any PCR, sequencing, or other primer of interest. In this report we examine the utility of this ET cassette technology by labeling seven different short tandem repeat (STR) specific primers with each of the four ET cassettes and analyzing the PCR products generated on a MegaBACE-1000 capillary array electrophoresis system. More than 60 amplicons were generated and successfully analyzed with the ET cassette-labeled primers. Both forward and reverse primers were labeled for multiplex PCR amplification and analysis. Single base pair resolution was achieved with all four ET cassettes. This ET cassette-primer labeling procedure is ideally suited for creating four-color fluorescent ET primers for STR and other DNA assays where large numbers of different loci are analyzed including sequencing, genetic identification, gene mapping, loss of heterozygosity testing, and linkage analysis.     

Orientational dynamics of T2 DNA during agarose gel electrophoresis: influence of gel concentration and electric field strength

The understanding, on a molecular level, of the mechanisms responsible for the improved separation in DNA gel electrophoresis when using modulated electric fields requires detailed information about conformational distribution and dynamics in the DNA/gel system. The orientational order due to electrophoretic migration ("electrophoretic orientation") is an interesting piece of information in this context that can be obtained through linear dichroism spectroscopy [M. Jonsson, B. Akerman, and B. Nordén, (1988) Biopolymers 27, 381-414]. The technique permits measurement of the orientation factor S of DNA (S = 1 corresponds to perfect orientation) within an electrophoretic zone in the gel during the electrophoresis. It is reported that the degree of orientation of T2 DNA [170 kilo base pairs (kpb)] is considerable (S = 0.17 in 1% agarose at 10 V/cm) compared to relatively modest orientations of short fragments found earlier (for 23-kbp DNA, S = 0.03 in 1% agarose at 10 V/cm), showing that large DNA coils are substantially deformed during the migration. Growth and relaxation dynamics of the orientational order of the T2 DNA are also reported, as functions of gel concentration (0.3-2%), electric field strength (0-40 V/cm), and pulse characteristics. The rise profile of the DNA orientation, when applying a constant field, is a nonmonotonic function that displays a pronounced overshoot, followed by a minor undershoot, before it reaches steady-state orientation (after 12 s in 1% agarose, 9 V/cm). The orientational relaxation in absence of field shows a multiexponential decay in a time region of some 10 s, when most of the DNA anisotropy has disappeared. A surprising phenomenon is a memory over minutes of the DNA/gel system to previous pulses: with two consecutive rectangular pulses (of the same polarity), the orientational overshoot and undershoot as a response to the second pulse are significantly reduced compared to the first pulse. The time required to recover 90% of their amplitudes is typically 1200 s (1% agarose, 9 V/cm), which may be compared to the time required to relax 90% of the DNA orientation, which is only 6 s. The major part of the over- and undershoot recovery is thus a reorganization of a system in which DNA is already randomly oriented. The different response amplitudes and relaxation times, including the amplitude and recovery time of the overshoot, of the orientational order of DNA in the electrophoretic gel have been studied as functions of gel concentration and field strength. The results are discussed against relevant theories of polymer dynamics.     

Electrohydrodynamically induced aggregation during constant and pulsed field capillary electrophoresis of DNA

We present a study aimed at understanding the factors affecting the separation of large DNA molecules by capillary electrophoresis in polymer solutions. In a first series of experiments, a systematic study of the effect of operational parameters on the development of an electrohydrodynamic instability resulting in DNA aggregation and spurious peaks in the electropherograms is presented. The results are discussed in regard to a recent theory of electrohydrodynamic instabilities in macroion suspensions, recently proposed by Isambert et al. Overall, the results provide strong support to the theory. Some situations of interest for applications, and not explicitly considered in the theory, such as asymmetric field pulsing and the use of polymer additives in the buffer, were also considered. Furthermore, robust optimized protocols for high resolution separation of DNA in the range of 100 base pairs to 160 kilobase pairs, are proposed. As predicted by the model, it is shown that using a concentrated isoelectric buffer (histidine) strongly reduces aggregation as compared to the use of a conventional buffer at the same concentration, and allows separation of DNA from 100 bp to 160 kbp in less than 10 min. We also present a systematic study of the dependence of the mobility vs DNA size, pulse frequency, and field strength. The results are discussed with respect to the Biased Reptation with Fluctuations model and a good agreement is obtained. Copyright 1999 John Wiley & Sons, Inc.     

Rapid DNA sequencing by horizontal ultrathin gel electrophoresis.

A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive thermal transfer methods commonly used. Bubble-free gels as thin as 25 microns can be routinely cast on this device. The application to these ultrathin gels of electric fields up to 250 volts/cm permits the rapid separation of multiple DNA sequencing reactions in parallel. When used in conjunction with 32P-based autoradiography, the DNA bands appear substantially sharper than those obtained in conventional electrophoresis. This increased sharpness permits shorter autoradiographic exposure times and longer sequence reads.