Capillary gel electrophoresis for rapid, high resolution DNA sequencing.

Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequencing instrument. Agreement of measured values for velocity, resolution and separation efficiency with theory, predicts further improvements will result from increased electric field strengths (higher voltages and shorter capillaries). Advantages of capillary gel electrophoresis for automatic DNA sequencing instruments and for genomic sequencing are discussed.     

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.     

Microcapillary reactors using solid-phase DNA sequencing for direct sample introduction into slab gels

Solid-phase micro-reactors have been prepared in glass capillaries for DNA sequencing applications using slab gel electrophoresis, which consisted of a fused silica capillary (i.d. = 100 microns; o.d. = 365 microns; length = 15 cm; volume = 1.2 microL) that contained a covalently bound biotin molecule. With the addition of streptavidin to the capillary, an anchoring site was produced for the tethering of biotinylated DNA sequencing templates to the wall of the capillary. Using a four-lane, single dye primer chemistry sequencing strategy, the individual tracts were prepared in the capillaries using cycle sequencing (20 thermal cycles) on a PCR-generated lambda-bacteriophage template (about 1000 bp). The dye label in this case was a fluorescent tag that displayed emission properties in the near-IR and could be processed on an automated sequencer. The read length was found to be 589 bases, which was determined primarily by the fractionating power of the gel. It was also found that the tethering system was very stable to typical cycle sequencing conditions, with the amount of tethered DNA lost amounting to 40% after 120 thermal cycles. The ability to use dye terminator chemistry was also investigated by using a near-IR dye-labeled terminator (ddGTP). It was found that the quality of the ladder that was generated was comparable to that obtained in a conventional sample preparation format. However, ethanol precipitation was required before gel loading to remove excess terminator.     

PCR template preparation for capillary DNA sequencing

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85% of the total number of bases had Phred scores of 15 or above.     

Fluorescence photobleaching in DNA sequencing

Advances in DNA-sequencing technology suggest many possibilities using either conventional or unconventional means. Several approaches offer conceivable improvements in running time by one hundred fold or more. A fluorescence photobleaching scheme in combination with ultra-thin gel slab or capillary electrophoresis which proposes to shorten the run time and to increase the resolution of multi-fragment DNA analysis is reviewed. The advantages and some of the yet to be resolved obstacles are discussed.     

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.     

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.     

Electrophoresis of DNA in ultrathin planar-format linear polyacrylamide

Linear or un-cross-linked polyacrylamides have been employed successfully in the field of capillary electrophoresis for the separation of nucleic acids. Typical acrylamide concentrations for those applications range from 3% to 14% (wt/vol), with consistencies ranging from virtually liquid to moderately viscous. Due to the absence of cross-links, and the relatively fluid nature of linear polyacrylamide at typically employed concentrations, its use in planar (slab) gel electrophoresis has been overlooked. We describe herein the application of ultrathin (100 μm) high-viscosity slabs of linear polyacrylamide to planar electrophoresis of nucleic acid fragments. The approach we describe is rapid and yields high-resolution separations of nucleic acid fragments in linear polyacrylamide supports. The mobilities of DNA fragments of various lengths in a range of concentrations of linear polymer are compared with those observed for conventional cross-linked gels. The reptative migration of larger DNA fragments in linear polymers is predictable from the models derived from work with cross-linked acrylamide and agarose. The migration of smaller fragments, however, is not entirely predicted by the Ogston model. The relative mobilities observed for very small DNA fragments are approximately half those predicted by the Ogston regimen.     

A DNA cycle sequencing reaction that minimizes compressions on automated fluorescent sequencers.

We recently demonstrated that most band compressions (>70%) on DNA sequencing gels result from the presence of a single sequence motif, 5'-YGN1-2AR-3', where Y and R indicate base-pairing pyrimidine and purine residues, respectively. This finding raised the possibility that the use of 7-deaza-dATP instead of dATP in chain termination sequencing reactions would resolve most of the band compressions. Thus, we examined the effects of 7-deaza-dATP on DNA sequencing using thermostable DNA polymerases. The results indicate that the replacement of dATP with 7-deaza-dATP in conventional cycle sequencing reactions can successfully eliminate most band compressions without sacrificing sequencing performance.     

Using a neural network for lane-tracking of DNA sequencing slab gels

High quality lane-tracking of gel images is the first task, and thus a prerequisite, for successful trace processing and base-calling of DNA sequencing slab gels. In most approaches, it is based on statistical calculations, for instance variance and co-variance analysis between neighboring pixel columns in the image. On the basis of these statistical calculations, Kohonen's self-organization neural network model was introduced. We have found that, using several well-structured input data, Kohonen's self-organization neural network model can be trained to fulfill our task of lane-tracking. Furthermore, the quality of lane-tracking could be improved compared to algorithmic approaches.     

Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis.

A simple system has been designed enabling ultrasensitive on-line detection of fluorescently labelled macromolecules, e.g. nucleic acids, proteins and peptides during electrophoretic separations in gels. An important application is the automated DNA sequence determination without radioactivity. Drying of gels, film exposure and handling are not necessary. A sulphydryl containing M13 sequencing primer has been synthesised and end-labelled in a reaction with fluorescein iodoacetamide. This is then used in the dideoxy reactions. In particular no moving parts or complicated software are required for data collection and analysis. Compared to our first automated device detection sensitivity has been improved by a factor of thirty to about 3 X 10(-18) mol per band. The resolution has increased to about 400 bases in 5 hours, with the possibility to read up to about 500 bases when they are properly labelled. Gels shorter than 20 cm may be used for resolution of about 300 bases. The single gel system may be upgraded for simultaneous running and reading of six or ten sequencing samples.     

Recent advances in DNA sequencing by End-Labeled Free-Solution Electrophoresis (ELFSE)

End-Labeled Free-Solution Electrophoresis (ELFSE) is a new technique that is a promising bioconjugate method for DNA sequencing (or separation) and genotyping by both capillary and microfluidic device electrophoresis. Because ELFSE enables high-resolution electrophoretic separation in aqueous buffer alone (i.e., without a polymer matrix), it eliminates the need to load viscous polymer networks into electrophoresis microchannels. To achieve microchannel DNA separations with high performance, ELFSE requires monodisperse perturbing entities (i.e., drag-tags), which create a large amounts of frictional drag when pulled behind DNA during free-solution electrophoresis, and which have other properties suitable for microchannel electrophoresis. In this article, the theoretical concepts of ELFSE and the required characteristics of the drag-tag molecules for the ultimate performance of ELFSE are reviewed. Additionally, the merits and limitations of current drag-tags are also discussed in the context of recent experimental data of ELFSE separation (or sequencing).     

高速DNA序列分析

高速DNA序列分析是人类基因组研究的关键技术．文章对高速DNA序列分析方法如阵列毛细管电泳、超薄层凝胶板电泳、质谱、杂交法、原子探针法、流动单分子荧光检测法等新进展进行了评论．     

发展中的DNA测序技术

发展中的ＤＮＡ测序技术赵晓娟刘金毅综述蔡有余琦祖和＊审校（中国医学科学院中国协和医科大学实验动物研究所北京）ＤＮＡ序列分析是基因工程和分子生物学领域最重要的技术之一,是了解基因结构和功能的基础。“人类基因组计划”（ｈｕｍａｎｇｅｎｏｍｅｐｒｏｊｅｃｔ）的实施,有力地推动了高速ＤＮＡ测序技术的发展。除经典的测序方法在技术环节上的不断改进外,近年来发展了一些全新的ＤＮＡ测序方法,如毛细管凝胶电泳...     

Vacuum blotting: a simple method for transferring DNA from sequencing gels to nylon membranes

We describe a vacuum blotting procedure for transferring DNA fragments from conventional polyacrylamide sequencing gels to nylon membranes. The method employs a combination of vacuum-assisted diffusion (effected by a standard gel drier) and an osmotic gradient (effected by over- and underlying filters presoaked in ammonium acetate). Fragments up to 310 nucleotides in length transfer at 40–60% efficiency within 90 min. When combined with indirect end-labelling, the method allows genomic sequencing of a single-copy gene of Saccharomyces cerevisiae employing as little as 5 μg DNA per lane.     

Minimal electrophoresis time for DNA sequencing.

This study presents a mathematical approach that allows one to determine the shortest electrophoresis time and migration path length required for DNA sequencing. The calculation was applied to the capillary electrophoresis of a DNA sequencing separation and showed that acceptable resolution could be obtained using a shorter path length than anticipated.     

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.     

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.     

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 systemic DNA sequencing strategy

A systematic DNA sequencing strategy is presented. Instead of the sequencing of randomly selected DNA fragments (the “shotgun” method), the nucleotide order is progressively determined along a DNA chain using the dideoxynucleotide termination sequencing system and the single-stranded bacteriophage vector M13 derivatives. The length of DNA along which the sequence data can be progressively read appears to be limited only by the insertion capacity of the vector. As an example of this strategy a recombinant replicative form with a 2327 nucleotide long HindIII fragment from the restriction enzyme digest of bacteriophage λ DNA was prepared. The replicative form of the recombinant was partially digested with DNAase I in the presence of Mn²⁺. As the replication origin of the phage vector was located near one end of the inserted DNA and the priming site of the vector at the other, the breaks outside the inserted DNA either destroyed the phage or removed the priming site. With the use of a unique restriction site close to the priming site, the breaks within the inserted DNA gave rise to a recombinant mixture with the inserted DNA fixed at one end and sequentially shortened at the other. Using the ddT reaction screening procedure, 11 recombinants were identified in which the inserted DNA varied in length by about 200 nucleotides. Sequencing these recombinants by the dideoxynucleotide sequencing system covered the whole 2327 nucleotides of the HindIII DNA fragment. The average number of nucleotides read from a gel was 210, which fell into the most readable region of a sequencing gel: the overlapping regions between two gels were of 33 to 48 nucleotides.