High-throughput automated DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory.

One of the aims of the facility is to develop and push the automated on-line DNA sequencing gel technology to its limit in sequence throughput, which may be somewhere around 100 kilobases of sequence per device per day. Key new developments were initiated and applied in operation on the European Molecular Biology Laboratory (EMBL) automated sequencer and its commercial version A.L.F. (Pharmacia). Sequencing speed was increased by a factor of 10-20, up to 1500 bases per hour per clone on ultrathin (about 100 microns) gels, while the resolution and reading length were extended to 1000 bases on gels with 50 cm separation length, using fluorescein-15-*dATP as the internal label. With our sequencing strategy, closing about 40% of the sequence with "walking" primers and F-15-*dATP as internal label, we sequenced both strands of a cosmid insert of 38.5 kb in length, each strand twice, in only 430 sequencing reactions and with average reading of 380 bases per reaction.     

High resolution-separation of DNA bands by electrophoresis with a long gel in a fluorescence-detection DNA sequencer.

A high-resolution separation of DNA bands is achieved by electrophoresis with a long gel in DNA base sequencing using fluorescence detection. We separate 760 and 761 base DNA fragments using the 93 cm migration electrophoresis optimized for the separation of DNA bands. A T7 DNA polymerase and an Mn++ buffer are used in sequencing reactions to obtain fluorescence peaks of uniform strength, and the peak areas in the spectrum are used for recognizing the peak number in a cluster of successive peaks. This method is successfully applied to the DNA fragment spectrum obtained by 93 cm migration electrophoresis, which results in a single-band differentiation of bands of 1040 base DNA.     

Improvement of base-calling in multilane automated DNA sequencing by use of electrophoretic calibration standards, data linearization, and trace alignment

We present a new method for the linearization and alignment of data traces generated by multilane automated DNA sequencing instruments. Application of this method to data generated with the Visible Genetics Open Gene DNA sequencing system (using MicroCel 700 gel cassettes, with a 25 cm separation distance) allows read lengths of > 1,000 nucleotides to be routinely obtained with high confidence and > 97% accuracy. This represents an increase of 10-15% in average read length, relative to data from this system that have not been processed in the fashion described herein. Most importantly, the linearization and alignment method allows usable sequence to be obtained from a fraction of 10-15% of data sets which, because of original trace misalignment problems, would otherwise have to be discarded. Our method involves adding electrophoretic calibration standards to the DNA sequencing fragments. The calibration standards are labeled with a dye that differs spectrally from the dye attached to the sequencing fragments. The calibration standards are identical in all the lanes. Analysis of the mobilities of the calibration standards allows correction for both systematic and random variation of electrophoretic properties between gel lanes. We have successfully used this method with two-dye and three-dye DNA sequencing instruments.     

Use of an automated capillary DNA sequencer to investigate the interaction of cisplatin with telomeric DNA sequences

The determination of the sequence selectivity of DNA-damaging agents is very important in elucidating the mechanism of action of anti-tumour drugs. The development of automated capillary DNA sequencers with fluorescent labelling has enabled a more precise method for DNA sequence specificity analysis. In this work we utilized the ABI 3730 capillary sequencer with laser-induced fluorescence to examine the sequence selectivity of cisplatin with purified DNA sequences. The use of this automated machine enabled a higher degree of precision of both position and intensity of cisplatin-DNA adducts than previously possible with manual and automated slab gel procedures. A problem with artefact bands was overcome by ethanol precipitation. It was found that cisplatin strongly formed adducts with telomeric DNA sequences.     

Separation and analysis of DNA sequence reaction products by capillary gel electrophoresis

This paper demonstrates the potential of capillary gel electrophoresis with laser induced fluorescence detection as a tool for DNA sequence determination. Both synthetic oligonucleotides and single-stranded phage DNA were utilized as templates in the standard chain termination procedure. Primer molecules were tagged at the 5' end with the fluorescent dye, JOE. First, baseline resolution of a dA extended primer from 18 to 81 bases long, a total of 64 fragments, was observed. A second synthetic template was designed to yield alternating stretches of dA and dT extensions of the primer. Thirdly, the sequence reaction products from a synthetic oligonucleotide template containing all four bases was analyzed in four independent runs, one for each of the four base-specific reactions. In all cases, the expected number and patterns of peaks were observed by capillary gel electrophoretic analysis. Finally, separation of sequence reaction products generated with single-strand M13mp18 phage DNA as template exhibited baseline resolution of fragments differing in length by a single nucleotide and from 18 to greater than 330 bases total length.     

CHASE, a charge-assisted sequencing algorithm for automated homology-based protein identifications with matrix-assisted laser desorption/ionization time-of-flight post-source decay fragmentation data

We describe CHASE, a novel algorithm for automated de novo sequencing based on the mass spectrometric (MS) fragmentation analysis of tryptic peptides. This algorithm is used for protein identification from sequence similarity criteria and consists of four steps: (1) derivatization of tryptic peptides at the N-terminus with a negatively charged reagent; (2) post-source decay (PSD) fragmentation analysis of peptides; (3) interpretation of the mass peaks with the CHASE algorithm and reconstruction of the amino acid sequence; (4) transfer of these data to software for protein identifications based on sequence homology (Basic Local Alignment Search Tool, BLAST). This procedure deduced the correct amino acid sequence of tryptic peptide samples and also was able to deduce the correct sequence from difficult mass patterns and identify the amino acid sequence. This allows complete automation of the process starting from MS fragmentation of complex peptide mixtures at low concentration (e.g. from silver-stained gel bands) to identification of the protein. We also show that if PSD data are collected in a single spectrum (instead of the segmented mode offered by conventional matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation), the complete workflow from MS-PSD data acquisition to similarity-based identification can be completely automated. This strategy may be applied to proteomic studies for protein identification based on automated de novo sequencing instead of MS or tandem MS patterns. We describe the Charge Assisted Sequencing Engine (CHASE) algorithm, the working protocol, the performance of the algorithm on spectra from MALDI-TOFMS and the data comparison between a TOF and a TOF-TOF instrument.     

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.     

Why can we not sequence thousands of DNA bases on a polyacrylamide gel?

Pulsed fields have been remarkably useful at extending the range of DNA molecular sizes that can be separated on agarose gels by controlling the field-induced molecular orientation that often limits the resolution of large molecules. Unfortunately, the same approach seems to be much less effective for DNA sequencing on polyacrylamide gels. We present an experimental and theoretical (modelling) study of DNA sequencing which shows that molecular orientation is indeed not the main limiting factor for sequencing devices that use moderate field intensities and polyacrylamide as a separating matrix. We examine the interplay between electric field intensity, molecular size and resolution, and we suggest different approaches to increase the resolution limit of standard and automated sequencing gels. The theoretical limits of high-field electrophoretic sequencing are also discussed. We conclude that new ideas will be needed to go beyond one kilobase.     

Unlimited increase in the resolution of DNA ladders

Fractionation of DNA ladders by gel electrophoresis is limited by the progressive compressing of the long DNA end of a ladder. Improvement in the resolution of this DNA is achieved by use of the following two-step electrophoresis. Initially, the DNA ladder is fractionated by conventional constant field agarose gel electrophoresis. Subsequently, gel electrophoresis is performed in the reverse direction by pulsing the electrical field (PFGE). A newly developed type of pulsing is used, which causes inversion of a double-stranded DNA ladder: the distance migrated increases as the length of the DNA molecule increases. Thus, the resolution of DNA bands continues to increase during the PFGE. These two stages of electrophoresis are serially repeated. Eventually, both the short and the long DNA ends of the ladder migrate out of the gel while a selected region of the ladder undergoes progressive increase in resolution during back-and-forth migration. Improved resolution of DNA bands is achieved, without a known limit.     

DNA sequencing

Determination of the sequence of DNA is one of the most important aspects of modern molecular biology. New sequencing methods currently being developed enable DNA sequence to be determined increasingly faster and more efficiently. One of the major advances in sequencing technology is the development of automated DNA sequencers. These utilize fluorescent rather than radioactive labels. A laser beam excites the fluorescent dyes, the emitted fluorescence is collected by detectors, and the information analyzed by computer. Robotic work stations are being developed to perform template preparation and purification, and the sequencing reactions themselves. Research is currently in progress to develop the technology of mass spectrometry for DNA sequencing. Success in this endeavor would mean that the gel electrophoresis step in DNA sequencing could be eliminated. A major innovation has been the application of polymerase chain reaction (PCR) technology to DNA sequence determination, which has led to the development of linear amplification sequencing (cycle sequencing). This very powerful yet technically simple method of sequencing has many advantages over conventional techniques, and may be used in manual or automated methods. Other recent innovations proposed recently to increase speed and efficiency include multiplex sequencing. This consists of pooling a number of samples and processing them as pools. After electrophoresis, the DNA is transferred to a membrane, and sequence images of the individual samples are obtained by sequential hybridizations with specific labeled oligonucleotides. Multiplex DNA sequencing has been used in conjunction with direct blotting electrophoresis to facilitate transfer of the DNA to a membrane. Chemiluminescent detection can also be used in conjunction with multiplex DNA sequencing to visualize the image on the membrane.     

Miniaturized Sequencing Gel System for Quick Analysis of DNA by Hydroxyl Radical Cleavage

We described a simple and quick miniaturized sequencing gel system for DNA analysis. Two major modifications were made to the previously reported miniaturized DNA sequencing gel system to achieve high-resolution hydroxyl radical cleavage analysis: including formamide in the miniaturized gel and providing uniform heating during electrophoresis. Our method enables one to reduce the cost for chemicals and to significantly reduce electrophoresis time. Furthermore, minimal gel handling simplifies the entire process. We show that the resolution of DNA fragments obtained by hydroxyl radical cleavage for the miniaturized gel is similar to that of a large conventional sequencing gel.     

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.     

A general approach to the analysis of errors and failure modes in the base-calling function in automated fluorescent DNA sequencing

We describe the analysis of errors and failure modes in the base-calling function in automated DNA sequencing, on instruments in which fluorescently-labeled Sanger dideoxy-sequencing ladders are detected via their times of migration past a fixed detector. A general approach entails the joint use of: (i) well-defined control samples such as M13mp18, and (ii) mathematical simulation of sequencing electropherograms, with the deliberate introduction of different types of distortion and noise. An algorithm, the electrophoretic trace simulator (ETS), is used to calculate electrophoresis traces corresponding to the output data stream of an automated fluorescent DNA sequencer. The ETS accepts a user-defined sequence of nucleotide bases (A, C, G, T) as input, and employs user-adjustable functions to compute the following critical parameters of an electropherogram: peak intensity, peak spacing, peak shape as a function of base number; background, noise, and spectral cross-talk correction (for a sequencer using multiple dyes). We use a combination of M13mp18 controls and simulated electropherograms to analyze two problems of considerable practical importance: (i) variation in electrophoretic migration rates between different lanes of a gel, and (ii) variation in signal intensity due to user-dependent loading artifacts. The issue of base-calling errors and failure modes, for electropherograms that contain noise and distortion, is addressed.     

Local conformation of glassy polystyrenes with different stereoregularity

The molecular structure of glassy polystyrenes with different stereoregularity was investigated by infrared, Raman, and solid state high resolution ¹³C NMR spectroscopies. The characteristic bands observed in infrared spectrum closely relates to the regular sequence length of conformational arrangement. The out-of-plane mode of phenyl ring in 500-600 cm⁻¹ region, which is one of the most conformational sensitive bands for polystyrene, provided no characteristic peaks concerned with longer regular sequence than four monomers. Raman spectrum showed that most of the bands providing different peaks among polystyrenes with different stereoregularity are assumed local phenyl ring modes. Solid state high resolution ¹³C NMR is available to estimate the gauche content in terms of analysis based on γ-gauche effect. We determined that the gauche content takes 25.0, 27.9, and 34.3% for glassy state of syndiotactic, atactic, and isotactic polystyrenes, respectively. This indicates that the conformational structure of sPS tends to take trans sequence. Since a large content of trans sequence would make the molecular dimension wide, the glassy sPS provided the smallest density for polystyrenes with different stereoregularity.     

Incorporation of guanosine gels into sieving matrices for length- and sequence-based separation of DNA in capillary electrophoresis

Sieving gels are used in capillary gel electrophoresis to resolve DNA strands of different lengths. For complex samples, however, such as those encountered in metagenomic analysis of microbial communities or biofilms, length-based separation may mask the true genetic diversity of the community since different organisms may contribute same-length DNA with different sequences. There is a need, therefore, for DNA separations based on both the length and sequence. Previous work has demonstrated the ability of guanosine gels (G-gels) to separate four single-stranded DNA 76-mers that differ by only a few A/G base substitutions. The goal of the present work is to determine whether G-gels could be combined with commercial sieving gels in order to simultaneously separate DNA based on both length and sequence. The results are given for the four 76-mers and for a standard dsDNA ladder. Commercial sieving gels were used alone and in combination with G-gels. For the 76-mers, the combined medium was less efficient than the G-gel alone but was able to achieve partial resolution. The combined medium was at least as effective as the sieving gel alone at resolving the denatured DNA ladder and showed indications of sequence-based resolution as well, as supported by MALDI-MS. The results show that the combined sieving gel/G-gel medium retains the selectivity of the individual media, providing a promising approach to simultaneous length- and sequence-based DNA separation for metagenomic analysis of complex systems.     

Maximum entropy image reconstruction of DNA sequencing gel autoradiographs

DNA sequencing gel autoradiographs become increasingly difficult to read as one moves up the gel, due to crowding and overlapping of the bands. Maximum entropy image reconstructions of the autoradiograph improve the ease with which crowded sequence data can be read, and extend the region of the gel in which reading is possible. Superior reconstructions are obtained by using nonuniform models based on the initial reconstructions.     

DNA sequencing in HydroLink matrices: extension of reading ability to greater than 600 nucleotides

All the systems for optimizing DNA sequencing published so far have introduced modifications regarding: (i) linearization of band migration via ionic strength gradients or wedge-shaped gels; (ii) automatization of band reading via introduction of fluorescent probes; (iii) direct blotting analysis; (iv) pulsed electric fields and (v) discontinuous buffer systems. In all these systems, DNA sequence reading with an accuracy of ca. 98% rarely exceeds a length of 350 bases. We have chosen, in order to increase the reading ability of a single gel, to manipulate the characteristics of the gel matrix. The Seq-HydroLink gel formation here reported allows optimal reading, from a single gel run, of at least 600 bases. In order to guarantee this reading ability in a single run, the upper and lower ends of the ladder are time-resolved, i.e. the same sample is applied to the gel matrix at three different time intervals. The present system represents an increase of at least 30% in reading ability as compared with any type of polyacrylamide gel formulation so far reported.     

人的基因组及DNA序列分析──过去、现去及将来（上）

人的基因组研究已成为生命科学前沿领域中最热门的课题之一。ＤＮＡ序列分析是基因组研究的关键技术．本文对人的基因组分析及其对ＤＮＡ序列分析的要求进行了论述．对ＤＮＡ序列分析方法如板凝胶电泳自放射显影法、板凝胶电泳激光荧光法、毛细管电泳激光荧光法、阵列毛细管凝胶电泳激光荧光法。超薄层板在胶电泳激光荧光法作了详细评论．并对正在开发的不用凝胶电泳分离的直接测序新技术和新方法，如质谱法、原子探针法（扫描隧道显微镜、原子力显微镜）、杂交法、流动单分子荧光检测法等进行了评论。     

Comparison of two different electrophoretic methods in studying the genetic diversity among plantains (Musa spp.) using ISSR markers

Inter simple sequence repeat markers were employed for the genotyping of 16 plantain ecotypes. Two different electrophoretic systems namely conventional gel electrophoresis (CVGE) and fully automated high‐resolution CGE were used to evaluate the genetic diversity. Comparative analysis indicated that all parameters related to marker informativeness were higher in CGE except polymorphic information content. But genetic diversity parameters like effective number of alleles, Nei's gene diversity (1973) and Shannon's information index showed higher values (1.52 ± 0.12, 0.34 ± 0.05 and 0.52 ± 0.05, respectively) in CVGE as against CGE (1.29 ± 0.04, 0.22 ± 0.02 and 0.38 ± 0.03, respectively) system. The unweighed pair group method with arithmetic averages was used to obtain the dendrogram for both analyses. The results of dendrogram and principal component analysis were found to be consistent in both systems except for some minor disagreements. The clone‐specific bands could be used in the identification and development of SCAR markers. Inter simple sequence repeat markers used in this study provided sufficient polymorphism and reproducible banding pattern for evaluating the genetic diversity of different plantain ecotypes. Lack of accuracy and consistency of the CVGE warrants the employment of high‐throughput CGE for diversity analysis as it provided better separation of bands with higher resolution.     

Adaption of a fragment analysis technique to an automated high-throughput multicapillary electrophoresis device for the precise qualitative and quantitative characterization of microbial communities

The analysis of microbial communities is of increasing importance in life sciences and bioengineering. Traditional techniques of investigations like culture or cloning methods suffer from many disadvantages. They are unable to give a complete qualitative and quantitative view of the total amount of microorganisms themselves, their interactions among each other and with their environment. Obviously, the determination of static or dynamic balances among microorganisms is of fast growing interest. The generation of species specific and fluorescently labeled 16S ribosomal DNA (rDNA) fragments by the terminal restriction fragment length polymorphism (T-RFLP) technique is a suitable tool to overcome the problems other methods have. For the separation of these fragments polyacrylamide gel sequencers are preferred as compared to capillary sequencers using linear polymers until now because of their higher electrophoretic resolution and therefore sizing accuracy. But modern capillary sequencers, especially multicapillary sequencers, offer an advanced grade of automation and an increased throughput necessary for the investigation of complex communities in long-time studies. Therefore, we adapted a T-RFLP technique to an automated high-throughput multicapillary electrophoresis device (ABI 3100 Genetic Analysis) with regard to a precise qualitative and quantitative characterization of microbial communities.