High-speed DNA Sequencing Research Articles

Nucleobase-functionalized graphene nanoribbons for accurate high-speed DNA sequencing.

We propose a water-immersed nucleobase-functionalized suspended graphene nanoribbon as an intrinsically selective device for nucleotide detection. The proposed sensing method combines Watson-Crick selective base pairing with graphene's capacity for converting anisotropic lattice strain to changes in an electrical current at the nanoscale. Using detailed atomistic molecular dynamics (MD) simulations, we study sensor operation at ambient conditions. We combine simulated data with theoretical arguments to estimate the levels of measurable electrical signal variation in response to strains and determine that the proposed sensing mechanism shows significant promise for realistic DNA sensing devices without the need for advanced data processing, or highly restrictive operational conditions.

Bioinformatics tools for discovery and functional analysis of single nucleotide polymorphisms.

With the high speed DNA sequencing of genome, databases of genome data continue to grow, and the understanding of genetic variation between individuals grows as well. Single nucleotide polymorphisms (SNPs), a main type of genetic variation, are increasingly important resource for understanding the structure and function of the human genome and become a valuable resource for investigating the genetic basis of disease. During the past years, in addition to experimental approaches to characterize specific variants, intense bioinformatics techniques were applied to understand effects of these genetic changes. In the genetics studies, one intends to understand the molecular basis of disease, and computational methods are becoming increasingly important for SNPs selection, prediction and understanding the downstream effects of genetic variation. The review provides systematic information on the available resources and methods for SNPs discovery and analysis. We also report some new results on DNA sequence-based prediction of SNPs in human cytochrome P450, which serves as an example of computational methods to predict and discovery SNPs. Additionally, annotation and prediction of functional SNPs, as well as a comprehensive list of existing tools and online recourses, are reviewed and described.

CMOS-based high-speed nanopore recording: signals and systems.

This paper considers the potential of CMOS-based nanopore measurement systems for high-speed molecular recording and DNA sequencing in particular. As such it approximates the signal fidelity limitations of a CMOS-based nanopore readout channel and discusses the means by which throughput could be increased through the use of a parallel CMOS readout system.

DNA fingerprinting in botany: past, present, future.

Almost three decades ago Alec Jeffreys published his seminal Nature papers on the use of minisatellite probes for DNA fingerprinting of humans (Jeffreys and colleagues Nature 1985, 314:67–73 and Nature 1985, 316:76–79). The new technology was soon adopted for many other organisms including plants, and when Hilde Nybom, Kurt Weising and Alec Jeffreys first met at the very First International Conference on DNA Fingerprinting in Berne, Switzerland, in 1990, everybody was enthusiastic about the novel method that allowed us for the first time to discriminate between humans, animals, plants and fungi on the individual level using DNA markers. A newsletter coined “Fingerprint News” was launched, T-shirts were sold, and the proceedings of the Berne conference filled a first book on “DNA fingerprinting: approaches and applications”. Four more conferences were about to follow, one on each continent, and Alec Jeffreys of course was invited to all of them. Since these early days, methodologies have undergone a rapid evolution and diversification. A multitude of techniques have been developed, optimized, and eventually abandoned when novel and more efficient and/or more reliable methods appeared. Despite some overlap between the lifetimes of the different technologies, three phases can be defined that coincide with major technological advances. Whereas the first phase of DNA fingerprinting (“the past”) was dominated by restriction fragment analysis in conjunction with Southern blot hybridization, the advent of the PCR in the late 1980s gave way to the development of PCR-based single- or multi-locus profiling techniques in the second phase. Given that many routine applications of plant DNA fingerprinting still rely on PCR-based markers, we here refer to these methods as “DNA fingerprinting in the present”, and include numerous examples in the present review. The beginning of the third phase actually dates back to 2005, when several novel, highly parallel DNA sequencing strategies were developed that increased the throughput over current Sanger sequencing technology 1000-fold and more. High-speed DNA sequencing was soon also exploited for DNA fingerprinting in plants, either in terms of facilitated marker development, or directly in the sense of “genotyping-by-sequencing”. Whereas these novel approaches are applied at an ever increasing rate also in non-model species, they are still far from routine, and we therefore treat them here as “DNA fingerprinting in the future”.

Prediction and Functional Analysis of Single Nucleotide Polymorphisms

With the high speed DNA sequencing of genome, databases of genome data continue to grow, and the understanding of genetic variation between individuals grows as well. Single nucleotide polymorphisms (SNPs), the most common type of genetic variation, are an increasingly important resource for understanding the structure and function of the human genome and become a valuable resource for investigating the genetic basis of disease. During the past years, in addition to experimental approaches to characterize specific variants, intense bioinformatics techniques were applied to understand the effects of these genetic changes. In the genetics studies, one intends to understand the molecular basis of disease, and computational methods are becoming increasingly important for SNPs selection, prediction and understanding the downstream effects of genetic variation. The review provides systematic information on the available resources and methods for SNPs discovery and analysis. We also report some new results on DNA sequence-based prediction of SNPs in human cytochrome P450, which serves as an example of computational methods to predict and discover SNPs. Additionally, annotation and prediction of functional SNPs, as well as a comprehensive list of existing tools and online recourses, are reviewed and described.

Gene tree discordance of wild and cultivated Asian rice deciphered by genome-wide sequence comparison

Although a large number of genes are expected to correctly solve a phylogenetic relationship, inconsistent gene tree topologies have been observed. This conflicting evidence in gene tree topologies, known as gene tree discordance, becomes increasingly important as advanced sequencing technologies produce an enormous amount of sequence information for phylogenomic studies among closely related species. Here, we aim to characterize the gene tree discordance of the Asian cultivated rice Oryza sativa and its progenitor, O. rufipogon, which will be an ideal case study of gene tree discordance. Using genome and cDNA sequences of O. sativa and O. rufipogon, we have conducted the first in-depth analyses of gene tree discordance in Asian rice. Our comparison of full-length cDNA sequences of O. rufipogon with the genome sequences of the japonica and indica cultivars of O. sativa revealed that 60% of the gene trees showed a topology consistent with the expected one, whereas the remaining genes supported significantly different topologies. Moreover, the proportions of the topologies deviated significantly from expectation, suggesting at least one hybridization event between the two subgroups of O. sativa, japonica and indica. In fact, a genome-wide alignment between japonica and indica indicated that significant portions of the indica genome are derived from japonica. In addition, literature concerning the pedigree of the indica cultivar strongly supported the hybridization hypothesis. Our molecular evolutionary analyses deciphered complicated evolutionary processes in closely related species. They also demonstrated the importance of gene tree discordance in the era of high-speed DNA sequencing.

Fabrication of nano structures in thin membranes with focused ion beam technology

In recent years, Focused Ion Beam (FIB) technology has emerged as an important tool for nanotechnology [V.J. Gadgil, F. Morrissey, Encyclopaedia of Nanoscience and Nanotechnology, vol. 1, American Science Publishers, ISBN: 1-58883-057-8, 2004, p101.]. In this paper, applications of focused ion beam technology to fabrication of nanostructures are presented. The structures are fabricated on free standing silicon nitride membranes. Nanopores are nanometer diameter holes used in bio medical research for high speed DNA sequencing [D.K. Stewart, L.A. Stern, G. Foss, G. Hughes and P. Govil, Proc. SPIE 21, 1990, 1263.]. FIB was used to mill nanopores in the membrane. The pores were further reduced using epitaxial deposition using electron beam, at a controlled rate. A STEM detector was used to monitor the pore in situ. Nanowires can be fabricated using shadow mask technique. The shadow mask for nanowires was fabricated using FIB. The mask was used to produce nanowires. Fabrication method and FIB process parameters for the fabrication are reported. Results of the nanopore fabrication are presented with STEM images. Results of the nanowire fabrication are presented. Various strategies employed to achieve the desired nanostructures are discussed.

High‐speed DNA sequencing by tube‐based capillary electrophoresis

We assessed the feasibility of high-speed DNA sequencing by tube-based capillary electrophoresis (TCE) with electrokinetic sample injections. We developed a water-circulated TCE system to control the capillary temperature precisely. Using this system and a ready-made sieving matrix at 50 degrees C, single-stranded DNA size marker fragments were separated at various pairs of the electric field strength, E, of 128-480 V/cm and the capillary effective length, L, of 100-360 mm. Assuming the read length (RL) is the fragment size at which the peak width equals the peak interval per base in obtained electropherograms, we estimated the values of RL (E, L), the RL at the pair (E, L). The points in ELz-space, (E, L, RL(E, L)), form a curved surface expressed by z = RL(E, L). Analyzing the contour lines of this curved surface, we determined the pairs of E and L providing target RLs of 300-500 bases within a minimum time. At a pair optimized for a 500-base RL (330 V/cm, 200 mm), one-color sequencing fragments were successfully separated up to 529 bases within 9.6 min. These results demonstrate that high-speed DNA sequencing comparable with that obtained by microfabricated chip-based capillary electrophoresis (MCE) can be achieved with TCE, which is more suitable in automation than MCE.

A Chip-Based Biosensor for the Functional Analysis of Single Ion Channels We thank E. Ermanntraut, L. Giovangrandi, T. Wohland, A. Brecht, M. Köhler, C. Bieri, D. Stamou, and R. Hovius for advice. This work was supported by the Swiss National Science Foundation (Priority Program for Biotechnology) and by an interdepartmental grant of the Swiss Federal Institute of Technology Lausanne (EPFL,

In this work the authors have shown that stable gigaohm seals over micrometer-sized holes can be obtained in a time-frame of seconds by the electrophoretic self-positioning of charged lipid membranes. For the first time, single-channel events were recorded on silicon microstructures. Self-integrating channel-forming peptides, such as alamethicin are expected to have applications in screening assays based on single-channel recordings, including high-speed DNA sequencing and metabolite detection. In the future, this method could be extended by either functional reconstitution of ion channels into giant vesicles or their fusion (small proteoliposomes) into preformed bilayers. The possibility of combining this type of planar setup with high numerical optics may also enable the simultaneous observation of elec. and optical signals, which would help to elucidate the coupling between ligand-binding and channel events of ionotropic receptors. [on SciFinder (R)]

Optimization of high-performance DNA sequencing on short microfabricated electrophoretic devices.

We have examined the parametric performance of short microfabricated electrophoresis devices that operate with a replaceable linear poly(acrylamide) (LPA) solution for the application of DNA sequencing. A systematic study is presented of the dependence of selectivity, separation efficiency, and resolution of sequencing fragments on buffer composition, LPA concentration, LPA composition, microdevice temperature, electric field, and device length. A specific optimization is made for DNA sequencing on 11.5-cm devices. Using a separation matrix composed of 3.0% (w/w) 10 MDa plus 1.0% (w/w) 50 kDa LPA, elevated microdevice temperature (50 degrees C), and 200 V/cm, high-speed DNA sequencing of 580 bases on standard M13mp18 was obtained in only 18 min with a base-calling accuracy of 98.5%. Read lengths of 640 bases at 98.5% accuracy were achieved in approximately 30 min by reducing the electric field strength to 125 V/cm. We believe that this constitutes matrix-limited performance for microdevices of this length using LPA sieving matrix and this buffer chemistry. In addition, it was confirmed, that shorter devices are rather impractical for production sequencing applications when LPA is used as sieving matrix.

DNA sequencing by capillary array electrophoresis and microfabricated array systems

To comply with the current needs for high-speed DNA sequencing analysis, several instruments and innovative technologies have been introduced by several groups in recent years. This review article discusses and compares the issues regarding high-throughput DNA sequencing by electrophoretic methods in miniaturized systems, such as capillaries, capillary arrays, and microchannels. Initially, general features of several capillary array designs (including commercial ones) will be considered, followed by similar analyses with microfabricated array electrophoretic devices and how they can contribute to the success of large sequencing projects.

DNA sequencing by capillary array electrophoresis and microfabricated array systems.

To comply with the current needs for high-speed DNA sequencing analysis, several instruments and innovative technologies have been introduced by several groups in recent years. This review article discusses and compares the issues regarding high-throughput DNA sequencing by electrophoretic methods in miniaturized systems, such as capillaries, capillary arrays, and microchannels. Initially, general features of several capillary array designs (including commercial ones) will be considered, followed by similar analyses with microfabricated array electrophoretic devices and how they can contribute to the success of large sequencing projects.

Optimization of high-speed DNA sequencing on microfabricated capillary electrophoresis channels.

DNA sequencing separations have been performed in microfabricated electrophoresis channels with the goal of determining whether high-quality sequencing is feasible with these microdevices. The separation matrix, separation temperature, channel length and depth, injector size, and injection parameters were optimized. DNA fragment sizing separations demonstrated that 50-micron-deep channels provide the best sensitivity for our detection configuration. One-color sequencing separations of single-stranded M13mp18 DNA on 3% linear polyacrylamide (LPA) were used to optimize the twin-T injector size, injection conditions, and temperature. The best one-color separations were observed with a 250-micron twin-T injector, an injection time of 60 s, and a temperature of 35 degrees C. The first 500 bases appeared in 9.2 min with a resolution of > 0.5, and the separation extended to 700 bases. The best four-color sequencing separations were performed using 4% LPA, a temperature of 40 degrees C, and a 100-micron twin-T injector. These four-color runs were complete in only 20 min, could be automatically base-called using BaseFinder to over 600 bp after the primer, and were 99.4% accurate to 500 bp. These results significantly advance the quality of microchip-based electrophoretic sequencing and indicate the feasibility of performing high-speed genomic sequencing with microfabricated electrophoretic devices.

ChemInform Abstract: Development of Analytical Technologies for Human Genomic DNA Using Capillary Electrophoresis and Their Applications

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Electrophore mass tag dideoxy DNA sequencing.

Toward a goal of dideoxy sequencing DNA utilizing electrophore labels, we prepared four electrophore-labeled DNA oligonucleotide primers. Each primer has a different electrophore and DNA sequence but a common glycol keto (alpha,beta-dihydroxyketo) release group. Cleavage of this latter group by either periodate oxidation or a thermal retroaldol reaction releases the electrophores for detection by mass spectrometry. Successful sequencing data with these primers was obtained by capillary electrophoresis on an ABI Model 310 after fluorescence dideoxy terminator cycle sequencing reactions were conducted. In a separate experiment, it was demonstrated that a cocktail of the four electrophore DNA primers could be detected as a dried sample spot by CO2 laser desorption/capillary collection/gas chromatography electron capture mass spectrometry. These results establish some feasibility for our long-term goal of high-speed multiplex electrophore mass tag dideoxy DNA sequencing. Ultimately we plan to use a higher number of electrophore mass tags and to rely on direct detection of the desorbed electrophores by electron capture time-of-flight mass spectrometry.

High-speed DNA sequencing in ultrathin slab gels

There have been many recent theoretical and technical advances in the sequencing of DNA in ultrathin slab gels. These include recent experimental progress in four areas: DNA polymerases; DNA template preparation; the separation and detection of DNA bands; and base-calling algorithms. The Zimm—Lumpkin theory of electrophoresis provides a new and improved framework for understanding the operation of sequencing gels.

Development of analytical technologies for human genomic DNA using capillary electrophoresis and their applications

Capillary electrophoretic systems equipped with a multi-color detectable laser-induced fluorescent DNA detector (CE-LIF) were developed. We examined the efficiency and the performance of the CE-LIF systems for the high-speed DNA sequencing and DNA diagnosis for human diseases. The effect of the gel composition, electric field strength, and capillary length on the separation of DNA sequencing reaction product was investigated in order to achieve high-speed DNA sequencing for large-scale sequencing in the Human Genome Project. The CE-LIF system is successfully applied to ultrafast cDNA sequencing for human and yeast genomes. Under optimum separation conditions, only 10 min is required to sequence 300 base DNA. A polymer solution of cellulose derivative was utilized as a sieving medium for the CE-LIF system and gave excellent resolution of polymerase chain reaction (PCR) amplified polymorphic loci on the human genome. The CE-LIF system is successfully applied to the DNA diagnosis for cancers through CA repeat analysis of human D8S 1218 locus, heart diseases through VNTR (variable number of tandem repeat) analysis of human apolipoprotein B gene and Alzheimer's disease through RFLP (restriction fragment length polymorphism) analysis of human apolipoprotein E gene with high-speed and high resolution. Capillary affinity gel electrophoresis was developed as a new technique for the recognition of the specific DNA base and/or sequence. This technology is also applicable to the characterization of binding properties of DNA based drugs. The principle, the theory, and the methods of capillary affinity gel electrophoresis are presented. This technique is applied to the determination of association constants between an affinity ligand and oligonucleotides. The great potential of capillary affinity gel electrophoresis for the detection of the mutation on DNA is illustrated.

High-Speed DNA Sequencing by Using Mixed Poly(ethylene oxide) Solutions in Uncoated Capillary Columns

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTHigh-Speed DNA Sequencing by Using Mixed Poly(ethylene oxide) Solutions in Uncoated Capillary ColumnsEliza N. Fung and Edward S. YeungCite this: Anal. Chem. 1995, 67, 13, 1913–1919Publication Date (Print):July 1, 1995Publication History Published online1 May 2002Published inissue 1 July 1995https://doi.org/10.1021/ac00109a002RIGHTS & PERMISSIONSArticle Views412Altmetric-Citations191LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (755 KB) Get e-Alerts Get e-Alerts

Optimization of Excitation and Detection Geometry for Multiplexed Capillary Array Electrophoresis of DNA Fragments

A new on-column excitation and detection geometry for multiplexed array capillary electrophoresis is evaluated. The detection limit is improved 100-fold for the same laser power because of more efficient coupling of the laser beam. The illumination is also more uniform over the entire array. Stray light and cross-talk are effectively suppressed by immersing the capillaries in water to roughly match the refractive index. A charge-coupled-device (CCD) camera combined with cutoff filters provides adequate sensitivity and speed for high-speed DNA sequencing based on sieving in a polymer-matrix solution.

High-speed and high-accuracy DNA sequencing by capillary gel electrophoresis in a simple, low cost instrument Two-color peak-height encoded sequencing at 40°C

A low-cost DNA sequencer was constructed based on a single helium-neon laser. The two-color peak-height encoded sequencing protocol, based on the use of T7 DNA polymerase in a manganese buffer, was used to generate samples. Two termination reactions were performed. In the first, a TAMRA (applied Biosystems)-labeled primer was extended in the presence of ddATP and ddCTP. The amounts of dideoxynucleotides were adjusted to produce a 3:1 peak height ratio. Similarly, a ROX (Applied Biosystems)-labeled primer was extended in the presence of ddGTP and ddTTP; the amounts of dideoxynucleotides was adjusted to produce a 3:1 peak height ratio. The pooled fragments were separated on a 4% T LongRanger gel operated at 39°C. Over 500 bases of sequence were generated in 50 min.