A novel thermogelling matrix for microchannel DNA sequencing based on poly‐<b><i>N</i></b>‐alkoxyalkylacrylamide copolymers

Kan, Cheuk‐Wai; Doherty, Erin A. S.; Barron, Annelise E.

doi:10.1002/elps.200305670

Cited by 20 publications

(21 citation statements)

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“…Hence, the chemical and physical structures of thermo-responsive polymer networks must be finely tuned to optimized their performance for DNA sequencing on chips and they may never yield read lengths quite as long as LPA does. Reprinted from [201], with permission.…”

Section: Thermo-responsive Polymer Matricesmentioning

confidence: 96%

“…Since then, a number of research groups have been involved in developing thermo-responsive polymers for dsDNA analysis [197,198] or single-color ssDNA separation [199], while the design of these "switchable-viscosity" networks for 4-color DNA sequencing has been mainly pursued by Barron et al [188,200,201]. Ideally, these matrices allow a decoupling of the capillary loading and DNA sieving performance, and if developed further, may be one of the key technological developments that enable the practical use of microfabricated devices for high-throughput sequencing and genotyping [188].…”

Section: Thermo-responsive Polymer Matricesmentioning

confidence: 99%

“…The above-mentioned three classes of "thermo-thickening" matrices have shown the ability to give high-resolution dsDNA separations. Very recently, thermo-gelling polymeric matrices based on copolymers of two different N-alkoxyalkylacrylamide monomers have been applied for 4-color DNA sequencing by CE [201]. A read length of around 600 bases, with base-calling accuracy of 98.5% was achieved with a thermo-gelling copolymer (M W , 2.5 MDa) composed of 90% w/w N-methoxyethylacrylamide (NMEA) and 10% w/w N-ethoxyethylacrylamide (NEEA).…”

Section: Thermo-responsive Polymer Matricesmentioning

confidence: 99%

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DNA sequencing and genotyping in miniaturized electrophoresis systems

et al. 2004

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Advances in microchannel electrophoretic separation systems for DNA analyses have had important impacts on biological and biomedical sciences, as exemplified by the successes of the Human Genome Project (HGP). As we enter a new era in genomic science, further technological innovations promise to provide other far-reaching benefits, many of which will require continual increases in sequencing and genotyping efficiency and throughput, as well as major decreases in the cost per analysis. Since the high-resolution size- and/or conformation-based electrophoretic separation of DNA is the most critical step in many genetic analyses, continual advances in the development of materials and methods for microchannel electrophoretic separations will be needed to meet the massive demand for high-quality, low-cost genomic data. In particular, the development (and commercialization) of miniaturized genotyping platforms is needed to support and enable the future breakthroughs of biomedical science. In this review, we briefly discuss the major sequencing and genotyping techniques in which high-throughput and high-resolution electrophoretic separations of DNA play a significant role. We review recent advances in the development of technology for capillary electrophoresis (CE), including capillary array electrophoresis (CAE) systems. Most of these CE/CAE innovations are equally applicable to implementation on microfabricated electrophoresis chips. Major effort is devoted to discussing various key elements needed for the development of integrated and practical microfluidic sequencing and genotyping platforms, including chip substrate selection, microchannel design and fabrication, microchannel surface modification, sample preparation, analyte detection, DNA sieving matrices, and device integration. Finally, we identify some of the remaining challenges, and some of the possible routes to further advances in high-throughput DNA sequencing and genotyping technologies.

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Section: Thermo-responsive Polymer Matricesmentioning

confidence: 96%

Section: Thermo-responsive Polymer Matricesmentioning

confidence: 99%

Section: Thermo-responsive Polymer Matricesmentioning

confidence: 99%

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DNA sequencing and genotyping in miniaturized electrophoresis systems

et al. 2004

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“…Current electrophoresis-based sequencing technology has been widely used in the laboratories around the world although this technology is complicated, expensive, and time-consuming. To overcome the limitations of the current electrophoresis-based sequencing technology [2]- [5], a variety of new DNA-sequencing methods have been investigated. Such approaches include sequencing by hybridization [6], mass spectrometry-based sequencing [7]- [9], sequence-specific detection of single-stranded DNA using engineered nanopores [10], and sequencing by ligation [11].…”

Section: Introductionmentioning

confidence: 99%

Vibrational Characteristics of Genetic Codons: 5$'$-GGX-3$'$ and 5$'$-XGG-3$'$ (${\rm X} = {\rm A}, {\rm C}$, and T)

Zhao

Woolard

2010

IEEE Sensors J.

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A theoretical study of the vibrational characteristics of six DNA codons (i.e., at the sequence forms 5 -GGX-3 and 5 -XGG-3 ; X = A, C, and T) in gas phase is presented.The simulation results show that the spectra of the codons in the near infrared regime can be classified into five semi-distinct spectral sub-regimes, namely: two end regions and one region that separates the two sequence identifier regions with features that arise primarily from coupling between the GG bases and other bases (T, C, and A). This study reveals a number of phenomenological trends that have scientific relevance to spectroscopic characterization of DNA molecules. For example, spectra in the high-frequency end-regions arise primarily from vibrational modes associated with the O-H bonds at the 5 end or 3 end of the codons and the spectra in the low-frequency end-regions are formed primarily by the vibrational modes of the backbone. Conversely, in the lower-frequency side, the sequence-identifier region is composed of the C-H bond stretch vibrations in the planes of the corresponding DNA bases, and in the higher-frequency side, sequence-identifier region is composed of the N-H bond stretch vibrations in the planes of the corresponding DNA bases. In addition, the sequence-identifier dividing region almost exclusively contains vibrational modes due to coupling between the G nucleotides and other bases. As will be shown, all the vibrational modes in sequence identifier regions are localized at the corresponding DNA bases and exhibit a definable dependence on the sequence form of the codons under study.

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“…Accurate high-throughput DNA sequencing methods are needed to explore the complete human genome sequence for applications in clinical medicine and health care. To overcome the limitations of the current electrophoresis-based sequencing technology (2)(3)(4)(5), a variety of new DNA-sequencing methods have been investigated. Such approaches include sequencing by hybridization (6), mass spectrometry-based sequencing (7)(8)(9), sequence-specific detection of single-stranded DNA using engineered nanopores (10), and sequencing by ligation (11).…”

mentioning

confidence: 99%

Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

Kim

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

187

160

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DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3-O-allyldNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3-O-allyl group on a DNA extension product, which is generated by incorporating 3-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pdcatalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner. DNA chip DNA sequencing is driving genomics research and discovery. The completion of the Human Genome Project has set the stage for screening genetic mutations to identify disease genes on a genome-wide scale (1). Accurate high-throughput DNA sequencing methods are needed to explore the complete human genome sequence for applications in clinical medicine and health care. To overcome the limitations of the current electrophoresis-based sequencing technology (2-5), a variety of new DNA-sequencing methods have been investigated. Such approaches include sequencing by hybridization (6), mass spectrometry-based sequencing (7-9), sequence-specific detection of single-stranded DNA using engineered nanopores (10), and sequencing by ligation (11). More recently, DNA sequencing by synthesis (SBS) approaches such as pyrosequencing (12), sequencing of single DNA molecules (13), and polymerase colonies (14) have been widely explored.The concept of DNA SBS was revealed in 1988 with an attempt to sequence DNA by detecting the pyrophosphate group that is generated when a nucleotide is incorporated in a DNA polymerase reaction (15). Pyrosequencing, which was developed based on this concept and an enzymatic cascade, has been explored for genome sequencing (16). However, there are inherent difficulties in this method for determining the number of incorporated nucleotides in homopolymeric regions of the template. Additionally, each of the four nucleotides needs to be added and detected separately, which increases the overall detection time. The accumulation of undegraded nucleotides and other components could also lower the accuracy of the method when sequencing a long DNA template. It is thus desirable to have a simple method t...

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A novel thermogelling matrix for microchannel DNA sequencing based on poly‐N‐alkoxyalkylacrylamide copolymers

Cited by 20 publications

References 35 publications

DNA sequencing and genotyping in miniaturized electrophoresis systems

DNA sequencing and genotyping in miniaturized electrophoresis systems

Vibrational Characteristics of Genetic Codons: 5$'$-GGX-3$'$ and 5$'$-XGG-3$'$ (${\rm X} = {\rm A}, {\rm C}$, and T)

Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

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