Biomedical subjects

Erin A S Doherty

Publications and source records attributed to Erin A S Doherty.

8 recordsLinked to original sources

PubMed2005

Effect of polymer matrix and glycerol on rapid single-strand conformation polymorphism analysis by capillary and microchip electrophoresis for detection of mutations in K-ras gene.

We present the rapid single-strand conformation polymorphism (SSCP) analysis by capillary and microchip electrophoresis to detect the mutations in K-ras gene. Parameters that might affect the analysis of mutation in K-ras gene, such as the polymer and the additive in the sieving matrix, have been studied systematically. Under the optimal conditions, the analysis of seven mutants of K-ras gene could be finished within 10 min by capillary electrophoresis (CE). Furthermore, with the wild-type gene as the inner standard, the analysis accuracy of mutations could be improved. In addition, by studying the properties of polymer solutions, the matrix suitable for microchip electrophoresis was found, and the detection of mutations in K-ras gene could be further shortened to 1 min.

Base Sequence↗

PubMed2004

Sparsely cross-linked "nanogel" matrixes as fluid, mechanically stabilized polymer networks for high-throughput microchannel DNA sequencing.

We have developed sparsely cross-linked "nanogels", subcolloidal polymer structures composed of covalently linked, linear polyacrylamide chains, as novel replaceable DNA sequencing matrixes for capillary and microchip electrophoresis. Nanogels were synthesized via inverse emulsion (water-in-oil) copolymerization of acrylamide and a low percentage (approximately 10(-4) mol %) of N,N-methylene bisacrylamide (Bis). Nanogels and nanogel networks were characterized by multiangle laser light scattering and rheometry, respectively, and tested for DNA sequencing in both capillaries and chips with four-color LIF detection. Typical nanogels have an average radius of approximately 230 nm, with approximately 75% of chains incorporating a Bis cross-linker. The properties and performance of nanogel matrixes are compared here to those of a linear polyacrylamide (LPA) network, matched for both polymer weight-average molar mass (M(w)) and the extent of interchain entanglements (c/c). At sequencing concentrations, the two matrixes have similar flow characteristics, important for capillary and microchip loading. However, because of the physical network stability provided by the internally cross-linked structure of the nanogels, substantially longer average read lengths are obtained under standard conditions with the nanogel matrix at a 98.5% accuracy of base-calling (for CE: 680 bases, an 18.7% improvement over LPA, with the best reads as long as 726 bases, compared to 568 bases for the LPA matrix). We further investigated the use of the nanogel matrixes in a high-throughput microfabricated DNA sequencing device consists of 96 separation channels densely fabricated on a 6-in. glass wafer. Again, preliminary DNA sequencing results show that the nanogel matrixes are capable of delivering significantly longer average read length, compared to an LPA matrix of comparable properties. Moreover, nanogel matrixes require 30% less polymer per unit volume than LPA. The addition of a small amount of low molar mass LPA or ultrahigh molar mass LPA to the optimized nanogel sequencing matrix further improves read length as well as the reproducibility of read length (RSD < 1.6%). This is the first report of a replaceable DNA sequencing matrix that provides better performance than LPA, in a side-by-side comparison of polymer matrixes appropriately matched for molar mass and the extent of interchain entanglements. These results could have significant implications for the improvement of microchip-based DNA sequencing technology.

Acrylic Resins↗

PubMed2004

Thermoresponsive N,N-dialkylacrylamide copolymer blends as DNA sieving matrices with a thermally tunable mesh size.

In an earlier study we showed that a blend of thermoresponsive and nonthermoresponsive hydroxyalkylcelluloses could be used to create a thermally tunable polymer network for double-stranded (ds) DNA separation. Here, we show the generality of this approach using a family of polymers suited to a wider range of DNA separations: a blended mixture of N,N-dialkylacrylamide copolymers with different thermoresponsive behaviors. A mixture of 47% w/w N,N-diethylacrylamide (DEA)/53% w/w N,N-dimethylacrylamide (DMA) (DEA47; thermoresponsive, transition temperature = 55 degrees C in water) and 30% w/w DEA/70% w/w DMA (DEA30; nonthermoresponsive, transition temperature > 85 degrees C in water) copolymers in the ratio of 1:5 w/w DEA47:DEA30 was used to separate a dsDNA restriction digest (PhiX174-HaeIII). We investigated the effects of changing mesh size on dsDNA separation, as controlled by temperature. We observed good DNA separation performance with the copolymer blend at temperatures ranging from 25 degrees C to 48 degrees C. The separation selectivity was evaluated quantitatively for certain DNA fragment pairs as a function of temperature. The results were compared with those obtained with a control matrix consisting only of the nonthermoresponsive DEA30. Different DNA fragment pairs of various sizes show distinct temperature-dependent selectivities. Over the same temperature range, no significant temperature dependence of selectivity is observed for these DNA fragment pairs in the nonthermoresponsive control matrix. Overall, the results show similar trends in the temperature dependency of separation selectivity to what was previously observed in hydroxyalkylcellulose blends, for the same DNA fragment pairs. Finally, we showed that a ramped temperature scheme enables improved separation in the blended copolymer matrix for both small and large DNA fragments, simultaneously in a single capillary electrophoresis (CE) run.

Acrylamide↗

PubMed2004

DNA sequencing and genotyping in miniaturized electrophoresis systems.

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.

Animals↗

PubMed2003

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

We have developed a novel class of thermogelling polymer networks based on poly-N-alkoxyalkylacrylamides, and demonstrated their use as DNA sequencing matrices for high-throughput microchannel electrophoresis in capillary arrays. Polymers and copolymers of N-ethoxyethylacrylamide (NEEA) and N-methoxyethylacrylamide (NMEA) were synthesized by aqueous-phase free-radical polymerization and characterized by tandem gel permeation chromatography-multi-angle laser light scattering. These copolymer matrices exhibit "re-entrant"-type volume phase transitions, forming entangled networks with high shear viscosity at low (< 20 degrees C) and high (> 35 degrees C) temperatures, and undergoing a "coil-to-globular", lower critical solution temperature (LCST)-like phase transition over an intermediate temperature range (20-35 degrees C). Hence, matrix viscosity is relatively low at room temperature (25 degrees C), and increases rapidly above 35 degrees C. The material properties and phase behavior of these thermogelling polymer networks were studied by steady-shear rheometry. These matrices are easily loaded into capillary arrays at room temperature while existing as viscous fluids, but thermogel above 35 degrees C to form transparent hydrogels via a thermo-associative phase transition. The extent of the intermediate viscosity drop and the final viscosity increase depends on the composition of the copolymers. DNA sequencing by capillary array electrophoresis with four-color laser-induced fluorescence (LIF) detection shows that these thermogelling networks provide enhanced resolution of both small and large DNA sequencing fragments and longer sequencing read lengths, in comparison to appropriate control (closely related, nonthermogelling) polymer networks. In particular, a copolymer comprised of 90% w/w NMEA and 10% w/w NEEA, with a molecular mass of approximately 2 MDa, delivers around 600 bases at 98.5% base-calling accuracy in 100 min of electrophoresis.

Acrylic Resins↗

PubMed2003

Sparsely cross-linked "nanogels" for microchannel DNA sequencing.

We have developed sparsely cross-linked "nanogels", sub-colloidal polymer structures composed of covalently linked, linear polyacrylamide chains, as novel DNA sequencing matrices for capillary electrophoresis. The presence of covalent cross-links affords nanogel matrices with enhanced network stability relative to standard, linear polyacrylamide (LPA), improving the separation of large DNA fragments. Nanogels were synthesized via inverse emulsion (water-in-oil) copolymerization of acrylamide and N,N-methylenebisacrylamide (Bis). In order to retain the fluidity necessary in a replaceable polymer matrix for capillary array electrophoresis (CAE), a low percentage of the Bis cross-linker (< 10(-4) mol%) was used. Nanogels were characterized by multiangle laser light scattering and rheometry, and were tested for DNA sequencing by CAE with four-color laser-induced fluorescence (LIF) detection. The properties and performance of nanogel matrices were compared to those of a commercially available LPA network, which was matched for both weight-average molar mass (Mw) and extent of interchain entanglements (c/c*). Nanogels presented in this work have an average radius of gyration of 226 nm and a weight-average molar mass of 8.8 x 10(6) g/mol. At concentrations above the overlap threshold, nanogels form a clear, viscous solution, similar to the LPA matrix (Mw approximately 8.9 x 10(6) g/mol). The two matrices have similar flow and viscosity characteristics. However, because of the physical network stability provided by the internally cross-linked structure of the nanogels, a substantially longer read length ( approximately 63 bases, a 10.4% improvement) is obtained with the nanogel matrix at 98.5% accuracy of base-calling. The nanogel network provides higher-selectivity separation of ssDNA sequencing fragments longer than 375 bases. Moreover, nanogel matrices require 30% less polymer per unit volume than LPA. This is the first report of a sequencing matrix that provides better performance than LPA, in a side-by-side comparison of polymer matrices matched for Mw and extent of interchain entanglements.

Acrylic Resins↗

PubMed2003

Microchannel wall coatings for protein separations by capillary and chip electrophoresis.

The necessity for microchannel wall coatings in capillary and chip-based electrophoretic analysis of biomolecules is well understood. The regulation or elimination of EOF and the prevention of analyte adsorption is essential for the rapid, efficient separation of proteins and DNA within microchannels. Microchannel wall coatings and other wall modifications are especially critical for protein separations, both in fused-silica capillaries, and in glass or polymeric microfluidic devices. In this review, we present a discussion of recent advances in microchannel wall coatings of three major classes--covalently linked polymeric coatings, physically adsorbed polymeric coatings, and small molecule additives. We also briefly review modifications useful for polymeric microfluidic devices. Within each category of wall coatings, we discuss those used to eliminate EOF, to tune EOF, to prevent analyte adsorption, or to perform multiple functions. The knowledgeable application of the most promising recent developments in this area will allow for the separation of complex protein mixtures and for the development of novel microchannel wall modifications.

Acrylic Resins↗

PubMed2002

Critical factors for high-performance physically adsorbed (dynamic) polymeric wall coatings for capillary electrophoresis of DNA.

Physically adsorbed (dynamic) polymeric wall coatings for microchannel electrophoresis have distinct advantages over covalently linked coatings. In order to determine the critical factors that control the formation of dynamic wall coatings, we have created a set of model polymers and copolymers based on N,N-dimethylacrylamide (DMA) and N,N-diethylacrylamide (DEA), and studied their adsorption behavior from aqueous solution as well as their performance for microchannel electrophoresis of DNA. This study is revealing in terms of the polymer properties that help create an "ideal" wall coating. Our measurements indicate that the chemical nature of the coating polymer strongly impacts its electroosmotic flow (EOF) suppression capabilities. Additionally, we find that a critical polymer chain length is required for polymers of this type to perform effectively as microchannel wall coatings. The effective mobilities of double-stranded (dsDNA) fragments within dynamically coated capillaries were determined in order to correlate polymer hydrophobicity with separation performance. Even for dsDNA, which is not expected to be a strongly adsorbing analyte, wall coating hydrophobicity has a deleterious influence on separation performance.

Acrylamides↗