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Richard A Mathies - One of the best experts on this subject based on the ideXlab platform.
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inline injection microdevice for attomole scale sanger DNA Sequencing
Analytical Chemistry, 2007Co-Authors: Robert G Blazej, Palani Kumaresan, Samantha A Cronier, Richard A MathiesAbstract:A new affinity-capture-based inline purification, concentration, and injection method is developed for microchip capillary electrophoresis (CE) and used to perform efficient attomole-scale Sanger DNA Sequencing separations. The microdevice comprises three axial domains for nanoliter-scale Sequencing sample containment, sample plug formation, and high-resolution capillary gel electrophoresis. Purified and concentrated inline sample plugs are formed by electrophoretically driving Sanger Sequencing extension fragments into an affinity-capture polymer network positioned within a CE separation channel. Extension fragments selectively hybridize and concentrate at the polymer interface while residual primer, nucleotides, and salts electrophorese out of the system. The plug is thermally released and injected into the CE channel by direct application of the separation voltage. To evaluate this system, 30 nL of Sequencing sample prepared from 100 amol (60 million molecules) of human mitochondrial hypervariable regi...
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microfluidic devices for DNA Sequencing sample preparation and electrophoretic analysis
Current Opinion in Biotechnology, 2003Co-Authors: Brian M Paegel, Robert G Blazej, Richard A MathiesAbstract:Abstract Modern DNA Sequencing ‘factories’ have revolutionized biology by completing the human genome sequence, but in the race to completion we are left with inefficient, cumbersome, and costly macroscale processes and supporting facilities. During the same period, microfabricated DNA Sequencing, sample processing and analysis devices have advanced rapidly toward the goal of a ‘Sequencing lab-on-a-chip’. Integrated microfluidic processing dramatically reduces analysis time and reagent consumption, and eliminates costly and unreliable macroscale robotics and laboratory apparatus. A microfabricated device for high-throughput DNA Sequencing that couples clone isolation, template amplification, Sanger extension, purification, and electrophoretic analysis in a single microfluidic circuit is now attainable.
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energy transfer primers a new fluorescence labeling paradigm for DNA Sequencing and analysis
Nature Medicine, 1996Co-Authors: Jingyue Ju, Alexander N Glazer, Richard A MathiesAbstract:Energy transfer primers: A new fluorescence labeling paradigm for DNA Sequencing and analysis
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ultra high speed DNA Sequencing using capillary electrophoresis chips
Analytical Chemistry, 1995Co-Authors: Adam T Woolley, Richard A MathiesAbstract:DNA Sequencing has been performed on microfabricated capillary electrophoresis chips. DNA separations were achieved in 50 x 8 microns cross-section channels microfabricated in a 2 in. x 3 in. glass sandwich structure using a denaturing 9% T, 0% C polyacrylamide sieving medium. DNA Sequencing fragment ladders were produced and fluorescently labeled using the recently developed energy transfer dye-labeled primers. Sequencing extension fragments were separated to approximately 433 bases in only 10 min using a one-color detection system and an effective separation distance of only 3.5 cm. Using a four-color labeling and detection format, DNA Sequencing with 97% accuracy and single-base resolution to approximately 150 bases was achieved in only 540 s. A resolution of greater than 0.5 was obtained out to 200 bases for both the one- and four-color separations. The prospects for enhancing the resolution and sensitivity of these chip separations are discussed. This work establishes the feasibility of high-speed, high-throughput DNA Sequencing using capillary array electrophoresis chips.
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ultra high speed DNA Sequencing using capillary electrophoresis chips
Analytical Chemistry, 1995Co-Authors: Adam T Woolley, Richard A MathiesAbstract:DNA Sequencing has been performed on microfabricated capillary electrophoresis chips. DNA separations were achieved in 50 x 8 {mu}m cross-section channels microfabricated in a 2 in. x 3 in. glass sandwich structure using a denaturing 9% T, 0% C polyacrylamide sieving medium. DNA Sequencing fragment ladders were produced and fluorescently labeled using the recently developed energy transfer dye-labeled primers. Sequencing extension fragments were separated to nearly 433 bases in only 10 min using a one-color detection system and an effective separation distance of only 3.5 cm. Using a four-color labeling and detection format, DNA Sequencing with 97% accuracy and single-base resolution to nearly 150 bases was achieved in only 540 s. A resolution of greater than 0.5 was obtained out to 200 bases for both the one- and four-color separations. The prospects for enhancing the resolution and sensitivity of these chip separations are discussed. This work establishes the feasibility of high-speed, high-throughput DNA Sequencing using capillary array electrophoresis chips. 22 refs., 4 figs.
Adam T Woolley - One of the best experts on this subject based on the ideXlab platform.
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ultra high speed DNA Sequencing using capillary electrophoresis chips
Analytical Chemistry, 1995Co-Authors: Adam T Woolley, Richard A MathiesAbstract:DNA Sequencing has been performed on microfabricated capillary electrophoresis chips. DNA separations were achieved in 50 x 8 microns cross-section channels microfabricated in a 2 in. x 3 in. glass sandwich structure using a denaturing 9% T, 0% C polyacrylamide sieving medium. DNA Sequencing fragment ladders were produced and fluorescently labeled using the recently developed energy transfer dye-labeled primers. Sequencing extension fragments were separated to approximately 433 bases in only 10 min using a one-color detection system and an effective separation distance of only 3.5 cm. Using a four-color labeling and detection format, DNA Sequencing with 97% accuracy and single-base resolution to approximately 150 bases was achieved in only 540 s. A resolution of greater than 0.5 was obtained out to 200 bases for both the one- and four-color separations. The prospects for enhancing the resolution and sensitivity of these chip separations are discussed. This work establishes the feasibility of high-speed, high-throughput DNA Sequencing using capillary array electrophoresis chips.
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ultra high speed DNA Sequencing using capillary electrophoresis chips
Analytical Chemistry, 1995Co-Authors: Adam T Woolley, Richard A MathiesAbstract:DNA Sequencing has been performed on microfabricated capillary electrophoresis chips. DNA separations were achieved in 50 x 8 {mu}m cross-section channels microfabricated in a 2 in. x 3 in. glass sandwich structure using a denaturing 9% T, 0% C polyacrylamide sieving medium. DNA Sequencing fragment ladders were produced and fluorescently labeled using the recently developed energy transfer dye-labeled primers. Sequencing extension fragments were separated to nearly 433 bases in only 10 min using a one-color detection system and an effective separation distance of only 3.5 cm. Using a four-color labeling and detection format, DNA Sequencing with 97% accuracy and single-base resolution to nearly 150 bases was achieved in only 540 s. A resolution of greater than 0.5 was obtained out to 200 bases for both the one- and four-color separations. The prospects for enhancing the resolution and sensitivity of these chip separations are discussed. This work establishes the feasibility of high-speed, high-throughput DNA Sequencing using capillary array electrophoresis chips. 22 refs., 4 figs.
Carl W Fuller - One of the best experts on this subject based on the ideXlab platform.
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templiphi 29 DNA polymerase based rolling circle amplification of templates for DNA Sequencing
BioTechniques, 2002Co-Authors: John Richard Nelson, Theresa L Giesler, Joseph W Farchaus, Shanmuuga Sundaram, Maria Ortizrivera, Lou P Hosta, Peter L Hewitt, Anthony J Mamone, Chockalingam Palaniappan, Carl W FullerAbstract:We have developed a novel, isothermal DNA amplification strategy that employs ⌽29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA Sequencing reactions. Th...
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Novel cyanine dye-labeled dideoxynucleoside triphosphates for DNA Sequencing.
Bioconjugate chemistry, 2002Co-Authors: R. Scott Duthie, Satyam Nampalli, Inta M. Kalve, Sui Bi Samols, Scott Hamilton, Inna Livshin, Mahesh Khot, Shiv Kumar, Carl W FullerAbstract:Single color cyanine dye-labeled (Cy 5.0 and Cy 5.5) dideoxynucleoside-5‘-triphosphates, or ‘terminators', containing different spacer lengths were synthesized and evaluated for efficacy in DNA Sequencing methods using a modified thermally stable DNA polymerase. The single color cyanine dye terminators were formulated into two separate sets of Sequencing mixes, one for Cy 5.0 and the other for Cy 5.5, and evaluated on different automated Sequencing platforms. Each set of mixes included two pyrimidine terminators with 17-atom linkers and two purine terminators with 10-atom linkers between the dye and the nucleotide. The two sets of cyanine dye-labeled terminators chosen for this cycle Sequencing study produced improved band patterns with band uniformity similar to that obtained with dye-primer Sequencing methods.
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fluorescence energy transfer dye labeled primers for DNA Sequencing and analysis
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Jingyue Ju, Carl W Fuller, Chihchuan Ruan, Alexander N Glazer, Richard A MathiesAbstract:Abstract Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA Sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA Sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical Sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA Sequencing and analysis methods.
Jingyue Ju - One of the best experts on this subject based on the ideXlab platform.
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first generation automated DNA Sequencing technology
Current protocols in molecular biology, 2011Co-Authors: Barton E. Slatko, Jingyue Ju, Jan Kieleczawa, Andrew F Gardner, Cynthia L Hendrickson, Frederick M AusubelAbstract:Beginning in the 1980s, automation of DNA Sequencing has greatly increased throughput, reduced costs, and enabled large projects to be completed more easily. The development of automation technology paralleled the development of other aspects of DNA Sequencing: better enzymes and chemistry, separation and imaging technology, Sequencing protocols, robotics, and computational advancements (including base-calling algorithms with quality scores, database developments, and sequence analysis programs). Despite the emergence of high-throughput Sequencing platforms, automated Sanger Sequencing technology remains useful for many applications. This unit provides background and a description of the “First-Generation” automated DNA Sequencing technology. It also includes protocols for using the current Applied Biosystems (ABI) automated DNA Sequencing machines. Curr. Protoc. Mol. Biol. 96:7.2.1-7.2.28. © 2011 by John Wiley & Sons, Inc. Keywords: dideoxy DNA Sequencing; thermal cycle Sequencing; BigDye terminator; high-throughput DNA Sequencing; genomics
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click chemistry to construct fluorescent oligonucleotides for DNA Sequencing
Journal of Organic Chemistry, 2003Co-Authors: Zengmin Li, Hameer Ruparel, Jingyue JuAbstract:“Click chemistry” 1,3-dipolar cycloaddition between alkynyl 6-carboxyfluorescein (FAM) and azido-labeled single-stranded (ss) DNA was carried out under aqueous conditions to produce FAM-labeled ssDNA in quantitative yield. The FAM-labeled ssDNA was successfully used as a primer to produce DNA Sequencing products with single-base resolution in a capillary electrophoresis DNA sequencer with laser-induced fluorescence detection.
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energy transfer primers a new fluorescence labeling paradigm for DNA Sequencing and analysis
Nature Medicine, 1996Co-Authors: Jingyue Ju, Alexander N Glazer, Richard A MathiesAbstract:Energy transfer primers: A new fluorescence labeling paradigm for DNA Sequencing and analysis
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fluorescence energy transfer dye labeled primers for DNA Sequencing and analysis
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Jingyue Ju, Carl W Fuller, Chihchuan Ruan, Alexander N Glazer, Richard A MathiesAbstract:Abstract Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA Sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA Sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical Sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA Sequencing and analysis methods.
Brian M Paegel - One of the best experts on this subject based on the ideXlab platform.
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microfluidic devices for DNA Sequencing sample preparation and electrophoretic analysis
Current Opinion in Biotechnology, 2003Co-Authors: Brian M Paegel, Robert G Blazej, Richard A MathiesAbstract:Abstract Modern DNA Sequencing ‘factories’ have revolutionized biology by completing the human genome sequence, but in the race to completion we are left with inefficient, cumbersome, and costly macroscale processes and supporting facilities. During the same period, microfabricated DNA Sequencing, sample processing and analysis devices have advanced rapidly toward the goal of a ‘Sequencing lab-on-a-chip’. Integrated microfluidic processing dramatically reduces analysis time and reagent consumption, and eliminates costly and unreliable macroscale robotics and laboratory apparatus. A microfabricated device for high-throughput DNA Sequencing that couples clone isolation, template amplification, Sanger extension, purification, and electrophoretic analysis in a single microfluidic circuit is now attainable.
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Microchip Bioprocessor for Integrated Nanovolume Sample Purification and DNA Sequencing
Analytical chemistry, 2002Co-Authors: Brian M Paegel, And Stephanie H. I. YeungAbstract:A microfabricated electrophoretic bioprocessor for integrated DNA Sequencing sample desalting, template removal, preconcentration, and CE analysis is presented. A low-viscosity gel capture matrix, containing an acrylamide−copolymerized oligonucleotide complementary to the 20-base sequence directly 3‘ of the M13−40 universal forward priming site, is introduced into the 60-nL capture chamber. Unpurified DNA Sequencing reaction products are electrophoretically driven through the chamber; extension products hybridize to the matrix, while contaminating buffering ions, Cl-, excess primer, and template DNA are unretained. Purification under optimized conditions is complete in only 120 s (binding temperature 50 °C, driving voltage 250 V). High-speed, integrated Sequencing analysis is accomplished by releasing the gel-purified duplex at 67 °C and directly injecting onto a 15.9-cm effective length CE microchannel. Electrophoretic resolution of the Sequencing products is complete in 32 min, producing a total of 560 ...
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High throughput DNA Sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor
Proceedings of the National Academy of Sciences, 2002Co-Authors: G. J. Wedemayer, J. R. Scherer, Charles A Emrich, Brian M PaegelAbstract:High throughput DNA Sequencing has been performed by using a microfabricated 96-channel radial capillary array electrophoresis (microCAE) microchannel plate detected by a 4-color rotary confocal fluorescence scanner. The microchannel plate features a novel injector for uniform sieving matrix loading as well as high resolution, tapered turns that provide an effective separation length of 15.9 cm on a compact 150-mm diameter wafer. Expanded common buffer chambers for the cathode, anode, and waste reservoirs are used to simplify electrode addressing and to counteract buffering capacity depletion arising from the high electrophoretic current. DNA Sequencing data from 95 successful lanes out of 96 lanes run in parallel were batch-processed with basefinder, producing an average read length of 430 bp (phred q > or = 20). Phred quality values were found to exceed 40 (0.01% probability of incorrectly calling a base) for over 80% of the read length. The microCAE system demonstrated here produces Sequencing data at a rate of 1.7 kbp/min, a 5-fold increase over current commercial capillary array electrophoresis technology. Additionally, this system permits lower reagent volumes and lower sample concentrations, and it presents numerous possibilities for integrated sample preparation and handling. The unique capabilities of microCAE technology should make it the next generation, high performance DNA Sequencing platform.