Multiplexed DNA sequencing-by-synthesis

Aksyonov, Sergei A.; Bittner, Michael; Bloom, Linda B.; Reha-Krantz, Linda J.; Gould, Ian R.; Hayes, Mark A.; Kiernan, Urban A.; Niederkofler, Eric E.; Pizziconi, Vincent; Rivera, Raul S.; Williams, D.; Williams, Peter W.

doi:10.1016/j.ab.2005.10.001

Cited by 21 publications

(13 citation statements)

References 21 publications

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“…If the fluorescein-dNTP was complementary to the bases in the polymerase extension system, the spots on the array fluoresced. 27 …”

Section: Polymerase Extension Reactionmentioning

confidence: 97%

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An Efficient Strategy for Sequencing-by-Synthesis

Gao¹,

Lü²,

Xu³

et al. 2010

J. Nanosci. Nanotech.

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Currently, the products of polymerase chain reaction (PCR) provide the templates for most of sequencing methods. RCA is a method for amplifying DNA. We report an approach for preparing sequencing templates based on rolling-circle amplification (RCA) to reduce the cost of sequencing when compared to the cost of conducting PCR. We describe a method using modified slides: acrylamide-modified slides, streptavidin-modified slides and aldehyde-modified slides. The products of RCA can be immobilized onto an acryl-modified slide surface. The Cy5-labeled deoxyribonucleoside triphosphate (dNTP) species are incorporated in extension reactions which can be read by determining the level of fluorescence. Then the fluorescence is deactivated using hydrogen peroxide (H2O2) as one of the oxidants. With the fluorescence deactivated, further extension and reading along the DNA chain is possible. Using this method, we successfully read a sequence of 27 bases. This strategy shows potential as a next-generation sequencing method.

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“…If the fluorescein-dNTP was complementary to the bases in the polymerase extension system, the spots on the array fluoresced. 27 …”

Section: Polymerase Extension Reactionmentioning

confidence: 97%

“…While uncomplicated, these methods have a high price tag. Williams et al 27 developed a photochemical bleaching reaction using the diphenyliodonium (DPI) with Ar laser light. They used a freshly prepared saturated solution of DPI which can react with photoexcited fluorescein and dissociates into phenyl iodide and a phenyl radical.…”

Section: Introductionmentioning

confidence: 99%

An Efficient Strategy for Sequencing-by-Synthesis

Gao¹,

Lü²,

Xu³

et al. 2010

J. Nanosci. Nanotech.

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“…Such "sequencing by synthesis" (SBS) techniques are intrinsically very interesting since native DNA polymerases can synthesize DNA at rates of up to 1000 bases/s; however, current methods proceed much more slowly since fresh reagents must be provided to the reactions, en masse, at each cycle. Fluorescently labeled-nucleotide SBS methods can involve chemically cleavable [70,71] or photocleavable [72][73][74][75] fluorescent groups covalently linked to the nucleotide to reveal its identity. Additionally, research groups have attempted to sequence DNA in real time at the single-molecule level using zero-mode waveguides [76] or molecular motors [77] to detect specific DNA polymerase activity with respect to labeled nucleotides.…”

Section: Massively Parallel Sequencing Systemsmentioning

confidence: 99%

What is the future of electrophoresis in large‐scale genomic sequencing?

et al. 2006

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Although a finished human genome reference sequence is now available, the ability to sequence large, complex genomes remains critically important for researchers in the biological sciences, and in particular, continued human genomic sequence determination will ultimately help to realize the promise of medical care tailored to an individual's unique genetic identity. Many new technologies are being developed to decrease the costs and to dramatically increase the data acquisition rate of such sequencing projects. These new sequencing approaches include Sanger reaction-based technologies that have electrophoresis as the final separation step as well as those that use completely novel, nonelectrophoretic methods to generate sequence data. In this review, we discuss the various advances in sequencing technologies and evaluate the current limitations of novel methods that currently preclude their complete acceptance in large-scale sequencing projects. Our primary goal is to analyze and predict the continuing role of electrophoresis in large-scale DNA sequencing, both in the near and longer term.

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“…The *G nucleotide is incorporated by the DNA polymerase only in spots where the template is the complimentary C. The slide is then rinsed to remove unincorporated fluorescent nucleotides and the array is imaged to record each spot where fluorescent *G nucleotides are incorporated. Then the fluorophore is attacked chemically to remove fluorescence and the process is repeated with another fluorescent nucleotide [183,184]. This method is a high-throughput system because 1000s of spots can be sequenced in parallel.…”

Section: Patents Deposited For Modified Dna Polymerasesmentioning

confidence: 99%

Recent Patents of Gene Sequences Relative to DNA Polymerases

Reha-Krantz

2008

DNAG

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Organisms with DNA genomes encode one or more DNA polymerases that are essential enzymes for chromosome replication, DNA repair and recombination. The ability of DNA polymerases to copy DNA templates has been exploited in a variety of in vitro reactions to sequence, amplify, mutate, label and recombine DNA, and in several other applications that are fundamental to molecular biology. Because natural DNA polymerases may have activities that interfere with in vitro applications or their substrate specificity is too narrow, DNA polymerases have been modified for specific applications. Patents are reviewed here on natural and variant DNA polymerases and their uses.

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Multiplexed DNA sequencing-by-synthesis

Cited by 21 publications

References 21 publications

An Efficient Strategy for Sequencing-by-Synthesis

An Efficient Strategy for Sequencing-by-Synthesis

What is the future of electrophoresis in large‐scale genomic sequencing?

Recent Patents of Gene Sequences Relative to DNA Polymerases

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