DNA sequencing and genotyping in miniaturized electrophoresis systems

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

doi:10.1002/elps.200406161

Cited by 112 publications

(81 citation statements)

References 233 publications

(229 reference statements)

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“…1 These techniques can be applied for micro-fluidic integrated gene analysis systems [2][3] demonstrating an overall reduction in size, reduced use of reagents, increased speed and accuracy of analysis. The field applications of such devices, however, are limited by power requirements imposed by highly resistive capillary columns.…”

Section: Introductionmentioning

confidence: 99%

Low Voltage Capillary Electrophoresis Using High Conductivity Agarose Nano-Platinum Composites

Bhattacharya¹,

Gangopadhyay²,

Chanda³

et al. 2008

sens lett

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Micro-channel based electrophoretic separation systems are commonly used in gene sequencing and analysis. A significant amount of research has focused on the development of fast and high resolution separation systems as part of miniaturized genotyping platforms. These devices are not suitable for field deployment due to their high voltage requirements for micro-channel based electrophoresis. In order to address this issue, we have developed and tested a new agarose gel doped with nanoplatinum for conducting capillary electrophoresis in micro capillaries at low electric field values. The platinum nanoparticles reduce the gel resistance of the agarose gels by 3-4 fold. We have further observed a faster movement of DNA band in this novel gel material allowing a reduction of the fractionating electric field within Poly dimethyl Siloxane (PDMS) capillaries. Impedance studies performed on this new gel material indicate a 1.34 times increase in the dielectric constant of the medium as a result of doping by nano-particles and a 37% reduction in the resistance. We believe that the higher conductivity of the medium and an increased dielectric constant of the medium result in increased ionic mobility within this material. We have also compared the stain mobility values in glass PDMS capillaries of a 1 kbp ladder, and a 750 bp dS DNA segment and have successfully obtained electrophoresis within the micro-channels at a lower electric field (25 V/cm).

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Section: Introductionmentioning

confidence: 99%

Low Voltage Capillary Electrophoresis Using High Conductivity Agarose Nano-Platinum Composites

Bhattacharya¹,

Gangopadhyay²,

Chanda³

et al. 2008

sens lett

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“…Recently developed microfabricated electrophoresis devices offer several advantages over conventional and capillary electrophoresis, including even higher separation speed and most importantly the capability of system integration and multiplexing. The speed of microchip electrophoresis is approximately tenfold higher than that of in CE and the price of multiplexed microfluidic devices are 10% of the cost of capillary arrays [9]. The introduction of plastic chips made the utilization of microdevices even more cost effective, allowing such large-scale applications as clinical genotyping more feasible, even with single use/disposable chips.…”

Section: Genome Variabilitymentioning

confidence: 99%

“…System integration led to the 'lab-on-a-chip' concept with such multiple functions as PCR amplification, restriction digestion, separation, fraction collection, etc [10]. The progress in the field was summarized in several reviews in the past few years [9,[11][12][13][14][15][16][17][18][19][20][21].…”

Section: Genome Variabilitymentioning

confidence: 99%

Genotyping with microfluidic devices

Szantai

Guttman

2006

Electrophoresis

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In the past few years, electrophoresis microchips have been increasingly utilized to interrogate genetic variations in the human and other genomes. Microfluidic devices can be readily applied to speed up existing genotyping protocols, in particular the ones that require electric field-mediated separations in conjunction with restriction fragment analysis, DNA sequencing, hybridization-based techniques, allele-specific amplification, heteroduplex analysis, just to list the most important ones. As a result of recent developments, microfabricated electrophoresis devices offer several advantages over conventional slab-gel electrophoresis, such as small sample volume requirement, low reagent consumption, the option of system integration and easy multiplexing. The analysis speed of microchip electrophoresis is significantly higher than that of any other electric field-mediated separation techniques. State-of-the-art microfluidic bioanalytical devices already claim their place in most molecular biology laboratories. This review summarizes the recent developments in microchip electrophoresis methods of nucleic acids, particularly for rapid genotyping, that will most likely play a significant role in the future of clinical diagnostics.

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“…Current state-of-the-art DNA sequencing technologies (7)(8)(9)(10)(11) to some extent address the accuracy and throughput requirements but suffer limitations with respect to cost and data quality. Thus, a new DNA sequencing approach is required to broaden the applications of genomic information in medical research and health care.…”

mentioning

confidence: 99%

Design and synthesis of a 3′- O -allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis

Ruparel

Bi²,

Li³

et al. 2005

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

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DNA sequencing by synthesis (SBS) offers an approach for potential high-throughput sequencing applications. In this method, the ability of an incoming nucleotide to act as a reversible terminator for a DNA polymerase reaction is an important requirement to unambiguously determine the identity of the incorporated nucleotide before the next nucleotide is added. A free 3 -OH group on the terminal nucleotide of the primer is necessary for the DNA polymerase to incorporate an incoming nucleotide. Therefore, if the 3 -OH group of an incoming nucleotide is capped by a chemical moiety, it will cause the polymerase reaction to terminate after the nucleotide is incorporated into the DNA strand. If the capping group is subsequently removed to generate a free 3 -OH, the polymerase reaction will reinitialize. We report here the design and synthesis of a 3 -modified photocleavable fluorescent nucleotide, 3 -O-allyl-dUTP-PC-Bodipy-FL-510 (PC-Bodipy, photocleavable 4,4-difluoro-4-bora-3␣,4␣-diaza-s-indacene), as a reversible terminator for SBS. This nucleotide analogue contains an allyl moiety capping the 3 -OH group and a fluorophore Bodipy-FL-510 linked to the 5 position of the uracil through a photocleavable 2-nitrobenzyl linker. Here, we have shown that this nucleotide is a good substrate for a DNA polymerase. After the nucleotide was successfully incorporated into a growing DNA strand and the fluorophore was photocleaved, the allyl group was removed by using a Pd-catalyzed reaction to reinitiate the polymerase reaction, thereby establishing the feasibility of using such nucleotide analogues as reversible terminators for SBS. 2-nitrobenzyl linker ͉ photocleavageT he completion of the Human Genome Project (1, 2) has led to an increased demand for high-throughput and rapid DNA sequencing methods to identify genetic variants for applications in pharmacogenomics (3), disease gene discovery (4, 5), and gene function studies (6). Current state-of-the-art DNA sequencing technologies (7-11) to some extent address the accuracy and throughput requirements but suffer limitations with respect to cost and data quality. Thus, a new DNA sequencing approach is required to broaden the applications of genomic information in medical research and health care. In this regard, DNA sequencing by synthesis (SBS) offers an alternative approach to possibly address the limitations of current DNA sequencing techniques. We have previously described the design of a parallel chip-based SBS system, which uses a self-priming DNA template covalently linked to the glass surface of a chip and four modified nucleotides (12-14). The nucleotides are modified such that they have a photocleavable fluorescent moiety attached to the base (5 position of pyrimidines, 7 position of purines) and a chemically cleavable group to cap the 3Ј-OH. When the correct nucleotide is incorporated in a DNA polymerase reaction, specific to the template sequence, the reaction is temporarily terminated because of the lack of a free 3Ј-OH group. After the fluorescent signal is detected ...

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DNA sequencing and genotyping in miniaturized electrophoresis systems

Cited by 112 publications

References 233 publications

Low Voltage Capillary Electrophoresis Using High Conductivity Agarose Nano-Platinum Composites

Low Voltage Capillary Electrophoresis Using High Conductivity Agarose Nano-Platinum Composites

Genotyping with microfluidic devices

Design and synthesis of a 3′- O -allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis

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