Extending chromosomal DNA in microstructures using electroosmotic flow

Terao, Kyohei; Kabata, Hiroyuki; Washizu, Masao

doi:10.1088/0953-8984/18/18/s11

Cited by 18 publications

(16 citation statements)

References 35 publications

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“…Single chromosomal DNA molecules are introduced into the channel after the microtools are washed with buffer solution. They are trapped stochastically at micropillars and extended by the ow as previously described [14,20,21] (Fig. 1b, c).…”

Section: Introductionsupporting

confidence: 63%

“…In this paper, we describe the development of technologies for targeted on-site cutting of single chromosomal DNA molecules. Chromosomal DNA molecules are readily fragmented by shear forces, making single-molecule analysis challenging [14]. The microtools described here provide a method for analyzing molecules through pinpoint enzymatic processing.…”

Section: Introductionmentioning

confidence: 99%

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On-Site Processing of Single Chromosomal DNA Molecules Using Optically Driven Microtools on A Microfluidic Workbench

Masuda

Takao

Shimokawa

et al. 2020

Preprint

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We developed optically driven microtools for processing single biomolecules using a microfluidic workbench composed of a microfluidic platform that functions under an optical microscope. The optically driven microtools have enzymes immobilized on their surfaces, which catalyze chemical reactions for molecular processing in a confined space. Optical manipulation of the microtools enables them to be integrated with a microfluidic device for controlling the position, orientation, shape of the target sample. Here, we describe the immobilization of enzymes on the surface of microtools, the microfluidics workbench, including its microtool storage and sample positioning functions, and the use of this system for on-site cutting of single chromosomal DNA molecules. We fabricated microtools by UV lithography with SU-8 and selected ozone treatments for immobilizing enzymes. The microfluidic workbench has tool-stock chambers for tool storage and micropillars to trap and extend single chromosomal DNA molecules. The DNA cutting enzymes DNaseI and DNaseII were immobilized on microtools that were manipulated using optical tweezers. The DNaseI tool shows reliable cutting for on-site processing. This pinpoint processing provides an approach for analyzing chromosomal DNA at the single-molecule level. The flexibility of the microtool design allows for processing of various samples, including biomolecules and single cells.

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Section: Introductionsupporting

confidence: 63%

Section: Introductionmentioning

confidence: 99%

On-Site Processing of Single Chromosomal DNA Molecules Using Optically Driven Microtools on A Microfluidic Workbench

Masuda

Takao

Shimokawa

et al. 2020

Preprint

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“…In recent years, DNA analysis based on the manipulation of single DNA molecules has attracted strong interest in many research fields, including next-generation DNA sequencing [1][2][3], optical mapping of genes [4][5][6] and DNA-protein interaction studies [7][8][9][10][11]. A number of DNA manipulation techniques have been proposed for these applications that use driving forces such as electroosmotic flow [12,13], dielectrophoretic force [14,15], electrophoretic force [16,17], optical tweezers [18][19][20] and flow in nanochannels [21][22][23]. Among these techniques, optical tweezers are suitable for manipulating single DNA molecules precisely [24] where a laser captures a microsphere and binds it chemically to a single DNA molecule to manipulate it.…”

Section: Introductionmentioning

confidence: 99%

Characterisation of optically driven microstructures for manipulating single DNA molecules under a fluorescence microscope

Terao

Masuda

Inukai

et al. 2016

IET nanobiotechnol.

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Optical tweezers are powerful tools for manipulating single DNA molecules using fluorescence microscopy, particularly in nanotechnology-based DNA analysis. We previously proposed a manipulation technique using microstructures driven by optical tweezers that allows the handling of single giant DNA molecules of millimetre length that cannot be manipulated by conventional techniques. To further develop this technique, the authors characterised the microstructures quantitatively from the view point of fabrication and efficiency of DNA manipulation under a fluorescence microscope. The success rate and precision of the fabrications were evaluated. The results indicate that the microstructures are obtained in an aqueous solution with a precision ∼50 nm at concentrations in the order of 10(6) particles/ml. The visibility of these microstructures under a fluorescence microscope was also characterised, along with the elucidation of the fabrication parameters needed to fine tune visibility. Manipulating yeast chromosomal DNA molecules with the microstructures illustrated the relationship between the efficiency of manipulation and the geometrical shape of the microstructure. This report provides the guidelines for designing microstructures used in single DNA molecule analysis based on on-site DNA manipulation, and is expected to broaden the applications of this technique in the future.

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“…The experiment showed that after cell lysis, short fragments of RNA were obtained immediately without removing impurities. Terao et al 16 fabricated microstructures that captured yeast cells while lysing the yeast cells with a lysis solution and stretching DNA. Hung and Chen 17 integrated atomic force microscope (AFM) and electro-osmosis and used the AFM's tip to lyse human cells that were immobilized to the glass surface and stretch DNA.…”

Section: Introductionmentioning

confidence: 99%

Developing microfluidics for rapid protoplasts collection and lysis from plant leaf

Hung

Chang

2012

Proceedings of the Institution of Mechanical Engineers, Part N:

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This study develops polydimethylsiloxane microfluidics to enable real-time collection and lysis of Phalaenopsis protoplasts and to analyze and compare the protoplast collecting efficiency of a concave sieving array with that of a convex-concave sieving array. Each set of microfluidics comprises a main flow channel and a protoplast sieving array with collecting channels. The protoplasts were isolated from Phalaenopsis leaves by an enzymatic breakdown of the cell walls and collected in side channels through microsieves. Finally, 1% w/v sodium dodecyl sulfate solution was injected into lysed protoplasts, and the DNA segment of the lysed protoplasts was released into the solution. The results indicate that the concave sieving array at the U-bend of the main flow channel causes the protoplasts to flow back into the main flow channel from the collecting channels. Thus, the protoplast collecting efficiency of the concave sieving array was lower in this study. Regarding the number and arrangement of protoplast microsieves, the convex-concave sieving array substantially restricts protoplasts from flowing back into the main flow channel, thereby increasing the collecting efficiency. Finally, DNA is released from lysed protoplasts after the injection of a lysis solution. DNA flow can be focused into a single stream to contact the rectangular microstructures in microchannels.

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Extending chromosomal DNA in microstructures using electroosmotic flow

Cited by 18 publications

References 35 publications

On-Site Processing of Single Chromosomal DNA Molecules Using Optically Driven Microtools on A Microfluidic Workbench

On-Site Processing of Single Chromosomal DNA Molecules Using Optically Driven Microtools on A Microfluidic Workbench

Characterisation of optically driven microstructures for manipulating single DNA molecules under a fluorescence microscope

Developing microfluidics for rapid protoplasts collection and lysis from plant leaf

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