Biomedical subjects

Yoshinobu Kohara

Publications and source records attributed to Yoshinobu Kohara.

6 recordsLinked to original sources

PubMed2004

High-speed DNA sequencing by tube-based capillary electrophoresis.

We assessed the feasibility of high-speed DNA sequencing by tube-based capillary electrophoresis (TCE) with electrokinetic sample injections. We developed a water-circulated TCE system to control the capillary temperature precisely. Using this system and a ready-made sieving matrix at 50 degrees C, single-stranded DNA size marker fragments were separated at various pairs of the electric field strength, E, of 128-480 V/cm and the capillary effective length, L, of 100-360 mm. Assuming the read length (RL) is the fragment size at which the peak width equals the peak interval per base in obtained electropherograms, we estimated the values of RL (E, L), the RL at the pair (E, L). The points in ELz-space, (E, L, RL(E, L)), form a curved surface expressed by z = RL(E, L). Analyzing the contour lines of this curved surface, we determined the pairs of E and L providing target RLs of 300-500 bases within a minimum time. At a pair optimized for a 500-base RL (330 V/cm, 200 mm), one-color sequencing fragments were successfully separated up to 529 bases within 9.6 min. These results demonstrate that high-speed DNA sequencing comparable with that obtained by microfabricated chip-based capillary electrophoresis (MCE) can be achieved with TCE, which is more suitable in automation than MCE.

Electrophoresis, Capillary↗

PubMed2003

Automated bead alignment apparatus using a single bead capturing technique for fabrication of a miniaturized bead-based DNA probe array.

We have developed an automated bead alignment apparatus for fabricating a bead-based DNA probe array inside a capillary. The apparatus uses 16 micro vacuum tweezers to extract single beads from among a large amount of beads in bead stock wells. It then manipulates single beads into the probe array capillaries. Single 100-microm-diameter beads were successfully extracted from the water-contained bead-stock well by the vacuum tweezers, which have inner and outer diameters of 50 and 150 microm. An interesting aspect is that unexpected extra beads adsorbed on the outer wall of the vacuum tweezers can be removed using the surface tension force between the water and the atmosphere. In testing the total performance of this apparatus, the DNA probe arrays with 10 sets of probe-conjugated beads and 2 plain beads were produced in the intended order in the capillaries. The time needed to align the 12 beads was 10 min, and the 16 bead arrays were fabricated simultaneously. After hybridization experiments using these fabricated DNA probe arrays, fluorescence from each bead was clearly observed.

Base Sequence↗

PubMed2003

Hybridization reaction kinetics of DNA probes on beads arrayed in a capillary enhanced by turbulent flow.

The hybridization reaction kinetics of DNA probes on beads arrayed in a capillary was investigated experimentally and theoretically by using fluid mechanical methods. A device was prepared to contain DNA probes conjugated on 103-microm-diameter beads that were queued in a 150-microm-diameter capillary. The hybridization experiments were performed by introducing sample into the capillary and moving it with a one-way or a reciprocal flow. From the relation between Reynolds number and the resistance coefficient of the system, we found that the flow in the system was turbulent and not laminar as has been said of other microfluidic devices. The reaction efficiency was estimated using a mass-transfer coefficient derived from Chilton-Colburn's analogy. The estimate agreed well with the experimental data. A diffusion equation under laminar assumption was also solved, but this estimated value was 4.0-10.4 times smaller than the experimental data. Using the device achieved a hybridization efficiency as high as approximately 90% in 10 min. It was concluded that the high hybridization performance of the device resulted from turbulent flow and that the flow compensated the slow molecular diffusion. Using this bead-included structure resulted in a rapid and effective reaction at the solid-liquid interface, and the device seems very promising for many future applications.

Base Sequence↗

PubMed2003

A bead-alignment device with a bead-sized microchamber on a rotating cylinder for fabrication of a miniaturized probe array.

We have developed a compact bead-alignment device with a bead-sized microchamber on a rotating cylinder. The cylinder fits inside a tube with bead-stock pipes containing different probe-conjugated beads and holes for bead-alignment capillaries. The cylinder rotates in the tube, and the microchamber transfers a single 100-microm-diameter bead from a pipe to one of the capillaries in 10 s. By using this process repeatedly, 'bead arrays', which are miniaturized DNA probe arrays in capillaries, were successfully fabricated.

Journal Article↗

PubMed2002

DNA probes on beads arrayed in a capillary, 'Bead-array', exhibited high hybridization performance.

A DNA analysis platform called 'Bead-array' is presented and its features when used in hybridization detection are shown. In 'Bead-array', beads of 100- micro m diameter are lined in a determined order in a capillary. Each bead is conjugated with DNA probes, and can be identified by its order in the capillary. This probe array is easily produced by just arraying beads conjugated with probes into the capillary in a fixed order. The hybridization is also easily completed by introducing samples (1-300 micro l) into the capillary with reciprocal flow. For hybridization detection, as little as 1 amol of fluorescent-labeled oligo DNA was detected. The hybridization reaction was completed in 1 min irrespective of the amount of target DNA. When the number of target molecules was smaller than that of probe molecules on the bead, 10 fmol, almost all targets were captured on the bead. 'Bead-array' enables reliable and reproducible measurement of the target quantity. This rapid and sensitive platform seems very promising for various genetic testing tasks.

Base Sequence↗

PubMed2002

Rapid multiplex single nucleotide polymorphism genotyping based on single base extension reactions and color-coded beads.

A single nucleotide polymorphism (SNP) typing method using color-coded beads is promising because it is easy to use and inexpensive. However, the present protocols are not suitable for clinical and diagnostic applications because they need centrifugation for bead-washing. Here, we developed a simplified protocol without a bead-washing procedure that enables SNP typing of PCR amplified fragments in only 30 min.

Journal Article↗