Takashi Anazawa

A capillary array gel electrophoresis system using multiple laser focusing for DNA sequencing

A very simple and highly sensitive capillary array gel electrophoresis system is constructed to analyze DNA fragments. On-column detection of DNA migration in a large number of gel-filled capillaries is carried out using side-entry laser irradiation and with a CCD camera, although it has been considered impossible because the irradiation laser is scattered strongly at the surfaces of the first few capillaries. By optimizing optical conditions, the laser beam can be focused repeatedly to irradiate all the capillaries held on a plate by working each capillary as a cylindrical convex lens. DNA sequencing samples migrating in 24 capillaries can simultaneously be analyzed with the system.

Slowing single-stranded DNA translocation through a solid-state nanopore by decreasing the nanopore diameter

To slow the translocation of single-stranded DNA (ssDNA) through a solid-state nanopore, a nanopore was narrowed, and the effect of the narrowing on the DNA translocation speed was investigated. In order to accurately measure the speed, long (5.3 kb) ssDNA (namely, ss-poly(dA)) with uniform length (±0.4 kb) was synthesized. The diameters of nanopores fabricated by a transmission electron microscope were controlled by atomic-layer deposition. Reducing the nanopore diameter from 4.5 to 2.3 nm slowed down the translocation of ssDNA by more than 16 times (to 0.18 μs base(-1)) when 300 mV was applied across the nanopore. It is speculated that the interaction between the nanopore and the ssDNA dominates the translocation speed. Unexpectedly, the translocation speed of ssDNA through the 4.5 nm nanopore is more than two orders of magnitude higher than that of double-stranded DNA (dsDNA) through a nanopore of almost the same size. The cause of such a faster translocation of ssDNA can be explained by the weaker drag force inside the nanopore. Moreover, the measured translocation speeds of ssDNA and dsDNA agree well with those calculated by molecular-dynamics (MD) simulation. The MD simulation predicted that reducing the nanopore diameter to almost the same as that of ssDNA (i.e. 1.4 nm) decreases the translocation speed (to 1.4 μs base(-1)). Narrowing the nanopore is thus an effective approach for accomplishing nanopore DNA sequencing.

A capillary-array electrophoresis system using side-entry on-column laser irradiation combined with glass rod lenses.

We have developed a simple and high‐throughput capillary‐array electrophoresis system that uses side‐entry on‐column laser irradiation. The number of capillaries in an array is generally limited by laser‐power attenuation along the array due to reflection and divergence. We overcame these problems by placing the capillaries in water and adding glass rod lenses between the capillaries. As a result, up to 45 capillaries could be simultaneously irradiated with a single laser beam and the fluorescence from all the capillaries could be detected with high sensitivity. We demonstrated the high throughput of 12 kbp/h with a 45 capillary array using this system.

Dual-view imaging system using a wide-range dichroic mirror for simultaneous four-color single-molecule detection.

A dual-view imaging system for simultaneous four-color single-molecule (SM) detection was developed. As for the detection procedure, four species of SM fluorophores, namely, Alexa 488, 555, 647, and 680, are immobilized on different slides and excited by evanescent-wave illumination. Fluorescence emitted from an SM fluorophore is split by a wide-range dichroic mirror (WR DM) in a dual-view optics and imaged as two SM fluorescence spots (SM spots) on an electron-multiplying charge-coupled device (EM-CCD) at 100 Hz. The transmittance of the WR DM changes gradually over the wavelength range of 500 to 700 nm so that the signal ratios of the two SM spots for the four fluorophore species differ. A method for classifying SM fluorophores into four species in accordance with their signal ratios was developed. It was used to classify 597 SM fluorophores at an accuracy of above 98% for all the species. This accuracy is comparable to that of a conventional four-color SM detection system. To classify four species, the conventional system disperses SM fluorescence with a prism and provides an elongated SM spot that uses more pixels of an EM-CCD chip than that of the developed system. The developed system can thus detect 1.5-fold more SM spots with the same-size EM-CCD chip, so it can achieve 1.5-fold higher throughput. Moreover, the developed system is based on a simple and practical approach, namely, replacing an ordinary dichroic mirror in a commercially available dual-view optics with a WR DM. This replacement transforms a dual-view imaging system for two-color detection into a system for four-color detection. The developed system is suitable for detection systems of next-generation DNA sequencers and DNA microarray-chip analyzers.

A novel side-gated ultrathin-channel nanopore FET (SGNAFET) sensor for direct DNA sequencing

A novel side-gated ultrathin-channel nanopore FET (SGNAFET), for fast and label-free DNA sequencing with high resolution and sensitivity, is proposed. The goal of the SGNAFET is to identify the four types of nucleotides in DNA by changes in the channel current of the SGNAFET. Aiming to reach that goal, a SGNAFET with channel thickness (tch.) of 2 or 4 nm was successfully operated and could detect DNA translocations through its nanopore on the basis of changes in its channel current.

Simultaneous four-color imaging of single molecule fluorophores using dichroic mirrors and four charge-coupled devices

We developed a total-internal-reflection (TIR) fluorescence microscopy using three dichroic mirrors and four charge-coupled devices (CCDs) to detect simultaneously four colors of single-molecule (SM) fluorophores. Four spectrally distinct species of fluorophores (Alexa 488, Cy3, Cy5, or Cy5.5) were each immobilized on a different fused silica slide. A species of fluorophores on the slide was irradiated simultaneously, by two excitation beams from an Ar ion laser (488 and 514.5 nm) and a diode laser (642 nm) through TIR on the slide surface. Fluorescence emitted from the fluorophores was spectrally resolved into four components by the dichroic mirrors, and four images were generated from them simultaneously and continuously, with the four CCDs at a rate of 10 Hz. A series of images was thus obtained with each CCD. Fluorescence spots for a species were observed mainly in the series of images recorded by its respective-color CCD. In the first image in the series, we picked out the spots as continuous pixel regions that had the values greater than a threshold. Then we selected only those spots that exhibited single-step photobleaching and regarded them as SM fluorescence spots. Pixel values of SM fluorescence spots widely differed. Some SM fluorophores had pixel values smaller than the threshold, and were left unpicked. Assuming the pixel values of SM fluorescence spots differed with a Gaussian profile, we estimated the ratios of unpicked fluorophores to be less than 20% for all the species. Because of the spectral overlaps between species, we also observed cross-talk spots into CCDs other than the respective-color CCDs. These cross-talk SM fluorescence spots can be mistaken for correct species. We thus introduced the classification method and classified SM fluorescence spots into correct species in accordance with two kinds of four-dimensional signal vectors. The error rates of fluorophore classification were estimated to be less than 3.2% for all the species. Our system is suitable for the biological studies that desire to simultaneously monitor the four colors of SM fluorophores.

Side-gated ultrathin-channel nanopore FET sensors

A side-gated, ultrathin-channel nanopore FET (SGNAFET) is proposed for fast and label-free DNA sequencing. The concept of the SGNAFET comprises the detection of changes in the channel current during DNA translocation through a nanopore and identifying the four types of nucleotides as a result of these changes. To achieve this goal, both p- and n-type SGNAFETs with a channel thicknesses of 2 or 4 nm were fabricated, and the stable transistor operation of both SGNAFETs in air, water, and a KCl buffer solution were confirmed. In addition, synchronized current changes were observed between the ionic current through the nanopore and the SGNAFETs drain current during DNA translocation through the nanopore.