JP2010284101A - Reactor vessel, parallel processing apparatus and sequencer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sequencer that has a more improved throughput. <P>SOLUTION: A power density of 10 times or more of fluorescence excitation intensity is achieved by using an inexpensive high-intensity LED light source. Consequently, the excitation light irradiation time in detection is made 1/10 or less to shorten the time required for detection. Consequently the throughput is improved by treating a plurality of three or more reaction substrates in parallel within a reaction time of a minimum unit which has been conventionally rate-controlling. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a fluorescence detection reaction control apparatus. More specifically, the present invention relates to a method and a nucleic acid sequence analyzer for decoding the base sequence of a nucleic acid such as DNA or RNA.

In the Human Genome Project with a budget of 3 billion dollars between 1990 and 2005, the easiest part (93% of the whole) can be read a few years earlier than planned. As you can see, the techniques and methods necessary for decoding were left as a legacy. Such technology has been further improved since then, and it is now possible to perform genome decoding at an accuracy of about 20 million dollars ($ 2 × 10 ⁷ ). Nonetheless, large amounts of base sequence decoding with this amount is limited to specialized decoding centers or large research projects with large budgets. However, if the sequencing cost is reduced, a larger number of larger genomes can be handled. For example, it becomes possible to compare the genomes of patients and healthy individuals, and as a result, the value of genome information is expected to be improved. As seen in Non-Patent Document 2, the acquisition of such basic data is expected to greatly contribute to the future development of tailor-made medicine.

Under the circumstances described above, two programs for “Innovative Genome Sequencing Technology” funded by the National Institutes of Health (NIH) will cost $ 100,000 for one human genome by 2009 ( $ 1 × 10 ⁵ ), and the goal is to make it $ 1000 ($ 1 × 10 ³ ) by 2014. This is the development of the so-called “$ 1000 genome” decoding technology.

As seen in Non-Patent Document 3, the next-generation sequencers of 454 Life Science, Solexa, and AB have been commercialized. These techniques have already achieved the cost of 1/10 to 1/100 of the prior art. In addition, the number of bases that can be measured by one analysis has reached 10 ⁹ orders. However, the improvement in cost of these sequencers has almost reached its peak, and it is expected that it will be difficult to achieve $ 1000 genome by using commercially available devices and systems.

Moreover, although the next-generation sequencer currently on the market can decode up to about 10 ⁹ base sequences, it takes a few days or more only for the sequence decoding runtime of a large amount of data corresponding to 10 ⁹ bases. . In order to make genome sequencing at the personal level a routine work in the medical field, it is necessary to speed up sequencing as well as cost.

  Patent Document 1 describes details of a system in a next-generation sequencer of AB company. The system includes a CCD camera, a fluorescence microscope, a movable stage, a Peltier flow cell, a temperature control device, a liquid operation device, and a dedicated computer. The AB company system has an Olympus epifluorescence microscope main body (installed sideways) as the main part, and includes an automatic autofocusing stage and a CCD camera. Four filter cubes in the rotary holder allow four color detection at various excitation and emission wavelengths. The device is designed so that the camera is kept in operation at all times. For this purpose, the next-generation sequencers of AB and Helicos employ a system in which two flow cells are mounted and a fluorescence image is acquired using the other flow cell during the reaction time of one flow cell.

JP 2008-528040 Gazette

Nature Reviews, vol5, pp335, 2004 “From complete human genome decoding to understanding“ human ””, pp253, Shohei Hattori, Toyo Shoten, 2005 Nature, vol.449, pp627, 2007

  As described in the background art above, in the next generation sequencing technology, further improvement in throughput is desired. However, biochemical reactions typified by extension reactions are rate-limiting in improving sequencing throughput. Both the extension reaction using polymerase and the ligation reaction using ligase require incubation of about 90 minutes until the reaction is completed. In order not to set this incubation time as dead time, AB and Helicos have installed a system with two flow cells and acquired a fluorescence image using another flow cell during the reaction time of one flow cell. Adopted. However, even in the above-described method, since the time required for the reaction of the minimum unit is still rate-limiting, it is difficult to further improve the throughput.

  In the next-generation sequencing technology, a laser with unstable behavior when the light source is turned on or off, or a high-power mercury lamp or Xe lamp is used. It was difficult to control the ON / OFF of these light sources themselves on the order of seconds. Therefore, in any of the above systems, it is necessary to block the excitation light with an expensive shutter during CCD data transfer.

  In addition, a glass slide with surface modification is widely used as a substrate for fluorescence detection. At the time of the experiment, the user needs to manually adsorb the beads on the slide glass. However, since the slide glass is exposed, careful handling is required, and improvement in usability is awaited.

  Moreover, in the conventional epi-illumination, uniform illumination called Koehler illumination is performed by converging excitation light perpendicularly to the center of the rear focal plane of the objective lens on a normal slide surface. After this, it becomes possible to achieve total reflection illumination on the surface of the slide glass by converging vertically after incidence on the edge of the focal plane.

  In the present invention, a low-priced high-intensity LED light source is used to achieve a power density that is 10 times or more that of fluorescence excitation by a conventionally used arc light source such as a mercury lamp. Thereby, the time required for detection is shortened by setting the excitation light irradiation time in detection to 1/10 or less. As a result, by processing three or more reaction substrates in parallel within the reaction time of the minimum unit, which has been the conventional rate-determining method, the throughput can be reduced without being limited to the reaction time of the minimum unit, which has been the conventional rate-determining method. It is possible to improve.

  Further, by using the LED, irradiation of excitation light can be controlled by current control without using a shutter. Therefore, it is possible to construct a shutterless system, and the cost can be reduced.

  Further, the slide glass is packaged to facilitate handling of the slide glass. Furthermore, it is possible to further reduce the burden on the user by preliminarily placing beads serving as a base point of light emission of the fluorescent dye in the packaged slide glass.

  The fluorescence measuring apparatus of the present invention uses a low-cost LED light source to shorten the time required for detection. As a result, the number of scans within the minimum unit reaction time is parallelized to three or more. As an effect of the present invention, a low-cost and high-throughput DNA sequencer system is realized.

  In addition, a shutterless system using LEDs is constructed to reduce the cost of the device.

  Further, the packaging of the slide glass brings about an effect of improving the operability for the user.

FIG. 3 is an explanatory diagram about packaging of a slide glass in Example 1. Explanatory drawing about the sequence reaction in Example 2. FIG. The absorption spectrum and fluorescence spectrum of the fluorescent dye used in Example 3. Explanatory drawing about the light source comprised from 2 or 4 LED in Example 3. FIG. Explanatory drawing about arrangement | positioning of the optical element used for LED illumination in Example 3. FIG. The emission spectrum of Royal Blue LED in Example 3. The emission spectrum of Warm White LED in Example 3. The transmission characteristic of the dichroic mirror for mixing the light emission from LED in Example 3. FIG. The transmission characteristic of the bandpass filter for 4 fluorescent dyes in Example 3. The transmission characteristic of the emission filter for 4 fluorescent dyes in Example 3. FIG. The transmission characteristics of the dichroic mirror for four fluorescent dyes in Example 3. Explanatory drawing about the epi-illumination fluorescent device in Example 3. FIG. 9 is a timing chart when the shutter is not used in the third embodiment. Explanatory drawing about the method of carrying out the parallel processing of the some slide glass in Example 4. FIG. The description about the method of carrying out the parallel processing of the some slide glass in Example 4. FIG. The description about the method of carrying out the parallel processing of the some slide glass in Example 4. FIG. Explanatory drawing about the packaging of the slide glass in Example 5. FIG. Explanatory drawing which fixes the bead which many DNA fragments couple | bonded to a slide glass. Explanatory drawing in case the slide glass by which the bead was previously arranged is packaged.

  As a first embodiment of the present invention, a method for fixing beads, to which a plurality of DNA fragments to be analyzed are bound, on a pre-packaged slide glass will be described below with reference to FIG. The packaged slide glass is formed by sandwiching the spacer 104 between the cover 103 and the slide glass 107 on the upper surface. In order to perform solution exchange smoothly, the thickness of the spacer 104 is preferably about 350 μm. On the upper surface of the cover 103, a septa 101 serving as a solution inlet and a septum 102 serving as an outlet are formed. By inserting an injection needle or the like into the septa 101, 102 and injecting the solution, the solution can be filled in the flow cell. In this case, the solution is injected using the septa 101 as an inlet. The septa 102 plays a role of venting air as an outlet. A large number of beads 108 having a plurality of probe oligos 109 immobilized therein exist in the solution. Since the beads 108 are chemically treated so as to form a covalent bond when they are brought into contact with the slide glass 107, the beads 108 are settled on the slide glass 107 and fixed by centrifuging the slide glass 107 filled with the solution. The

  When the slide glass 107 that has not been packaged is used, there is a risk that the user may inadvertently touch the surface of the slide glass 107. Further, since the chemical state of the beads 108 fixed on the slide glass 107 changes when dried, the subsequent sequencing reaction does not proceed smoothly. Therefore, since the surface of the slide glass 107 to which the beads 108 are bonded does not dry, it is necessary to always exist in the aqueous solution. Therefore, by using the packaged slide glass 107 described in this embodiment, it is possible to prevent the glass 108 from contacting the slide glass 107 and drying the beads 108 on the slide glass 107. Further, by using the pre-packaged slide glass 107, the beads 108 can be fixed on the slide glass 107 with good operability.

  Next, as a second embodiment of the present invention, a sequence reaction will be described below with reference to FIG. A reaction solution is injected through the septa 211, 212 onto the surface of the slide glass in the flow cell 213. Each reaction solution contains four types of nucleotides and polymerases labeled with different fluorescent dyes. Each nucleotide is FAM-dCTP214, Cy3-dATP215, y5-dCTP216, Cy5-dCTP217, respectively. The concentration of each nucleotide is 200 nM. In addition, the salt concentration, the magnesium concentration, and the pH of the reaction solution are optimized so that the extension reaction can be performed efficiently. The measurement region 218 is a visual field capable of measuring the objective lens on the sample surface. Therefore, in order to measure the entire surface of the slide glass, it is necessary to scan at least the number obtained by dividing the entire area of the slide glass by the area of the measurement region 218. Before starting the reaction, the DNA fragment 206 whose probe sequence is to be decoded and the probe oligo 205 are hybridized. A polymerase is contained in the reaction solution, and only one base of fluorescent nucleotide complementary to the DNA fragment 206 is incorporated. The extension of the second base does not occur because the fluorescent dye of the first base is bulky and causes steric hindrance for the incorporation of the second base. After one base is incorporated, the floating fluorescent nucleotide is removed by washing, and then fluorescence measurement is performed. In order to carry out the reaction of the minimum unit thereafter, a step of dissociating the fluorescent dye from the base with the dissociation solution after the fluorescence measurement is essential. This step can reset the steric hindrance of the fluorescent dye in the minimum unit reaction. This allows the reaction to continue. The fluorescent nucleotide is again fed into the flow cell and the reaction is repeated, whereby a sequential sequence of the DNA fragments 206 becomes possible.

  Next, as a third embodiment of the present invention, a sequence reaction measurement using an LED as a light source for exciting a fluorescent dye was used and will be described below with reference to FIGS. 3, 4, 5, 6, 7 and 8. explain. As described in Example 2, four kinds of fluorescent dyes are used for the sequence reaction. More specific examples of four types of fluorescent dye sets include FAM, Cy3, Texas Red, and Cy5 as shown in FIG. 3A and 3B show the absorption and emission characteristics of FAM, Cy3, Texas Red, and Cy5. FIG. 3A shows that the optimum excitation wavelengths of FAM, Cy3, Texas Red, and Cy5 are 495, 512, 590, and 650 nm. In order to excite these four fluorescent dyes, it is conceivable to use two LEDs as shown in FIG. More specifically, one is a Royal Blue LED, which is used for FAM excitation. The other is a Warm-White LED that can generate excitation light over a wide wavelength range, and is used for excitation of Cy3, Texas Red, and Cy5. The light emission characteristics of these two LEDs are shown in FIGS. 6 (a) and 6 (b). The Royal Blue LED 401 has a small spectral width, has an emission center wavelength near 450 nm, and can efficiently excite the FAM fluorescent dye. The warm-white LED 402 has a wide spectrum width close to that of a white light source, and can efficiently excite Cy3, Texas Red, and Cy5. The output described in the catalog of each LED is 515 mW, 180 lumen. In addition, FIG. 6D shows the wavelength characteristics of a band-pass filter for allowing a wavelength component optimum for dye excitation to pass through from the light emitted from these LEDs. When the four dyes are excited, these band pass filters are sequentially placed in front of the LED as the light source, whereby the optimum excitation light can be selected for each dye.

  The LEDs 401 and 402 emit light when a current of 700 mA flows. The element size of the LEDs 401 and 402 is 1 mm square, but the apparent element size is 1.39 mm square for the bullet lens.

  The arrangement of the optical elements when performing LED illumination will be described below with reference to FIG. The light emitted from the LED 121 is collected by the aspheric lens 122, passes through the bandpass filter 123, and forms an image on the pupil plane of the objective lens 124. Koehler illumination is achieved when the image of the LED 121 is placed on the pupil of the objective lens 124. Koehler illumination is an illumination method that can uniformly illuminate the sample surface even when the light source itself has uneven brightness. Light emitted from a position where an image is present passes through and illuminates the sample surface at an angle corresponding to the emitted position. With the above configuration, the fluorescent dye 126 fixed on the slide glass 125 can be illuminated uniformly without depending on luminance unevenness of the LED light source.

  More detailed sizes and positions of the optical elements will be described below. The aspherical lens 122 has a diameter of 16 mm, a front focal length (BFL) of 11.2 mm, a rear focal length (BFL) of 7.8 mm, and a thickness of 6 mm. In addition, although the light emitting surface of the LED is 1 mm square, since the bullet lens covers the LED, the apparent size of the LED is 1.39 mm. When a 20 × objective lens (NA 0.45) and a condensing lens with a focal length of 180 mm are used, the focal length of the objective lens is 180/20 = 9 mm. The angle at which the objective lens can collect light from the beads emitted from the sample surface is sinθ = NA / n = 0.45 / 1 = 0.45 according to the formula NA = nsinθ. Therefore, the diameter of the pupil of the objective lens is 2 × 9 mm × 0.45 = 8.1 mm. Therefore, it is necessary to construct an optical system that images a 1.39 mm LED to 8.1 mm or less. In this case, the magnification of the optical system is 8.1 / 1.39 = 5.8 times. If it is desired to create a 5.8 times magnified image using a specific spherical lens with a front focal length of 11.2 mm, the position where the object should be placed can be calculated. As a conclusion, if the distance from the LED fixed surface to the image side surface of the aspheric lens on the optical axis is 15.8 mm, the distance at which the LED image is formed from the image side surface of the aspheric lens is unique. It is determined to be 76.0 mm. Therefore, Koehler illumination is achieved by installing the pupil plane of the objective lens at this position.

Next, the power density capable of condensing the light emitted from the LED on the sample surface is estimated below. As an example, a description will be given using a Royal Blue LED used for FAM excitation. The output of this LED is a catalog value of 515 mW. The light of 515 mW can be condensed by the aspherical lens 122 by 1/3 or more. After passing through the aspherical lens 122, a wavelength band suitable for FAM excitation is selected by installing a bandpass filter for FAM (shown in FIG. 6D) on the optical path for condensing. The amount of energy selected by the bandpass filter depends on the width of the bandpass filter, but when the bandpass filter of FIG. Further, the ratios of energy amounts lost when passing through the band pass, the dichroic mirror, and the objective lens are 95%, 90%, and 90%, respectively. The power density of light finally reaching the sample surface of the slide glass is 515 mW × 1/3 × 0.3 × 0.95 × 0.9 × 0.9 = 35.7 mW. Since the illumination range of the 20 × objective lens is 1 mm or less, the final power density is 35.7 mW / mm ² or more. The power density in Cy3, Texas Red, and Cy5 can also be calculated by estimating the amount of light when the Warm-White LED spectrum shown in FIG. 6B is passed through the bandpass filter of Cy3, Texas Red, and Cy5. The resulting power densities are 15.0, 15.7, and 14.8 mW / mm ² for Cy3, Texas Red, and Cy5, respectively. Since this value is 10 times or more larger than the power density of 1.5 mW / mm ² which is generally required for observation of fluorescent dyes, it is possible to acquire a signal with a shorter exposure time than in the past.

  Next, an illumination light source using LEDs will be described with reference to FIG. The LEDs 401 and 402 are fixed to heat sinks 403 and 404, respectively, for releasing generated heat into the atmosphere. More specifically, as shown in FIG. 5, the generated heat is monitored by the thermistor 129 and is blown by the fan 128 to efficiently dissipate the heat to the outside. Further, the deterioration of the LED 121 can be monitored by using the photodiode 130. In FIG. 4, the light emitted from the LEDs 401 and 402 is collected by the aspheric lenses 406 and 407 having NA of 0.7 or more. Light emitted from the LEDs 401 and 402 is guided to the same optical path by the dichroic mirror 408. FIG. 6C shows the spectral characteristics of the dichroic mirror used in this example. Since this dichroic mirror rises steeply in the vicinity of 500 nm, the light from the LED 401 is reflected, and the light from the LED 402 is allowed to go straight so that the light from the two LEDs can be mixed efficiently. The mixed light is reflected by the mirror 409 and irradiated onto the measurement object. In FIG. 4B, four LEDs 410, 411, 412, and 413 optimized for the excitation wavelengths of four color fluorescent dyes are used. While it is possible to obtain a stronger signal than in (a), it is necessary to prepare a larger number of dichroic mirrors and mirrors than in (a), which causes a problem of high cost. Therefore, description using two LEDs will be given below.

  Further, a fluorescence measuring apparatus for four fluorescent dyes will be described with reference to FIG. The LED light source 501 is driven from a power source 519 through a drive circuit 518 to emit light. The light emitted from the LED light source 501 in which the LEDs 401 and 402 are stored travels to the cubes 504, 505, 506, and 507 held by the rotating turret 502. The cubes 504, 505, 506, and 507 are equipped with the band pass filter 521, the dichroic mirror 522, and the emission filter 523 shown in FIGS. 6D, 6E, and 6F, respectively. The optical filters in cubes 504, 505, 506, and 507 are optimized for FAM, Cy3, Texas Red, and Cy5, respectively. The rotating turret 502 is placed on the optical path by rotating a cube suitable for the fluorescent dye to be measured. Light emitted from the LEDs 401 and 402 is reflected upward by the dichroic mirror 522 through the bandpass filter 521, condensed by the objective lens 509, and irradiated onto the beads fixed on the slide glass. The fluorescent dye incorporated on the beads with the extension reaction is excited and emits fluorescence. A part of the fluorescence emitted isotropically on the slide glass is again collected by the objective lens 509 and becomes parallel light. Further, the fluorescence passes through the dichroic mirror 522 and the emission filter 523, passes through the condenser lens 517, and forms an image on the detection surface of the CCD 516. Along with the extension reaction, the fluorescent dye is taken in on the fixed beads on the flow cell, and emits one color fluorescence corresponding to the extended base among the four colors. Hereinafter, the case of FAM will be described as an example. Along with the extension reaction, the LED 401 is turned on and the LED 402 is turned off to excite the captured FAM-dCTP. The rotating turret rotates the cube 504 and guides it to the optical path. As a result, excitation light optimal for FAM is irradiated onto the sample surface, and only the beads incorporating FAM-dCTP emit light. This fluorescent image is acquired and stored by the CCD camera 520. Next, in order to acquire fluorescent images of Cy3, Texas Red, and Cy5, the LED 401 is turned off and the LED 402 is turned on. In order to excite Cy3, a cube 505 is guided to the optical path. As a result, the excitation light optimal for Cy3 is irradiated on the sample surface, and only the beads incorporating Cy3-dATP emit light. In order to focus immediately before acquiring the fluorescent image, the objective lens 509 is driven in the Z direction by the objective lens autofocus motor 508 to perform autofocus.

  The LEDs 401 and 402 can be easily turned on / off by the drive circuit 518. The light source of the conventional sequencer was an arc light source used for a laser or a mercury lamp or a xenon lamp. However, the behavior of these light sources when the light source was turned on / off was unstable. Therefore, when a laser or arc light source is used as the light source, it is necessary to block the excitation light by using an expensive shutter when transferring the CCD. This state is shown in FIG. Since the LEDs 401 and 402 can be easily turned on / off by the drive circuit 518, a shutterless system can be constructed as shown in FIG. 8B, and the cost of the apparatus can be reduced.

  The slide glass 524 has a size of 25 × 75 mm, and the reaction region to which the beads are bonded has a size of 20 × 60 mm. The objective lens 509 used for measurement is a 20 × objective lens with 22 fields of view. Therefore, the size of the measurable area is about 770 um square. Therefore, in order to measure all the reaction areas of the slide glass 524, it is necessary to move the slide glass 524 and acquire an image. In order to cover a 20 × 60 mm area with a measurement of 770 um square, the slide glass 524 needs to be moved about 2100 times. In this case, the slide glass 524 is moved by the XY stage.

  Next, as a fourth embodiment of the present invention, parallelization of sequence reaction measurement using an LED as a light source for exciting a fluorescent dye will be described below with reference to FIGS. 9, 10, and 11.

  In general, the reaction time required for extension of one base is as long as about 90 minutes. In order to make effective use of the base extension time in the conventional next-generation sequencer, 2 slide glass can be scanned during 90 minutes when the extension reaction is performed with one slide glass. Many are designed so that a single slide glass can be mounted. Specific examples include LifeTech's SOLiD and Helicos' Heliscope.

In the present invention, illumination was performed using LEDs, and power densities of FAM, Cy3, Texas Red, and Cy5 were 35.7, 15.0, 15.7, and 14.8 mW / mm ² . This is a power density that is 10 times or more of 1.5 mW / mm ² of the power density necessary for exciting the fluorescent dye using a normal mercury lamp or the like. This means that the time during which light is purely irradiated can be shortened to 1/10 or less. Reducing the exposure time leads to a significant reduction in scanning time. On the other hand, shortening the reaction of the minimum unit, which is a chemical reaction, causes a reduction in the reaction efficiency, so that shortening is difficult. Therefore, to improve the throughput, it is determined by how many slides can be processed in parallel during 90 minutes of the reaction of the smallest unit. As shown in the table of FIG. 9B, when the ratio of the time required for reaction and scanning is 1: 1, two slides can be processed in parallel. What should be noted here is that since there is only one objective lens and CCD camera, scanning cannot be performed in parallel, but the reaction of the smallest unit can be performed in parallel because the solution is only filled in the flow cell.

  If the ratio of reaction to scan time is 2: 1, three slides can be processed in parallel. Similarly, when the ratio of the time required for reaction and scanning is 3: 1, parallel processing of four slides is possible. If this is generalized and the ratio of the time required for reaction and scanning is n: 1, parallel processing of n + 1 slides is possible.

  FIG. 9A shows a case where four slide glasses 704, 705, 706, and 707 are simultaneously processed in parallel. In this case, an XY stage capable of simultaneously mounting four slide glasses is required. The scanning of the slide glasses 704, 705, and 706 is completed while the extension reaction is progressing on the slide glass 707, and then the process proceeds to the extension reaction of the slide glass 706. The glass slides 704, 705, and 707 are scanned during this extension reaction. By repeating the above procedure, a plurality of slide glasses can be processed in parallel, and the throughput can be improved four times in this case.

  In FIG. 10, the same processing is performed for 10 slide glasses. The case where 10 slide glasses are processed simultaneously in parallel is shown. In this case, an XY stage capable of simultaneously mounting 10 slide glasses is required. While the extension reaction is progressing on the slide glass 804, scanning of the other slide glass is completed, and then the process proceeds to the extension reaction of the slide glass 805. During this time, scanning is performed except for the slide glass 805. By repeating the above procedure, parallel processing of 10 slide glasses is possible, and throughput can be improved.

  FIG. 11 illustrates an embodiment in which a slide glass is installed on an annular stage and a plurality of slide glasses are processed simultaneously in parallel. Eight flow cells are installed on the annular stage. A slide glass is held in the flow cell. The reaction solution is fed into the flow cell 911 via pipettes 907 and 908. The annular stage rotates to the position of the adjacent flow cell every reaction time / (8-1) ≈13 minutes. Pipettes 912 and 913 wash the reaction solution with a washing solution immediately before scanning. When the flow cell 910 is scanned, the annular stage is driven in the X and Y directions, and the fluorescence signal emitted from the beads fixed on the slide glass in the flow cell 910 can be acquired for the entire slide surface. A temperature control device 906 required for the primer dissociation reaction and the like is installed. Since it is an annular stage, it is not necessary to install eight temperature control devices for the eight slide glasses 804. This is because the slide glass 804 performs a circular motion, so that any slide glass 804 sequentially passes through the temperature control device 906. The same effect is obtained with respect to the number of pipettors installed. Therefore, by adopting the annular stage, it is possible to eliminate the active driving of the pipettors 907, 908, 912, and 913 and to reduce the number of temperature control devices 906 to one. Further, the annular stage can always rotate at the same speed. Since it always rotates at a steady speed without stepping, the stability of the apparatus is improved.

  Next, as a fifth embodiment, a fluorescence enhancement method using total reflection illumination will be described below with reference to FIG. As shown in FIG. 12A, in the normal epifluorescence method, excitation light enters the sample surface perpendicularly through the objective lens. On the other hand, in total reflection illumination, an evanescent field is formed at the interface between the slide glass and the solution by causing the excitation light to enter the interface between the slide glass and the solution at an angle larger than the critical angle. The area is illuminated. Therefore, fluorescence is generated from the sample irradiated with the excitation light only in this thickness range. As shown in FIG. 12B, it is known that the light intensity at the interface increases with respect to the incident light intensity as the incident angle approaches the critical angle. At the critical angle, the enhancement factor is four times. Even in the fluorescence detection after the sequence reaction, it is possible to increase the amount of fluorescence emitted from the vicinity of the boundary surface by using total reflection illumination instead of the conventional epi-illumination method. At present, beads having a diameter of 1 μm are used, but with the demand for higher throughput, higher density of beads is required, and as a result, bead miniaturization is required. In particular, since the enhancement effect is obtained in the region of 100 nm or less in total reflection illumination, it is effective to use beads of about 100 nm. In this case, a fluorescence enhancement effect four times that of epi-illumination can be obtained. It is also effective to use 2 um beads for epi-illumination. Currently, fluorescent signals are acquired using 1 um beads, but the diameter of the beads that match the instrument function is 2 um. More specifically, when 3 pixels of a CCD camera are used to disassemble a 1 um bead, in consideration of the effect of an image blurring in the periphery, 5 pixels are actually used for a 1 um bead. When 2 um beads are used, the beads are already large enough to prevent bleeding like 1 um beads. Therefore, it takes about 5-6 pixels to resolve both 1 um beads and 2 um beads. Therefore, there is almost no difference between 1 um and 2 um beads from the viewpoint of image size. On the other hand, since the surface area of 1 um beads and 2 um beads is four times larger, the amount of DNA fragments to be bound is also four times larger, and an increase in the amount of fluorescence that can be obtained is expected. In addition, as shown in FIG. 13, beads 108 having a diameter of 500 μm to which a large number of DNA fragments are bonded are fixed to a slide glass 112. Since there are many minute voids inside the beads 108, the beads 108 are flattened when pressed against the beads 108 by a centrifugal process, and a disk-shaped spot 113 is formed. The disk-shaped spot has a diameter of 1 μm and a height of 0.1 μm. When total reflection illumination is performed in this state, the fluorescence emitted from the beads 108 can be included in the evanescent field, and the fluorescence enhancement close to four times that of the epi-illumination method is confirmed.

  As a sixth embodiment, a case where a slide glass in which beads are arranged in advance is packaged will be described below with reference to FIG. As described in Example 1, conventionally, externally adjusted beads were fixed by being poured into a slide glass immediately before measurement. However, it is more desirable for the measurer that the beads 1111 are fixed to the surface of the slide glass 1112 in advance. As a result, the operator only needs to inject the adjusted sample into the flow cell, and the work of performing chemical treatment using beads on the outside can be omitted. Further, since the beads are not flowed into the flow path, it becomes possible to avoid clogging of the flow path. In the future, it is expected that the density of the beads will be further increased. Therefore, it is desirable to package the slide glass 1112 in which the beads 1111 are arranged in a lattice shape. The present embodiment is the same as the first embodiment except that beads 1111 are arranged in a lattice pattern on the surface of the slide glass 1112.

  The subject of the present invention can be expressed as follows. In order to improve the throughput of next-generation sequencing technology, biochemical reactions represented by single-base extension reactions are rate-limiting. In both the extension reaction using polymerase and the ligation reaction using ligase, incubation of about 90 minutes is required until the reaction of the minimum unit is completed. In order not to set this incubation time as dead time, AB and Helicos employ two flow cells and adopt a system that uses one flow cell to acquire fluorescence images during the reaction time of one flow cell. is doing. However, since the time required for the reaction of the smallest unit is still rate-determined even in the above-described method, it is difficult to further improve the throughput.

  The means for solving this problem can be expressed as follows. In the present invention, a low-cost high-intensity LED light source is used to achieve a power density of 10 times or more the fluorescence excitation intensity. Thereby, the time required for detection is shortened by setting the excitation light irradiation time in detection to 1/10 or less. As a result, it is possible to improve throughput by processing three or more reaction substrates in parallel within the reaction time of the minimum unit, which has conventionally been rate-limiting. Further, by using the LED, irradiation of excitation light can be controlled by current control without using a shutter. Therefore, it is possible to construct a shutterless system, and the cost can be reduced. Further, the slide glass is packaged to facilitate handling of the slide glass. Furthermore, it is possible to further reduce the burden on the user by preliminarily placing beads serving as a base point of light emission of the fluorescent dye in the packaged slide glass.

  The effect of the present invention can be expressed as follows. The fluorescence measuring apparatus of the present invention uses a low-cost LED light source to shorten the time required for detection. As a result, the number of scans within the minimum unit reaction time is parallelized to three or more. As an effect of the present invention, a low-cost and high-throughput DNA sequencer system is realized. In addition, a shutterless system using LEDs is constructed to reduce the cost of the device. Further, the packaging of the slide glass brings about an effect of improving the operability for the user.

101, 102, 211, 212 Septa 103 Cover 104 Spacers 107, 112, 125, 524, 704, 705, 706, 707, 804, 805, 904, 910, slide glass 108, 111 beads 109 probe oligo 113 disc-shaped spot 121, 401, 402, 410, 411, 412, 413 LED
122, 418, 419, 420, 421 Aspherical lens 123, 521 Bandpass filter 124, 509, 703, 803, 903 Objective lens 126 Fluorescent dye 127, 403, 404, 414, 415, 416, 417 Heat sink 128 Fan 129 Thermistor 130 Photodiode 206 DNA fragment 213 Flow cell 214 FAM-dCTP
215 Cy3-dATP
216 Texas Red-dCTP
217 Cy5-dCTP
218 Measurement area 408, 423, 424, 425, 522 Dichroic mirror 409, 426 Mirror 501 LED light source 502, 701, 801, 901 Rotating turret 504, 505, 506, 507 Cube 508, 702, 802, 902 For objective lens autofocus Motor 516 CCD camera 517 Condensing lens 518 Drive circuit 519 Power supply 520 PC
523 Emission filter 525, 526, 907, 908, 912, 913 Liquid feeding tube 905 Annular stage 906 Temperature control device

Claims (22)

  The distance between the two substrates is constant, the substrate holding at least two holes for injecting the aqueous solution, the light-transmitting substrate that has been subjected to a surface treatment that strongly binds to the fine particles contained in the injected aqueous solution, and And a spacer for holding the substrate and the spacer sandwiched between two substrates and bonded together.   2. The reaction container according to claim 1, wherein a plurality of amplified nucleic acid fragments are bound to the surface of the fine particles.   3. The reaction container according to claim 2, wherein the sequence analysis of the nucleic acid fragments is performed by analyzing the sequence of the nucleic acid fragments based on fluorescence signals of four or more colors from the fine particles bound on the substrate.   4. The reaction container according to claim 3, wherein the spacer has a thickness of 350 ± 50 μm.   5. The reaction container according to claim 4, wherein a size of the light-transmitting substrate is 25 × 75 mm.   6. The reaction container according to claim 5, wherein the fine particles have a diameter of 1 μm.   A substrate for holding at least two holes for injecting an aqueous solution, a substrate having a light transmission property in which a plurality of fine particles are combined, a spacer for keeping the distance between the two substrates constant, and the spacers as two substrates A reaction vessel formed by sandwiching and bonding together.   8. The reaction container according to claim 7, wherein the fine particles are randomly bonded on the light-transmitting substrate.   8. The reaction container according to claim 7, wherein the fine particles are bonded to the light-transmitting substrate in a lattice shape.   10. The reaction container according to claim 9, wherein the fine particles have a size of 1 um and a lattice spacing of 3 um.   Hold n reaction containers, and complete the measurement of the reaction containers n-1 automatically while the reaction of the minimum unit of one reaction container is completed, and sequentially proceed the reaction automatically A parallel processing device that improves measurement throughput by a factor of n.   12. The apparatus according to claim 11, wherein the reaction proceeds in the container according to claim 1 or 7 and is a sequence reaction on a substrate surface, and a time required for a reaction of a minimum unit of the sequence is 90 minutes. A sequencer characterized in that the number of fluorescent dyes used in the sequence reaction is four or more.   The apparatus according to claim 12, wherein the light source used for the fluorescence image measurement is two or more types of LEDs, and the fluorescence detection method is an epifluorescence detection method through an objective lens, and is a band optimized for the fluorescent dye. A sequencer characterized in that a cube equipped with a pass filter, a dichroic mirror, and an emission filter is rotationally controlled by a rotating turret, performs autofocus, and a sensor used for fluorescence detection is a two-dimensional CCD camera.   14. The sequencer according to claim 13, wherein the LED light source is turned on and off from a circuit so that light for fluorescence excitation is not required to be blocked and the sequencer is shutterless. The sequencer according to claim 14, wherein the outputs of the LED light sources are all 15 mW / mm ² or more.   16. The apparatus according to claim 15, wherein one of the two types of LED light sources is a Royal Blue color LED and the other is a Warm-White color LED, and the light emitted from the two types of LED light sources is mixed by a dichroic mirror, A sequencer characterized by irradiating with the same.   17. The apparatus according to claim 16, wherein the light emitted from the LED is condensed by an aspheric lens having a high aperture angle, and an image of the LED is formed on the rear focal plane of the objective lens, thereby forming a substrate surface of the reaction vessel. A sequencer characterized by performing Koehler illumination, which is uniform illumination with the illumination unevenness of the light source removed.   18. The sequencer according to claim 17, wherein the reaction container is scanned over the entire surface of the reaction container by sequentially moving the reaction container by the size of the measurement field of view of the objective lens by an XY stage.   19. The apparatus according to claim 18, wherein the XY stage for moving the reaction vessel includes a θ stage for rotating the reaction vessel in an annular manner, and sequentially performing solution exchange and temperature adjustment of the reaction vessel with rotation. Sequencer.   18. The apparatus according to claim 17, wherein the LED is imaged in the vicinity of the outer periphery of the rear focal plane of the objective lens, whereby fluorescence is enhanced four times by total reflection illumination near the critical angle on the substrate surface of the reaction vessel. Sequencer to do.   21. The sequencer according to claim 20, wherein fluorescence is enhanced four times by using 100 nm beads to place all fluorescent dyes bound to the beads in a fluorescence enhancement field.   6. The reaction container according to claim 5, wherein the fine particle has a diameter of 2 μm, and has a fluorescence enhancement effect due to a 4-fold increase in DNA fragment binding sites compared to the 1 μm fine particle.

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