JP5271980B2 - Nucleic acid analyzer and nucleic acid analysis method - Google Patents

Description

  The present invention relates to an apparatus for analyzing nucleic acids (DNA, RNA) using chemiluminescence.

  For DNA base sequence determination, capillary DNA sequencers using the Sanger method are most widely used. This device greatly contributed to the human genome project that reads the entire sequence of 3 billion base pairs of human genomic DNA. Human genome sequence analysis was completed in 2003, but there are still comparative genomic analyzes aimed at analyzing the functions of microorganisms and plants and gene function analysis. The demand for scale DNA sequencing is still growing. On the other hand, in order to spread DNA sequencers for gene diagnosis, general laboratories, etc., it is considered necessary to provide performance such as low cost and easy operability.

  In order to satisfy the above-mentioned demands, several DNA sequencing methods using a method different from the Sanger method have been proposed. One of them is a method (pyrosequence method) for determining a base sequence by detecting pyrophosphoric acid (PPi) produced during DNA elongation using chemiluminescence (Patent Document 1). In this method, first, a primer is hybridized to a target DNA strand, introduced into a reaction solution containing a complementary strand synthase (such as polymerase), and dNTP (dATP, dCTP, dGTP, dTTP) is added to the reaction solution one by one in order to synthesize complementary strands. In addition to polymerase, ATP sulfurylase, adenosine 5 'phosphosulfate (APS), luciferase, luciferin, and dNTP-degrading enzyme (apyrase) are present in the reaction solution. When the complementary strand synthesis reaction occurs, the DNA complementary strand is elongated and PPi is generated as a by-product. This PPi is added to APS by the action of ATP sulfurylase to produce ATP. Luminescence is produced from the ATP and luciferin by the action of luciferase. By detecting this light, it can be determined whether the added complementary strand synthesis substrate has been incorporated into the DNA strand. The dNTP that has not undergone complementary strand synthesis is degraded by an apyrase contained in the reaction solution. By sequentially performing the above steps with four types of dNTPs, the base sequence of the complementary strand can be determined. Since dATP, which is one of dNTPs, emits light by luciferin and luciferase, background light increases, leading to deterioration in measurement and analysis accuracy. Nyren et al. Solved the above problem by using dATPαS, which is an analog of dATP, instead of dATP (Patent Document 2). APS also causes background light because light is generated by luciferin and luciferase. Kanbara et al. Solved the above problem by using adenosine monophosphate (AMP), phosphoenolpyruvate (PEP), and pyruvate phosphate dikinase PPDK instead of APS and ATP sulfurylase (Patent Document 3).

  As described above, the pyrosequencing method makes it possible to determine the base sequence without using a light source such as a laser. Therefore, compared to a DNA sequencer using the Sanger method, the apparatus configuration is simplified, and it is inexpensive and downsized. It becomes easy. In fact, Kanbara et al. Have developed a small DNA sequencer using this pyrosequencing method (Non-patent Document 1).

  On the other hand, the pyrosequencing method has a problem derived from the coexistence of complementary strand synthesis reaction and dNTP decomposition reaction. If there are many dNTP-degrading enzymes, all dNTPs are degraded before complementary strand synthesis, and some unreacted DNA strands are present in the reaction solution. In addition, when dNTP-degrading enzyme is small, dNTP degradation is slowed down, so that when unresolved dNTP is introduced into the reaction solution, undegraded dNTP remains, and several DNA strands for which complementary strand synthesis proceeds at one time. It will be present in the reaction solution. In either case, the degree of extension varies between DNA strands, leading to a decrease in the accuracy of DNA strand sequence analysis. In order to solve these problems, Kanbara et al. Have proposed a method of spatially separating a luminescence reaction including a complementary strand synthesis reaction and a dNTP decomposition reaction (separation reaction method) (Patent Document 4).

  FIG. 1 shows a configuration for spatially separating a luminescent reaction including a complementary strand synthesis reaction and a dNTP decomposition reaction described in Patent Document 4. A dispenser 303 for dispensing a reagent (for example, dNTP) and a reaction vessel 101 containing a reaction solution are arranged one above the other (the dispenser 303 is located on the reaction vessel 101), and a holding member for holding a DNA chain 401 is arranged between the dispenser 303 and the reaction vessel 101, and the measurement is performed by repeatedly moving the holding member 401 in and out of the reaction solution 102 by the vertical movement of the holding member 401. The holding member 401 contains a DNA strand hybridized with the primer and a polymerase, and the reaction solution 102 contains a dNTP-degrading enzyme and a reagent involved in a luminescence reaction, and holds dNTP when the holding member 401 is outside the reaction solution 102. It introduce | transduces into the member 401, a complementary strand synthesis reaction is performed, and when the holding member 401 exists in the reaction liquid 102, a luminescent liquid reaction and dNTP decomposition reaction are performed. By the above method, the complementary strand synthesis reaction and dNTP decomposition reaction / luminescence reaction are spatially separated.

Patent No.3533223 Patent No. 3510272 JP 2007-97471 A JP 2009-247297

Electrophoresis, 22, 3497-3504, 2001

  In the configuration of Patent Document 4, a membrane having a diameter of 3 mm is cited as an example of the holding member 401. The holding member 401 is fixed to a moving table (here, fixed pin 403) which is a means for fixing and moving up and down. The fixing position of the fixing pin 403 to the holding member 401 is the dispensing surface 402 side (upper side) where the dNTP of the holding member 401 is dispensed. The holding member 401 comes close to a distance of about 0.5 mm from the dNTP dispensing position of the dispenser 303 in order to dispense dNTP with high accuracy. Therefore, the fixing pin 403 and the dispenser 303 interfere with each other. The dispenser 303 has a reservoir 301 having a capacity of 200 μL corresponding to four types of dNTPs, and a capillary 302 is provided below each reservoir 301. There is a 1 mm through hole 304 in the center of the four reservoirs 301, and the fixing pin 403 is passed through the through hole 304. Therefore, the diameter of the fixing pin 403 is 1 mm or less. On the other hand, considering the height of the reservoir 301 necessary for holding a certain amount (several tens of μL or more) of dNTP and the piping portion for supplying pressure to the reservoir 301, the length of the fixing pin 403 is 20 mm or more. Necessary.

  From the above, in order to prevent interference between the dispenser 303 and the fixing pin 403, the fixing pin 403, which is a fixing / moving means of the holding member 401, is a bar having a diameter of 1 mm or less and a length of 20 mm or more, and is very fragile (easy to break). ), The shape is difficult to handle.

  Further, when attaching the holding member 401 and the fixing pin 403 to the apparatus, it is necessary to pass the fixing pin 403 through the through hole 304 of the dispenser 303.

  In order to improve the strength of the fixing pin 403, it is conceivable to increase the diameter. For example, it is possible to attach a fixing pin 403 having a diameter of 3 mm to a membrane having a diameter of 4 mm (holding member 401), but since the area for dispensing dNTP is reduced, the dispensing accuracy is lowered. Although the above problem can be solved by enlarging the area of the membrane (holding member 401), it is necessary to enlarge the reaction vessel 101 in order to put a large membrane (holding member 401), and the amount of the reaction reagent increases accordingly. This increases the reagent cost.

  In order to solve the above problems, as a nucleic acid analyzer, a sample holding member including a sample holding unit for a nucleic acid sample, and a dispenser for supplying a complementary strand synthesis substrate that performs a complementary strand synthesis reaction with the nucleic acid sample to the sample holding unit, A reaction part holding a reaction solution containing a luminescent enzyme that immerses the sample holding part and reacts with the product of the complementary chain synthesis reaction to emit light and a degradation enzyme of the complementary chain synthesis substrate, and light that detects luminescence by the luminescent enzyme A detector, a rotation mechanism that supports the sample holding member, is provided between the dispenser and the reaction unit, and rotates so that the surface of the sample holding unit facing the dispenser faces the reaction unit; and dispensing A controller for controlling dispensing by the container and rotation by the rotation mechanism.

  In addition, as an analysis method using a sample holding member in which a nucleic acid sample is fixed to a sample holding unit, the sample holding unit faces a dispenser that supplies a complementary strand synthesis substrate that performs a complementary strand synthesis reaction with the nucleic acid sample. A step of supplying a complementary strand synthesis substrate from the dispenser to the sample holder, and a surface of the sample holder that faces the dispenser reacts with the product of the complementary strand synthesis reaction to emit light. And rotating the sample holding member so as to face the reaction part holding the reaction solution containing the reaction solution containing the complementary strand synthesis substrate, immersing the sample holding part in the reaction solution, and detecting luminescence by the luminescent enzyme The process of carrying out.

  With the above configuration, the holding member that holds the sample holding portion does not interfere with the dispenser, and the sample holding member can be made strong. In addition, the sample holding unit and the holding member can be easily attached.

The example of the separation reaction system for demonstrating a subject. An example of the separation reaction system by this invention. Pyrosequence reaction mode schematic. An example of the schematic diagram of the analyzer by this invention. The rotation mechanism part periphery enlarged view. An example of a rotation mechanism drive process. An example of a membrane system. Sequence operation flowchart. Sectional drawing of the stick and stick holder in the example using a magnetic bead. Fig. 2 is a conceptual diagram of fixation of magnetic beads on a stick dispensing surface. Sectional drawing of the stick and stick holder using a magnetic circuit. Sectional drawing of a magnet rotation type stick and a stick holder. Sectional drawing of a magnet movement type stick and a stick holder. Sectional drawing of an electromagnet-type stick and a stick holder. Sectional drawing of the stick and stick holder in the Example using a Sepharose bead. An example of the schematic diagram of a reaction plate type | mold analyzer. 7 shows a rotation mechanism driving process in Embodiment 4. Schematic of magnetic circuit structure.

  FIG. 2 shows a structure in which the fixing portion of the fixing pin 404 is on a different surface from the dispensing surface 402 of the holding member 401 and the dispensing surface 402 can be immersed in the reaction solution 102 after the reagent is dispensed. In order to realize this, the fixing pin 404 is configured to be capable of rotating. Specifically, the fixing pin 404 is fixed to the back surface of the dispensing surface 402 of the holding member 401. After the reagent is dispensed onto the dispensing surface 402, the fixing pin 404 rotates and the dispensing surface 402 faces the reaction container 101 side (lower side). Thereafter, the fixing pin 404 is lowered, and the holding member 401 is immersed in the reaction liquid 102 in the reaction vessel 101. The fixing pin 404 and the holding member 401 may be the same. For example, a structure in which a nucleic acid (DNA or RNA) is fixed to one end surface of a rod-shaped fixing pin and the surface is a dispensing surface may be used. The rotational movement and vertical movement of the fixing pin 404 may be performed simultaneously. Furthermore, as shown in FIG. 16, when the reaction solution 102 is placed on the reaction plate, the fixing pin 404 may be rotated only without moving up and down.

  In the separation reaction method using such a configuration, since the fixing pin for fixing the holding member does not interfere with the dispenser, the thickness of the fixing pin can be equal to or larger than that of the holding member. In addition, the holding member / fixing pin can be easily attached.

  Hereinafter, a specific configuration will be described according to an embodiment.

  In this example, the base sequence to be measured is determined using a pyrosequencing method. Regarding the pyro sequence, the principle of reaction and sequencing described in Patent Document 2 was adopted. FIG. 3 shows a schematic diagram of the reaction mode. In the pyrosequencing reaction, the target DNA whose sequence is to be determined is made into a single strand (ssDNA), the primer is hybridized, DNA polymerase is added, and four types of dNTPs (dATP, dCTP, dGTP, dTTP) are sequentially added to the reaction solution. In addition, a complementary strand synthesis reaction is carried out, PPi produced as a by-product during the synthesis of the complementary strand is converted to ATP by AMP, PEP and PPDK, and ATP and luciferin are reacted in the presence of luciferase to emit light. Excess dNTP is degraded by apyrase. In this embodiment, AMP, PEP, and PPDK are used to convert PPi to ATP, but a system using ATP sulfurylase and APS may be used.

  FIG. 4 shows an overview of the analyzer of the present invention. This apparatus includes a reaction storage unit, a light detection unit, a reagent dispensing unit, and a rotation mechanism unit. These components are covered with a casing so as to be shielded from light.

In the reaction container 106, four reaction vessels 101 having a diameter of 5 mm and a depth of 10 mm are arranged in a row at a pitch of 9 mm. The diameter, depth, number and pitch of the reaction vessel 101 are arbitrary and are not limited. Further, the reaction vessels 101 may be arranged in a plurality of rows. Moreover, the reaction accommodating part 106 may be in the form of a combination of a commercially available reaction container (for example, Nunc-Immuno ^™ Modules; CAT No. 473539) and a holder for fixing it. The pyrosequence reaction may be performed at a temperature higher than room temperature (28 ° C. to 35 ° C.). Therefore, a temperature control means 105 is connected to the reaction storage unit 106 in order to efficiently control the temperature of the reaction solution. In this embodiment, a Peltier element is used as the temperature control means 105, but a heater or the like may be used. Further, in order to stir the reaction liquid, a stirring means 104 is connected to the reaction storage unit 106. In this embodiment, a motor having an eccentric cam on the rotating shaft is used as the agitating means 104, the eccentric cam is applied to a groove dug in the reaction accommodating portion 106, and the motor is rotated to rotate the reaction accommodating portion 106. Is configured to vibrate. The operation of the temperature control unit 105 and the stirring unit 104 is controlled by an H8 microcomputer 501 (HD64F3052BF25, Renesas Technology).

  The photodetecting portion is entirely covered with a conductive casing 205 made of a conductive material. Four photo detectors 201 are accommodated in the conductive housing 205, and the photo detectors 201 are arranged in a 1: 1 relationship below the reaction vessels 101. Although a photodiode (Si photodiode S1133-1, Hamamatsu Photonics) is used as the photodetector 201, light emitted from the four reaction vessels 101 may be detected by one photodetector using a line sensor or a CCD. . A glass substrate 202 having a transparent electrode layer (here, ITO is used) is disposed on the back surface between the photodetector 201 and the bottom of the reaction container 106, and the transparent electrode layer is disposed on the conductive casing 205. Electrically connected and connected to the installation potential of the device. A signal from the photodetector 201 is amplified by an amplifier 203 (opA129UB, Texas Instruments) incorporated in the conductive housing 205, and is amplified by a multiplexer 204 (MAX4051ACESE, Maxim Integrated Products) disposed outside the conductive housing 205. The signals from the two photodetectors 201 are time-divided and A / D converted, processed by the H8 microcomputer 501, and output to a personal computer (PC).

The reagent dispensing unit includes a dispenser 303, a dispenser holder 305, and a pressure piping block 306. The dispenser 303 is integrated with four reservoirs 301 capable of accommodating 50 μL of reagents (here, 4 types of dNTPs), and each reservoir 301 has a capillary 302 (outer diameter 370 μm, inner diameter 50 μm). . The dNTP accommodated in the reservoir 301 is pressurized from the upper surface of the reservoir 301 and dispensed from the end surface of the capillary 302. Each dispenser 303 is held by a dispenser holder 305 so as to face each reaction vessel 101. A pressure pipe block 306 is connected to the upper surface of the dispenser 303 in order to pressurize and dispense dNTPs. In the pressure pipe block 306, a flow path 307 is formed so that pressure can be simultaneously applied to each of the reservoirs 301 in which four types of dNTPs are stored, and to the reservoirs 301 in which the same dNTPs are stored in the four dispensers 303. Has been. Ultimately, the pressure pipe block 306 is aggregated into four flow paths on the back surface. Four piping tubes 308 are connected to the four flow paths at the back of the pressure piping block 306, and all the piping tubes 308 are connected to one pressure supply source 502. Here, the CO ₂ high-pressure gas tank is used as the pressure supply source 502, but other means may be used. An electromagnetic valve 309 is provided between each piping tube 308 and the pressure supply source 502. The solenoid valve 309 is connected to the H8 microcomputer 501 and controls the opening / closing timing of the solenoid valve 309 to control the dispensing timing and amount of each dNTP.

  The rotation mechanism unit includes a stick 405, a stick holder 406, and a rotation drive unit 407, and is disposed between the reagent dispensing unit and the reaction storage unit. In this embodiment, a rod-like structure (stick) in which the holding member for fixing the DNA strand and the fixing pin for fixing the holding member are integrated is used. FIG. 5 shows an enlarged view around the rotating mechanism. The stick 405 has a diameter of 3 mm, a length of 15 mm, and is made of polyetheretherketone (PEEK). The diameter and length are not particularly limited as long as they can be introduced into the reaction vessel 101. The material is not limited to PEEK. The dispensing surface 402 of the stick 405 is surface-treated so that the DNA strand can be fixed. Specifically, the dispensing surface 402 was coated with avidin. The four sticks 405 are fixed to the stick holder 406 so as to be 1: 1 with respect to each dispenser 303 and each reaction vessel 101. The stick 405 is configured to be detachable from the stick holder 406. As a detachable configuration, a ball plunger built in the stick holder 406 and a stick groove 408 provided on the side of the stick were used.

  The rotation drive unit 407 includes a holder fixing plate 409, an arm 410, a linear guide 416, and a vertical motor 419. The stick holder 406 is fixed to the rotation driving unit 407 via a holder fixing plate 409. When the stick holder 406 is fixed to the holder fixing plate 409, the magnet holder 412 can be easily attached and detached. In addition, the mounting position was secured by using guide pins 411. The holder fixing plate 409 is connected to the arm 410 so as to be rotatable about the rotation shaft 415. An eccentric cam 413 is fixed to one end of the rotation shaft 415, and a pinion 417 is fixed to the other end. . The arm 410 is fixed to the linear guide 416 and is driven up and down by a vertical motor 419. The vertical motor 419 is connected to the H8 microcomputer 501 and controls the vertical movement of the rotation drive unit 407 via the vertical motor 419.

  FIG. 6 shows a driving process of the rotation mechanism unit 407. The dNTP accommodated in the reservoir 301 is dispensed on the dispensing surface 402 of the stick 405, and left still for several seconds for the extension reaction (FIG. 6 (a)). The arm 410 is lowered by the vertical motor 419. At that time, since the arm 410 descends while the teeth of the pinion 417 engage with the teeth of the rack 418 attached to the apparatus housing, the stick holder 406 rotates around the rotation shaft 415 (FIG. 6B). When the arm 410 is lowered by a predetermined distance, the teeth of the pinion 417 are disengaged from the teeth of the rack 48, and the rotational movement of the stick holder 406 and the stick 405 is stopped. Using the eccentric cam 413 and the stopper 414, the rotation of the stick holder 406 and the stick 405 is completely stopped. By this operation, the dispensing surface 402 of the stick 405 is rotated 180 degrees from the time when the dNTP is dispensed. At this time, the dispensing surface 402 of the stick 405 has not yet entered the reaction vessel 101 (FIG. 6C). The arm 410 further descends and stops moving downward at a position of 1 mm from the inner bottom surface of the reaction vessel 101, and the dispensing surface 402 is immersed in the reaction solution in the reaction vessel 101 (FIG. 6 (d)). When the arm 410 is raised, the operation is the reverse of the above process.

  The stick holder 406 moves up without rotating until the teeth of the pinion 417 come into contact with the teeth of the rack 418. When the teeth of the pinion 417 and the rack 418 come into contact with each other, the stick holder 406 moves up and moves up. Ascending, the rotary motion is stopped with the dispensing surface 402 facing upward, and dNTP is dispensed. At this time, the distance between the dispensing surface 402 and the capillary 302dNTP discharge end of the dispenser 303 was set to 1 mm. Pyrosequence reaction is performed by repeating the above process. In this embodiment, the dispensing surface 402 is rotated using the pinion 417 and the rack 418. However, a small motor is connected to the rotary shaft 415, and the small motor is connected to the H8 microcomputer 501 to control the small motor. Thus, the dispensing surface 402 may be rotated.

  Here, an example in which the arm 410 is moved up and down by the up and down motor 419 is shown. However, since the relative position between the stick 405 and the reaction vessel 101 may be controlled, for example, a mechanism in which the reaction vessel 101 moves up and down. May be provided.

  In this embodiment, the holding member for fixing the DNA strand and the fixing pin for fixing the holding member are used as an integrated stick. However, the holding member and the fixing pin may be divided and removed. An example in which a membrane is used as a holding member is shown (FIG. 7). Here, SAM2 (R) Biotin Capture Membrane (V7861, Promega) having a diameter of 3 mm was used as the membrane 420. The membrane 420 is coated with streptavidin by a covalent bond. A pin 421 having a diameter of 1 mm and a length of 5 mm is fixed to a surface different from the dispensing surface 402 of the membrane 420, and the pin 421 is fixed to the stick holder 406 via the pin holder 422. The diameter and length of the pins 421 are not particularly limited as long as they can be introduced into the reaction vessel 101. However, when the membrane 420 is immersed in the reaction solution, the pin 421 is made as thin as possible in consideration of the strength so that the reaction solution can be efficiently transmitted from the surface opposite to the dispensing surface 402 to the membrane. Better. The pin holder 422 and the stick holder 406 are detachable. The stick holder 406 is fixed to the rotation drive unit 407 as in the case of the stick in which the holding member and the fixing pin are integrated. Even when the holding member and the fixing pin for fixing the holding member are separated by the above method, the fixing pin does not interfere with the dispenser, so that it can be shortened even if the diameter of the fixing pin is small, that is, can be strengthened.

Hereinafter, the method of the pyro sequence reaction of the DNA to be measured will be described with reference to the flowchart of FIG. First, a single-stranded DNA (ssDNA) subject to sequence analysis labeled with biotin at the 5 ′ end is hybridized with a primer (601). The primer-ssDNA complex is dropped onto the dispensing surface 402 and allowed to stand for 5 minutes to fix the primer-ssDNA complex to the dispensing surface 402 via a biotin-avidin bond (602). The dispensing surface 402 of the stick 405 is washed with 2 × C buffer (composition: 120 mM Tricine, 4 mM EDTA, 40 mM MgAc ₂ ) (603). Next, polymerase (Klenow) is dropped onto the dispensing surface 402 and allowed to stand for 5 minutes to bind to the primer-ssDNA complex immobilized on the dispensing surface 402 (604). The fixation of the polymerase to the primer-ssDNA complex may be performed in step 608 in which the polymerase is introduced into the reaction solution and the following dispensing surface 402 is immersed in the reaction solution for 1 minute. The stick 405 to which the primer-ssDNA complex is fixed is attached to the stick holder 406, and the stick holder 406 is attached to the apparatus (605). All the operations are performed outside the apparatus. Four types of dNTPs are introduced into the respective reservoirs 301 of the dispenser 303, the reaction solution is introduced into the reaction vessel 101, and the dispenser 303 and the reaction vessel 101 are attached to the apparatus (606). The composition of the reaction solution is, for example, 60 mM Tricine, 2 mM EDTA, 20 mM MgAc, 15 mM / μL PPDK, 60 μM luciferase, 400 μM luciferin, 80 μM PEP · 3Na, 400 μM so that the enzyme for light emission and dNTP-degrading enzyme are filled. AMP, 1 nL / μL BSA, 200 μM DTT, 1.2 mU / μL apyrase were used.

  The vertical motor 419 is driven to rotate and lower the stick 405 (607), and the dispensing surface 402 is immersed in the reaction solution for 1 minute (608). By the immersion step, ATP and PPi contained in the primer-ssDNA complex and the polymerase can be decomposed to prevent the background light from rising. Next, the stick 405 was rotated and raised (609), 0.5 μL of dNTP was dropped onto the dispensing surface 402, and left for 6 seconds for the complementary strand synthesis reaction (610). The amount of about 0.5 μL depends on the rotational speed of the stick 405, but the dNTP solution does not spill from the dispensing surface 402 even if the stick 405 is rotated by the surface tension of the liquid. The dispensing amount need not be limited to 0.5 μL, but it should be less than or equal to the amount that does not spill during the rotational movement of the stick 405. After the stick is allowed to stand for 6 seconds, the stick 405 is rotated and lowered (611), and the dispensing surface 402 is immersed in the reaction solution (612). Immediately after the dispensing surface 402 is immersed in the reaction solution, the motor which is the stirring means 104 is driven to stir the reaction solution and measure luminescence (613). At this time, a luminescence reaction by PPDK and luciferase and an excessive dNTP decomposition reaction by apyrase are performed. The presence / absence and intensity of the luminescence are confirmed, and it is determined whether complementary strand synthesis has been performed by the dispensed dNTPs and how many bases have been synthesized. After stirring is performed for 114 seconds, the stirring operation is stopped, the stick 405 is rotated and raised (614), the dispensing surface 402 is disposed immediately below the dispenser 303, and the next dNTP is dispensed. The base sequence to be measured can be determined by repeating the 610 to 614 operations by repeating the dNTP dispensing (610) to the dispensing surface 402 and the rotation / lifting operation (614) of the stick 405.

  In this example, DNA was immobilized on the holding member using a biotin-avidin bond, but the immobilization method is not limited thereto. For example, a method may be used in which biotin is labeled on the polymerase, the biotin-labeled polymerase is fixed to a holding member, and a primer-ssDNA complex is bound to the biotin-labeled polymerase.

  In the complementary strand synthesis reaction of DNA, the efficiency of the complementary strand synthesis reaction may be significantly reduced due to higher-order structure formation such as self-hybridization of the primer-ssDNA complex. In order to solve the above problem, a temperature adjusting (30 ° C. to 40 ° C.) mechanism is provided on the dispensing surface 402 of the stick 405, SSB (single-strand binding protein) or DMSO as a denaturing agent may be added to the reaction solution. good.

  In this embodiment, the base sequence to be measured by the pyrosequencing method is determined by using magnetic beads as the holding member. The reaction process other than the stick and the apparatus configuration other than the stick holder and the method for fixing the DNA strand to the holding member is the same as in Example 1.

  FIG. 9 shows a cross-sectional view of a columnar stick and a stick holder for fixing magnetic beads on which DNA strands are fixed. The stick 405 has a cylindrical shape with a diameter of 4 mm and a length of 15 mm, with one end face closed. Although a cylindrical configuration is shown here, the present invention is not limited to this. The material used was PEEK. The diameter, length, and material of the stick 405 are not limited to this. However, regarding the material, if all magnetic material is used, the magnetic beads are fixed to the entire stick 405, so that a non-magnetic material is preferable. Only one end face that closes the cylinder may be a magnetic material, and the other surface may be a non-magnetic material.

  The magnetic shield sheet 426 was covered on the inner side surface of the stick 405 so that the magnetic beads were not fixed to the side surface of the stick 405. Further, a helicopter 447 is provided around the dispensing surface 402 so that the magnetic beads do not move to the side surface of the stick 405 by stirring the reaction solution when the dispensing surface is present in the stirring reaction solution. gave. In addition to the above method, a method using a magnetic circuit (FIG. 18) is also conceivable as a measure for preventing the magnetic beads from moving to the stick side. In place of the magnet 423 of FIG. 9, a magnetic body (for example, 78 permalloy) cap 450 having a closed cylindrical structure with a magnet 449 fixed to the inner bottom surface is used, so that the magnetic field is the same as that of FIG. It is formed on the upper surface of the magnet 449 and is not formed on the side surface of the magnetic body cap 450. Therefore, even without the magnetic shield sheet 426 and the helicopter 447, the magnetic beads are stably fixed only to the dispensing surface of the stick 405.

  As in the first embodiment, the stick 405 and the stick holder 406 are configured to be detachable using the ball plunger 425 and the stick groove 408 provided on the side surface of the stick 405. The stick holder 406 is provided with a magnet fixing pin 424 having a magnet 423 fixed at the tip so that the stick holder 406 is included in the cylinder of the stick 205. By pressing a surface of the magnet fixing pin 424 different from the surface on which the magnet 423 is fixed with the spring 427, the magnet 423 always presses the inner bottom surface 430 of the stick, and the magnetic field strength of the outer bottom surface of the stick (that is, the dispensing surface 402) is reproduced. It was set as the structure from which a property is acquired. Further, a protrusion 428 is formed at one end of the magnet fixing pin 424, and the protrusion 428 interferes with the receiving surface 429 inside the stick holder 406, so that the magnet fixing pin 424 does not come off the stick holder 406.

  A method for fixing the DNA strand to be measured to the magnetic beads and a method for fixing the DNA-fixed magnetic beads to the stick 405 will be described. The DNA to be measured was labeled with biotin 602 at the 5 ′ end using PCR, and the biotin 602 labeled DNA was immobilized on magnetic beads 605 (DynaBeads (R) M-280 Streptavidin, Invitrogen) labeled with avidin 604 (FIG. 10 (a)). The magnetic beads 605 are 2.8 μm in diameter and hydrophobic beads, but other beads may be used. In addition, although biotin-avidin bond is used as a means for fixing the DNA strand and the magnetic beads, it may be fixed by other methods. Next, 0.2N NaOH is added to the magnetic beads 605 on which the DNA strands are fixed, and alkali denaturation is performed. After the alkali denaturation, the ssDNA 601 fixed to the magnetic beads 605 is prepared by separating the magnetic beads fixed chains and the free chains contained in the supernatant using a magnetic stand. A 4-fold amount (2 pmol) of primer 603 is added to ssDNA 601 (0.5 pmol) immobilized on magnetic beads 605, and primer 603 is hybridized to ssDNA 601 to prepare a primer-ssDNA complex. After the hybridization, a polymerase (Klenow) is added to the solution containing the magnetic beads 605 and allowed to stand for 5 minutes to bind the polymerase to the primer-ssDNA complex. The solution containing the magnetic beads 605 is dropped onto the dispensing surface 402 of the stick 405 attached to the stick holder 406, and the magnetic beads are fixed to the dispensing surface 402 (FIG. 10B). After the fixing operation, the excess solution is removed and attached to the apparatus, and a sequence process equivalent to that in Example 1 is performed.

  When only one magnet shown in FIG. 9 is used, the magnetic beads form a lump at the center of the dispensing surface 402. Depending on the amount of the magnetic beads, the ratio of the height to the area of the fixed region of the magnetic beads may increase, and the penetration of dNTP into the center of the magnetic bead mass may be slow. In such a case, the efficiency of the complementary strand synthesis reaction of the DNA strand at the center of the magnetic bead block is reduced, leading to a decrease in sequence analysis accuracy due to non-uniformity of the complementary strand synthesis reaction between the DNA strands. As means for preventing the above problem, it is conceivable to use a ring magnet 432 as shown in FIG. As a result, the magnetic beads are dispersed and fixed on the outer periphery of the dispensing surface 402, the ratio of the height to the area of the fixed region of the magnetic beads is reduced, and dNTPs can penetrate into the magnetic beads more quickly and evenly. The heterogeneity of complementary strand synthesis can be suppressed. The ring shape is preferably along the inner side surface of the stick 205. In addition to the above method, it is also conceivable to improve the efficiency of dNTP penetration into the magnetic beads by stirring the magnetic beads fixed on the dispensing surface.

  FIG. 12 shows a sectional view of a magnet rotating type stick and a stick holder. The configuration other than the magnet, the magnet fixing pin, and the spring is the same as that shown in FIG. A small motor 435 is built in the stick holder 436, a shaft 434 is connected to the shaft of the motor 435, and a small magnet 433 is fixed to the tip of the shaft 434. The small magnet 433 is sized to have a diameter that is at least smaller than the inner diameter of the stick 405. And as a site | part fixed, it is good to make it the small magnet 433 move away from the center axis | shaft of a stick. When the stick 405 is attached to the stick holder 436, the shaft 434 and the magnet 433 are disposed in the stick 405. By rotating the motor 435 after dNTP dispensing, the magnet 433 rotates inside the stick 405, and the magnetic beads fixed to the dispensing surface 402 also rotate. The efficiency of permeation of dNTPs into the magnetic beads can be improved by stirring by rotating the magnetic beads. A method is also conceivable in which dNTPs easily penetrate into the center of the magnetic bead mass by loosening the magnetic field strength to loosen the degree of accumulation (fixed degree) of the magnetic beads that are firmly accumulated.

  FIG. 13 shows a magnet moving type stick and a cross-sectional view of the stick. The configuration other than the magnet, the magnet fixing pin, and the spring is the same as that shown in FIG. The stick holder 440 is provided with a shaft 439. A spring 438 is connected to the tip of the shaft 439, and a magnet 437 is fixed to one end of the spring 438. When the stick 405 is attached to the stick holder 440, the shaft 439, the spring 438, and the magnet 437 are configured to enter the stick 405. The extension direction of the spring 438 is the central axis direction of the stick 405. When the dispensing surface 402 faces upward, the spring 438 is contracted by the weight of the magnet 437 and the magnet 437 is separated from the dispensing surface 402. When the magnet 437 is separated from the dispensing surface 402, the magnetic field strength is weakened, the degree of magnetic bead accumulation is reduced, and dNTPs easily penetrate into the center of the magnetic bead block. When the dispensing surface 402 is directed downward, the magnet 437 is pressed against the bottom surface of the stick by the spring 438, so that the magnetic beads are fixed to the dispensing surface 402 and the magnetic beads are prevented from falling into the reaction vessel 101. In addition to the above method, there is a method of using an electromagnet as a method of weakening the magnetic field strength during dNTP dispensing.

  FIG. 14 shows a cross-sectional view of an electromagnetic stick and a stick holder. The configuration other than the magnet, the magnet fixing pin, and the spring is the same as that shown in FIG. The stick holder 440 is provided with a magnetic shaft 441 (here, an iron shaft), and an electric wire with an insulating coating (here, nichrome wire 442) is wound around the magnetic shaft 441. The nichrome wire 442 is connected to a power source whose ON / OFF is controlled by the H8 microcomputer 501. When dNTP is dispensed onto the dispensing surface 402 of the stick 450, the power is turned off so that no magnetic field is generated from the magnetic material shaft 441. By the above operation, the magnetic field strength is weakened, the degree of magnetic bead accumulation is relaxed, and dNTP easily penetrates into the center of the magnetic bead block. When the stick 405 is rotated or lowered, the power is turned on, a magnetic field is generated from the magnetic shaft 441, and the magnetic beads are fixed to the dispensing surface 402. The operation prevents the magnetic beads from diffusing into the reaction solution when the dispensing surface 402 is immersed in the reaction solution. Further, when the dispensing surface 402 is immersed in the reaction solution, the power is turned off to diffuse the magnetic beads into the reaction solution, and the reaction solution of PPi generated by complementary strand synthesis or excess dNTP The diffusion efficiency may be improved. In this case, when the dispensing surface 402 is taken out from the reaction solution, it is necessary to turn on the power and re-fix the magnetic beads dispersed in the reaction solution to the dispensing surface 402.

  As described above, when the present invention is used, the fixing pin arrangement on the back surface of the dispensing surface does not interfere with the dispenser. Therefore, in addition to suppressing the vulnerability of the fixing pin and improving the ease of installation. Various shapes of magnets can be used, and the reaction efficiency can be improved.

  In this example, the base sequence to be measured by the pyrosequencing method is determined using sepharose beads as the holding member. The reaction process other than the stick and the apparatus configuration other than the stick holder and the method for fixing the DNA strand to the holding member is the same as in Example 1.

FIG. 15 shows a cross-sectional view of a stick and a stick holder used when Sepharose beads are used as a holding member. Sepharose beads 444 are introduced into one end face of the stick 443 and confined with a filter cap 445 made of a membrane filter. Sepharose beads can be easily introduced when they have recesses as shown in FIG. As the Sepharose beads 444, streptavidin-coated beads having an average particle size of 34 μm (Streptavidin Sepharose High Performance Lab Pack and Hitrap Streptavidin HP, GE Healthcare) were used. As a membrane filter of the filter cap 445, a mesh size of 12 μm, manufactured by polycarbonate (ISOPORE ^™ MEMBRANE FILTERS, MILLIPORE) was used. Since the mesh size of the membrane filter is smaller than the size of the Sepharose beads 444, the dNTP solution penetrates into the filter cap 445, but the Sepharose beads 444 cannot move outside the filter cap 445. As the membrane filter, one having a large number of holes may be used, and at that time, the size of the holes may be smaller than the size of the beads.

  A method for fixing DNA to Sepharose beads 444 will be described. Sepharose beads 444 are washed with a buffer using a column, 5′-end biotin-labeled double-stranded DNA (dsDNA) is added to Sepharose beads 444, and dsDNA is immobilized on Sepharose beads 444. The Sepharose beads 444 are then washed with buffer using a column to wash away unbound dsDNA. 0.2N NaOH is added to denature, and the Sepharose beads 444 are washed with a buffer to produce ssDNA fixed to the Sepharose beads 444. Sepharose beads 444 with the ssDNA obtained in the above-described steps are introduced into a stick 443 and a filter cap 445 is attached to the tip of the stick 443. Thereafter, the stick 443 is attached to the stick holder 446, the stick holder 446 is attached to the apparatus, and a sequence process equivalent to that in the first embodiment is performed.

  In this embodiment, the position of the stick dispensing surface is controlled only by rotational driving, and the base sequence to be measured by the pyrosequencing method is determined. The apparatus configuration and reaction process other than the rotation mechanism and reaction container are the same as those in the first embodiment.

  FIG. 16 shows an overview of the analyzer used in this embodiment. In this embodiment, a reaction plate 107 is used in place of the reaction vessel constituting the reaction storage unit. Although stainless steel is used as the material of the reaction plate 107, it is not limited thereto. Further, the surface of the reaction plate 107 is subjected to a hydrophobic treatment. The reaction plate 107 is provided with a detection window 108 made of an optically transparent material. Quartz glass is used as the material of the detection window 108, but not limited as long as it is optically transparent. The upper end surface of the detection window 108 in FIG. 16 is subjected to hydrophilic treatment. Therefore, when a reaction solution equivalent to that in Example 1 is dropped onto the reaction plate 107, a reaction droplet 109 is formed around the detection window 108. By using an optically transparent material for the reaction plate 107, the detection window 108 may not be used. In this case, it is desirable to perform hydrophilic treatment only at the predetermined position and perform hydrophobic treatment on the other surfaces so that the reaction droplet 109 is maintained at the predetermined position. The hydrophilic treatment and the hydrophobic treatment may be performed so that the hydrophilic treatment portion has a higher hydrophilicity with respect to the reaction solution than the hydrophobic treatment portion.

  A temperature control means 105 is connected to the reaction plate 107, and the temperature of the reaction droplet can be adjusted via the reaction plate. Although the Peltier is used as the temperature control means 105, it is not limited thereto. The reaction plate 107 was disposed on the glass substrate 202 so that the detector 201 was positioned immediately below the detection window 108.

  Similar to the first embodiment, the rotation mechanism unit includes a stick 405, a stick holder 406, and a rotation drive unit 407, and is arranged between the reagent dispensing unit and the reaction storage unit. The stick 405 and the stick holder 406 are the same as those in the first embodiment. Also, the method of attaching the stick 405 and the stick holder 406 to the rotation drive unit 407 is the same as that of the first embodiment. The rotation drive unit 407 includes a holder fixing plate 409, an arm 410, and a motor 448. The holder fixing plate 409 is connected to the arm 410 so as to be rotatable about the rotation shaft 415, and a motor 448 is connected to one end of the rotation shaft 415. The motor 448 is connected to the H8 microcomputer 501 and controls the rotational movement of the rotation drive unit 407 via the motor 448.

  FIG. 17 shows a driving process of the rotation mechanism unit 407. The dNTP accommodated in the reservoir 301 is dispensed onto the dispensing surface 402 of the stick 405, and left still for several seconds for the extension reaction (FIG. 17 (a)). Next, the stick holder 406 is rotated by the motor 448 via the holder fixing plate 409 about the rotation shaft 415. By this operation, the dispensing surface 402 of the stick 405 rotates 180 degrees from the time when dNTP is dispensed, and the dispensing surface 402 contacts the reaction droplet 109 on the reaction plate 107 (FIG. 17B). In order to efficiently diffuse PPi and excess dNTP generated by the complementary strand synthesis reaction into the reaction droplet 109, the stick 405 is moved by the motor 448 when the dispensing surface 402 is brought into contact with the reaction droplet 109 (see FIG. 17). In b), it was rotated slightly from side to side. The reaction plate 107 may be vibrated to efficiently diffuse PPi generated by the complementary strand synthesis reaction and excess dNTP into the reaction droplet 109. When dispensing dNTPs onto the dispensing surface 402, the operation is the reverse of the above process.

  By the motor 448, the stick holder 406 is rotated 180 degrees around the rotation shaft 415 so that dNTP is dispensed. At this time, the distance between the dispensing surface 402 and the capillary 302dNTP discharge end of the dispenser 303 was set to 1 mm. Pyrosequence reaction is performed by repeating the above process.

101: Reaction vessel, 102: Reaction liquid, 103: Groove, 104: Stirring means, 105: Temperature control means, 106: Reaction container, 107: Reaction plate, 108: Detection window, 109: Reaction droplet, 201: Light Detector: 202: Glass substrate, 230: Amplifier, 204: Multiplexer, 205: Conductive housing, 301: Reservoir, 302: Capillary, 303: Dispenser, 304: Through hole, 305: Dispenser holder, 306: Pressure piping block, 307: flow path, 308: piping tube, 309: solenoid valve, 401: holding member, 402: dispensing surface, 403: fixed pin, 404: fixed pin, 405: stick, 406: stick holder, 407 : Rotation drive unit, 408: Stick groove, 409: Holder fixing plate, 410: Arm, 411: Guide pin, 412: Magnet, 13: Eccentric cam 414: Stopper 415: Rotating shaft 416: Linear guide 417: Pinion 418: Rack 419: Vertical motor 420: Membrane 421: Pin 422: Pin holder 423: Magnet 424: Magnet fixing pin, 425: Ball plunger, 426: Magnetic shield, 427: Spring, 428: Projection, 429: Receiving surface, 430: Bottom surface of stick, 432: Ring type magnet, 433: Magnet, 434: Shaft, 435: Motor 436: Stick holder, 437: Magnet, 438: Spring, 439: Shaft, 440: Stick holder, 441: Magnetic material shaft, 442: Nichrome wire, 443: Stick, 444: Sepharose beads, 445: Filter cap, 446: Stick holder, 447: Helicopter 448: motor 449: magnet 450: magnetic cap, 501: H8 microcontroller 502: a pressure source, 601: ssDNA, 602: biotin, 603: primer, 604: avidin, 605: magnetic beads

Claims (17)

A sample holding member comprising a sample holding portion for a nucleic acid sample;
A dispenser for supplying a complementary strand synthesis substrate that performs a complementary strand synthesis reaction with the nucleic acid sample, to the sample holder;
A reaction unit that holds a reaction solution including a luminescent enzyme that immerses the sample holding unit and reacts with a product of the complementary strand synthesis reaction to emit light; and a degrading enzyme of the complementary strand synthesis substrate;
A photodetector for detecting luminescence by the luminescent enzyme;
A rotation mechanism that supports the sample holding member, is provided between the dispenser and the reaction unit, and rotates so that a surface of the sample holding unit facing the dispenser faces the reaction unit. When,
A nucleic acid analyzer comprising: a controller that controls dispensing by the dispenser and rotation by the rotation mechanism.   The nucleic acid analyzer according to claim 1, further comprising an up / down mechanism that moves the sample holding member so as to control a relative distance between the sample holding unit and the reaction unit.   The nucleic acid analyzer according to claim 2, wherein the rotation mechanism includes a rotation member that rotates as the sample holding member moves by the vertical mechanism.   The nucleic acid analyzer according to claim 1, wherein the sample holding member has a columnar structure having the sample holding portion.   The nucleic acid analyzer according to claim 1, wherein the sample holding member is a member to which a membrane as the sample holding unit is fixed.   2. The nucleic acid analyzer according to claim 1, further comprising a holder for fitting the sample holding member, wherein the sample holding member is supported by the rotating mechanism via the holder.   The nucleic acid analyzer according to claim 2, wherein the reaction unit includes a container that holds the reaction solution.   The nucleic acid analyzer according to claim 1, wherein the reaction unit is a plate having a hydrophilic region for holding the reaction solution and a hydrophobic region having a lower hydrophilicity than the hydrophilic region.   5. The nucleic acid analyzer according to claim 4, wherein the sample holding member includes a cylindrical member having the sample holding portion as a bottom surface, and a magnetic body inside the cylindrical member.   The nucleic acid analyzer according to claim 9, wherein an inner surface of the cylindrical member includes a magnetic shield.   The nucleic acid analyzer according to claim 9, wherein the magnetic body is a ring-shaped magnet along an inner surface of the cylindrical member.   The magnetic body is a magnet having a diameter smaller than an inner diameter of the cylindrical member, and is fixed by a fixing member so that a central axis of the magnet is different from a central axis of the cylindrical member. The nucleic acid analyzer according to claim 9, further comprising a rotation mechanism that rotates along the inner periphery of the cylindrical member.   The cylindrical member includes a spring that expands and contracts in a direction of a central axis of the cylindrical member, and the magnetic body is a magnet placed between the sample holder and the spring. The nucleic acid analyzer according to claim 9.   The magnetic body is an electromagnet wound with an electric wire, and the control unit turns off the power when the sample holding unit faces the dispenser, and when the sample holding unit faces the reaction unit. The nucleic acid analyzer according to claim 9, wherein the nucleic acid analyzer is controlled to be turned on.   2. The nucleic acid analyzer according to claim 1, wherein the sample holding unit includes a recess that holds a bead on which a sample to be measured is fixed, and a membrane that covers the recess and has a smaller hole than the bead. . An analysis method using a sample holding member in which a nucleic acid sample to which a polymerase is bound is fixed to a sample holding part,
Facing the sample holder to a dispenser that supplies a complementary strand synthesis substrate that undergoes a complementary strand synthesis reaction with the nucleic acid sample;
Supplying a complementary strand synthesis substrate from the dispenser to the sample holder;
A reaction part for holding a reaction solution containing a luminescent enzyme that emits light by reacting with a product of a complementary strand synthesis reaction, and a surface of the sample holding part facing the dispenser; and a decomposing enzyme of the complementary strand synthesis substrate; Rotating the sample holding member to face each other;
Immersing the sample holder in the reaction solution;
A nucleic acid analysis method comprising: detecting luminescence by the luminescent enzyme. The nucleic acid analysis according to claim 16, further comprising a step of immersing the sample holder in the reaction solution before the step of supplying a complementary strand synthesis substrate from the dispenser to the sample holder. Method.

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