WO2012017629A1 - Analyseur d'acide nucléique et procédé d'analyse d'acide nucléique - Google Patents

Analyseur d'acide nucléique et procédé d'analyse d'acide nucléique Download PDF

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Publication number
WO2012017629A1
WO2012017629A1 PCT/JP2011/004307 JP2011004307W WO2012017629A1 WO 2012017629 A1 WO2012017629 A1 WO 2012017629A1 JP 2011004307 W JP2011004307 W JP 2011004307W WO 2012017629 A1 WO2012017629 A1 WO 2012017629A1
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Prior art keywords
nucleic acid
reaction
sample
sample holding
stick
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PCT/JP2011/004307
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English (en)
Japanese (ja)
Inventor
友幸 坂井
神原　秀記
白井　正敬
徹 土生
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株式会社日立製作所
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0241—Drop counters; Drop formers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples

Definitions

  • the present invention relates to an apparatus for analyzing nucleic acids (DNA, RNA) using chemiluminescence.
  • 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.
  • a complementary strand synthase such as polymerase
  • ATP sulfurylase In addition to polymerase, ATP sulfurylase, adenosine 5 'phosphosulfate (APS), luciferase, luciferin, and dNTP-degrading enzyme (apyrase) are present in the reaction solution.
  • APS adenosine 5 'phosphosulfate
  • luciferase luciferin
  • luciferin luciferin
  • dNTP-degrading enzyme apyrase
  • apyrase dNTP-degrading enzyme
  • the dNTP that has not undergone complementary strand synthesis is degraded by an apyrase contained in the reaction solution.
  • the base sequence of the complementary strand can be determined.
  • 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 dATP ⁇ S, 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.
  • AMP adenosine monophosphate
  • PEP phosphoenolpyruvate
  • PPDK pyruvate phosphate dikinase
  • 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).
  • 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.
  • 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.
  • a reagent for example, dNTP
  • 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
  • the complementary strand synthesis reaction and dNTP decomposition reaction / luminescence reaction are spatially separated.
  • 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 ⁇ L corresponding to four types of dNTPs, and a capillary 302 is provided below each reservoir 301.
  • the length of the fixing pin 403 is 20 mm or more. Necessary.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the holding member that holds the sample holding portion does not interfere with the dispenser, and the sample holding member can be made strong.
  • 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.
  • 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 liquid 102 after the reagent is dispensed.
  • 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).
  • 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.
  • 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.
  • the fixing pin 404 may be rotated only without moving up and down.
  • the thickness of the fixing pin can be equal to or larger than that of the holding member.
  • the holding member / fixing pin can be easily attached.
  • the base sequence to be measured is determined using the pyrosequencing method.
  • the principle of reaction and sequencing described in Patent Document 2 was adopted.
  • FIG. 3 shows a schematic diagram of the reaction mode.
  • the target DNA whose sequence is to be determined is made into a single strand (ssDNA)
  • the primer is hybridized
  • DNA polymerase is added
  • four types of dNTPs dATP, dCTP, dGTP, dTTP
  • a complementary strand synthesis reaction is performed, PPi generated 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.
  • 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.
  • reaction container 106 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.
  • the reaction storage unit 106 may have a shape in which a commercially available reaction container (for example, Nunc-Immuno TM Modules; CAT No. 473539) and a holder for fixing the same are combined.
  • the pyrosequencing reaction may be performed at a temperature higher than room temperature (28 ° C. to 35 ° C.).
  • a temperature control means 105 is connected to the reaction storage unit 106 in order to efficiently control the temperature of the reaction solution.
  • a Peltier element is used as the temperature control means 105, but a heater or the like may be used.
  • a stirring means 104 is connected to the reaction storage unit 106.
  • a motor having an eccentric cam on the rotation 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 so that the reaction accommodating portion 106 is rotated. 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 photodetection unit is entirely covered with a conductive casing 205 made of a conductive material.
  • a photo detector 201 is accommodated in the conductive housing 205, and the photo detectors 201 are arranged in a 1: 1 relationship below the reaction vessels 101.
  • a photodiode Si photodiode S1133-01, Hamamatsu Photonics
  • the 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 optical detector 201 is amplified by an amplifier 203 (opA129UB, Texas Instruments) built 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 ⁇ L of reagents (here, 4 types of dNTPs), and a capillary 302 (outer diameter 370 ⁇ m, inner diameter 50 ⁇ m) is provided at the bottom of each reservoir 301. .
  • 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.
  • 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.
  • 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.
  • 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 arranged between the reagent dispensing unit and the reaction storage unit.
  • a rod-like structure 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.
  • 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.
  • 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.
  • 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.
  • the stick holder 406 rotates around the rotation shaft 415 (FIG. 6B).
  • 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.
  • the rotation of the stick holder 406 and the stick 405 is completely stopped.
  • the dispensing surface 402 of the stick 405 is rotated 180 degrees from the time when the dNTP is dispensed.
  • 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)).
  • 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.
  • the holding member for fixing the DNA strand and the fixing pin for fixing the holding member are an integrated stick, but the holding member and the fixing pin may be divided and removed.
  • FIG. 7 An example in which a membrane is used as a holding member is shown (FIG. 7).
  • 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.
  • 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.
  • 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.
  • ssDNA single-stranded DNA
  • primer 601
  • the primer-ssDNA complex is dropped onto the dispensing surface 402 and allowed to stand for 5 minutes, and the primer-ssDNA complex is fixed 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).
  • 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.
  • the composition of the reaction solution is, for example, 60 mM Tricine, 2 mM EDTA, 20 mM MgAc, 15 mM / ⁇ L PPDK, 60 ⁇ M luciferase, 400 ⁇ M luciferin, 80 ⁇ M PEP ⁇ 3Na, 400 ⁇ M so that the enzyme for light emission and dNTP-degrading enzyme are filled.
  • AMP 1 nL / ⁇ L BSA, 200 ⁇ M DTT, 1.2 mU / ⁇ L 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).
  • ATP and PPi contained in the primer-ssDNA complex and polymerase can be decomposed to prevent an increase in background light.
  • the stick 405 was rotated and raised (609), 0.5 ⁇ L 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 ⁇ L 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 ⁇ L, but it should be less than or equal to the amount that does not spill during the rotational movement of the stick 405.
  • the stick 405 is rotated and lowered (611), and the dispensing surface 402 is immersed in the reaction solution (612).
  • 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.
  • 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.
  • DNA was immobilized on the holding member using a biotin-avidin bond
  • the immobilization method is not limited thereto.
  • a method may be used in which biotin is labeled on a polymerase, the biotin-labeled polymerase is fixed to a holding member, and a primer-ssDNA complex is bound to the biotin-labeled polymerase.
  • the efficiency of the complementary strand synthesis reaction may be significantly reduced due to the formation of higher order structures such as self-hybridization of the primer-ssDNA complex.
  • a temperature adjustment (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.
  • the base sequence to be measured by the pyrosequencing method is determined 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.
  • 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 surface. In place of the magnet 423 of FIG.
  • 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.
  • 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, as in the first embodiment.
  • 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.
  • the magnet 423 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.
  • 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® M-280 Streptavidin, invitrogen) labeled with avidin 604 (FIG. 10 (a)).
  • the magnetic beads 605 are 2.8 ⁇ m in diameter and hydrophobic beads, but other beads may be used.
  • the biotin-avidin bond is used as a means for fixing the DNA strand and the magnetic beads, but it may be fixed by other methods.
  • 0.2N NaOH is added to the magnetic beads 605 on which the DNA strands are fixed to denature with alkali.
  • 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.
  • a polymerase Klenow
  • 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.
  • the magnetic beads form a lump in the center of the dispensing surface 402.
  • 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.
  • 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.
  • a ring magnet 432 as shown in FIG.
  • 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 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
  • the shaft 434 and the magnet 433 are disposed in the stick 405.
  • 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 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.
  • the extension direction of the spring 438 is the central axis direction of the stick 405.
  • the spring 438 is contracted by the weight of the magnet 437 and 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.
  • 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.
  • 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.
  • the power is turned off so that no magnetic field is generated from the magnetic material shaft 441.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 beads having a mean particle size of 34 ⁇ m coated with streptavidin (Streptavidin Sepharose High Performance Lab Pack and Hitrap Streptavidin HP, GE Healthcare) were used.
  • a mesh size of 12 ⁇ m, manufactured by polycarbonate (ISOPORE TM 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.
  • a membrane filter having a large number of holes may be used, and at that time, a pore size smaller than that of the beads is used.
  • 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 prepare 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.
  • the position of the stick dispensing surface is controlled only by rotational drive, 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 example.
  • a reaction plate 107 is used in place of the reaction vessel constituting the reaction storage unit.
  • stainless steel is used as the material of the reaction plate 107, it is not limited thereto.
  • 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.
  • a reaction droplet 109 is formed around the detection window 108.
  • the detection window 108 may not be used.
  • 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.
  • the 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.
  • 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.
  • the rotation mechanism unit is composed of a stick 405, a stick holder 406, and a rotation drive unit 407 as in the first embodiment, 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.
  • 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)).
  • the stick holder 406 is rotated by the motor 448 via the holder fixing plate 409 about the rotation shaft 415.
  • 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).
  • the stick 405 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.
  • dispensing dNTPs onto the dispensing surface 402 the operation is the reverse of the above process.
  • the stick holder 406 is rotated 180 degrees around the rotating shaft 415 by the motor 448 so that the 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.

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  • Physics & Mathematics (AREA)
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  • Apparatus Associated With Microorganisms And Enzymes (AREA)
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Abstract

La présente invention concerne un dispositif pour analyser un acide nucléique via la détection optique de l'extension d'acide nucléique, comprenant : un distributeur pour alimenter un substrat pour la synthèse de brin complémentaire dans un échantillon d'acide nucléique ; une partie de réaction contenant une solution de réaction contenant une luciférase, qui réagit avec un produit formé par la synthèse de brin complémentaire et induit une émission de lumière, et une enzyme digérant le substrat pour la synthèse de brin complémentaire ; et un mécanisme tournant qui est positionné entre le distributeur et la partie de réaction et fait tourner un porte-échantillon de sorte que le plan du porte-échantillon faisant face au distributeur tourne de manière à faire face à la partie de réaction.
PCT/JP2011/004307 2010-08-06 2011-07-29 Analyseur d'acide nucléique et procédé d'analyse d'acide nucléique WO2012017629A1 (fr)

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JP2010176918A JP5271980B2 (ja) 2010-08-06 2010-08-06 核酸分析装置及び核酸分析方法

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Cited By (6)

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IT201800006621A1 (it) * 2018-06-25 2019-12-25 Dispositivo robotizzato per il carico e la movimentazione di una pluralita' di elementi di ricopertura utilizzabili in un processo di estrazione di molecole, in particolare acidi nucleici
WO2021046036A1 (fr) * 2019-09-04 2021-03-11 Illumina, Inc. Enceintes et joints magnétiques correspondants
NL2024325B1 (en) * 2019-09-04 2021-04-13 Illumina Inc Enclosures and corresponding magnetic joints
US11446662B2 (en) * 2016-03-28 2022-09-20 Hitachi, Ltd. System for capturing single cell-derived biomolecules
JP7343124B2 (ja) 2018-03-02 2023-09-12 プソマーゲン， インコーポレイテッド 磁場及びデバイスを使用したハイスループットな粒子ハンドリングのための方法及びシステム
RU2813898C1 (ru) * 2019-09-04 2024-02-19 Иллумина, Инк. Корпуса и соответствующие магнитные соединения

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JP2005052148A (ja) * 2004-10-05 2005-03-03 Hitachi Ltd 発光検出装置
JP2009247297A (ja) * 2008-04-08 2009-10-29 Hitachi Ltd Ｄｎａ分析装置

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Publication number Priority date Publication date Assignee Title
JP2005052148A (ja) * 2004-10-05 2005-03-03 Hitachi Ltd 発光検出装置
JP2009247297A (ja) * 2008-04-08 2009-10-29 Hitachi Ltd Ｄｎａ分析装置

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11446662B2 (en) * 2016-03-28 2022-09-20 Hitachi, Ltd. System for capturing single cell-derived biomolecules
JP7343124B2 (ja) 2018-03-02 2023-09-12 プソマーゲン， インコーポレイテッド 磁場及びデバイスを使用したハイスループットな粒子ハンドリングのための方法及びシステム
IT201800006621A1 (it) * 2018-06-25 2019-12-25 Dispositivo robotizzato per il carico e la movimentazione di una pluralita' di elementi di ricopertura utilizzabili in un processo di estrazione di molecole, in particolare acidi nucleici
EP3588094A1 (fr) * 2018-06-25 2020-01-01 MASMEC S.p.A. Dispositif robotisé pour le chargement et le traitement d'une pluralité d'éléments de revêtement utilisable dans un processus d'extraction moléculaire, en particulier d'acides nucléiques
WO2021046036A1 (fr) * 2019-09-04 2021-03-11 Illumina, Inc. Enceintes et joints magnétiques correspondants
NL2024325B1 (en) * 2019-09-04 2021-04-13 Illumina Inc Enclosures and corresponding magnetic joints
US11160199B2 (en) 2019-09-04 2021-10-26 Illumina, Inc. Enclosures and corresponding magnetic joints
TWI764247B (zh) * 2019-09-04 2022-05-11 美商伊路米納有限公司 外殼與相應的磁性接頭及相關之方法與系統
US11647617B2 (en) 2019-09-04 2023-05-09 Illumina, Inc. Enclosures and corresponding magnetic joints
RU2813898C1 (ru) * 2019-09-04 2024-02-19 Иллумина, Инк. Корпуса и соответствующие магнитные соединения

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