JP4693851B2 - Base sequence determination method and base sequence determination system - Google Patents

Description

  The present invention relates to a base sequence determination method for determining the base sequence of a nucleic acid, and a base sequence determination system for realizing the base sequence determination method.

  The human genome is composed of about 3 billion genetic codes (bases). The ongoing "Human Genome Project" will unravel all of this genetic code (base sequence). In these circumstances, it has become clear that there are many genetic code (base sequence) differences among individuals. Differences in nucleotide sequences (polymorphisms) in the human genome are single nucleotide polymorphisms (SNPs) in which one base is replaced by another base, deletions or insertions ranging from one base to several thousand bases, Although it is classified into the difference in the number of repetitions (VNTR, microsatellite polymorphism), at present, single nucleotide polymorphism (SNP) is attracting attention among these polymorphisms. A single nucleotide polymorphism (SNP) is the difference in a single base in the DNA base sequence, which is the smallest unit of human personality, such as being weak against alcohol and being easy to work with. is there. It is suggested that about 3 million single nucleotide polymorphisms (SNPs) in the human genome of 3 billion base pairs (1 to 500-1000 base pairs) to 10 million, and a specific protein cannot be made. , Making something different from others has brought differences such as individual differences (constitution) and racial differences. In research on individual differences in genes in humans, single nucleotide polymorphisms are analyzed, susceptibility to diseases and response to drugs are investigated, and custom-made medical treatment such as administration of drugs with few side effects that are suitable for humans is possible It is said that analysis of single nucleotide polymorphisms (SNPs) is underway. If it is a plant, it is possible to elucidate the mechanism of resistance to diseases and pests inherent in the plant and enhance its function.

The reason why single nucleotide polymorphisms (SNPs) are attracting attention is the growing interest in the relationship between diseases and SNPs due to the fact that analysis of many SNPs has become possible due to improved analysis techniques. The research has a wide range of purposes, including disease-related genes, analysis of individual differences in drug metabolism, and chronic diseases. About some drug metabolism and lipid metabolism, the relation with SNP has also become clear. It is expected that the relationship between SNPs and these will gradually become clear in the future.
Molecular biological techniques, such as SNP analysis, involve performing a vast number of operations on a very large number of samples. They are often complex and time consuming and generally require high accuracy. Many techniques are limited in their application due to lack of sensitivity, specificity or reproducibility.

For example, problems with sensitivity and specificity have so far limited the practical application of nucleic acid hybridization. “Hybridization” refers to nucleic acid hybridization or nucleic acid hybrid molecule formation, and is used as a method for examining the primary structure of a nucleic acid, that is, the homology of a base sequence, or detecting a nucleic acid having a homologous base sequence. It is done. Single-stranded nucleic acids can form hydrogen bonds between complementary base pairs, that is, between adenine (A) and thymine (T), or between guanine (G) and cytosine (C). The property of forming a double-stranded nucleic acid is used. Nucleic acid hybridization analysis generally involves detecting a very small number of specific target nucleic acids (DNA or RNA) with a probe from a large number of non-target nucleic acids. In order to maintain high specificity, hybridization is usually performed under the most stringent conditions achieved by various combinations of temperature, salt, detergent, solvent, chaotropic agent and denaturing agent. It is associated with the very complexity of the DNA in most samples, particularly human genomic DNA samples. If a sample consists of a vast number of sequences that closely resemble a specific target sequence, even the most unique probe sequence will cause multiple partial hybridizations with non-target sequences. There may also be unfavorable hybridization kinetics between the probe DNA and its specific target (sample DNA). Even under the best conditions, most hybridization reactions are performed with relatively low concentrations of probe DNA and target molecules (sample DNA). In addition, the probe DNA often competes with the complementary strand to the sample DNA. There is also the problem that high levels of non-specific background signals are caused by the affinity of probe DNA for almost any substance. Thus, these problems individually or in combination cause loss of nucleic acid hybridization sensitivity and specificity.
Under such circumstances, the present inventors have already determined, for example, the magnitude of each signal in order to determine SNP (single nucleotide polymorphism) (homo / hetero determination of two types of bases). A method for determining a significant difference by t-test has been proposed (see Patent Document 1). In the method described in Patent Document 1, hetero determination is not performed unless the two types of signal values almost completely match, but this is not possible in actual measurement.

As described above, there has conventionally been an algorithm for determining the base sequence of a nucleic acid, but there has been a problem in determination accuracy.
JP 2004-125777 A

  An object of the present invention is to provide a base sequence determination method and a base sequence determination system that are highly accurate in determination and can be determined in accordance with the actual situation.

  In the first aspect of the present invention, (a) a plurality of first detection electrodes to which each of the first probe DNAs can be fixed is disposed corresponding to the plurality of first detection electrodes. A plurality of first control electrodes to which a first control DNA having a different base sequence can be fixed, a plurality of second control DNAs to which a second probe DNA having a different base sequence from each of the first control electrode, the first probe DNA, and the first control DNA can be fixed An electrode and a plurality of second control electrodes which are arranged corresponding to the plurality of second detection electrodes and to which a second control DNA having a base sequence different from that of the first probe DNA and the second probe DNA can be respectively fixed. A step of injecting a chemical solution containing sample DNA into the chip cartridge; (b) a step of introducing an insertion agent into the chip cartridge; First detection signals from a plurality of first detection electrodes, first negative control signals from a plurality of first control electrodes, and second detection signals from a plurality of second detection electrodes obtained by introducing an intercalating agent Measuring a second negative control signal from each of the plurality of second control electrodes as a current-voltage characteristic of an electrochemical current, and (d) obtaining a slope of a baseline of the current-voltage characteristic, respectively, A step of determining whether each current-voltage characteristic is normal or abnormal based on the slope of the baseline; and (e) each corresponding to the first detection signal, the first negative control signal, the second detection signal, and the second negative control signal. Subtract the respective background currents from the current-voltage characteristics of each to obtain each as a true peak current value And a degree, and summarized in that a nucleotide sequence determination method characterized by identifying the nucleotide sequence of the sample DNA to the true current value.

  In the second aspect of the present invention, (a) a plurality of first detection electrodes to which each of the first probe DNAs can be fixed is arranged corresponding to the plurality of first detection electrodes, and the first probe DNA is a base. A plurality of first control electrodes to which first control DNAs having different sequences can be respectively fixed, and a plurality of second detection electrodes to which second probe DNAs having different base sequences from the first probe DNA and the first control DNA can be respectively fixed. A chip provided with a plurality of second control electrodes arranged corresponding to the plurality of second detection electrodes and capable of fixing a second control DNA having a base sequence different from that of the first probe DNA and the second probe DNA, respectively. A cartridge; and (b) a plurality of first detection electrodes, a plurality of first control electrodes, a plurality of second detection electrodes, and a plurality of second controls. A measurement system for measuring current through the poles, (c) a liquid supply system for supplying a chemical solution to the chip cartridge, (d) a control mechanism for controlling the measurement system and the liquid supply system, and (e) this control. Via a mechanism and a measurement system, a first detection signal from a plurality of first detection electrodes, a first negative control signal from a plurality of first control electrodes, a second detection signal from a plurality of second detection electrodes, Second negative control signals from a plurality of second control electrodes are respectively acquired as current-voltage characteristics, and the slopes of the baselines of the current-voltage characteristics are obtained, and the respective current-voltages are determined by the slopes of the respective baselines. A current waveform determination module that determines normality / abnormality of characteristics and excludes abnormal detection signals from calculation targets; a first detection signal; a first negative control signal; a second detection signal; A computer including a peak current detection module that subtracts each background current from each current-voltage characteristic corresponding to two negative control signals and obtains each as a true peak current value. The gist is that it is an array determination system. The first control DNA and the second control DNA may be the same or different.

  ADVANTAGE OF THE INVENTION According to this invention, the base sequence determination method and base sequence determination system which can perform presence / absence determination of a nucleic acid and SNP homo / hetero type | mold determination with high precision according to the actual condition can be provided.

  Next, embodiments of the present invention will be described with reference to the drawings. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, it should be noted that the drawings are schematic, and the relationship between the thickness and the planar dimensions, the ratio of the thickness of each layer, and the like are different from the actual ones. Therefore, specific thicknesses and dimensions should be determined in consideration of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings. Further, the following embodiments exemplify apparatuses and methods for embodying the technical idea of the present invention, and the technical idea of the present invention includes the material, shape, structure, The layout is not specified as follows. The technical idea of the present invention can be variously modified within the technical scope described in the claims.

(Base sequence determination system)
As shown in FIG. 7, the base sequence determination system according to the embodiment of the present invention includes a chip cartridge 11, a measurement system 12 electrically connected to the chip cartridge 11, and a flow path provided in the chip cartridge 11. And a temperature control mechanism 14 for controlling the temperature of the liquid supply system 13 and the chip cartridge 11 physically connected via the interface unit. As shown in FIG. 1, the measurement system 12 of FIG. 7 feeds back (negative feedback) the voltages of the reference electrodes 561 and 562 with respect to the input of the counter electrode 502, thereby allowing various conditions such as electrodes and solutions in the cell. This is a three-electrode potentiostat that applies a desired voltage to the solution regardless of fluctuations, and is connected to the terminals C, R, and W of the detection chip 21. The detection chip 21 in FIG. 1 is provided in the chip cartridge 11 in FIG.

  As shown in FIG. 2, the detection chip 21 includes a working electrode 551 that is an SNP1 detection electrode, a working electrode 552 that is a SNP2 detection electrode, a working electrode 553 that is a control electrode, and these working electrodes 551, 552, and 553. An electrode unit in which reference electrodes 561 and 562 and a counter electrode 502 are arranged on a detection chip is used. That is, the measurement system 12 changes the voltage of the counter electrode 502 so that the voltages of the reference electrodes 561 and 562 with respect to the working electrodes 551, 552, and 553 are set to certain predetermined characteristics, and is based on the electrochemical reaction of the insertion agent. The current (hereinafter referred to as “electrochemical current”) is measured electrochemically.

  In the base sequence determination method according to the embodiment of the present invention, first, a base sequence complementary to a target base sequence (sample DNA) 581, 582, 583 (see FIGS. 9 to 11) targeted for base sequence determination. Are immobilized on the working electrodes 551 and 552, respectively. That is, as shown in FIG. 3A, the working electrode 551 has a base sequence GACTC... Complementary to the target base sequence (sample DNA) 581 having the base sequence CTGAG. This is an electrode on which a probe DNA 571 having a. In FIG. 3A, the SNP = “G” detection electrode with the base at the SNP position as the third C from the bottom.

  3 (b), the working electrode 552 has a base sequence GAATC, which is complementary to the target base sequence (sample DNA) 582 having the base sequence CTTAG... Shown in FIG. This is an electrode on which a probe DNA 572 having. FIG. 3B also shows an SNP = "T" detection electrode in which the base at the SNP position is the third A from the bottom.

  On the other hand, as shown in FIG. 3 (c), the working electrode 553 is a probe DNA (negative control DNA) having a base sequence CAGTG... That is not complementary to the target base sequences (sample DNA) 581 and 582. ) 573 is a control electrode to be immobilized. These working electrodes 551, 552, and 553 function as electrodes that detect the reaction current in the cell. Note that the types of probe DNAs 571, 572, and 573 immobilized on the working electrodes 551, 552, and 553 are merely examples, but the types of probe DNA immobilized on the working electrodes 551, 552, and 553 are in principle 1 One.

  The counter electrode 502 is an electrode that supplies a current to the cell by applying a predetermined voltage between the working electrodes 551, 552, and 553. The reference electrodes 561 and 562 are electrodes that feed back the electrode voltage to the counter electrode 502 in order to control the voltage between the reference electrodes 561 and 562 and the working electrodes 551, 552, and 553 to a predetermined voltage characteristic. The voltage by the counter electrode 502 is controlled, and the electrochemical current can be detected with high accuracy regardless of various detection conditions in the cell.

As shown in FIG. 1, the measurement system 12 of the base sequence determination system according to the embodiment of the present invention includes a voltage pattern generation circuit 510 that generates a voltage pattern for detecting a current flowing between electrodes. The pattern generation circuit 510 is connected to the inverting input terminal of the inverting amplifier 512 (OP _c ) for reference voltage control of the reference electrodes 561 and 562 through the wiring 512b. The voltage pattern generation circuit 510 is a circuit that generates a voltage pattern by converting a digital signal input from the control mechanism 15 shown in FIG. 7 into an analog signal, and includes a DA converter. A resistor R _s is connected to the wiring 512b. The non-inverting input terminal of the inverting amplifier 512 is grounded, and the wiring 502a is connected to the output terminal. The wiring 512b on the inverting input terminal side of the inverting amplifier 512 and the wiring 502a on the output terminal side are connected by the wiring 512a. The wiring 512a, the protection circuit 500 is provided comprising a feedback resistor R _ff and a switch SW _f. The line 502a is connected to the terminal C of the detection chip 21. The terminal C is connected to the counter electrode 502 on the detection chip 21. When a plurality of counter electrodes 502 are provided, the terminal C is connected in parallel to each. Thereby, a voltage can be simultaneously applied to the plurality of counter electrodes 502 by one voltage pattern. The line 502a, the switch SW ₀ performing on-off control of voltage applied to the terminal C is provided.

  The protection circuit 500 provided in the inverting amplifier 512 is configured such that an excessive voltage is not applied to the counter electrode 502. Therefore, an excessive voltage is applied at the time of measurement, and the solution is electrolyzed, so that stable measurement is possible without affecting the detection of the electrochemical current of the desired intercalating agent.

The terminal R of the detection chip 21 is connected to the non-inverting input terminal of the voltage follower amplifier (OP _r ) 513 by the wiring 503a. The inverting input terminal of the voltage follower amplifier 513 is short-circuited by the wiring 513b and the wiring 513a connected to the output terminal. The wiring 513b is provided with a resistance _{Rf, and} is connected between the resistance of the wiring 512b and the intersection of the wiring 512a and the wiring 512b. As a result, a voltage obtained by feeding back the voltages of the reference electrodes 561 and 562 to the voltage pattern generated by the voltage pattern generation circuit 510 is input to the inverting amplifier 512, and the output of the counter electrode 502 is based on the output obtained by inverting and amplifying such a voltage. Control the voltage.

The terminal W of the detection chip 21 is connected to the inverting input terminal of the trans-impedance amplifier (OP _w ) 511 through the wiring 501a. The non-inverting input terminal of the trans-impedance amplifier 511 is grounded, and the wiring 511b and the wiring 501a connected to the output terminal are connected by the wiring 511a. Resistance R _w is provided in the wiring 511a. If the voltage at the output terminal O of the transimpedance amplifier 511 is V _w and the current is I _w :
V _w = I _w · R _w (1)
It becomes. The electrochemical signal obtained from the terminal O is output to the control mechanism 15 shown in FIG. Since there are a plurality of working electrodes 551, 552, and 553, a plurality of terminals W and terminals O are provided corresponding to the working electrodes 551, 552, and 553, respectively. Outputs from the plurality of terminals O are switched by a signal switching unit described later, and are subjected to AD conversion, whereby electrochemical signals from the working electrodes 551, 552, and 553 can be acquired almost simultaneously as digital values. A circuit such as the trans-impedance amplifier 511 between the terminal W and the terminal O may be shared by the plurality of working electrodes 551, 552, and 553. In this case, a signal switching unit for switching the wiring from the plurality of terminals W may be provided in the wiring 501a.

  As shown in FIGS. 4 and 5, the chip cartridge 11 constituting the base sequence determination system of FIG. 7 constitutes a cassette including a cassette upper lid 711, a cassette lower lid 712, a packing 713 (seal member), and a substrate 714. . The inner surfaces of the cassette upper lid 711 and the cassette lower lid 712 are opposed to each other, and the packing 713 and the substrate 714 are fixed between the cassette upper lid 711 and the cassette lower lid 712 in a narrowed state. From the outer surface to the inner surface of the cassette upper lid 711, nozzle insertion holes 722 and 723 having a substantially circular cross section are formed so as to penetrate therethrough. The inner diameters of the nozzle insertion holes 722 and 723 are set to be slightly larger than the outer diameters of the inlet and outlet ports 752 and 753 and the nozzles 707 and 708 of the valve unit 705 in FIG. 6, for example, 3.2 mm. Degree. As shown in FIG. 4, electrical connector ports 724 and 725 having a substantially rectangular cross section are formed so as to penetrate from the outer surface to the inner surface of the cassette upper lid 711. An electrical connector described later is inserted into each of the electrical connector ports 724 and 725 and used. Further, a seal detection hole 726 is formed on the outer surface so as to penetrate the inner surface. The seal detection hole 726 is used for detecting the presence or absence of a seal. In other words, the chemical solution (sample) is injected into the cassette (detection chip) 21 with the seal applied from the surface of the seal detection hole 726 on the outer surface of the cassette (detection chip) 21 to the surfaces of the electrical connector ports 724 and 725. The seal is peeled off after the chemical solution (sample) is injected into the cassette (detection chip) 21, and the presence or absence of the seal is detected. By injecting the chemical solution (sample) with the seal attached to the cassette (detection chip) 21, even if the chemical solution (sample) solution is accidentally dropped on the electrical connector port 724 or 725, the seal Since it is covered, the liquid does not enter the actual port 724 or 725 so that there is no fear of causing a problem such as an electrical short circuit.

  As shown in FIG. 5, a substrate positioning groove having a predetermined depth and substantially the same cross-sectional shape as that of the substrate 714 is provided on the inner surface side of the cassette upper lid 711. Is surrounded by the inner surface. The substrate positioning groove is formed so as to overlap the nozzle insertion holes 722 and 723 and the electrical connector ports 724 and 725. By inserting the substrate 714 in accordance with the substrate positioning groove, the substrate 714 can be positioned and disposed on the cassette upper lid 711. The depth of the substrate positioning groove is formed to be substantially the same as the thickness of the substrate 714.

  As shown in FIG. 5, a packing positioning groove that is deeper than the substrate positioning groove is provided so as to overlap the substrate positioning groove on the inner surface side of the cassette upper lid 711, and the periphery of the packing positioning groove is the substrate positioning groove. Surrounded by The packing positioning groove is formed so as to overlap the nozzle insertion holes 722 and 723. By fitting the packing 713 in accordance with the packing positioning groove, the packing 713 can be positioned and arranged on the cassette upper lid 711. The depth of the packing positioning groove with respect to the substrate positioning groove is formed to be substantially the same as the thickness of packing 713 described later. Therefore, the depth of the packing positioning groove with respect to the inner surface is formed to be substantially the same as the thickness of the packing 713 plus the thickness of the substrate 714.

  Four screw holes 727a, 727b, 727c, and 727d are provided on the peripheral edge of the inner surface of the cassette upper lid 711. With these screw holes 727a to 727d, the cassette upper lid 711 and the cassette lower lid 712 can be screwed. Two cassette positioning holes 728 a and 728 b are provided on the peripheral edge of the inner surface of the cassette upper lid 711. By positioning the cassette (detection chip) 21 with the cassette positioning holes 728a and 728b aligned with two positioning pins provided on the slide stage, the cassette (detection chip) 21 is positioned relative to the slide stage. Can be arranged.

  Further, a seal detection hole 746 is formed through the outer surface. Further, the seal detection hole 746 of the cassette lower lid 712 is formed at a position communicating with the seal detection hole 726 of the cassette upper lid 711 in a state where the cassette upper lid 711 and the cassette lower lid 712 are fixed. Thus, when the cassette upper lid 711 and the cassette lower lid 712 are fixed, a seal detection hole 726 that penetrates from the cassette upper lid 711 to the cassette lower lid 712 is provided, and detection light is irradiated to the seal detection hole 726. By doing so, the presence or absence of the seal can be determined.

  Four screw holes 747 a, 747 b, 747 c, and 747 d are provided in the peripheral portion of the outer surface of the cassette lower lid 712. The cassette lower lid 712 can be fixed to the cassette upper lid 711 by screwing the screw holes 747a to 747d and the corresponding screw holes 727a to 727d provided in the cassette upper lid 711. Two cassette positioning holes 748 a and 748 b are provided on the peripheral edge of the outer surface of the cassette lower lid 712. Cassette positioning holes 728a and 728b of the cassette upper lid 711 pass through the cassette positioning holes 748a and 748b, respectively. The positioning of the cassette (detection chip) 21 with respect to the slide stage is performed by two positionings of two positioning pins provided on the slide stage and a cassette upper lid 711 passing through the cassette positioning holes 748a and 748b of the cassette lower lid 712. The structure is defined by the holes 728a and 728b. The cassette lower lid 712 is further provided with a cassette type discriminating hole 749, and the type of the cassette (detection chip) 21 can be discriminated by the presence or absence of the cassette type discriminating hole 749. Note that the type discrimination can be automatically performed depending on whether or not a cassette type discrimination pin (not shown) is pushed down. The push-down state of the cassette type determination pin (not shown) is detected by the control mechanism 15.

  Even if a cassette (detection chip) 21 that is not provided with the cassette type discrimination hole 749 is used, the same measurement can be performed only by different types of the cassette (detection chip) 21 to be discriminated. Alternatively, it can be designed so that the control mechanism 15 displays a warning on the display unit (not shown) that the type of the cassette (detection chip) 21 is different and does not proceed to the measurement process. Alternatively, the cassette type discriminating pin (not shown) is a fixed pin, and the cassette (detection chip) 21 not provided with the cassette type discriminating hole 749 cannot be mounted, so that an erroneous cassette is obtained. The (detection chip) 21 may not be set.

  The packing 713 is substantially rectangular and has a predetermined thickness, and has a plate-like portion formed by cutting out four corners thereof, is located on both sides of the long side on the main surface of the plate-like portion, and It consists of a cylindrical inlet port 752 and outlet port 753 provided near the center of the short side. Openings are provided at the distal ends of the inlet port 752 and the outlet port 753. From the opening to the plate-like portion, a flow path is provided in a direction perpendicular to the main surface of the plate-like portion at the axis of the inlet port 752 and the outlet port 753. As shown in FIG. 5, a bent groove is formed on the rear surface of the plate-like portion from the position where the feed port 752 is formed to the position where the feed port 753 is formed. The groove constitutes a flow path. The grooves are formed so as to advance alternately a plurality of times, and by making the turning point of the groove a curve with a predetermined curvature, it is possible to suppress the retention of chemicals and air generated when a corner of the turning point is provided. .

    As shown in FIG. 4, an electrode unit 761 and pads 762 and 763 are formed on the main surface of the substrate 714. As shown in FIG. 2, the electrode unit 761 is a three-electrode system unit including a combination of a counter electrode 502, working electrodes 551, 552, 553, and reference electrodes 561, 562. Probe DNAs 571, 572, and 573 are immobilized on the working electrodes 551, 552, and 553 of the electrode unit 761. The electrode unit 761 and the pad 762, and the electrode unit 761 and the pad 763 are connected by wiring (not shown). FIG. 4 illustrates the case where the electrode unit 761 to which the probe DNA is fixed and the pads 762 and 763 are formed on the same surface with respect to the substrate 714. However, the pads 762 and 763 are formed on the substrate. When the electrode unit 761 is formed on the surface opposite to the surface on which the electrode unit 761 is formed, the valve unit 705 is on the upper side with respect to the cassette (detection chip) 21 and the probe unit 710 is on the lower side. Will be installed. In that case, the valve unit 705 and the probe unit 710 are inevitably formed integrally.

  The arrangement of the electrode units 761 is formed at the packing arrangement position and in accordance with the bent flow path forming position. Thereby, when the packing 713 and the substrate 714 are narrowly fixed in a state of being positioned on the cassette upper lid 711 and the cassette lower lid 712, a bent channel is formed by the bent groove and the surface of the substrate 714, In addition, the electrode unit 761 is exposed on the surface of the flow path. That is, on the electrode unit 761, a gap is provided by a bent groove, and a bent flow path is formed by this gap. In this state, the seal between the packing 713 and the substrate 714 is held.

  First, the packing 713 is fitted into the packing positioning groove so that the feeding port 752 and the sending port 753 are inserted through the nozzle insertion holes 722 and 723 in accordance with the packing positioning groove on the inner surface of the cassette upper lid 711. Next, the substrate 714 is positioned and arranged in the substrate positioning groove so that the main surface thereof, that is, the surface on which the electrode unit 761 and the pads 762 and 763 are formed faces the cassette upper lid 711 side. Next, the cassette lower lid 712 is placed on the cassette upper lid 711 so that the inner surface 742 faces the cassette upper lid 711 and the positions of the screw holes 747a to 747d and the screw holes 727a to 727d correspond to each other. . Then, screws 770a to 770d are screwed into the screw holes 747a to 747d and the screw holes 727a to 727d. As a result, the cassette upper lid 711 and the cassette lower lid 712 are screwed together, and the packing 713 and the substrate 714 are tightly fixed between the cassette upper lid 711 and the cassette lower lid 712, thereby completing the cassette (detection chip) 21. In this completed state, a bent flow path is formed from the nozzle insertion hole 722 to the nozzle insertion hole 723.

  4 and 5 show an example in which the cassette upper lid 711 and the cassette lower lid 712 are fixed by screwing, but the present invention is not limited to this. For example, an engaging method using uneven members may be used.

  FIG. 6 is a diagram showing an overall configuration of the valve unit 705 provided in the liquid feeding system 13 constituting the base sequence determination system according to the embodiment of the present invention. In FIG. 6, the configuration of the probe unit is omitted, but the probe unit is integrally formed in the valve unit 705, and these valve unit and probe unit are simultaneously connected by the valve unit / probe unit drive mechanism. Driven. For example, two electrical connectors are arranged at a predetermined interval on a probe unit made of a glass epoxy substrate or the like. At the tip of the electrical connector, a plurality of convex electrodes are arranged in a matrix with the same arrangement as the pads on the substrate 714, and these convex electrodes are connected to the pads 762, 763 on the substrate 714 shown in FIG. By contact, electrical connection between the substrate 714 and the probe unit is ensured. Moreover, wiring is provided in the electrical connector, and each convex electrode and the control mechanism 15 are electrically connected by this wiring.

  The valve unit / probe unit drive mechanism is driven by an instruction from the control mechanism 15. The valve unit / probe unit drive mechanism has a drive direction in the up and down direction. As a result, the nozzles 707 and 708 and the electrical connector 703 are lowered with respect to the upper part of the cassette (detection chip) 21 on the slide stage side, so that the nozzles 707 and 708 are moved into the nozzle insertion holes 722 and The electrical connector 703 is positioned at the electrical connector ports 724 and 725. Thereby, the liquid supply system and the flow path in the cassette (detection chip) 21 communicate with each other. Further, the electrical connector is positioned on the pad of the cassette (detection chip) 21, and the pad and the electrical connector are electrically connected.

  The valve unit 705 is used with the valve body 781 connected and fixed. The valve body 781 is provided with two-way solenoid valves 403, three-way solenoid valves 413, 423, and 433, and the valve body 782 is provided with three-way solenoid valves 441 and 445. The valve body 781 is made of, for example, PEEK resin. In addition, when the valve body 781 is produced separately and they are joined together, for example, PTFE is used as a packing at the joint portion. Therefore, in both valve bodies 781, the material of the portion in contact with the chemical solution is made of PEEK and PTFE. The valve body 781 is provided with a cavity having a substantially constant cross-sectional shape. This cavity functions as piping for connecting between solenoid valves to be described later, packing 713, and the like. A nozzle 707 and a nozzle 708 communicate with a cavity provided in the valve body 782. The nozzle 707 and the nozzle 708 are made of PEEK resin.

  The three-way solenoid valve 413 switches between air and milli-Q water and supplies it to the downstream three-way solenoid valve 423. The three-way solenoid valve 423 switches the buffer and the air or milli-Q water from the three-way solenoid valve 413 and supplies them to the three-way solenoid valve 433 on the downstream side. The three-way solenoid valve 433 switches the intercalating agent and the air, milli-Q water, or buffer from the three-way solenoid valve 423 and supplies them to the valve body 782 on the downstream side. The three-way solenoid valve 441 switches between supply of air or chemical liquid from the valve body 781 to the nozzle 707 or supply to the three-way solenoid valve 445 via a bypass pipe. The three-way solenoid valve 445 switches between supply of air or chemical liquid from the three-way electromagnetic valve 441 or delivery of chemical liquid or air from the cassette (detection chip) 21 via the nozzle 708.

  In the valve unit 705 shown in FIG. 6, in order to send the buffer into the cassette (detection chip) 21, the three-way solenoid valves 423, 441, 445 and the liquid feed pump 454 may be turned on. As a result, the buffer is sucked up, and the chemical solution is switched to the nozzle 707 side, and is sucked out from the nozzle 707 to the cassette (detection chip) 21 and further from the cassette (detection chip) 21 to the nozzle 708, via the three-way solenoid valve 445. Can be drained. In order to send milli-Q water into the cassette (detection chip) 21, the three-way solenoid valve 413 may be turned on instead of the three-way solenoid valve 423. In order to feed the insertion agent into the cassette (detection chip) 21, the three-way solenoid valve 433 may be turned on instead of the three-way solenoid valve 423. In order to supply air into the cassette (detection chip) 21, the three-way solenoid valve 403 may be turned on and all of the three-way solenoid valves 413, 423, and 433 may be turned off. The internal capacity of the hollow pipe provided in the valve body 781 of the valve unit 705 is about 100 μL including the internal volume of the valve. Unlike this embodiment, when each three-way valve is connected by a tube to form the same flow, an internal volume of about 500 μL is required, but the reagent amount can be greatly reduced compared to this. it can. Further, the internal capacity between the valve unit 705 and the cassette (detection chip) 21 is 100 μL or more in an example different from the present embodiment, but can be greatly reduced to 10 μL in the present embodiment. With such a structure, the amount of solution or air that flows in the cassette (detection chip) 21 unintentionally after the reagent switching can be greatly reduced. As a result, variations in reaction and measurement are reduced, and the reproducibility of the results is greatly improved.

  Further, a chemical solution swinging device (not shown) is provided, and the chemical solution in the chip cassette can be swung. It is effective to shake the chemical solution in (1) a hybridization step between sample DNA and probe DNA, (2) a washing step, and (3) an intercalating agent supply step. By shaking the sample DNA in the hybridization step (1), the hybridization efficiency can be improved and the hybridization time can be shortened. In addition, (2) the washing time can be shortened by improving the efficiency of peeling off nonspecifically adsorbed DNA by oscillating the buffer solution in the washing step. Further, (3) by shaking the insertion agent in the insertion agent supply step, the uniformity of the concentration of the insertion agent is improved, and the adsorption uniformity of the insertion agent is also improved. The ratio can be improved. Even if the chemical solution swinging step is applied to all of the steps (1) to (3) or applied to a part of the steps, the effect of the chemical solution swinging can be obtained.

  Although partly mentioned at the beginning, the base sequence determination system according to the embodiment of the present invention includes a measurement unit 10, a control mechanism 15 connected to the measurement unit 10, and a control mechanism 15 as shown in FIG. And a computer 16 connected to the computer. The measurement unit 10 includes a chip cartridge 11, a measurement system 12 that is electrically connected to the chip cartridge 11, and a liquid supply system 13 that is physically connected to a flow path provided in the chip cartridge 11 via an interface unit. And a temperature control mechanism 14 for controlling the temperature of the chip cartridge 11.

  As shown in FIG. 8, the computer 16 shown in FIG. 7 determines whether or not the target nucleic acid is present and the input device 304 that accepts input of data and commands from the operator, or the SNP A central processing unit (CPU) 300 that determines which type is one of the two types, whether it is a homo type or a hetero type, an output unit 305 that outputs a determination result, and a display The apparatus 306 includes a data storage unit (not shown) that stores predetermined data necessary for base sequence determination, and a program storage unit (not shown) that stores a base sequence determination program.

  The CPU 300 includes a noise removal module 301, a current waveform determination module 302, a peak current detection module 310, an abnormal data exclusion module 320, a presence / absence determination module 330, and a type determination module 340.

The noise removal module 301 performs smoothing by using the “simple moving average method” from the current measured via the SNP1 detection electrode 551, the SNP2 detection electrode 552, and the control electrode 553 shown in FIG. , Remove noise. For smoothing, for example, a simple moving average method as shown in FIG. 15 may be used. The “simple moving average method” literally averages several actual values, and averages time-series data as shown in FIG. 15A by paying attention to its regularity. In FIG. 15, the moving average value section is m = 5, and the data after 5 points of each data are divided by 5:
y [n] = (x [n] + x [n + 1] + x [n + 2] + x [n + 3] + x [n + 4]) / 5 (2)
Is the moving average value shown in FIG. With the moving average, fluctuations as shown in FIG. 15A are leveled (smoothed) as shown in FIG. 15B, and it becomes easy to analyze the overall tendency.

  The current waveform determination module 302 follows the procedure shown in the flowchart of FIG. 16 to measure current waveforms (current-voltage) measured via the SNP1 detection electrode 551, the SNP2 detection electrode 552, and the control electrode 553 shown in FIG. Characteristic) baseline slopes are obtained, the normality / abnormality of each detection signal (current waveform) is determined based on the respective baseline slopes, and abnormal detection signals are excluded from computation.

  The peak current detection module 310 includes a voltage value calculation unit 311, a baseline approximation unit 312, and a peak current value calculation unit 313. According to the procedures shown in the flowcharts of FIGS. 18 and 19, the SNP1 detection electrode 551 and the SNP2 detection By subtracting the background current from the current (detection signal) measured via the electrode 552 and the control electrode 553, the peak value (peak current) of the true electrochemical current (true detection signal) caused by the intercalating agent 591 Value).

The voltage value calculation unit 311 converts the waveform of the current (i) -voltage (v) characteristic of the electrochemical current measured by the chip cartridge 11 into the voltage value (v) according to the procedure shown in steps S221 to S223 of FIG. The voltage value V _pk1 and the current value I _pk1 at the point where the differential value (di / dv) “zero- _crosses ” in the range of the predetermined lower limit value V1 to the upper limit value V2 are _determined for each electrode unit 761. Calculation is performed for each current-voltage characteristic (see FIG. 20). The “zero cross” point is a point at which the differential value (di / dv) of the electrochemical current changes from a positive value to a negative value, or a point at which the negative value changes from a positive value to a voltage value that gives a current peak. This corresponds to V _pk1 and current value I _pk1 . FIG. 20 shows a voltage value V _pk1 and a current value I _{pk1 at} which the differential value (di / dv) changes from a negative value to a positive value as the voltage value increases. When there are an odd number of “zero cross values”, the center value is _used as the voltage value V _pk1 , and when there are an even number of values, the value closest to the center value is _used as the voltage value V _pk1 .

The baseline approximating unit 312 calculates the baseline (background) of the current-voltage characteristics of the electrochemical current according to the procedure shown in steps S224 to S228 of FIG. 18 and the current-voltage of each electrode unit 761. The characteristics are approximated (see FIGS. 21 and 22). Peak current value calculating unit 313, following the procedure of step S229~S230 of FIG. 19, a current of the electrochemical current - the true peak current values I _pk2 in the voltage characteristic curve, the zero-crossing current voltage value calculating unit 311 is calculated It is obtained by subtracting the background (baseline) current value I _bg calculated by the baseline approximation unit 312 from the value I _pk1 .

The abnormal data exclusion module 320 uses the series of steps shown in the flowchart of FIG. 26 to calculate the peak value (peak current value) of the true electrochemical current (true detection signal) calculated by the peak current detection module 310. Exclude abnormal data from the group. That is, the abnormal data exclusion module 320 is a group obtained from a plurality of SNP1 detection electrodes 551, SNP2 detection electrodes 552, and control electrodes 553 respectively included in a plurality of electrode units 761 arranged on the substrate 714 shown in FIG. The data that does not satisfy the predetermined standard is excluded as an abnormal value from the data of the current value I _pk2 of each group.
The presence / absence determination module 330 determines whether or not the target nucleic acid exists in accordance with a series of procedures shown in the flowchart shown in FIG.

  The type determination module 340 determines whether the SNP type is one of SNP = “G” and SNP = “T”, whether it is a G / G homotype, Whether it is a mold or a T / T homo-type is determined by a series of procedures shown in the flowcharts shown in FIGS.

  The CPU 300 is connected to the voltage determination voltage range storage unit 351, the allowable tilt range storage unit 352, the peak search voltage value range storage unit 353, the zero cross value storage unit 354, the inflection point storage unit 355, and the intersection voltage via the bus 303. Storage unit 356, offset voltage storage unit 357, baseline current value storage unit 358, average value / standard deviation storage unit 360, CV value storage unit 361, SLL storage unit 362, ESLL storage unit 363, HUL storage unit 364, MSL storage A unit 365, an SLR storage unit 366, an absolute value logarithm storage unit 367, an HLL storage unit 368, and a classification result storage unit 369 are connected.

  The waveform determination voltage range storage unit 351 calculates the slope of the baseline of the current (detection signal) measured by the current waveform determination module 302 via the SNP1 detection electrode 551, the SNP2 detection electrode 552, and the control electrode 553. “Range lower limit voltage VLo” and “Range upper limit voltage VHi (VLo <VHi)” are stored as the range to be performed. The inclination allowable range storage unit 352 is configured so that the current waveform determination module 302 has “inclination lower limit (Coef Lo)” and “inclination upper limit (Coef Hi)” as parameters that define the allowable inclination of the baseline inclination of the detection signal. Remember.

The peak search voltage value range storage unit 353 stores a predetermined peak search voltage value range [V1, V2] as a setting parameter, and the voltage value calculation unit 311 stores the peak search voltage value range [V1, V2]. Readable. Since the current peak indicated by the current-voltage characteristic of the electrochemical current appears in a substantially constant voltage range when the measurement conditions are fixed, the peak search voltage value range [V1, V2] is determined as a setting parameter. The zero cross value storage unit 354 stores the “zero cross value (zero cross voltage value V _pk1 , zero cross current value I _pk1 )” calculated by the voltage value calculation unit 311 for every electrode unit 761 on the substrate 714 shown in FIG. Organize and store.

The inflection point storage unit 355 stores the inflection point voltage V _ifp necessary for the calculation of the baseline approximation unit 312. As shown in FIG. 21, the “inflection point voltage V _ifp ” is a voltage in which the differential value is minimized by tracing the voltage in the negative direction (decreasing the voltage) from the zero-cross voltage value V _pk1 that gives the current peak. is there. The intersection voltage storage unit 356 stores the intersection voltage V _crs . As shown in FIG. 22, the “intersection voltage V _crs ” is a voltage given by the intersection of the current-voltage characteristic curve of the electrochemical current and the tangent line of the current-voltage characteristic curve. As shown in FIG. 22, the offset voltage storage unit 357 starts the crossing voltage V _crs as the starting point, and sets the offset voltage V _ofs retroactive (decreasing the voltage) by the offset value defined as the setting parameter. Remember.

The baseline current value storage unit 358 stores the background (baseline) current value I _bg necessary for the calculation of the peak current value calculation unit 313. As shown in FIG. 22, “background (baseline) current value I _bg ” is a zero-cross voltage value calculated by the voltage value calculation unit 311 in the approximate primary expression of the baseline calculated by the baseline approximation unit 312. This is a current value obtained by substituting V _pk1 .

The average value / standard deviation storage unit 360 includes the average value X ₁ of the current of the SNP1 detection electrode 551 calculated by the abnormal data exclusion module 320, the presence / absence determination module 330, and the type determination module 340, and the SNP1 detection electrode. The average value X _nc1 of the current of the control electrode (NC1) 553 corresponding to 551, the average value X ₂ of the current of the SNP2 detection electrode 552, and the average value of the current of the control (NC2) electrode 553 corresponding to the SNP2 detection electrode 552 X _nc2 , standard deviation σ ₁ of the peak current value from the SNP 1 detection electrode 551, standard deviation σ _nc , σ _nc1 or σ _{cmp1 of} the corresponding peak current value from the control electrode 553, peak current from the SNP 2 detection electrode 552 standard deviation sigma ₂ values, the standard deviation of the peak current value from the corresponding control electrode 553 sigma _{nc, nc2} or sigma _cmp2, further, the difference in the mean-values _{(X 1 -X nc), (} X 1 -X nc1), (X 2 -X nc2), the sum of the standard deviations _{(σ 1 + σ nc),} (σ 1 + Σ _nc1 ) (σ ₁ + σ _cmp1 ), (σ ₂ + σ _cmp2 ), the ratio of the difference between the mean values and the standard deviation (X ₁ −X _nc ) / (σ ₁ + σ _nc ), etc. These averages _{X 1, X nc1, X 2} , X nc2 and standard deviation _{σ 1, σ nc, σ nc1} , σ cmp1, σ 2, σ nc2, is like sigma _cmp2, abnormal-data eliminating module 320, the presence judgment module 330 and the type determination module 340 are read at any time in response to a calculation request.

The CV value storage unit 361 includes various CV values CV (0), CV (1), CV (2) calculated by the abnormal data exclusion module 320 and a reference CV value CV ( %), CV value correction coefficient dCV / CV, and the like. The “CV (variation coefficient) value” is obtained by multiplying the value obtained by dividing the standard deviation of a target group of data by the corresponding average value by 100 and displaying the percentage. Since variance and variation have sample units, the degree of variation between the two sample groups cannot be compared. The SLL storage unit 362 stores a signal increase determination criterion SLL (+/−) required for the calculation of the presence / absence determination module 330 and a signal increase determination criterion SLL (M) required for the calculation of the type determination module 340. . The signal increase determination criterion SLL (Signal Lower Limit) is a setting parameter that provides a selection criterion (determination criterion) for a signal increase determination algorithm for the control electrode 553. FIG. 31 shows a conceptual image showing various setting parameters serving as criteria for determination. The vertical axis represents the difference (X ₁ −X _nc1 ) between the average value X ₁ of the current of the SNP ₁ detection electrode 551 and the average value X _nc1 of the current of the control electrode (NC 1) 553 corresponding to the SNP ₁ detection electrode 551, The amount of signal increase on the SNP1 detection electrode 551 side is shown. The horizontal axis represents the difference (X ₂ −X _nc2 ) between the average value X ₂ of the current of the SNP ₂ detection electrode 552 and the average value X _nc2 of the current of the control electrode (NC 2) 553 corresponding to the SNP ₂ detection electrode 552. The amount of signal increase on the SNP2 detection electrode 552 side is shown. The signal increase amount determination criterion SLL is shown as two lines orthogonal to each other in a region relatively close to the origin. The ESLL storage unit 363 stores an effective variation coefficient ESLL necessary for calculation by the presence / absence determination module 330 and the type determination module 340. The effective fluctuation coefficient ESLL (Effective Signal Lower Limit) is a setting parameter that gives a lower limit for determining how many times the signal increase amount is with respect to the standard deviation σ.

The HUL storage unit 364 stores a log signal ratio determination criterion HUL necessary for the calculation of the type determination module 340. The logarithmic signal ratio determination criterion HUL (Hetero Type Upper Limit) is a setting parameter that gives an upper limit of determination of the signal ratio in the case of the hetero type, and ideally abs ((X ₁ −X _nc1 ) / (X ₂ − X _nc2 )) = 1, that is, Log ₁₀ (1) = 0. In the conceptual image showing various setting parameters which are the determination criteria shown in FIG. 31, the diagonal line passing through the origin is an ideal hetero type, and two logarithms are used as lines defining the regions on both sides of the diagonal line. A signal ratio criterion HUL is shown. The MSL storage unit 365 stores setting parameters MSL necessary for the operations of the presence / absence determination module 330 and the type determination module 340. The setting parameter MSL (Minimum Signal Level) is the minimum signal amount used for determination when there is no increase in the current signal (or when data is missing), and is used to determine whether there is a device (hardware) malfunction. This is the reference value for the current value. The setting parameter MSL is set to a relatively small value, for example, a current value in the range of about 0 to 100 nA, as shown by the broken lines in FIGS. 24 (a) to (c) and FIGS. 25 (d) to (f). Keep it. The setting parameter MSL is determined in advance and stored in the MSL storage unit 365. The SLR storage unit 366 stores a significance determination reference magnification SLR necessary for the calculation of the type determination module 340. Significance determination reference magnification SLR (Signal Lower Ratio) is a setting parameter that gives a lower limit of significance determination when the amount of increase in one type of current signal is small. In the conceptual image showing various setting parameters serving as the determination criteria shown in FIG. 31, the significance determination reference magnification SLR is conceptually shown as two line segments orthogonal to each other that define a small rectangle near the origin. .

The absolute value logarithm storage unit 367 is Log ₁₀ which is the absolute value of the ratio of the difference between the average values on the SNP1 detection electrode 551 side and the average value difference on the SNP2 detection electrode 552 side necessary for the calculation of the type determination module 340. A certain R value is stored. The HLL storage unit 368 stores logarithmic signal ratio determination criteria (+ HLL, −HLL) necessary for the calculation of the type determination module 340. A log signal ratio determination criterion HLL (Homo Type Lower Limit) is a setting parameter that gives a lower limit of determination of the signal ratio in the case of the homo type. In the conceptual image showing various setting parameters as determination criteria shown in FIG. 31, the logarithmic signal ratio determination criterion HLL extends from the origin defining the regions in the vicinity of the horizontal axis and the vertical axis corresponding to the ideal homo-type. Shown as diagonal lines in the book.

  The classification result storage unit 369 stores various classification results classified by the presence / absence determination module 330 and the type determination module 340.

  Although not shown, an interface is connected to the CPU 300 via a bus 303, and data is transmitted to and received from the control mechanism 15 shown in FIG. 7 via this interface through a local bus (not shown). It can be carried out.

  In FIG. 8, the input device 304 includes a keyboard, a mouse, a light pen, a flexible disk device, or the like. A base sequence determination executor can specify input / output data and set a setting parameter, an allowable error value, and an error level from the input device 304. Furthermore, it is possible to set the form of output data and the like from the input device 304, and it is also possible to input an instruction to execute or stop the calculation. In addition, the output device 305 and the display device 306 can be configured by, for example, a printer device and a display device, respectively. The display device 306 displays input / output data, determination results, determination parameters, and the like. A data storage unit (not shown) stores input / output data, determination parameters, a history thereof, data being calculated, and the like.

  As described above, according to the base sequence system according to the embodiment of the present invention, the presence / absence determination of nucleic acids and the SNP homo / hetero type determination can be performed with high accuracy in accordance with the actual situation.

(Base sequence determination method)
The base sequence determination method according to the embodiment of the present invention will be described using the flowchart shown in FIG. It should be noted that the base sequence determination method described below is an example, and it is possible to implement it by various other base sequence determination methods including this modification. In any case, FIG. A chemical solution (sample) containing the sample DNAs 581, 582, 583 is injected into the chip cartridge 11 on which the probe DNAs 571, 572, 573 as shown in FIG. It is fundamental to obtain a current-voltage characteristic of an electrochemical current as shown in FIG. 13 using the measurement system 12 through an electrochemical reaction by introduction. From the current-voltage characteristics of the electrochemical current, a peak current value corresponding quantitatively to the hybridization reaction of each probe DNA 571, 572, 573 is calculated. Then, the calculated peak current value is statistically processed, thereby determining the presence or absence of the nucleic acid or determining the type of the single nucleotide polymorphism of the nucleic acid.

  Prior to the description of the flowchart shown in FIG. 14, first, hybridization of probe DNA and sample DNA will be described with reference to FIGS. 9 to 11. For the hybridization step, the chip cartridge 11 shown in FIGS. 4 and 5 may be used.

  FIG. 9 shows a case where the sample DNA 581 is a target base sequence (sample DNA with SNP = “G”) having the base sequence CTGAG. As shown in FIG. 9 (a), the working electrode (SNP1 detection electrode) 551 on which the probe DNA 571 having the base sequence GACTC... Is immobilized is the target base having the base sequence CTGAG. Since the sequence (sample DNA of SNP = "G") 581 and the sequence are perfectly matched, a double strand is formed. However, if even one base has a different sequence, a double strand is not formed. Therefore, as shown in FIG. 9B, a working electrode (for detecting SNP2) on which a probe DNA 572 having a base sequence GAATC. The electrode) 552 cannot form a double strand with the target base sequence (sample DNA of SNP = "G") 581. Also, if the sequences are completely different, it is naturally impossible to form a double strand, so that probe DNA (negative control DNA) 573 having the base sequence CAGTG... Is immobilized as shown in FIG. The working electrode (control electrode) 553 thus formed cannot form a double strand with the target base sequence (sample DNA of SNP = “G”) 581.

  FIG. 10 shows a case where the sample DNA 582 is a target base sequence (sample DNA with SNP = "T") having the base sequence CTTAG. Similarly, the working electrode (SNP2 detection electrode) 552 on which the probe DNA 572 having the base sequence GAATC... Is immobilized by the hybridization process using the chip cartridge 11 as shown in FIG. Since the sequence is perfectly matched with the target base sequence (sample DNA of SNP = "T") 582 having the base sequence CTTAG ..., a double strand is formed. However, even if one base has a different sequence, a double strand is not formed. Therefore, as shown in FIG. 10A, a working electrode (for detecting SNP1) on which a probe DNA 571 having a base sequence GACTC. The electrode) 551 cannot form a double strand with the target base sequence (sample DNA with SNP = "T") 582. If the sequences are completely different, naturally a double strand cannot be formed. As shown in FIG. 10 (c), the probe DNA (negative control DNA) 573 having the base sequence CAGTG. The working electrode (control electrode) 553 thus formed cannot form a double strand with the target base sequence (sample DNA with SNP = "T") 582.

  In FIG. 11, the sample DNA 583 has a target base sequence (SNP = "G" sample DNA) having a base sequence CTGAG ... and a target base sequence (SNP = "T") having a base sequence CTTAG ... In the case of a heterotype with the sample DNA). Similarly, as shown in FIG. 11A, the working electrode (SNP1 detection electrode) 551 on which the probe DNA 571 having the base sequence GACTC... G / T hetero target base sequence (SNP = "G / T" hetero sample DNA) 583 having both base sequence CTAGAG ... and base sequence CTTAG ... perfectly matches the sequence As a result, a double strand is formed. 11B, the working electrode (SNP2 detection electrode) 552 on which the probe DNA 572 having the base sequence GAATC... Is immobilized has the base sequence CTTAG. Since the G / T hetero target base sequence (SNP = "G / T" hetero sample DNA) 583 and the sequence are perfectly matched, a double strand is formed. However, if the sequences are completely different, it is natural that double strands cannot be formed. Therefore, as shown in FIG. 11 (c), the probe DNA (negative control DNA) 573 having the base sequence CAGTG. The working electrode (control electrode) 553 thus formed cannot form a double strand with the G / T hetero target base sequence (sample DNA of SNP = “G / T” hetero) 583.

  When different from the configuration shown in FIG. 4 and FIG. 5, that is, when a planar packing is mounted on a substrate (chip) and a flow path is formed in the cassette (chip cartridge), the cassette ( The flow path in the (detection chip) 21 becomes longer, and the amount of unnecessary reagent increases. In addition, when a chemical solution (sample) is injected into the cassette (detection chip) 21, since the flow path exists long in the cassette (detection chip) 21 as well as on the substrate, the chemical solution is applied to unnecessary portions other than the substrate. (Sample) flows in and is wasted. In addition, the adhesion of the cassette (detection chip) 21 to the packing is difficult, and a leak occurs between the packing and the cassette (detection chip) 21, often causing a problem of liquid feeding. With the configuration as in this embodiment, the amount of unnecessary reagent is reduced, and the adhesion between the packing, the substrate and the cassette (detection chip) 21 is increased, and the stability of liquid feeding is increased.

  FIG. 12 shows the insertion agent 591 in each of the hybridized working electrodes 551, 552, 553 using the target base sequence (sample DNA) 581 having the base sequence CTGAG. The state where is introduced. As shown in FIG. 12 (a), a working electrode (SNP1 detection electrode) 551 on which a probe DNA 571 having a base sequence GACTC... Is immobilized is a target base having a base sequence CTGAG. Since the sequence (sample DNA) 581 and the sequence are perfectly matched to form a double strand, the intercalating agent 591 binds to the double-stranded DNA. However, as shown in FIG. 12B, the working electrode (SNP2 detection electrode) 552 on which the probe DNA 572 having the base sequence GAATC... Is immobilized is the target base sequence (sample DNA) 581. Since the double strand cannot be formed, the intercalating agent 591 cannot be bound. As shown in FIG. 12 (c), the working base (control electrode) 553 to which the probe DNA (negative control DNA) 573 having the base sequence CAGTG. DNA) 581 cannot form a double strand, and therefore intercalator 591 cannot bind.

  FIG. 13 shows the electrochemical current from the intercalating agent 591 bound to the double-stranded DNA hybridized to the probe DNA 571, 572, 573 immobilized on each working electrode 551, 552, 553, or double-stranded DNA. It is a figure which shows the relationship between the electric current and voltage in case the insertion agent 591 was not able to couple | bond together. A curve (a) in FIG. 13 corresponds to FIG. That is, the curve (a) shows the electrochemical when the probe DNA 571 and the target base sequence (sample DNA) 581 are perfectly matched to form a double strand, and the intercalating agent 591 is bound to this double-stranded DNA. This is a current-voltage characteristic of a typical current, and shows a large current peak. However, the curve (b) in FIG. 13 corresponds to FIG. 12 (b), and the probe DNA 572 and the target base sequence (sample DNA) 581 cannot form a double strand, and the insertion agent 591 cannot bind. It is a current-voltage characteristic and shows a peak of a small current value compared to the curve (a). Moreover, the curve (c) of FIG. 13 corresponds to FIG. 12 (c), and the negative control DNA 573 and the target base sequence (sample DNA) 581 cannot form a double strand, and the insertion agent 591 cannot bind. This is a current-voltage characteristic, and shows a peak of a smaller current value than that of the curve (b). The small current value peaks observed in FIGS. 13B and 13C are currents caused by the insertion agent 591 slightly adsorbed on the surfaces of the electrodes 552 and 553, as shown in FIGS. 12B and 12C. is there.

Although the introduction has become longer, the base sequence determination method according to the embodiment of the present invention will be described using the flowchart shown in FIG.
(A) First, a chemical solution (sample) containing sample DNA is injected into the chip cartridge 11 to cause a hybridization reaction or the like, and a current-voltage characteristic due to an electrochemical reaction due to introduction of an intercalating agent is measured using the measurement system 12. Measure for each electrode. FIG. 4 schematically shows a large number of electrode units 761 on the substrate 714. The SNP1 detection electrodes 551 and SNP2 to which the probe DNAs 571, 572, and 573 are respectively immobilized are associated with the large number of electrode units 761. A large number of detection electrodes 552 and control electrodes 553 exist as equivalent electrodes, and a large amount of data is acquired correspondingly. In step S101, the noise removal module 301 removes noise by leveling (smoothing) each measured group of data for each electrode on which the probe DNAs 571, 572, and 573 are immobilized. For smoothing, as already described, a simple moving average method as shown in FIG. 15 may be used.
(B) Next, in step S102, the current waveform determination module 302 obtains the slope of the baseline of the current waveform (current-voltage characteristics) measured for each electrode, and the respective slopes of the respective baselines The normal / abnormal of the detection signal (current waveform) is determined, and the abnormal detection signal is excluded from the calculation target. Details of the processing of the current waveform determination module 302 in step S102 will be described later with reference to the flowchart of FIG.

(C) Next, in step S103, the peak current detection module 310 detects the peak value (peak current value) of the detection signal measured for each electrode. Details of the processing of the peak current detection module 310 in step S103 will be described later with reference to the flowcharts of FIGS. 18 and 19. In the processing in step S103, the electrochemical current from the intercalating agent 591 as shown in FIG. From this, the background current other than this is subtracted, and the peak value (peak current value) of the true detection signal is obtained as a group of data for each equivalent electrode.

(D) After the background current component is removed in step S103, the signal processing in step S104 is performed on each of the group of data. That is, in step S104, the abnormal data exclusion module 320 excludes abnormal data from each group of data. Details of the processing of the abnormal data exclusion module 320 in step S104 will be described later with reference to the flowchart of FIG.

(E) Next, in step S105, the presence / absence determination module 330 determines the presence / absence of a nucleic acid, or the type determination module 340 performs a single nucleotide polymorphism (SNP) type determination of the nucleic acid. Details of the processes of the presence / absence determination module 330 and the type determination module 340 in step S105 will be described later with reference to the flowcharts of FIGS.

  According to the base sequence determination method according to the embodiment of the present invention shown in the flowchart of FIG. 14, even when there is an abnormality in the chip cartridge 11 or the measurement system 12, or when there is a variation in data, etc. Exclude abnormal data, it can be determined whether there is exactly a certain nucleic acid, what type of SNP is, and whether it is homozygous or heterozygous. Hereinafter, each step of the flowchart shown in FIG. 14 will be specifically described.

[Step S102: Determination of Normal / Abnormal Current Waveform]
The signal (current waveform) of the electrochemical current in the chip cartridge 11 measured by the measurement system 12 of FIG. 1 has a current-voltage characteristic waveform as shown in FIG. A voltage peculiar to a substance (insertion agent) that generates an electric signal has a waveform of current-voltage characteristics having a peak shape as shown in FIG. In FIG. 17, two types of current-voltage characteristics of data 1 and data 2 are shown. However, the current-voltage characteristics of data 2 indicate the slope of the baseline indicated by the current-voltage characteristics of data 1. The slope of the baseline is larger, and in the current-voltage characteristics of data 2, the peak shape is unclear and shows a shoulder (shoulder) change.

  Ideally, the current-voltage characteristic of the electrochemical current has a current value of substantially “zero” below the voltage that generates the peak current. However, when some trouble occurs in the substrate 714, the current-voltage characteristic with the inclined base line is often obtained as shown in the data 2. In the case of data 2, since the peak current value cannot be detected correctly, it is necessary to eliminate it as “abnormal” in step S102 of FIG.

Details of the processing of the current waveform determination module 302 in step S102 are as shown in the flowchart of FIG.
(a) First, in step S201, a range for calculating the baseline slope of the current waveform (current-voltage characteristic) measured for each electrode is extracted and set. In this range extraction, the range lower limit voltage VLo and the range upper limit voltage VHi (VLo <VHi) are set as setting parameters using the input device 304 and stored in the waveform determination voltage range storage unit 351. Further, “inclination lower limit value (Coef Lo)” and “inclination upper limit value (Coef Hi)” are set as parameters for defining the allowable inclination range of the baseline, and stored in the allowable inclination range storage unit 352.

  (b) Next, in step S202, the current waveform determination module 302 reads the range lower limit voltage VLo and the range upper limit voltage VHi stored in the waveform determination voltage range storage unit 351, and in this setting range, An approximate expression for the slope is derived. The range lower limit voltage VLo and the range upper limit voltage VHi are parameters that define a region for calculating the slope of the baseline, and are voltage values between the range lower limit voltage VLo and the range upper limit voltage VHi, and are measured for each electrode. A straight line (baseline) is approximated by a least square approximation to the waveform of the current-voltage characteristics.

  (c) Next, in step S203, the current waveform determination module 302 sets the readout lower limit voltage VLo and the range upper limit voltage VHi for each of the current waveforms (current-voltage characteristics) measured for each electrode, as starting points. The start point and end point are substituted into the approximate expression derived in step S202, and the baseline slope (b) of the current waveform (current-voltage characteristic) measured for each electrode is calculated. To do.

  (d) Next, in step S204, the current waveform determination module 302 reads “inclination lower limit (Coef Lo)” and “inclination upper limit (Coef Hi)” from the inclination allowable range storage unit 352, and in step S203. Then, it is determined whether the baseline inclination (b) calculated for each electrode exists between “inclination lower limit (Coef Lo)” and “inclination upper limit (Coef Hi)”.

  (e) In step S204, the slope (b) of the baseline of the current waveform (current-voltage characteristic) measured at a specific electrode is expressed as “slope lower limit (Coef Lo)” and “slope upper limit (Coef Hi)”. ”Is determined as“ normal waveform ”, and the process proceeds to step S103 in FIG. When it is determined in step S204 that the current waveform (current-voltage characteristics) measured at a specific electrode is outside the range of “inclination lower limit (Coef Lo)” and “inclination upper limit (Coef Hi)” In step S205, the current waveform determination module 302 determines that the current waveform (current-voltage characteristic) measured at the electrode is an “abnormal waveform”. In step S205, the current waveform ( “Error determination” is made for the current-voltage characteristics), and the display device 306 and the output device 305 are made to output them.

  The process of step S103 illustrated in FIG. 14 is performed for all the electrode units 761 on the substrate 714 illustrated in FIG.

[Step S103: Detection of Peak Current Value]
Details of the processing of the peak current detection module 310 in step S103 will be described with reference to the flowcharts of FIGS. In step S103, the step of detecting each net peak current value from the waveform of the current-voltage characteristic of each electrode unit 761 measured by the measurement system 12 calculates the voltage value that gives the current peak in steps S221 to S223. A step of approximating the baseline in steps S224 to S228 (see FIGS. 21 and 22); and a step of calculating a peak current value in steps S229 to S230 (see FIG. 23). , Consisting of a procedure performed for each current-voltage characteristic of each electrode unit 761:
(A) The current peak indicated by the current-voltage characteristic of the electrochemical current measured by the chip cartridge 11 appears in a substantially constant voltage range. For this reason, in step S221, the peak search voltage value range [V1, V2] is stored in the peak search voltage value range storage unit 353 as a setting parameter in advance using the input device 304 shown in FIG. That is, as shown in FIG. 20, the peak current search of the electrochemical current is performed in the range of the lower limit value V1 to the upper limit value V2. First, in step S222, the voltage value calculation unit 311 of the peak current detection module 310 differentiates the waveform of the current (i) -voltage (v) characteristic of the electrochemical current with respect to the voltage value (v). In step S223, the voltage value calculation unit 311 determines the voltage value _Vpk1 and the current value at the point where the differential value (di / dv) of the electrochemical current “zero _crosses ” in the range of the lower limit value V1 to the upper limit value V2. I _pk1 is calculated (see FIG. 20). The “zero cross” point is a point at which the differential value (di / dv) of the electrochemical current changes from a positive value to a negative value, or a point at which the negative value changes from a positive value to a voltage value that gives a current peak. This corresponds to V _pk1 and current value I _pk1 . FIG. 20 shows a voltage value V _pk1 and a current value I _{pk1 at} which the differential value (di / dv) changes from a negative value to a positive value as the voltage value increases. When there are an odd number of “zero cross values”, the center value is _used as the voltage value V _pk1 , and when there are an even number of values, the value closest to the center value is _used as the voltage value V _pk1 . The “zero cross value (zero cross voltage value V _pk1 , zero cross current value I _pk1 )” calculated in step S 223 is stored in the zero cross value storage unit 354 for every electrode unit 761 on the substrate 714 shown in FIG. And store.

(B) In step S224, the baseline approximation unit 312 of the peak current detection module 310 _{traces the} voltage in the negative direction (decreases the voltage) from the zero-cross voltage value _Vpk1 that gives the current peak, and the result is shown in FIG. As described above, the voltage at which the differential value is minimum is defined as the inflection point voltage V _ifp . The inflection point voltage V _ifp is stored in the inflection point storage unit 355. Then, the baseline approximation unit 312 _reads the zero cross voltage value V _pk1 and the inflection point voltage V _ifp from the zero cross value storage unit 354 and the inflection point storage unit 355, respectively, and in step S225, a tangent to the current-voltage characteristic curve. A linear formula for finding the starting point of:
y = ax + b (3)
The obtained between the zero-cross voltage value V _pk1 and the inflection point voltage V _ifp. For example, the current-voltage characteristic waveform data between the zero cross voltage value V _pk1 and the inflection point voltage V _ifp is linearly approximated as shown in Equation (3) by the least square method. In the example shown in FIG. 21, the coefficient a = −1.397 × 10 ⁻¹⁰ and the constant b = 3.396 × 10 ⁻⁸ are obtained, and in the example shown in FIG. 22, the coefficient a = −2.899 × 10 ^{− 11} and a constant b = 4.504 × 10 ⁻⁹ . Further, in the baseline approximation unit 312 step S226, the current - to obtain the intersection voltage V _crs give intersection of the approximate line of the waveform and expressions voltage characteristics (3) As shown in FIG. 22, the intersection voltage V _crs Stored in the intersection voltage storage unit 356. In step S227, the baseline approximating unit 312 sets the offset voltage V _ofs obtained by tracing back the voltage in the negative direction (decreasing the voltage) by the offset value specified as the setting parameter starting from the intersection voltage V _crs. As shown in FIG. The obtained offset voltage V _ofs is stored in the offset voltage storage unit 357. Further, the baseline approximating unit 312 reads the offset voltage V _ofs from the offset voltage storage unit 357 and the intersection voltage V _crs from the intersection voltage storage unit 356, and in step S228, between the offset voltage V _ofs and the intersection voltage V _crs . As shown in FIG. 23, an approximate linear expression that is a background tangent (baseline) of the waveform data of the current-voltage characteristics is obtained by the method of least squares. The approximate linear expression can be expressed in the same form as the expression (3). In the linear approximation shown in FIG. 23, the coefficient a = −6.072 × 10 ⁻¹² and the constant b = −5.902 × 10 ^{−9 of the} linear expression Desired.

(C) Thereafter, the peak current value calculation unit 313 of the peak current detection module 310 reads the zero cross voltage value V _pk1 from the zero cross value storage unit 354. In step S229, the peak current value calculation unit 313 substitutes the zero-cross voltage value _Vpk1 for the approximate primary expression of the baseline obtained in step S228 to obtain the background (baseline) current value I _bg . The background (baseline) current value I _bg is stored in the baseline current value storage unit 358. Further, the peak current value calculation unit 313 reads the zero cross current value I _pk1 indicating the peak of the waveform of the current-voltage characteristic from the zero cross value storage unit 354, and in step S230, from the zero cross current value I _pk1 , the background (base The current value I _bg of the line) is subtracted as shown in equation (4):
I _pk2 = abs (I _pk1 −I _bg ) (4)
Equation (4) is calculated based on each SNP1 detection electrode (SNP = "G" detection electrode) 551, SNP2 detection electrode (SNP = "T" detection electrode) 552, and control electrode 553 of each electrode unit 761. By applying to the current-voltage characteristics, the true current value I _pk2 is calculated from the respective electrodes 551, 552, _{553 of} each electrode unit 761.

[Step S104: Exclude Abnormal Data]
As described above, in step S103 of FIG. 14, the peak current detection module 310 determines the background from the zero-cross current value _Ipk1 indicating the peak of each current-voltage characteristic by each electrode unit 761 measured by the measurement system 12. (Baseline) current value I _bg is subtracted, and each SNP1 detection electrode (SNP = "G" detection electrode) 551, SNP2 detection electrode (SNP = "T" detection electrode) of each electrode unit 761 For each of the control electrodes 553, the respective net current values _Ipk2 are calculated and classified in various determination modes. Modes A to F classified by the collective bar graphs shown in FIGS. 24A to 24C and FIGS. 25D to 25F show typical examples of such detection signals. . In each figure showing the modes A to F, the current value I _pk2 indicated as “control” at the left end is a value measured by the plurality of control electrodes 553, and the current value I _pk2 indicated as “T” in the center. Is a value measured by a plurality of SNP2 detection electrodes (SNP = "T" detection electrode) 552, and a current value _Ipk2 indicated by "G" on the right end is a plurality of SNP1 detection electrodes (SNP = This is a value measured by “G” detection electrode) 551.

However, in the base sequence determination method according to the embodiment of the present invention, it cannot be immediately classified into the modes A to F shown in FIGS. 24 (a) to (c) and FIGS. 25 (d) to (f). . For example, when only one of a plurality of equivalent SNP1 detection electrodes (SNP = "G" detection electrode) 551 has an abnormally large value or a small value, a plurality of equivalent SNP2 detection electrodes (SNP = " If only one of the T ″ detection electrodes) 552 shows an abnormally large value or a small value, the determination algorithm will be confused unless these abnormal values are excluded. That is, as shown in FIG. 4, the peak current value I _pk2 obtained from a plurality of equivalent electrode units 761 arranged on the substrate 714 is used as it is, and the process of step S105 in FIG. Problems may arise when determining whether or not the SNP type is one of the two types, whether it is a homo type or a hetero type is there.

Therefore, in step S104 of FIG. 14, before proceeding to step S105, the abnormal data excluding module 320 obtains current values obtained from all the electrodes defined by the electrode units 761 arranged on the substrate 714 shown in FIG. The procedure of the flowchart shown in FIG. 26 is _performed for I _{pk2 in} units of groups, and abnormal values are eliminated on a predetermined basis:
(A) In step S301, the abnormal data exclusion module 320 _{employs the} current value _Ipk2 obtained from all the electrodes defined in the group as data. A group unit is defined for each equivalent electrode. That is, it is defined for each SNP1 detection electrode (SNP = "G" detection electrode) 551, SNP2 detection electrode (SNP = "T" detection electrode) 552, and control electrode 553 of each of the plurality of electrode units 761. The

(B) Next, for the first group, the abnormal data exclusion module 320 calculates a standard deviation and an average value of the current values I _pk2 obtained from all the electrodes defined in the first group. In step S302, the first group CV value (0) is obtained by dividing the standard deviation of the first group by the average value of the first group. The abnormal data exclusion module 320 reads the reference CV value CV (%) defined as the setting parameter from the CV value storage unit 361 and compares it with the first CV value CV (0) in step S303. When the first CV value CV (0) is smaller than the reference CV value CV (%), all the current values I _pk2 of the group are set as normal values, and the process proceeds to the subsequent determination algorithm (step S105 in FIG. 14). Similarly for the other groups, the procedure from step S302 to step S303 is repeated for all groups, and abnormal values are excluded.

(C) In step S303, if the first CV value CV (0) exceeds the reference CV value CV (%), the first CV value CV (0) is stored in the CV value storage unit 361 and then abnormal. The data exclusion module 320 proceeds to step S304, excludes the minimum value of the series of data of the group, and again _calculates the standard deviation and the average value of the current value _Ipk2 obtained from all other electrodes defined in the group. calculate. In step S304, the standard deviation excluding the minimum value is divided by the average value excluding the minimum value to obtain the second CV value CV (1), and the process proceeds to step S305.

(D) In step S305, the abnormal data exclusion module 320 reads the first CV value CV (0) and the CV value correction coefficient dCV / CV from the CV value storage unit 361, and sets the CV value correction coefficient dCV to the first CV value CV (0). A magnitude comparison between the value corrected (multiplied) by / CV and the second CV value CV (1) is performed:
(CV (0)) * (dCV / CV)> CV (1) (4)
(E) When the value obtained by correcting (multiplying) the first CV value CV (0) by the CV value correction coefficient dCV / CV becomes smaller than the second CV value CV (1) in step S305, the minimum value is excluded. Since it was not appropriate, the process proceeds to step S321. In step S321, the abnormal data exclusion module 320 restores the minimum value excluded from the series of data of the group, and this time, the maximum value of the series of data of the group is excluded and defined again in the group. The standard deviation and average value of the current value I _pk2 obtained from all the other electrodes are calculated. In step S321, the standard deviation excluding the maximum value is divided by the average value excluding the maximum value to obtain the third CV value CV (2), and the process proceeds to step S322.

(F) In step S322, the abnormal data exclusion module 320 reads the first CV value CV (0) and the CV value correction coefficient dCV / CV from the CV value storage unit 361, and uses the first CV value CV (0) as the CV value correction coefficient dCV. A magnitude comparison between the value corrected (multiplied) by / CV and the third CV value CV (2) is performed:
(CV (0)) * (dCV / CV)> CV (2) (5)
(E) If the value obtained by correcting (multiplying) the first CV value CV (0) by the CV value correction coefficient dCV / CV in step S322 becomes smaller than the third CV value CV (2), the excluded value is abnormal. Since it can be determined that the value is a value, the process proceeds to step S325, and an error set is made in which the maximum value excluded in step S321 is “not subject to calculation”. The decision will be made excluding.

  (F) If the value obtained by correcting (multiplying) the first CV value CV (0) by the CV value correction coefficient dCV / CV in step S322 is not smaller than the third CV value CV (2), it is excluded. Since it can be determined that there is more power data, the process proceeds to step S323, where the data excluding the maximum value excluded in step S321 is defined as new data, and the process proceeds to step S324. In step S324, an error set in which the maximum value excluded in step S321 is “not subject to calculation” is set, the process returns to step S302, and the above procedure is repeated.

  (G) Similarly, if the value obtained by correcting (multiplying) the first CV value CV (0) by the CV value correction coefficient dCV / CV is not smaller than the second CV value CV (1) in step S305. In step S306, data excluding the minimum value excluded in step S304 is defined as new data, and the process proceeds to step S307. In step S324, an error set that sets the minimum value excluded in step S304 as “not subject to calculation” is set, the process returns to step S302, and the above procedure is repeated.

  Outliers are excluded by repeating the above routine for all groups.

The abnormal data exclusion module 320 makes an “error determination” when the current value I _pk2 obtained from the electrodes is not subject to calculation, and _causes the display device 306 and the output device 305 to output it. In subsequent steps, all because of the calculation to the true current value I _pk2, unless otherwise specified, it will be described as a "true current value I _pk2" simply "current value".

[Select from two types of algorithms]
Step S105 in FIG. 14 includes two types of algorithms as shown in FIG. For this reason, first, in step S332, the process proceeds to an algorithm flow for determining whether or not a certain nucleic acid is present, whether the SNP type is one of the two types, or is a G / G homotype. It is determined whether to proceed to an algorithm flow for determining whether there is a G / T hetero type or a T / T homo type.

[Step S105-1: Determination of Presence of Nucleic Acid]
If it is determined in step S332 in FIG. 27 that the process proceeds to an algorithm flow for determining whether or not a certain nucleic acid is present, the presence / absence determination module 330 determines in accordance with the procedure of the flowchart shown in FIG.

  FIG. 2 shows an electrode unit in which the SNP1 detection electrode 551, the SNP2 detection electrode 552, the control electrode 553, the reference electrodes 561, 562, and the counter electrode 502 are arranged on the detection chip. In the case of the algorithm for determining the SNP1, either the SNP1 detection electrode 551 or the SNP2 detection electrode 552 may be used. That is, on the substrate 714 shown in FIG. 4, a plurality of electrode units 761 each having one of the SNP1 detection electrode 551 and the SNP2 detection electrode 552 as a detection electrode (working electrode) are arranged. Here, description will be made assuming that the detection electrode (working electrode) for detecting genotype 1 is the SNP1 detection electrode 551 shown in FIG.

(A) In step S341, the presence / absence determination module 330 determines an average value (X ₁ ) of current values from a plurality of detection electrodes (working electrodes) 551 to be determined and an average of current values from the corresponding control electrode 553. The value (X _nc ) is calculated and stored in the average value / standard deviation storage unit 360.

(B) Further, in step S342, the presence / absence determination module 330 determines the standard deviation (σ ₁ ) of the current value from the plurality of detection electrodes (working electrodes) 551 to be determined, and the current from the control electrode 553 corresponding thereto. The standard deviation (σ _nc ) of the value is calculated and stored in the average value / standard deviation storage unit 360.

(C) Next, in step S343, it is determined whether there is any data abnormality due to a device (hardware) failure. That is, the presence / absence determination module 330 reads the setting parameter MSL from the MSL storage unit 365. The setting parameter MSL is set to a relatively small value, for example, a current value in the range of about 0 to 100 nA, as shown by the broken lines in FIGS. 24 (a) to (c) and FIGS. 25 (d) to (f). Keep it. The setting parameter MSL is determined in advance and stored in the MSL storage unit 365. In step S343, the presence / absence determination module 330 calculates the average value X ₁ of the currents of the plurality of detection electrodes 551 to be determined and the average value X _nc of the currents of the control electrodes 553 corresponding to the average value / standard deviation. It reads sequentially from the memory | storage part 360, and each confirms whether it is larger than the setting parameter MSL. In step S343, if at least _one of the average current value X _{1 of} the plurality of detection electrodes 551 to be determined and the average current value X _nc of the corresponding control electrode 553 is equal to or less than the setting parameter MSL, Since the measurement abnormality due to the device (hardware) failure is considered, the type determination is not performed, and the determination result is classified and stored in the classification result storage unit 369 in step S435 as shown in FIG. “Undecided (hardware error)” is displayed to indicate that “determination is impossible”. Figure 25 is (f), as a mode F, a current value SNP1 average X ₁ of the current detecting electrode (SNP = "G" detecting electrodes) 551, corresponds to the case where smaller than the set parameter MSL I _pk2 However, the SNP1 detection electrode (SNP = "G" detection electrode) 551 may be replaced with a plurality of detection electrodes 551 to be determined.

(D) In step S343, the average current value X _{1 of} the plurality of detection electrodes 551 to be determined and the average current value X _nc of the corresponding control electrode 553 are not less than the set parameter MSL. The process proceeds to step S344. Then, the presence / absence determination module 330 calculates the average value / standard deviation of the standard deviation (σ _nc ) of the current value from the control electrode 553 and the standard deviation (σ ₁ ) of the current value from the detection electrode (working electrode) 551. Reading from the storage unit 360, the sum (σ ₁ + σ _nc ) of the standard deviation (σ ₁ ) of the current value from the detection electrode (working electrode) 551 and the standard deviation (σ _nc ) of the current value from the control electrode 553 is obtained. In step S344, it is confirmed whether or not the sum of standard deviations (σ ₁ + σ _nc ) is not “zero”. When the sum of standard deviations (σ ₁ + σ _nc ) is “zero”, the calculation in the next step S345 cannot be performed. In step S351, the determination result is classified and stored in the classification result storage unit 369. Then, the display device 306 displays that the target nucleic acid is “automatic determination is impossible”. In step S341, if the sum of standard deviations (σ ₁ + σ _nc ) is not “zero”, the difference between the average values (X ₁ −X _nc ) and the sum of standard deviations (σ ₁ + σ _nc ) is calculated as the average value / standard deviation. The data is stored in the storage unit 360, and the process proceeds to step S345.

(E) In step S345, the presence / absence determination module 330 calculates the average value (X ₁ ) of the current value from the detection electrode (working electrode) 551 and the average value (X _nc ) of the current value from the corresponding control electrode 553. The difference between the average value (X ₁ ) of the current value from the detection electrode (working electrode) 551 and the average value (X _nc ) of the current value from the control electrode 553 (X ₁ -X _nc ). Further, in step S345, the presence / absence determination module 330 reads the standard deviation sum (σ ₁ + σ _nc ) from the average value / standard deviation storage unit 360, and calculates the standard deviation sum of the average value differences (X ₁ −X _nc ). (σ ₁ + σ _nc) against determining the ratio _{(X 1 -X nc) / (} σ 1 + σ nc). And the ratio Y ₁ of the difference between the average values and the standard deviation:
Y ₁ = (X ₁ −X _nc ) / (σ ₁ + σ _nc ) (6)
Is stored in the average value / standard deviation storage unit 360, and the process proceeds to step S346.

(F) In step S346, the presence / absence determination module 330 reads the average value difference (X ₁ −X _nc ) from the average value / standard deviation storage unit 360, and stores the signal increase determination criterion SLL (+/−) in the SLL. Read from the unit 362. In step S346, the magnitude relationship between the average value difference (X ₁ −X _nc ) and the signal increase determination criterion SLL (+/−) is compared. If it is determined in step S346 that the signal increase amount determination criterion SLL (+/−) or less, the current value of the electrochemical current of the target nucleic acid is not observed from the detection electrode (working electrode) 551. In step S350, the determination result is classified and stored in the classification result storage unit 369, and the determination of “-(none)” is displayed on the display device 306. On the other hand, if it is larger than the signal increase amount criterion SLL (+/−) in step S346, the process proceeds to step S347.

(G) In step S347, the presence / absence determination module 330 calculates the ratio Y ₁ = (X ₁ −X _nc ) / (σ ₁ + σ _nc ) between the average value difference and the standard deviation as the average value / standard deviation storage unit 360. The effective variation coefficient ESLL is read from the ESLL storage unit 363. In step S347, the magnitude relationship between the ratio Y ₁ = (X ₁ −X _nc ) / (σ ₁ + σ _nc ) of the sum of the difference between the average values and the standard deviation is compared with the effective variation coefficient ESLL. If it is determined in step S347 that it is smaller than the effective variation coefficient ESLL, the presence of the target nucleic acid is determined to be “unclear”, and the determination result is classified and stored in the classification result storage unit 369 in step S349. The determination of “unclear” is displayed on the display device 306. On the other hand, if the effective variation coefficient ESLL is greater than or equal to step S347, it is determined that the target nucleic acid is “obviously”, and the determination result is classified and stored in the classification result storage unit 369 and displayed in step S348. “+ (OK)” is displayed on the device 306. If the average value difference (X ₁ −X _nc ) is larger than the signal increase determination criterion SLL (+/−) in step S346, the determination of “+ (OK)” is estimated, but immediately “ “+ (OK)” is not displayed, and it is determined in step S347 whether or not there is variation in the current data.

[Step S105-2: SNP Type Determination]
In step S332 of FIG. 27, the SNP type is one of the two types of SNP = “G” and SNP = “T”, is it a G / G homotype, or G / T When it is determined that the process proceeds to an algorithm flow for determining whether it is a hetero type or a T / T homo type, the type determination module 340 determines in accordance with the procedure of the flowcharts shown in FIGS.

  The modes A to F shown in FIGS. 24 (a) to 24 (c) and FIGS. 25 (d) to 25 (f) are representative detection signals that are determined by the type determination module 340 according to the flowcharts shown in FIGS. 29 and 30. Examples (modes) are classified. The mode A shown in FIG. 24A is a standard detection mode, but the mode B shown in FIG. 24B is obtained from the SNP2 detection electrode (SNP = "T" detection electrode) 552. This is a detection mode that indicates a sample in which the signal is too small to expect an intentional signal increase. On the other hand, mode C shown in FIG. 24C shows a sample in which the signal from the SNP1 detection electrode (SNP = "G" detection electrode) 551 is too small to expect an increase in the intended signal. Detection mode.

  In addition, mode D shown in FIG. 25D is a signal from the SNP1 detection electrode (SNP = "G" detection electrode) 551 and the signal from the SNP2 detection electrode (SNP = "T" detection electrode) 552. This is a detection mode in which both signals are too small to be judged and excluded as abnormal data. Further, in the mode E shown in FIG. 25E, the signal from the SNP1 detection electrode (SNP = "G" detection electrode) 551 and the signal from the SNP2 detection electrode (SNP = "T" detection electrode) 552 are used. This is a detection mode in which the signal is much smaller than in mode D and cannot be determined at the bio level and is excluded as abnormal data. Further, in the mode F shown in FIG. 25 (f), the signal from the SNP1 detection electrode (SNP = "G" detection electrode) 551 is abnormally small, and conversely, the SNP2 detection electrode (SNP = "T" detection) This is a detection mode in which the signal from the electrode 552 is abnormally large and a hardware level problem is estimated and excluded as abnormal data.

The type determination module 340 is classified into the modes A to F shown in FIGS. 24A to 24C and FIGS. 25D to 25F in the procedure of the flowcharts shown in FIGS. However, for example, it is determined whether the base at a certain SNP position is a G / G homotype, a G / T heterotype, or a T / T homotype.
(A) First, in step S361, the type determination module 340 determines whether the number of targets is two. Since two types of SNP = "G" and SNP = "T" are to be determined, the initial setting is wrong in the first place when the number of targets is 0, 1, 3, and so on, as shown in FIG. As described above, in step S 436, the determination result is classified and stored in the classification result storage unit 369, and “setting error” is displayed on the display device 306.

(B) If it is determined in step S361 that the number of targets is two, the type determination module 340 proceeds to step S362. In step S362, the type determination module 340 determines the average value X ₁ of the current values of the SNP1 detection electrode (SNP = "G" detection electrode) 551 and the current of the control electrode (NC1) 553 corresponding to the SNP1 detection electrode 551. average mean X _nc1, SNP2 current value detecting electrodes (SNP = "T" detecting electrode) 552 of the current value of the average value X _2, SNP2 control corresponding to the detection electrode 552 (NC2) electrodes 553 of the value The value X _nc2 is calculated.

Although FIG. 2 shows the common control electrode 553 corresponding to the SNP1 detection electrode 551 and the SNP2 detection electrode, the control electrode (NC1) corresponding to the SNP1 detection electrode 551 and the control corresponding to the SNP2 detection electrode 552 are shown. (NC2) may be provided separately. The calculated average value X ₁ of the current value of the SNP1 detection electrode 551, the average value X _nc1 of the current value of the control electrode (NC1) 553 corresponding to the SNP1 detection electrode 551, and the average value of the current value of the SNP2 detection electrode 552 The average value X _nc2 of the current value of the control (NC2) electrode 553 corresponding to the X ₂ , SNP2 detection electrode 552 is stored in the average value / standard deviation storage unit 360.

(C) In step S363, the type determination module 340 determines the standard deviation σ ₁ of the current value of the SNP1 detection electrode 551, the standard deviation σ _nc1 of the current value of the control electrode (NC1) 553 corresponding to the SNP1 detection electrode 551, The standard deviation σ ₂ of the current value of the SNP ₂ detection electrode 552 and the standard deviation σ _nc2 of the current value of the control (NC2) electrode 553 corresponding to the SNP ₂ detection electrode 552 are calculated. The standard deviation σ _{1 of} the calculated current value of the SNP1 detection electrode 551, the standard deviation σ _nc1 of the current value of the control electrode (NC1) 553 corresponding to the SNP1 detection electrode 551, and the standard deviation of the current value of the SNP2 detection electrode 552 The standard deviation σ _nc2 of the current value of the control (NC2) electrode 553 corresponding to the σ ₂ , SNP2 detection electrode 552 is stored in the average value / standard deviation storage unit 360.

(D) Next, the type determination module 340 reads the setting parameter MSL from the MSL storage unit 365. The setting parameter MSL is set to a relatively small value, for example, a current value in the range of about 0 to 100 nA, as shown by the broken lines in FIGS. 24 (a) to (c) and FIGS. 25 (d) to (f). Keep it. The setting parameter MSL is determined in advance and stored in the MSL storage unit 365. Then, in step S364, the type determination module 340 causes the average value X ₁ of the current value of the SNP1 detection electrode 551 and the average value X _nc1 and SNP2 of the current value of the control electrode (NC1) 553 corresponding to the SNP1 detection electrode 551. The average value X ₂ of the electric current flow of the detection electrode 552 and the average value X _nc2 of the current value of the control (NC2) electrode 553 corresponding to the SNP2 detection electrode 552 are sequentially read from the average value / standard deviation storage unit 360, It is confirmed whether each is larger than the setting parameter MSL. In step S364, if the average value X ₁ of the current value of the SNP1 detection electrode 551, the average value X _nc1 of the current value of the control electrode (NC1) 553 corresponding to the SNP1 detection electrode 551, the current of the SNP2 detection electrode 552, If at least one of the average value X _{2 of} the values and the average value X _nc2 of the current value of the control (NC 2) electrode 553 corresponding to the SNP ₂ detection electrode 552 is less than the set parameter MSL, it is due to a device (hardware) malfunction. Since the measurement abnormality is considered, the type determination is not performed, and as shown in FIG. 30, the determination result is classified and stored in the classification result storage unit 369 in step S435, and “undecided (hardware error ) ”Is displayed to indicate that“ determination is impossible ”. In FIG. 25 (f), as mode F, current value I corresponding to the case where average value X ₁ of the current value of SNP 1 detection electrode (SNP = “G” detection electrode) 551 is smaller than setting parameter MSL. The distribution of _pk2 is shown.

(E) Next, the type determination module 340 reads the signal increase amount determination criterion SLL (M) from the SLL storage unit 362. SLL (M) is a setting parameter that gives a selection criterion for a determination algorithm of a signal increase amount for the control electrode 553. Then, in step S365, the type determination module 340 calculates the average value X _nc1 of the current value of the control electrode (NC1) 553 corresponding to the average value X ₁ of the current value of the SNP1 detection electrode 551 as an average value / standard deviation storage unit. 360, a difference (X ₁ −X _nc1 ) between the average value X ₁ of the current value of the SNP1 detection electrode 551 and the average value X _nc1 of the current value of the control electrode (NC1) 553 is determined, and the signal increase amount criterion The magnitude relationship with SLL (M) is compared, and the average value difference (X ₁ −X _nc1 ) on the _{SNP 1} detection electrode 551 side is stored in the average value / standard deviation storage unit 360. Further, in step S365, the type determination module 340 calculates the average value X _nc2 of the current value of the control electrode (NC2) 553 corresponding to the average value X ₂ of the current value of the SNP2 detection electrode 552 and the average value / standard deviation storage unit. 360, the difference (X ₂ −X _nc2 ) between the average value X ₂ of the current value of the SNP2 detection electrode 552 and the average value X _nc2 of the current value of the control electrode (NC2) 553 is determined, and the signal increase criterion The magnitude relationship with SLL (M) is compared, and the average value difference (X ₂ −X _nc2 ) on the SNP 2 detection electrode 552 side is stored in the average value / standard deviation storage unit 360. In step S365, the difference between the average values on the SNP1 detection electrode 551 side (signal increase amount) (X ₁ −X _nc1 ) and the difference between the average values on the SNP2 detection electrode 552 side (signal increase amount) (X ₂ −X _nc2 ). ) Are smaller than SLL (M), there is a possibility that the chip or the sample (biosample) is abnormal. As shown in FIG. 30, the classification result storage unit 369 determines in step S434. The results are classified and stored, and “undecided (sample error)” is displayed on the display device 306, indicating that “determination is impossible”. In FIG. 25 (e), as mode E, an average value difference (X ₁ −X _nc1 ) on the SNP1 detection electrode 551 side and an average value difference (X ₂ −X _nc2 ) on the _SNP2 detection electrode 552 side are shown. The distribution of the current value I _pk2 corresponding to the case where both are smaller than SLL (M) is shown. In step S365, at least _one of the difference between the average values on the SNP1 detection electrode 551 side (X ₁ −X _nc1 ) and the average value difference on the SNP2 detection electrode 552 side (X ₂ −X _nc2 ) is SLL (M). If larger, the process proceeds to step S366.

(F) In step S366, the type determination module 340 determines the signal increase determination criterion SLL (M) from the SLL storage unit 362, and the difference between the average value / standard deviation storage unit 360 and the average value on the SNP2 detection electrode 552 side ( X ₂ −X _nc2 ) is read out, and the magnitude relationship between the average value difference (X ₂ −X _nc2 ) and the signal increase determination criterion SLL (M) is compared. When the difference (X ₂ −X _nc2 ) in the average value on the SNP 2 detection electrode 552 side is smaller than the signal increase amount determination criterion SLL (M), this is the difference in the average value on the SNP 1 detection electrode 551 side (X ₁ − X _nc1 ) is only larger than SLL (M), and since it becomes a candidate to be a homozygous SNP1 base (G), the process proceeds to step S371. If it is determined in step S366 that the average difference (X ₂ −X _nc2 ) on the _SNP2 detection electrode 552 side is larger than the signal increase determination criterion SLL (M), the process proceeds to step S367.

(G) In step S367, the type determination module 340 reads the average value difference (X ₁ −X _nc1 ) on the SNP1 detection electrode 551 side from the average value / standard deviation storage unit 360, and the average value difference (X ₁ -X _nc1 ) and the signal increase determination criterion SLL (M) are compared in magnitude. When the difference (X ₁ −X _nc1 ) on the _{SNP 1} detection electrode 551 side is smaller than the signal increase amount determination criterion SLL (M), this is the difference between the average values on the SNP 2 detection electrode 552 side (X ₂ − Only X _nc2 ) is larger than SLL (M), and since it becomes a candidate to be a homozygous form of SNP2 base (T), the process proceeds to step S373. In step S367, if the difference (X ₁ −X _nc1 ) in the average value on the SNP1 detection electrode 551 side is larger than the signal increase determination criterion SLL (M), the difference in the average value on the SNP1 detection electrode 551 side (X ₁₋ X _nc1 ) and the difference between the average values (X ₂ −X _nc2 ) on the _SNP2 detection electrode 552 side are both larger than the signal increase determination criterion SLL (M), and the process proceeds to step S368. It moves to hetero judgment.

(H) The type determination module 340 reads the average value difference (X ₂ −X _nc2 ) on the SNP 2 detection electrode 552 side from the average value / standard deviation storage unit 360, and in step S 368, the NP 1 detection electrode 551 side The ratio (X ₁ −X _nc1 ) / (X ₂ −X _nc2 ) of the average value difference (X ₁ −X _nc1 ) to the average value difference (X ₂ −X _nc2 ) on the SNP 2 detection electrode 552 side is obtained. . Further, in step S368, an R value that is Log ₁₀ of the absolute value of this ratio (X ₁ −X _nc1 ) / (X ₂ −X _nc2 ) is calculated:
R = Log ₁₀ (abs ((X ₁ −X _nc1 ) / (X ₂ −X _nc2 ))) (7)
Then, the calculated R value is stored in the absolute value logarithm storage unit 367, and the process proceeds to step S368.

(I) In step S369, the type determination module 340 defines the average value X _nc1 of the current of the control electrode (NC1) 553 corresponding to the SNP1 detection electrode 551 as X _cmp1, and stores it in the average value / standard deviation storage unit 360. Store. Further, the standard deviation σ _nc1 of the current of the control electrode (NC1) 553 corresponding to the SNP1 detection electrode 551 is _set to σ _cmp1, and the average value X _nc2 of the current of the control (NC2) electrode 553 corresponding to the SNP2 detection electrode 552 is calculated. and X _cmp2, defined as SNP2 control corresponding to the detection electrode 552 (NC2) standard deviation sigma _nc2 of the current of the electrode 553 sigma _cmp2, and stores each average value, standard deviation memory 360, the process proceeds to step S401. The type determination module 340 reads the log signal ratio determination criterion (+ HLL) from the HLL storage unit 368, and compares the magnitude relationship between the R value and the log signal ratio determination criterion (+ HLL) in step S401. In step S401, if the R value is equal to or greater than the logarithmic signal ratio criterion (+ HLL) (if it is determined to be inside the diagonal line labeled HLL close to the vertical axis in FIG. 31 and the vertical axis), SNP1 A homotype of the base (G) is estimated, and the process proceeds to step S402. If the R value is smaller than the log signal ratio criterion (+ HLL) in step S401, the process proceeds to step S411. In step S411, the magnitude relation between the R value and the log signal ratio determination criterion (-HLL) is compared. If it is determined in step S411 that the R value is equal to or less than the logarithmic signal ratio criterion (-HLL) (if it is determined that it is inside the diagonal line labeled HLL close to the horizontal axis and the horizontal axis in FIG. 31). , A homozygous SNP 2 base (T) is estimated, and the process proceeds to step S412. If it is determined in step S411 that the R value is greater than the log signal ratio criterion (-HLL), that is, the R value is between the log signal ratio criterion (-HLL) and (+ HLL), SNP1 / SNP2, Since it becomes a G / T hetero-type candidate, the process proceeds to step S421. In step S421, the log signal ratio determination criterion HUL is read from the HUL storage unit 364, and the magnitude relationship between the absolute value of the R value and the log signal ratio determination criterion HUL is compared. If it is determined in step S421 that the absolute value of the R value is equal to or smaller than the logarithmic signal ratio determination criterion HUL (if it is determined to be inside the hatched area labeled with two HULs in FIG. 31), SNP1 / SNP2 The hetero type is estimated, and the process proceeds to step S422. If it is determined in step S421 that the absolute value of the R value is larger than the logarithmic signal ratio determination criterion HUL (if it is determined to be outside the hatched area labeled with two HULs in FIG. 31), FIG. As shown, the determination result is classified and stored in the classification result storage unit 369 in step S430, “automatically determined (homo or hetero)” is displayed on the display device 306, and “determination is impossible”. Indicates.

(J) Now, let us move on to step S371. As already described, if the difference (X ₂ −X _nc2 ) in the average value on the SNP2 detection electrode 552 side is smaller than the signal increase determination criterion SLL (M) in step S366, the homozygous form of the SNP1 base (G) The process proceeds to step S371. In step S371, the type determination module 340 defines the average value X ₂ of the current of the SNP 2 detection electrode 552 as X _cmp1 and the standard deviation σ ₂ of the current of the SNP 2 detection electrode 552 as σ _cmp1 , respectively. The data is stored in the standard deviation storage unit 360, and the process proceeds to step S372. In step S372, the type determination module 340 reads the significance determination reference magnification SLR from the SLR storage unit 366. Typing module 340, further, the average value and the standard deviation memory mean X ₁ 360 from SNP1 current value of the detection electrodes 551, reads out the X _cmp1 that determined in step S371, the average value X ₁ and the average value X _cmp1 Ratio (= magnification) X ₁ / X _cmp1 is obtained and compared with the significance criterion magnification SLR. If the average value ratio X ₁ / X _cmp1 is less than or equal to the significance determination reference magnification SLR in step S372, it is determined that the current increase amount of the SNP1 detection electrode 551 is not sufficient, and classification is performed in step S433 as shown in FIG. The determination result is classified and stored in the result storage unit 369, and “undecided (small signal)” is displayed on the display device 306, indicating that “determination is impossible”. FIG. _25D shows the distribution of the current value I _pk2 corresponding to the case where the current increase amount of the SNP1 detection electrode (SNP = “G” detection electrode) 551 is not sufficient as the mode D. If the average ratio X ₁ / X _cmp1 is larger than the significance determination reference magnification SLR in step S372, a homozygous SNP1 base (G) is estimated, and the process proceeds to step S402. Although the value of the counterpart taking X ₁ and a ratio, in step S372, although the X _cmp1 (= X _2), it also makes sense to partner taking the ratio of the X _nc1. That is, in the case of a SNP1 base (G) homozygous candidate, X ₁ / X _nc1 may be compared in size with SLR.

(K) Next, step S373 will be described. As already described, when the difference (X ₁ −X _nc1 ) of the average value on the SNP1 detection electrode 551 side is smaller than the signal increase determination criterion SLL (M) in step S367, the SNP2 base (T) homozygous type The process proceeds to step S373. In step S373, the average value X ₁ of the current value of the SNP1 detecting electrodes 551 is defined as X _cmp2, it defines a standard deviation sigma ₁ of the current values of the SNP1 detecting electrode 551 and the sigma _cmp2, respectively mean, standard deviation The data is stored in the storage unit 360, and the process proceeds to step S374. In step S374, the type determination module 340 reads the significance determination reference magnification SLR from the SLR storage unit 366. Furthermore, the typing module 340, the average value and the average value X ₂ of the current value of the standard deviation memory 360 from the SNP2 detecting electrodes 552, reads out the X _cmp2 that determined in step S371, the average value X ₂ and the average value X _cmp2 Ratio (= magnification) X ₂ / X _{cmp 2} is obtained and compared with the significance criterion magnification SLR. If the average value ratio X ₂ / X _cmp2 of less significant criterion magnification SLR in the step S374, the current increase of SNP2 detecting electrodes 552 is insufficient, as shown in FIG. 30, in step S433, the classification The determination result is classified and stored in the result storage unit 369, and “undecided (small signal)” is displayed on the display device 306, indicating that “determination is impossible”. Is greater than the ratio X ₂ / X _cmp2 significant criterion magnification SLR mean values in step S374, the estimated homo-type SNP2 base (T), the process proceeds to step S412. Although the value of the counterpart taking X ₂ and the ratio, in step S372, although the X _cmp2 (= X _1), it also makes sense to partner taking the ratio of the X _nc2. That is, in the case of a SNP 2-base (T) homozygous candidate, X ₂ / X _nc2 may be compared in size with SLR.

(L) Here, the description proceeds to step S402. In step S402, the type determination module 340 calculates the average value / standard deviation of the standard deviation (σ ₁ ) of the current value of the SNP1 detection electrode 551 and the standard deviation (σ _cmp1 ) of the corresponding comparison target current value determined in step S369. Reading from the storage unit 360, the sum (σ ₁ + σ _cmp1 ) of the standard deviation (σ ₁ ) of the current value of the SNP1 detection electrode 551 and the standard deviation (σ _cmp1 ) of the comparison target current value is obtained. Furthermore, in step S402, it is confirmed whether or not the sum of standard deviations (σ ₁ + σ _cmp1 ) is not “zero”. When the sum of standard deviations (σ ₁ + σ _cmp1 ) is “zero”, the calculation in the next step S403 cannot be performed, and therefore the determination result is classified and stored in the classification result storage unit 369 in step S406. Then, the display device 306 displays “automatic determination impossible”. In step S402, if the sum of standard deviations (σ ₁ + σ _cmp1 ) is not “zero”, the process proceeds to step S403.

(M) In step S403, the type determination module 340 reads the average value difference (X ₁ −X _cmp1 ) from the average value / standard deviation storage unit 360, and the standard deviation of the average value difference (X ₁ −X _cmp1 ). the sum (σ ₁ + σ _cmp1) against the ratio of the _{(X 1 -X cmp1) / (} σ 1 + σ cmp1). And the ratio Y ₁ of the difference between the average values and the standard deviation:
Y ₁ = (X ₁ −X _cmp1 ) / (σ ₁ + σ _cmp1 ) (8)
Is stored in the average value / standard deviation storage unit 360, and the process proceeds to step S404.

(N) In step S404, the type determination module 340 calculates the ratio Y ₁ = (X ₁ −X _cmp1 ) / (σ ₁ + σ _cmp1 ) of the average value difference and the standard deviation as the average value / standard deviation storage unit 360. The effective variation coefficient ESLL is read from the ESLL storage unit 363. In step S404, the magnitude relationship between the ratio Y ₁ = (X ₁ −X _cmp1 ) / (σ ₁ + σ _cmp1 ) of the difference between the average values and the standard deviation is compared with the effective variation coefficient ESLL. If it is determined in step S404 that it is smaller than the effective variation coefficient ESLL, it is determined that the presence of the homozygous SNP1 base (G) is “unclear”, and the determination result is stored in the classification result storage unit 369 in step S407. The data is classified and stored, and the determination of “G / G homo” is unclear ”is displayed on the display device 306. On the other hand, if the effective variation coefficient ESLL is greater than or equal to step S404, it is determined that the SNP1 base (G) homotype is “obviously”, and the determination result is classified into the classification result storage unit 369 in step S405. Then, the display device 306 displays ““ G / G homo ”OK”.

(O) Next, the description proceeds to step S412. Typing module 340, in step S412, the average value and the standard deviation standard deviation of the corresponding current values to be compared with (sigma _cmp2) that defines the standard deviation (sigma ₂₎ and step S369 of the current value of the SNP2 detecting electrode 552 from the storage unit 360, the sum (σ ₂ + σ _cmp2) of the standard deviation of the compared current values and the standard deviation (sigma ₂₎ of the current value of the SNP2 detecting electrode 552 (σ _cmp2). Furthermore, in step S412, it is confirmed whether or not the sum of standard deviations (σ ₂ + σ _cmp2 ) is not “zero”. When the sum of standard deviations (σ ₂ + σ _cmp2 ) is “zero”, the calculation in the next step S413 cannot be performed, and therefore the determination result is classified and stored in the classification result storage unit 369 in step S416. Then, the display device 306 displays “automatic determination impossible”. If the sum of standard deviations (σ ₂ + σ _cmp2 ) is not “zero” in step S412, the process proceeds to step S413.

(P) In step S413, the type determination module 340 reads the average value difference (X ₂ −X _cmp2 ) from the average value / standard deviation storage unit 360, and the standard deviation of the average value difference (X ₂ −X _cmp2 ). The ratio (X ₂ −X _{cmp 2} ) / (σ ₂ + σ _{cmp 2} ) to the sum of (σ ₂ + σ _{cmp 2} ) is obtained. And the ratio Y ₂ of the difference between the average values and the standard deviation:
Y ₂ = (X ₂ −X _{cmp 2} ) / (σ ₂ + σ _{cmp 2} ) (9)
Is stored in the average value / standard deviation storage unit 360, and the process proceeds to step S414.

(Q) In step S 414, the type determination module 340 calculates the ratio Y ₂ = (X ₂ −X _{cmp 2} ) / (σ ₂ + σ _{cmp 2} ) as the average value / standard deviation storage unit 360. The effective variation coefficient ESLL is read from the ESLL storage unit 363. Then, in step S414, the average value of the difference and the sum of the standard deviation ratio _{Y 2 = (X 2 -X cmp2} ) / (σ 2 + σ cmp2), compares the magnitude relationship between the effective coefficient of variation ESLL. If it is determined in step S414 that it is smaller than the effective variation coefficient ESLL, it is determined that the presence of the homozygous SNP 2 base (T) is “unclear”, and the determination result is classified in the classification result storage unit 369 in step S417. The display device 306 displays “T / T homo” unclear ”determination. On the other hand, if the effective variation coefficient ESLL is greater than or equal to step S414, it is determined that the SNP 2-base (T) homotype is “obviously”, and the determination result is classified into the classification result storage unit 369 in step S415. The data is stored, and “T / T homo” OK ”is displayed on the display device 306.

(R) Now, let us move on to step S422. In step S422, the type determination module 340 calculates the average value / standard deviation of the standard deviation (σ ₁ ) of the current value of the SNP1 detection electrode 551 and the standard deviation (σ _cmp1 ) of the corresponding comparison target current value determined in step S369. Reading from the storage unit 360, the sum (σ ₁ + σ _cmp1 ) of the standard deviation (σ ₁ ) of the peak current value from the SNP1 detection electrode 551 and the standard deviation (σ _cmp1 ) of the comparison target current value is obtained. Furthermore, in step S422, it is confirmed whether or not the sum of standard deviations (σ ₁ + σ _cmp1 ) is not “zero”. When the sum of standard deviations (σ ₁ + σ _cmp1 ) is “zero”, the calculation in the next step S423 cannot be performed. Therefore, the determination result is classified and stored in the classification result storage unit 369 in step S432. Then, the display device 306 displays “automatic determination impossible”. If the sum of standard deviations (σ ₁ + σ _cmp1 ) is not “zero” in step S422, the process proceeds to step S423.

(S) In step S423, the type determination module 340 reads the average value difference (X ₁ −X _cmp1 ) from the average value / standard deviation storage unit 360, and the standard deviation of the average value difference (X ₁ −X _cmp1 ). A ratio (X ₁ −X _cmp1 ) / (σ ₁ + σ _cmp1 ) to the sum of (σ ₁ + σ _cmp1 ) is obtained (see formula (8)). Then, the ratio Y ₁ of the difference between the average values and the standard deviation is stored in the average value / standard deviation storage unit 360, and the process proceeds to step S424.

In (t) step S424, the typing module 340, the corresponding reference standard deviation of the target current value (sigma _cmp2) the average value determined by the standard deviation (sigma ₂₎ and step S369 of the current value of the SNP2 detecting electrode 552 and reading from the standard deviation memory 360, the sum (σ ₂ + σ _cmp2) of the standard deviation of the compared current values and the standard deviation (sigma ₂₎ of the current value of the SNP2 detecting electrode 552 (σ _cmp2). In step S424, it is confirmed whether or not the sum of standard deviations (σ ₂ + σ _cmp2 ) is not “zero”. If the sum of standard deviations (σ ₂ + σ _cmp2 ) is “zero”, the calculation in the next step S425 cannot be performed. Then, the display device 306 displays “automatic determination impossible”. If the sum of standard deviations (σ ₂ + σ _cmp2 ) is not “zero” in step S424, the process proceeds to step S425.

(U) In step S425, the type determination module 340 reads the average value difference (X ₂ −X _cmp2 ) from the average value / standard deviation storage unit 360, and the standard deviation of the average value difference (X ₂ −X _cmp2 ). The ratio (X ₂ −X _{cmp 2} ) / (σ ₂ + σ _{cmp 2} ) to the sum of (σ ₂ + σ _{cmp 2} ) is obtained. Then, the ratio Y ₂ (see equation (9)) of the difference between the average values and the standard deviation is stored in the average value / standard deviation storage unit 360, and the process proceeds to step S426.

(V) In step S426, the type determination module 340 calculates the ratio Y ₁ = (X ₁ −X _cmp1 ) / (σ ₁ + σ _cmp1 ) of the average value difference and the standard deviation as the average value / standard deviation storage unit 360. The effective variation coefficient ESLL is read from the ESLL storage unit 363. In step S426, the magnitude relationship between the ratio Y ₁ = (X ₁ −X _cmp1 ) / (σ ₁ + σ _cmp1 ) of the difference between the average values and the standard deviation is compared with the effective variation coefficient ESLL. In step S 426, the type determination module 340 obtains the ratio Y ₂ = (X ₂ −X _{cmp 2} ) / (σ ₂ + σ _{cmp 2} ) between the average value difference and the standard deviation from the average value / standard deviation storage unit 360. reading, in step S426, the average value of the difference and the sum of the standard deviation ratio _{Y 2 = (X 2 -X cmp2} ) / (σ 2 + σ cmp2), compares the magnitude relationship between the effective coefficient of variation ESLL. If it is determined in step S426 that either _one of Y ₁ and Y ₂ is smaller than the effective variation coefficient ESLL, it is determined that the presence of the G / T heterotype is “unclear”, and in step S429, the classification result storage unit The determination result is classified and stored in 369, and the determination of ““ G / T heterology ”unclear” is displayed on the display device 306. On the other hand, if Y ₁ and Y ₂ are simultaneously greater than or equal to the effective variation coefficient ESLL in step S426, it is determined that the G / T heterotype is “obviously”, and the determination result is stored in the classification result storage unit 369 in step S427. Are classified and stored, and "" G / T hetero "OK" is displayed on the display device 306.

As can be understood from the above description, according to the base sequence method according to the embodiment of the present invention,
Even when there is an abnormality in the chip cartridge 11 or the measurement system 12 or when there is a variation in data, it is possible to accurately determine whether or not a certain nucleic acid exists by excluding such abnormal data. . Further, according to the base sequence method according to the embodiment of the present invention, if there is an error in the initial setting, it is determined as “setting error”, and if there is an abnormality in the chip or the sample (biosample), “undecided (sample error) If the device (hardware) has a defect, the abnormal data can be excluded by performing a process such as “undecided (hardware error)”. In addition, if the signal cannot be determined due to signal variation, or if the signal strength is weak or unclear due to other causes, it is “unclear”, “automatically undecided (homo or hetero)”, “undecided (small signal)” , “Automatic judgment impossible”, ““ 1/1 homo ”unclear”, “2/2 homo” unclear ”,“ “1/2 hetero” unclear ”, etc. It is. For this reason, according to the nucleotide sequence method according to the embodiment of the present invention, ““ 1/1 homo ”OK”, ““ 2/2 homo ”are used appropriately depending on various measurement environments and situations (actual conditions). It is possible to determine with high accuracy whether the SNP type is a homo type or a hetero type, such as “OK”, “1/2 hetero” OK ”, etc. . Here, what is conveniently expressed as “1” or “2” is actually displayed as “A”, “T”, “G”, “C” or the like.

(Base sequence determination program)
The series of determination operations shown in FIG. 14, FIG. 16, FIG. 18-19, and FIG. 26-30 are performed as shown in FIG. 8 by a program of an algorithm equivalent to that shown in FIG. The base sequence determination system shown can be controlled and executed. This program may be stored in a program storage device (not shown) of a computer system constituting the base sequence determination system of the present invention. The program can be stored in a computer-readable recording medium, and the recording medium can be read into the program storage device of the base sequence determination system to execute the series of determination operations of the present invention. Here, the “computer-readable recording medium” means a medium capable of recording a program such as an external memory device of a computer, a semiconductor memory, a magnetic disk, an optical disk, a magneto-optical disk, and a magnetic tape. To do. Specifically, a “flexible disk, CD-ROM, MO disk, cassette tape, open reel tape, memory card, hard disk, removable disk, etc. are included in the“ computer-readable recording medium ”.

  For example, the main body of the base sequence determination system can be configured to incorporate or externally connect a flexible disk device (flexible disk drive) and an optical disk device (optical disk drive). A flexible disk is inserted into the flexible disk drive, and a CD-ROM is inserted into the optical disk drive through the insertion slot, and the program stored in these recording media is sequenced by performing a predetermined read operation. It can be installed in a program storage device constituting the determination system. Further, by connecting a predetermined drive device, for example, a ROM as a memory device used in a game pack or the like, or a cassette tape as a magnetic tape device can be used. Furthermore, this program can be stored in a program storage device via an information processing network such as the Internet.

(Other embodiments)
As described above, the present invention has been described according to the above-described embodiments. However, it should not be understood that the description and drawings constituting a part of this disclosure limit the present invention. From this disclosure, various alternative embodiments, examples and operational techniques will be apparent to those skilled in the art.

  In the above description of the above-described embodiment, a method for determining whether it corresponds to the G type, the T type, or the G / T type is shown. However, the A type, the G type, the C type, and the T type are shown. Of course, the present invention can be applied to the determination of any two of these types or their heterogeneity. Further, as can be understood from the description of the above embodiment, it is not always necessary to acquire measurement data for the four types of groups of A type, G type, C type, and T type, and two possible bases of the SNP are considered. It is also possible to obtain only about two groups.

  As described above, the present invention naturally includes various embodiments not described herein. Therefore, the technical scope of the present invention is defined only by the invention specifying matters according to the scope of claims reasonable from the above description.

It is a circuit diagram explaining an example of the measurement system which comprises the base sequence determination system which concerns on embodiment of this invention. It is a typical top view explaining the structure of the detection chip which makes a part of measurement system of FIG. FIG. 3A is a schematic diagram for explaining a state in which a probe DNA having the base sequence GACTC... Is immobilized on the SNP = “G” detection electrode, and FIG. FIG. 3 (c) is a schematic diagram for explaining a state in which a probe DNA having a base sequence GAATC... Is immobilized on the "T" detection electrode. It is a schematic diagram explaining the state by which probe DNA (negative control DNA) which has was fixed. It is a typical assembly bird's-eye view explaining an example of the structure of the chip cartridge used for the base sequence determination system concerning an embodiment of the invention. FIG. 5 is an assembly bird's-eye view of the chip cartridge according to the embodiment of the present invention, showing FIG. 4 upside down. It is a schematic diagram which shows the whole structure of the valve unit with which the liquid feeding system which comprises the base sequence determination system which concerns on embodiment of this invention is equipped. It is a conceptual block diagram explaining an example of the base sequence determination system which concerns on embodiment of this invention. It is a conceptual block diagram explaining an example of the structure of the computer which comprises the base sequence determination system which concerns on embodiment of this invention. FIG. 9 (a) shows a state in which double-stranded strands are formed because the probe DNA of the SNP1 detection electrode and the sample DNA of SNP = “G” are completely matched in base sequence. FIG. 9C shows a state in which the probe DNA of the SNP2 detection electrode and the sample DNA of SNP = “G” cannot form a double strand, FIG. 9C shows the sample of the negative control DNA of the control electrode and SNP = “G” It is a schematic diagram which shows the state which cannot form a double strand with DNA. 10A shows a state where the probe DNA of the SNP1 detection electrode and the sample DNA of SNP = “T” cannot form a double strand, and FIG. 10B shows the probe DNA and SNP of the SNP2 detection electrode. FIG. 10C shows a state in which the negative control DNA of the control electrode and the sample DNA of SNP = “T” cannot form a double strand. It is a schematic diagram shown. FIG. 11A shows a state in which the probe DNA of the SNP1 detection electrode and the sample DNA of SNP = “G / T” heterogeneous form a double strand, and FIG. 11B shows the probe of the SNP2 detection electrode. FIG. 11C shows a state in which the DNA and SNP = “G / T” heterogeneous sample DNA form a double strand. FIG. 11C shows the negative control DNA of the control electrode and the SNP = “G / T” heterogeneous sample DNA. It is a schematic diagram which shows the state which cannot form a double strand. FIG. 12A shows a state in which the probe DNA of the electrode for detecting SNP1 and the sample DNA of SNP = “G” form a double strand, and therefore the state where the intercalating agent is bound to the double strand is shown in FIG. FIG. 12 (c) shows a state where the probe DNA of the electrode for SNP2 detection and the sample DNA of SNP = “G” cannot form a double strand, so that an intercalator cannot bind to the double strand. FIG. 4 is a schematic diagram showing a state in which an intercalator cannot bind to a double strand because the negative control DNA of the control electrode and the sample DNA of SNP = “G” cannot form a double strand. Curve (a) corresponds to FIG. 12 (a), in which the probe DNA of the SNP1 detection electrode and the sample DNA of SNP = "G" form a double strand, and the insertion agent is bound to the double strand. This is a current-voltage characteristic of the electrochemical current of FIG. Curve (b) corresponds to FIG. 12 (b), and the probe DNA of the SNP2 detection electrode and the sample DNA of SNP = "G" cannot form a double strand, and the intercalator cannot bind to the double strand. Current-voltage characteristics, and shows a peak of a small current value as compared with the curve (a). The curve (c) in FIG. 13 corresponds to FIG. 12 (c), and the negative control DNA of the control electrode and the sample DNA of SNP = “G” cannot form a double strand, and are inserted into the double strand. This is a current-voltage characteristic when the agent cannot be bonded, and shows a peak with a smaller current value as compared with the curve (b). It is a flowchart explaining the flow as a whole of the base sequence determination method which concerns on embodiment of this invention. It is a schematic diagram explaining the leveling (smoothing) by a simple moving average method. In the base sequence determination method according to the embodiment of the present invention, each current waveform (current−) is determined by the slope of each baseline of the current waveform (current-voltage characteristics) of the electrochemical current measured for each electrode. It is a flowchart explaining an example of the method of determining normality / abnormality of a voltage characteristic. Two types of current-voltage characteristics of data 1 and data 2 are shown as examples of electrochemical current signals in the chip cartridge. The slope of the baseline indicated by the current-voltage characteristic of data 2 is larger than the slope of the baseline indicated by the current-voltage characteristic of data 1. In the base sequence determination method according to the embodiment of the present invention, the true current detection signal is obtained by subtracting each background current from the current waveform (current-voltage characteristics) of the electrochemical current measured for each electrode. It is a flowchart explaining an example of the method of calculating | requiring a peak value (peak current value). Following the procedure of the flowchart shown in FIG. 18, the flow of the procedure of the method for obtaining the peak value (peak current value) of the true detection signal from the current waveform (current-voltage characteristics) measured for each electrode will be described. It is a flowchart for. In the base sequence determination method according to the embodiment of the present invention, in order to subtract each background current from the current waveform (current-voltage characteristics) of the electrochemical current measured for each electrode, the electrochemical current It is a schematic diagram explaining the method of calculating | requiring the point in which the differential value (di / dv) of "is zero-crossed". In the base sequence determination method according to the embodiment of the present invention, in order to subtract each background current from the current waveform (current-voltage characteristics) of the electrochemical current measured for each electrode, the current-voltage characteristics It is a schematic diagram explaining the method of carrying out the linear approximation of the tangent to a curve. FIG. 22 is an enlarged view of FIG. 21. In the base sequence determination method according to the embodiment of the present invention, it is a schematic diagram for explaining a method for obtaining a true detection signal (peak current value) by subtracting a corresponding background current from a peak current at zero cross voltage. The true detection signal (peak current value) obtained by the method shown in FIG. 23 is obtained for each of a plurality of control electrodes, a plurality of SNP2 detection electrodes (SNP = "T" detection electrode), and a plurality of SNP1 detection electrodes ( It is a collective bar graph which shows the determination mode arranged and classified for every SNP = "G" detection electrode). The mode A shown in FIG. 24A is a standard detection mode, and the mode B shown in FIG. 24B is a detection mode indicating a sample when the signal from the SNP2 detection electrode is very small. The mode C shown in FIG. 24C is a detection mode indicating a sample when the signal from the SNP1 detection electrode is very small. 23 shows a determination mode in which true detection signals (peak current values) obtained by the method shown in FIG. 23 are arranged and classified for each of a plurality of control electrodes, a plurality of SNP2 detection electrodes, and a plurality of SNP1 detection electrodes. It is a collective bar graph. Mode D shown in FIG. 25 (d) is a detection mode in which the signal from the SNP1 detection electrode and the signal from the SNP2 detection electrode are both too small to be determined and excluded as abnormal data. The mode E shown in e) is a detection mode in which the signal from the SNP1 detection electrode and the signal from the SNP2 detection electrode are much smaller than in the mode D, cannot be determined at the bio level, and are excluded as abnormal data. The mode F shown in FIG. 25 (f) is a detection mode in which a signal from the SNP1 detection electrode is abnormally small, a hardware level problem is estimated, and is excluded as abnormal data. 5 is a flowchart for explaining an example of a method of excluding abnormal data from a group of peak current value data calculated for each electrode in the base sequence determination method according to the embodiment of the present invention. In the base sequence determination method according to the embodiment of the present invention, whether to proceed to an algorithm flow for determining whether or not a certain nucleic acid is present, which of the two types of SNPs is a homotype It is a flowchart explaining an example of the method of determining whether it progresses to the flow of the algorithm which determines whether it is or hetero type. It is a flowchart explaining an example of the algorithm which determines whether a certain nucleic acid exists in the base sequence determination method which concerns on embodiment of this invention. In the nucleotide sequence determination method according to the embodiment of the present invention, which type of SNP is one of the two types of SNP = "G" and SNP = "T", and is it a homotype? It is a flowchart explaining an example of the algorithm which determines whether it is a hetero type (the 1). In the nucleotide sequence determination method according to the embodiment of the present invention, which type of SNP is one of the two types of SNP = "G" and SNP = "T", and is it a homotype? FIG. 6 is a flowchart for explaining an example of an algorithm for determining whether a hetero type is used (No. 2). It is a conceptual image figure which shows the various setting parameters used as the reference | standard regarding determination in the base sequence determination method which concerns on embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Measurement unit 11 ... Chip cartridge 12 ... Measurement system 13 ... Liquid feeding system 14 ... Temperature control mechanism 15 ... Control mechanism 16 ... Computer 21 ... Detection chip 300 ... CPU
DESCRIPTION OF SYMBOLS 301 ... Noise removal module 301, ... Noise removal module 302 ... Current waveform determination module 303 ... Bus 304 ... Input device 305 ... Output device 306 ... Display device 310 ... Peak current detection module 311 ... Voltage value calculation unit 312 ... Baseline approximation unit 313 ... Peak current value calculation unit 320 ... Abnormal data exclusion module 330 ... Presence determination module 340 ... Type determination module 351 ... Voltage determination voltage range storage unit 352 ... Permissible range storage unit 353 ... Peak search voltage value range storage unit 354 ... Zero cross Value storage unit 355 ... Inflection point storage unit 356 ... Intersection voltage storage unit 357 ... Offset voltage storage unit 358 ... Baseline current value storage unit 360 ... Average value / standard deviation storage unit 361 ... CV value storage unit 362 ... SLL storage unit 363... ESLL storage unit 364 HUL storage unit 365 ... MSL storage unit 366 ... SLR storage unit 367 ... absolute value logarithm storage unit 368 ... HLL storage unit 369 ... classification result storage unit 403, 413, 423, 433, 441, 445, 454 ... electromagnetic valve 454 ... send Liquid pump 500 ... Protection circuit 501a, 502a, 503a, 511a, 511b, 512a, 512b, 513a, 513b ... Wiring 502 ... Counter electrode 510 ... Voltage pattern generation circuit 511 ... Transimpedance amplifier 513 ... Inverting amplifier 513 ... Voltage follower amplifier 551 ... SNP1 detection electrode (working electrode)
552... SNP2 detection electrode (working electrode)
553 ... Control electrode (working electrode)
561, 562 ... Reference electrode 571, 572, 573 ... Probe DNA
581, 582, 583 ... sample DNA
591: Insertion agent 703 ... Electrical connector 705 ... Valve unit 707, 708 ... Nozzle 710 ... Probe unit 711 ... Cassette upper lid 712 ... Cassette lower lid 713 ... Packing 714 ... Substrate 722, 723 ... Nozzle insertion hole 724, 725 ... Electrical connector Port 726 ... seal detection hole 727a to 727d, 728a, 728b, 747a to 747d, 748a, 748b ... hole 742 ... inner surface 746 ... seal detection hole 749 ... hole for cassette type discrimination 752, 753 ... delivery port 756, 757 ... Flow path 761 ... Electrode unit 762, 763 ... Pad 781, 782 ... Valve body 789 ... Cassette type discrimination pin Rff ... Feedback resistance Rf, Rw, Rs ... Resistance

Claims (6)

A plurality of first detection electrodes and the first probe DNA is fixed respectively, are arranged corresponding to the first detection electrode of the plurality of, and the first control DNA in which the first nucleotide sequence and the probe DNA are different respectively fixed Corresponding to a plurality of second detection electrodes, a plurality of second detection electrodes each having a plurality of first control electrodes, a second probe DNA having a base sequence different from that of the first probe DNA and the first control DNA. The chemical solution containing the sample DNA is injected into a chip cartridge having a plurality of second control electrodes to which a second control DNA having a base sequence different from that of the first probe DNA and the second probe DNA is fixed. And a process of
Introducing an intercalating agent into the chip cartridge;
First detection signals from the plurality of first detection electrodes, first negative control signals from the plurality of first control electrodes, and from the plurality of second detection electrodes obtained by introducing the intercalating agent. Measuring a second detection signal and a second negative control signal from the plurality of second control electrodes, respectively, as current-voltage characteristics of an electrochemical current;
Obtaining a slope of the baseline of each current-voltage characteristic, and determining normality / abnormality of each current-voltage characteristic based on the slope of each baseline;
By subtracting the respective background currents from the respective current-voltage characteristics corresponding to the first detection signal, the first negative control signal, the second detection signal, and the second negative control signal, each of which is a true peak. Whether or not the sample DNA matches the sequence of at least one of the first probe DNA or the second probe DNA and forms a double strand according to the true current value. And determining the base sequence of the sample DNA. A plurality of first detection electrodes to which the first probe DNA can be fixed, and a first control DNA that is arranged corresponding to the plurality of first detection electrodes and has a base sequence different from that of the first probe DNA is fixed. A plurality of possible first control electrodes, a plurality of second detection electrodes to which a second probe DNA having a base sequence different from that of the first probe DNA and the first control DNA can be respectively fixed, and the plurality of second detection electrodes A chip cartridge comprising a plurality of second control electrodes, each of which is arranged corresponding to an electrode and to which a second control DNA having a base sequence different from that of the first probe DNA and the second probe DNA can be fixed; 1 detection electrode, the plurality of first control electrodes, the plurality of second detection electrodes, and the plurality of second control electrodes. A measuring system for measuring current through the poles;
A liquid feeding system for feeding a chemical to the chip cartridge;
A control mechanism for controlling the measurement system and the liquid feeding system;
Via the control mechanism and the measurement system, a first detection signal from the plurality of first detection electrodes, a first negative control signal from the plurality of first control electrodes, and a plurality of second detection electrodes. Second detection signal and second negative control signal from the plurality of second control electrodes are obtained as current-voltage characteristics, respectively, and the slopes of the baselines of the current-voltage characteristics are obtained, respectively. A current waveform determination module that determines whether each current-voltage characteristic is normal or abnormal and excludes an abnormal detection signal from a calculation target, the first detection signal, the first negative control signal, and the second detection signal. The respective background currents are subtracted from the respective current-voltage characteristics corresponding to the second negative control signal. There are, nucleotide sequence determination system, comprising a computer including a peak current detection module for acquiring each a true peak current value. The peak current detection module is
A voltage value calculation unit that performs a differential operation on each current value of the current-voltage characteristic with a voltage value, and calculates a voltage value at which a differential curve obtained by the differential operation crosses zero;
A baseline approximation unit that linearly approximates each baseline of the current-voltage characteristics;
A current value at the zero-crossing voltage value is subtracted from the current value of the baseline approximated to the voltage value to obtain a true peak current value in a curve indicated by the current-voltage characteristic. The base sequence determination system according to claim 2, comprising: An abnormal data exclusion module that excludes abnormal data from a data group of true peak current values calculated by the peak current detection module;
Existence determination module that determines whether or not the base of the target nucleic acid exists using a data group of true peak current values from which abnormal data has been excluded, or a type determination module that identifies single nucleotide polymorphisms of sample DNA The base sequence determination system according to claim 2, further comprising:   The step of obtaining the true peak current value is
  Differentially calculating each current value of the current-voltage characteristics with a voltage value, and calculating a zero crossing voltage value of a differential curve obtained by the differential operation;
  Linearly approximating each baseline of the current-voltage characteristics;
  Subtracting the current value of the baseline approximated to the voltage value from the current value at the zero-crossing voltage value to obtain a true peak current value in the curve indicated by the current-voltage characteristic
  The base sequence determination method according to claim 1, comprising:   Excluding abnormal data from the true peak current value data group;
  A step of determining whether or not a base of a nucleic acid of interest exists using a data group of true peak current values from which abnormal data has been excluded, or identifying a single nucleotide polymorphism of the sample DNA
  The base sequence determination method according to claim 1, further comprising:

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