Capillary electrophoresis device and capillary electrophoresis method

Abstract

A capillary electrophoresis apparatus injects a sample containing a first component and a second component into a capillary tube, performs electrophoretic separation of the injected first component and second component, measures light emission from the first component and light emission from the second component induced by irradiation of light onto the capillary tube by using a detector, and performs electrophoresis of the sample containing the first component and the second component. The signal intensity of the first component and the signal intensity of the second component are acquired by means of the signal intensity of the first component and the signal intensity of the second component, and the concentration range of the first component contained in the sample includes a concentration range in which the signal intensity of the first component is proportional to the concentration of the first component. The signal intensity of the first component is lower than the saturation signal intensity of the detector and reaches a concentration range of the saturation signal intensity by deviating from the proportion, and the capillary electrophoresis device is configured to detect the signal intensity of the first component based on the ratio of the signal intensity of the first component to the signal intensity of the second component. The ratio of the concentration of the first component relative to the concentration of the second component in the sample is quantified.

Description

TECHNICAL FIELD

[0001] The present application relates to a capillary electrophoresis device and a capillary electrophoresis method. BACKGROUND

[0002] Instrument analysis in analytical chemistry is analysis using an instrument that performs nuclear magnetic resonance spectroscopy, absorption spectroscopy, Raman spectroscopy, fluorescence spectroscopy, mass spectrometry, chromatography, electrophoresis analysis, and the like.

[0003] As shown in Non-Patent Literature 1 and Non-Patent Literature 2, in these instrument analyses, external standard method (absolute calibration curve method) or internal standard method (internal standard method) is often used to quantify unknown concentrations of an analysis object contained in a sample.

[0004] The external standard method is a method of quantifying unknown concentrations of an analysis object contained in a sample using a standard sample containing an analysis object of various known concentrations, and preparing a calibration curve of the concentration of the analysis object versus the signal intensity in advance. Generally, it is assumed that the signal intensity is proportional to the concentration of the analysis object, that is, the increase in the signal intensity with respect to the concentration of the analysis object is linear, and that the rate of increase in the signal intensity with respect to the concentration of the analysis object is constant.

[0005] However, in the case where the increase in the signal intensity with respect to the concentration of the analysis object is nonlinear, for example, even in the case where the rate of increase in the signal intensity with respect to the concentration of the analysis object decreases together with the concentration of the analysis object, the external standard method can function. However, in the external standard method, the quantitative accuracy is lowered due to the influence of the matrix effect of the sample, the influence of the deviation in the injection amount of the sample into the analysis instrument.

[0006] In contrast, the internal standard method is a method of reducing these influences and improving the quantitative accuracy. This method uses a standard sample containing an analysis object of various known concentrations and an internal standard of a known concentration, and prepares a calibration curve of the concentration ratio of the analysis object with respect to the internal standard versus the signal intensity ratio of the analysis object with respect to the internal standard in advance, to quantify unknown concentrations of the analysis object contained in a sample.

[0007] Here, it is necessary to be able to measure the signal intensities of the internal standard and the analysis object independently. As shown in Non-Patent Literature 1 and Non-Patent Literature 2, in the internal standard method, it is necessary to make the respective signal intensities proportional to the concentrations of the analysis object and the internal standard, that is, the respective signal intensities linearly increase with respect to the concentrations of the analysis object and the internal standard.

[0008] On the other hand, in a case where the respective signal strengths are nonlinear with respect to the increase in the concentration of the analysis object and the internal standard, the internal standard method does not function. That is, it is not possible to reduce the influence of the matrix effect of the sample, the influence of the bias in the injection amount of the sample into the analysis instrument, and improve the quantitative accuracy. Furthermore, as pointed out in Non-Patent Literature 2, it is known that the quantitative accuracy is rather reduced.

[0009] A more specific description is given of a case where capillary electrophoresis analysis by laser-induced fluorescence measurement quantifies the concentration C(tg) of a fluorescently labeled DNA fragment, which is an analysis object, contained in a sample. In this specification, a coefficient for a mathematical expression or a subscript for a variable is sometimes indicated in parentheses. For example, in a case where a subscript tg is added to a variable C, it is sometimes expressed as C(tg) and sometimes as C tg .

[0010] In FIG. 1 of Patent Literature 1, a capillary electrophoresis device that performs parallel processing of four capillary electrophoresis analyses is used, and one of the capillaries is used to perform electrophoresis analysis. A fluorescently labeled DNA fragment other than the fluorescently labeled DNA fragment that is an analysis object can also be contained in the sample. In addition, a salt (ion) other than the DNA fragment contained in the sample is removed as much as possible in advance using ethanol precipitation, column purification.

[0011] First, a part of the sample is injected from the sample injection end of the capillary by electric field injection. In general, the amount of the DNA fragment injected is proportional to the electric field strength E and the time T of the electric field injection, and the concentration C(tg) of the DNA fragment in the sample.

[0012] Next, the injected DNA fragment moves toward the sample elution end of the capillary by electrophoresis while being separated by the base length. At this time, the DNA fragment passing through the measurement point on the capillary by electrophoresis is irradiated with a laser beam, and the fluorescent body labeled on the DNA fragment emits fluorescence.

[0013] The emitted fluorescence is measured sequentially by an image sensor, and its time series is given to an electropherogram. A peak corresponding to the analysis object DNA fragment is obtained on the electropherogram.

[0014] The signal strength S(tg) of the analysis object DNA fragment is generally expressed by the area of the peak of the analysis object DNA fragment, but is expressed by the height of the peak of the analysis object DNA fragment when the width of the peak is considered to be constant. The signal strength S(tg) of the DNA fragment that is an analysis object is proportional to the amount of the analysis object DNA fragment injected into the capillary, and therefore a proportional coefficient is set to K(tg), which is the following expression.

[0015] [Equation 1]

[0016] S tg = Ktg • E • T • C tg (Formula 1)

[0017] Here, E represents the effective electric field strength in the sample in the vicinity of the sample injection end of the capillary at the time of electric field injection, and T represents the time of electric field injection.

[0018] When E and T are fixed, (Formula 1) indicates that the signal strength S(tg) of the analysis target DNA fragment is proportional to the concentration C(tg) of the analysis target DNA fragment in the sample, and becomes a calibration curve of the external standard method. Using (Formula 1), the unknown concentration C(tg) of the analysis target contained in the sample can be quantified from the measured signal strength S(tg) of the analysis target DNA fragment.

[0019] On the other hand, in the internal standard method, a DNA fragment that is an internal standard of known concentration is mixed in the sample and capillary electrophoresis analysis is performed. The internal standard DNA fragment is also fluorescently labeled. The wave peak of the analysis target DNA fragment and the wave peak of the internal standard DNA fragment are independently observed on the electropherogram. The signal strength S(st) of the internal standard DNA fragment is proportional to the amount of the internal standard DNA fragment injected into the capillary, and thus the proportional coefficient is set to K(st), which is the following formula.

[0020] [Formula 2]

[0021] S st = K st • E • T • C st (Formula 2)

[0022] (Formula 2) indicates that the signal strength S(st) of the internal standard DNA fragment is proportional to the concentration C(st) of the internal standard DNA fragment in the sample. The electric field injection of the analysis target DNA fragment and the internal standard DNA fragment in the sample is performed together, and thus the electric field strength E and the time T of the electric field injection of (Formula 1) and (Formula 2) are the same. Therefore, by taking the ratio of (Formula 1) to (Formula 2), the following formula can be obtained.

[0023] [Formula 3]

[0024]

[0025] (Formula 3) indicates that the signal strength ratio S(tg) / S(st) of the analysis target DNA fragment with respect to the internal standard is proportional to the concentration ratio C(tg) / C(st) of the analysis target DNA fragment with respect to the internal standard in the sample, and becomes a calibration curve of the internal standard method.

[0026] Using (Math. 3), the concentration ratio C(tg) / C(st) of the analyte DNA fragment in the sample with respect to the internal standard can be quantified from the measured signal intensity ratio S(tg) / S(st) of the analyte DNA fragment with respect to the internal standard. This is synonymous with quantifying the unknown concentration Ctg of the analyte DNA fragment in the sample, since the concentration C(st) of the internal standard DNA fragment in the sample is known.

[0027] In the external standard method based on (Math. 1), for example, if the electric field strength E of the electric field injection deviates by ±10%, it is known from (Math. 1) that this deviation directly leads to a decrease in the quantitative accuracy of the concentration of the analyte DNA fragment. In contrast, in the internal standard method based on (Math. 3), the electric field strength E of the electric field injection is not included in (Math. 3), so this deviation does not affect the quantitative accuracy of the concentration of the analyte DNA fragment. This is because the effect of the deviation of the electric field strength E in (Math. 1) is the same as that of the electric field strength E in (Math. 2), and by taking the ratio of (Math. 1) to (Math. 2), their effects are eliminated. The above is the reason why the quantitative accuracy of the analyte based on the internal standard method can be improved compared to the external standard method.

[0028] Prior Art Documents

[0029] Patent Documents

[0030] Patent Document 1: International Publication No. 2023 / 007567

[0031] Non-Patent Documents

[0032] Non-Patent Document 1: Harvey, David. Modern analytical chemistry. Vol. 1. New York: McGraw-Hill, 2000.

[0033] Non-Patent Document 2: A. K. Hewavitharana (2009) Internal Standard-Friend or Foe?, Critical Reviews in Analytical Chemistry, 39:4, 272-275 SUMMARY

[0034] PROBLEMS TO BE SOLVED BY THE INVENTION

[0035] In the case where the signal intensity is not proportional to the concentration of the analyte, the reason why the external standard method can function but the internal standard method cannot function is examined.

[0036] As an example, following Non-Patent Literature 2, if the signal intensity of the analysis object is represented by a quadratic function of the concentration, L(tg) and K(tg) are taken as coefficients, and the following equation is obtained.

[0037] [Equation 4]

[0038] S tg = L tg · (E · T · C tg ) 2 + K tg · E · T · C tg (Equation 4)

[0039] The constant term of the quadratic function is set to zero so that S(tg) = 0 when C(tg) = 0. In addition, in the case where L(tg) = 0, (Equation 4) is the same as (Equation 1).

[0040] In the external standard method, (Equation 4) is the calibration curve. As in the case of (Equation 1), using (Equation 4), the unknown concentration C(tg) of the analysis object contained in the sample can be quantified from the signal intensity S(tg) of the measured analysis object DNA fragment.

[0041] On the other hand, likewise, if the signal intensity of the internal standard is represented by a quadratic function of the concentration, L(st) and K(st) are taken as coefficients, and the following equation is obtained.

[0042] [Equation 5]

[0043] S st = L st · (E · T · C st ) 2 + K st · E · T · C st (Equation 5)

[0044] The constant term of the quadratic function is set to zero so that S(tg) = 0 when C(tg) = 0. In addition, in the case where L(tg) = 0, (Equation 4) is the same as (Equation 1).

[0045] At this time, unlike (Math. 3), even if the ratio of (Math. 4) to (Math. 5) is taken, the equation is not simplified, and the signal intensity ratio S(tg) / S(st) of the analysis target DNA fragment with respect to the internal standard cannot be expressed by the concentration ratio C(tg) / C(st) of the analysis target DNA fragment with respect to the internal standard. Furthermore, unlike (Math. 3), the influence of the electric field strength E and the time T of the residual electric field injection cannot be eliminated. Therefore, the quantitative accuracy of the analysis target of the internal standard method does not improve compared to the external standard. Furthermore, the quantitative accuracy is lower than the external standard method based on (Math. 4). As described in Non-Patent Literature 2, if the signal intensity and the concentration of either of the analysis target and the internal standard are not proportional, the quantitative accuracy based on the internal standard method decreases.

[0046] The above describes the case where the signal intensity of the analysis target and the internal standard is expressed by a quadratic function of the respective concentrations, but the same applies to any function other than a linear function with a constant term of zero. As described above, in the case where the signal intensity and the concentration of the analysis target are not proportional, the internal standard method cannot be used to improve the quantitative accuracy.

[0047] On the other hand, in Patent Literature 1, it is proposed to improve the dynamic range of the fluorescence measurement of a capillary electrophoresis device by optimizing the binning conditions according to the noise conditions of the image sensor. According to Patent Literature 1, it is expected that the concentrations of DNA fragments in a sample in a wider concentration range than before can be quantified.

[0048] However, it was actually found that, when the concentration of the analysis target DNA fragment contained in the sample is low, a signal intensity proportional to the concentration is obtained, but if the concentration of the analysis target DNA fragment contained in the sample becomes high, the signal intensity saturates with respect to the concentration. In addition, the saturated signal intensity is lower than the signal intensity at which the image sensor saturates, and thus it is clear that the cause is not the saturation of the image sensor.

[0049] This phenomenon is a new problem that becomes clear by using a capillary electrophoresis device having a wider dynamic range than before. When the concentration of the analysis target DNA fragment contained in the sample is low, a signal intensity proportional to the concentration of the analysis target DNA fragment can be obtained, and thus the internal standard method can be used to improve the quantitative accuracy of the analysis target. However, when the concentration of the analysis target DNA fragment contained in the sample is high, a signal intensity not proportional to the concentration of the analysis target DNA fragment is obtained, and thus the internal standard method cannot be used to improve the quantitative accuracy of the analysis target.

[0050] The present application was made to solve such a problem, and aims to provide a capillary electrophoresis device and a capillary electrophoresis method in which the quantitative accuracy of the analysis target can be improved using the internal standard method.

[0051] Means for solving the problem

[0052] In one example of the capillary electrophoresis device of the present application,

[0053] The capillary electrophoresis device performs the following processes:

[0054] injecting a sample containing a first component and a second component into a capillary;

[0055] performing electrophoretic separation of the injected first component and second component; and

[0056] measuring, with a detector, luminescence from the first component and luminescence from the second component induced by irradiation of light onto the capillary, thereby obtaining signal intensity of the first component and signal intensity of the second component,

[0057] The concentration range of the first component contained in the sample includes, in addition to the concentration range in which the signal intensity of the first component is proportional to the concentration of the first component, a concentration range in which the signal intensity of the first component is lower than the saturation signal intensity of the detector with respect to the concentration of the first component, and the concentration reaches the saturation signal intensity deviating from the proportionality,

[0058] The capillary electrophoresis device quantifies the ratio of the concentration of the first component to the concentration of the second component in the sample based on the ratio of the signal intensity of the first component to the signal intensity of the second component.

[0059] In one example of the capillary electrophoresis device of the present application,

[0060] The capillary electrophoresis device performs the following processes:

[0061] injecting a sample containing a first component and a second component into a capillary;

[0062] performing electrophoretic separation of the injected first component and second component; and

[0063] measuring, with a detector, luminescence from the first component and luminescence from the second component induced by irradiation of light onto the capillary, thereby obtaining signal intensity of the first component and signal intensity of the second component,

[0064] The concentration range of the first component contained in the sample includes, in addition to the concentration range in which the signal intensity of the second component is constant with respect to the concentration of the first component, a concentration range in which the signal intensity of the second component decreases deviating from the constancy with respect to the concentration of the first component,

[0065] The capillary electrophoresis device quantifies a ratio of a concentration of the first component to a concentration of the second component in the sample based on a ratio of a signal intensity of the first component to a signal intensity of the second component.

[0066] In one example of the capillary electrophoresis method of the present application,

[0067] The capillary electrophoresis method includes the steps of:

[0068] injecting a sample containing a first component and a second component into a capillary;

[0069] performing electrophoretic separation of the injected first component and second component; and

[0070] measuring, with a detector, luminescence from the first component and luminescence from the second component induced by irradiation of light onto the capillary, thereby obtaining a signal intensity of the first component and a signal intensity of the second component,

[0071] The concentration range of the first component contained in the sample includes, in addition to a concentration range in which the signal intensity of the first component is proportional to the concentration of the first component, a concentration range in which the signal intensity of the first component is lower than the saturation signal intensity of the detector with respect to the concentration of the first component and deviates from the proportionality to reach the saturation signal intensity.

[0072] The capillary electrophoresis method includes the steps of:

[0073] The capillary electrophoresis device quantifies a ratio of a concentration of the first component to a concentration of the second component in the sample based on a ratio of a signal intensity of the first component to a signal intensity of the second component.

[0074] In one example of the capillary electrophoresis method of the present application,

[0075] The capillary electrophoresis method includes the steps of:

[0076] injecting a sample containing a first component and a second component into a capillary;

[0077] performing electrophoretic separation of the injected first component and second component; and

[0078] measuring, with a detector, luminescence from the first component and luminescence from the second component induced by irradiation of light onto the capillary, thereby obtaining a signal intensity of the first component and a signal intensity of the second component,

[0079] The concentration range of the first component contained in the sample includes, in addition to the concentration range in which the signal intensity of the second component is constant with respect to the concentration of the first component, a concentration range in which the signal intensity of the second component decreases with respect to the concentration of the first component deviating from constant,

[0080] The capillary electrophoresis method includes the following steps:

[0081] The concentration ratio of the first component to the second component in the sample is quantified based on the ratio of the signal intensity of the first component to the signal intensity of the second component.

[0082] Inventive Effects

[0083] By using the new internal standard method found in the present application, DNA fragments contained in a wider concentration range than ever before can be quantified with high accuracy.

[0084] The above-mentioned problems, structures, and effects are clarified by the following description of embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0085] Figure 1A is a structure of a capillary electrophoresis device of an embodiment of the present application.

[0086] Figure 1B is a structure of a capillary electrophoresis method of an embodiment of the present application.

[0087] Figure 2 is a schematic view of electric field injection.

[0088] Figure 3 is an electropherogram of a sample of various concentrations.

[0089] Figure 4 is a relationship between the concentration of a sample and the fluorescence intensity (1 thereof).

[0090] Figure 5 is a relationship between the concentration of a sample and the fluorescence intensity (2 thereof).

[0091] Figure 6 is an electropherogram of a sample in which two size standards are mixed at varying ratios (1 thereof).

[0092] Figure 7 is an electropherogram of a sample in which two size standards are mixed at varying ratios (2 thereof).

[0093] Figure 8 is a relationship between the concentration of a sample and the fluorescence intensity and the fluorescence intensity ratio of two DNA fragments (1 thereof).

[0094] Figure 9is a relationship of the concentration of the sample to the fluorescence intensity of the 3 kinds of DNA fragments.

[0095] Figure 10 is a relationship of the concentration of the sample to the fluorescence intensity of the 2 kinds of DNA fragments and the fluorescence intensity ratio thereof (its 2).

[0096] Figure 11 is a relationship of the concentration of the sample to the fluorescence intensity ratio of the 2 kinds of DNA fragments. DETAILED DESCRIPTION

[0097] [PRINCIPLE]

[0098] In the above-described capillary electrophoresis analysis, the reason for the phenomenon that the signal intensity is proportional to the concentration of the analysis target DNA fragments included in the sample when the concentration of the analysis target DNA fragments included in the sample is low, and the signal intensity is saturated with respect to the concentration when the concentration of the analysis target DNA fragments included in the sample is high, was investigated.

[0099] If it is not the reason for saturation of the image sensor, other considered reasons are self-quenching of the fluorophore at the measurement point. In general, it is known that if the concentration of the fluorophore becomes very high, self-quenching of the fluorophore occurs, the rate of increase of the fluorescence intensity decreases depending on the concentration of the fluorophore, and the fluorescence intensity reaches saturation. In addition, it is also known that when the concentration of the fluorophore further increases, the fluorescence intensity sometimes turns to decrease.

[0100] In order to verify the presence or absence of self-quenching in the above-described capillary electrophoresis analysis, samples in which the analysis target was a plurality of DNA fragments and the concentration of each was different were analyzed using the above-described capillary electrophoresis apparatus. When the concentration of the DNA fragments of the sample was increased, the total concentration of the plurality of DNA fragments was increased while the concentration ratio of the plurality of DNA fragments in the sample was maintained.

[0101] The plurality of DNA fragments were spatially separated by capillary electrophoresis, independently detected by laser-induced fluorescence measurement at the measurement point, and respectively assigned different peaks on the electropherogram.

[0102] The DNA fragments having a high concentration in the sample were assigned a high-intensity peak, and the DNA fragments having a low concentration in the sample were assigned a low-intensity peak. When the total concentration of the plurality of analysis target DNA fragments included in the sample was low, a signal intensity proportional to the concentration of each DNA fragment was obtained. In contrast, when the total concentration of the plurality of analysis target DNA fragments included in the sample was high, a signal intensity not proportional to the concentration of each DNA fragment was obtained. That is, regardless of the magnitude of the intensity of the peak on the electropherogram corresponding to the concentration in the sample, a change from a state in which the "signal intensity is proportional to the concentration of the DNA fragments in the sample" to a state in which the "signal intensity is not proportional to the concentration of the DNA fragments in the sample" was simultaneously produced.

[0103] The degree of self-quenching of the fluorescent substance should depend on the concentration of the fluorescent substance at the measurement point on the capillary. However, the above-described change occurs regardless of the concentration of the fluorescent substance at the measurement point on the capillary. From the above, the cause of the present phenomenon is not self-quenching of the fluorescent substance. Therefore, in the above-described capillary electrophoresis analysis, the concentration of the DNA fragments and the fluorescent substance labeled to the DNA fragments at the measurement point of the capillary does not reach the concentration at which self-quenching occurs. Of course, the phenomenon also does not occur due to saturation of the image sensor, and therefore the cause of the phenomenon is not saturation of the image sensor. Therefore, in the above-described capillary electrophoresis analysis, the concentration of the DNA fragments and the fluorescent substance labeled to the DNA fragments at the measurement point of the capillary does not reach the concentration at which saturation of the image sensor occurs.

[0104] Therefore, as described below, the cause of the present phenomenon is elucidated by the research of the present application. The present phenomenon is first discovered in the research of the present application, and the cause thereof is also first discovered in the research of the present application. Further, while the present phenomenon is allowed, based on the discovered cause, a new internal standard method is designed independently, and high-precision quantitative analysis of the analysis object can be performed.

[0105] [Embodiment 1]

[0106] Figure 1A is a structural view of a capillary electrophoresis apparatus. The capillary electrophoresis apparatus is widely used as an analysis apparatus for performing DNA sequence, DNA fragment analysis. By using four capillaries 1, different samples can be analyzed in each capillary 1.

[0107] Figure 1B is a structure of a capillary electrophoresis method. By the capillary electrophoresis method having the procedures of (1) to (8) of Figure 1B , one capillary electrophoresis analysis is performed. By repeatedly performing the procedures of (1) to (8), multiple capillary electrophoresis analyses can be performed.

[0108] (1) First, the sample injection end 2 of the four capillaries 1 is immersed in the negative electrode side buffer 6, and the sample elution end 3 is connected to the positive electrode side buffer 7 via the polymer solution 8 inside the pump block 10.

[0109] (2) Next, the valve 11 of the pump block 10 is closed, and the piston of the syringe 12 connected to the pump block 10 is pressed, whereby the polymer solution 8 inside is pressurized, and the polymer solution 8 is filled from the sample elution end 3 toward the sample injection end 2 inside each capillary 1.

[0110] (3) Next, the valve 11 is opened, the sample injection end 2 of each of the four capillaries 1 is dipped into a different sample 9, and a constant voltage is applied between the cathode 4 and the anode 5 by the power source 13, whereby a portion of the different sample 9 (containing at least the first component and the second component) is electrokinetically injected from the sample injection end 2 into each capillary 1. Thus, the sample 9 containing the first component and the second component is injected into the capillary.

[0111] (4) After that, the sample injection end 2 of each of the four capillaries 1 is dipped into the cathode-side buffer 6, and a high voltage is applied between the cathode 4 and the anode 5 by the power source 13, whereby capillary electrophoresis is started. The DNA fragments labeled with the fluorescent substance are electrophoresed from the sample injection end 2 toward the sample elution end 3. Thus, the injected first component and the second component are electrophoretically separated.

[0112] (5) In parallel, the position of each capillary 1 after electrophoresis for a constant distance from the sample injection end 2 is set as a measurement point 16, and the laser beam 14 oscillated from the laser light source 15 is irradiated to each measurement point 16. Here, the coating of each capillary 1 near the measurement point 16 is removed in advance, each capillary 1 near the measurement point 16 is arranged on the same plane, and the laser beam 14 is condensed and introduced from the side of the arrangement plane along the arrangement plane.

[0113] (6) Then, the DNA fragments labeled with the fluorescent substance are excited by the irradiation of the laser beam 14 when passing through the measurement point 16, and emit fluorescence. That is, the intensity of the fluorescence emitted from the four measurement points 16 changes over time as the electrophoresis proceeds.

[0114] (7) Next, the fluorescence emitted from each measurement point 16 is measured by the detector 17. That is, the luminescent fluorescence from the first component and the luminescent fluorescence from the second component induced by the irradiation of the laser beam onto the capillary 1 are measured by the detector 17, whereby the signal intensity of the first component and the signal intensity of the second component are obtained. An electropherogram as time-series data of these signal intensities is obtained, and analysis of the sample 9 injected into each capillary 1 is performed. The detector 17 includes a spectrometer and an image sensor (not shown), and can simultaneously and independently perform spectrometric measurement of the luminescent fluorescence from the four measurement points 16. Thus, luminescent fluorescence from a plurality of fluorescent substances can be identified.

[0115] (8) Finally, the ratio of the concentration of the first component to the concentration of the second component in the sample is quantified based on the ratio of the signal intensity of the first component to the signal intensity of the second component. This quantification can be performed by the following verification method based on the description of the [principle] above.

[0116] Figure 2Electrode injection of the sample 9 in capillary electrophoresis analysis is schematically shown. The sample injection end 2 of the 1 capillary 1 filled with a polymer solution 8 is immersed in the sample 9 in solution form, Figure 2 (a) shows the state before the electrode injection, Figure 2 (b) shows the state after the electrode injection. Figure 2 After (b), the sample injection end 2 is immersed in the cathode side buffer, and the electrophoresis is started.

[0117] The sample 9 contains a first DNA fragment 18 (a first component), a second DNA fragment 19 (a second component) and a negative ion 20 other than the DNA fragment as a negative ion. The first DNA fragment 18 and the second DNA fragment 19 as the analysis object are both DNA fragments labeled with a fluorescent substance. The base length of the first DNA fragment 18 is different from that of the second DNA fragment 19. The concentrations of the first DNA fragment 18 and the second DNA fragment 19 in the sample 9 are different.

[0118] As the specific example described later, the sample 9 can contain a size standard, and the second DNA fragment 19 can be a DNA fragment contained in the size standard. In addition, the sample 9 can contain a PCR product, and the first DNA fragment 18 can be a DNA fragment as the PCR product or a DNA fragment from the PCR product. In addition, the sample 9 can contain a single base extension product, and the first DNA fragment 18 can be a first DNA fragment as the single base extension product, and the second DNA fragment 19 can be a second DNA fragment as the single base extension product.

[0119] In this example, as the simplest example of a plurality of DNA fragments, two DNA fragments are mainly handled, but the same investigation can of course be made for any number of DNA fragments of three or more.

[0120] In Figure 2 , the cation and the cathode are omitted. The solvent of the sample 9 is pure water or formamide. The sample 9 is previously removed of the negative ion (salt) other than the DNA fragment as much as possible by ethanol precipitation or the like, but cannot be set to zero.

[0121] In the state of (a) of Figure 2 , the sample injection end 2 of the capillary 1 is set as the cathode side, and the sample elution end 3 is set as the anode side, and the electrode injection is performed by applying a voltage to both ends of the capillary 1 in such a manner that the value of the voltage x time is constant. For example, a constant voltage for a constant time can be applied. Thereby, the state of (b) of Figure 2 is obtained. As Figure 2As shown in (b), a portion of the negative ions, i.e., the first DNA fragments 18, the second DNA fragments 19, and the negative ions other than the DNA fragments 20, in the sample 9 is injected into the inside of the capillary 1 from the sample injection end 2 of the capillary 1. Hereinafter, the injection amounts of the respective negative ions at this time are estimated.

[0122] The current I flowing when a constant voltage V is applied between the negative electrode and the positive electrode is substantially determined by the resistance R of the capillary 1 filled with the polymer solution 8, I « V / R. This is because the resistance R(i) between the negative electrode 4 Figure 1A ) and the sample injection end 2 of the capillary 1, and the resistance R(o) between the sample elution end 3 Figure 1A ) of the capillary and the positive electrode 5 Figure 1A ) are sufficiently small compared with R (R » R(i), R(o)). That is, the total resistance R(i) + R + R(o) between the negative electrode 4 and the positive electrode 5 is approximately equal to R (R(i) + R + R(o) « R).

[0123] Therefore, the current I flowing when a constant voltage V is applied between the negative electrode and the positive electrode hardly changes regardless of the composition of the sample, for example, whether the sample is pure water or a high ionic concentration solution. Therefore, "application of a constant voltage between the negative electrode and the positive electrode" is sometimes expressed as "application of a constant voltage to both ends of the capillary".

[0124] In addition, the current I flowing between the negative electrode and the sample injection end of the capillary, the current I flowing in the capillary, and the current I flowing between the sample elution end of the capillary and the positive electrode are equal to the continuity of the current.

[0125] Here, the current I flowing between the negative electrode and the sample injection end of the capillary at the time of the electric field injection, i.e., the current I flowing in the sample, is borne by the negative ions injected into the capillary if the positive ions are ignored for simplicity. Therefore, the total amount of the negative ions injected into the capillary by the electric field injection of a constant voltage applied to both ends of the capillary for a constant time is constant regardless of the composition of the sample.

[0126] The effective electric field strength in the sample near the sample injection end of the capillary at the time of the electric field injection is set to E, the time of the electric field injection is set to T, and the inner cross-sectional area of the capillary is set to A. If the mobility of the negative ions other than the DNA fragments in the sample is set to μ(0) and the concentration is set to C(0), the injected molecule number J(0) of the negative ions other than the DNA fragments based on the electric field injection is the following equation.

[0127] [Equation 6]

[0128] J0= E · T · A · μ0· C0 (Equation 6)

[0129] The mobility indicates the moving speed of each negative ion per unit electric field strength.

[0130] In the case where there are various negative ions other than the DNA fragments, the average of the mobility and the concentration thereof is set to μ(0) and C(0). If the average charge amount of each molecule of the negative ions other than the DNA fragments is set to q(0), the injection charge amount Q(0) of the negative ions other than the DNA fragments based on the electric field injection is the following equation.

[0131] [Equation 7]

[0132] Q0 = q0 · J0 = E · T · A · q0 · μ0 · C0 (Equation 7)

[0133] In addition, when the mobility of the first DNA fragments and the second DNA fragments in the sample is set to μ, the concentration of the first DNA fragments is set to C(1), and the concentration of the second DNA fragments is set to C(2), the injection molecule number J(1) of the first DNA fragments and the injection molecule number J(2) of the second DNA fragments based on the electric field injection are the following equations.

[0134] [Equation 8]

[0135] J1 = E · T · A · μ · C1 (Equation 8)

[0136] [Equation 9]

[0137] J2 = E · T · A · μ · C2 (Equation 9)

[0138] Here, the reason why the mobility of the first DNA fragments and the second DNA fragments is made equal is because the mobility of the DNA fragments in the sample is constant regardless of the base length in a solution where there is no molecular sieve effect.

[0139] When the average charge amount of each molecule of the first DNA fragments and the second DNA fragments is set to q(1) and q(2), the injection charge amount Q(1) and Q(2) of the first DNA fragments and the second DNA fragments based on the electric field injection are the following equations.

[0140] [Equation 10]

[0141] Q1 = q1 · J1 = E · T · A · q1 · μ · C1 (Equation 10)

[0142] [Equation 11]

[0143] Q2 = q2 · J2 = E · T · A · q2 · μ · C2 (Equation 11)

[0144] When the total concentration of the first DNA fragments and the second DNA fragments in the sample is set as C = C(1) + C(2), the total number of injected molecules J of the first DNA fragments and the second DNA fragments based on the electric field injection is the following equation.

[0145] [Equation 12]

[0146] J = J1+ J2= E · T · A · μ · C (Equation 12)

[0147] Here, in the case where the average charge amount of each molecule of the first DNA fragments and the second DNA fragments is equal, and can be approximated as q = q(1) = q(2), the total injected charge amount Q of the first DNA fragments and the second DNA fragments based on the electric field injection is the following equation.

[0148] [Equation 13]

[0149] Q = q · J = E · T · A · q · μ · C (Equation 13)

[0150] Alternatively, in the case where the ratio of the concentrations C(1) and C(2) of the first DNA fragments and the second DNA fragments in the sample with respect to the total concentration C of the first DNA fragments and the second DNA fragments is constant, by defining q = (q(1) · C(1) + q(2) · C(2)) / (C(1) + C(2)), (Equation 13) can also be obtained.

[0151] In (Equation 6) to (Equation 13), E, T, and A are the same values. Since the total of the injected charge amounts of the negative ions per unit time at the time of the electric field injection is equal to the current I, the following equation is obtained.

[0152] [Equation 14]

[0153]

[0154] That is,

[0155] [Equation 15]

[0156]

[0157] In (Equation 14) and (Equation 15), as described above, since I is constant, the electric field injection is a competitive process between a plurality of negative ions contained in the sample, meaning that the injected charge amount of each negative ion is distributed according to the product of the charge amount, the mobility, and the concentration of each negative ion. In the case where the contribution of the positive ions to the current is taken into account, the contribution rate of the negative ions to the current is multiplied by the right side of (Equation 14).

[0158] If (Equation 15) is used, (Equation 8) to (Equation 11) can be modified as follows.

[0159] [Equation 16]

[0160]

[0161] [Equation 17]

[0162]

[0163] [Equation 18]

[0164]

[0165] [Equation 19]

[0166]

[0167] Further, in a case where q = q(l) = q(2) can be approximated as described above, (Equation 12) and (Equation 13) can be deformed as follows.

[0168] [Equation 20]

[0169]

[0170] [Equation 21]

[0171]

[0172] Alternatively, in a case where the ratio of the concentrations C(l) and C(2) of the first DNA fragment and the second DNA fragment in the sample with respect to the total concentration C of the first DNA fragment and the second DNA fragment is constant, (Equation 20) and (Equation 21) can also be obtained by defining q = (q(l) · C(l) + q(2) · C(2)) / (C(l) + C(2)).

[0173] On the other hand, with respect to the signal intensities S(l) and S(2) of the wave peaks of the first DNA fragment and the second DNA fragment on the electropherogram obtained by capillary electrophoresis, if the respective sensitivity coefficients are set to m(l) and m(2), then according to (Equation 8) and (Equation 9), the following equation is obtained.

[0174] [Equation 22]

[0175] S1 = m1 · J1 = E · T · A · m1 · μ · C1 (Equation 22)

[0176] [Equation 23]

[0177] S2 = m2 · J2 = E · T · A · m2 · μ · C2 (Equation 23)

[0178] If (Equation 15) is used, the following equation is obtained.

[0179] [Formula 24]

[0180]

[0181] [Formula 25]

[0182]

[0183] In addition, the sensitivity coefficient includes the average number of labeled fluorophores per molecule of the DNA fragment, the excitation efficiency of the fluorophore, the quantum yield of the fluorophore, the light collection efficiency of the luminescent fluorescence, the sensitivity of the image sensor, and the like.

[0184] In addition, in a case where the sensitivity coefficients of the first DNA fragment and the second DNA fragment can be approximated as m(l) and m(2) are equal, m = m(l) = m(2) is assumed, and the total signal intensity S of the wave peaks of the first DNA fragment and the second DNA fragment on the electropherogram obtained by capillary electrophoresis becomes the following formula according to (Formula 12).

[0185] [Formula 26]

[0186] S = m · J = E · T · A · m · μ · C (Formula 26)

[0187] Alternatively, in a case where the ratio of the concentrations C(l) and C(2) of the first DNA fragment and the second DNA fragment in the sample with respect to the total concentration C of the first DNA fragment and the second DNA fragment is constant, (Formula 26) can also be obtained by defining m = (m(l) · C(l) + m(2) · C(2)) / (C(l) + C(2)).

[0188] When using (Formula 15) to approximate q = q(l) = q(2), the following formula is obtained.

[0189] [Formula 27]

[0190]

[0191] Alternatively, in a case where the ratio of the concentrations C(l) and C(2) of the first DNA fragment and the second DNA fragment in the sample with respect to the total concentration C of the first DNA fragment and the second DNA fragment is constant, (Formula 27) can also be obtained by defining q = (q(l) · C(l) + q(2) · C(2)) / (C(l) + C2).

[0192] (Formulae 16 to 21, Formula 24, Formula 25, and Formula 27) can be expressed by the following formula in which a and b are constants and y is a function of x.

[0193] [Formula 28]

[0194]

[0195] For example, when y = J(l), x = C(l), a = I · T / q(l), and b = (q(0) · μ(0) · C(0) + q(2) · μ · C(2)) / (q(l) · μ), (Formula 16) becomes (Formula 28). (Formula 28) indicates that y is proportional to x (y = a / b x) when x is smaller than b, and indicates that y saturates with respect to x and gradually approaches a constant value a when x is larger than b.

[0196] That is, (Formula 16) indicates that J(l) is proportional to C(l) when C(l) is smaller than b = (q(0) · μ(0) · C(0) + q(2) · μ · C(2)) / (q(l) · μ), and indicates that J(l) saturates with respect to C(l) and gradually approaches a constant value when C(l) is larger than b = (q(0) · μ(0) · C(0) + q(2) · μ · C(2)) / (q(l) · μ).

[0197] On the other hand, (Formula 8) indicates a relationship in which J(l) is proportional to C(l), and thus (Formula 8) and (Formula 16) appear to be contradictory. However, in fact, E included in (Formula 8) varies depending on C(l), and thus J(l) is not necessarily proportional to C(l) in (Formula 8). Therefore, (Formula 8) and (Formula 16) do not contradict each other but coexist.

[0198] Specifically, in (Formula 15), I is constant, and thus E decreases when C(l) increases in a state in which C(0) and C(2) are constant. E is the effective electric field strength in the sample in the vicinity of the sample injection end of the capillary at the time of electric field injection, and can vary depending on the composition of the sample even if the electric field strength averaged over the electric field applied to both ends of the capillary is constant.

[0199] Likewise, (Math. 17) indicates that J(2) is proportional to C(2) in the case where C(2) is small, and that J(2) is saturated with respect to C(2) in the case where C(2) is large, gradually approaching a constant value. (Math. 18) indicates that Q(1) is proportional to C(1) in the case where C(1) is small, and that Q(1) is saturated with respect to C(1) in the case where C(1) is large, gradually approaching a constant value. (Math. 19) indicates that Q(2) is proportional to C(2) in the case where C(2) is small, and that Q(2) is saturated with respect to C(2) in the case where C(2) is large, gradually approaching a constant value. (Math. 20) indicates that J is proportional to C in the case where C is small, and that J is saturated with respect to C in the case where C is large, gradually approaching a constant value. (Math. 21) indicates that Q is proportional to C in the case where C is small, and that Q is saturated with respect to C in the case where C is large, gradually approaching a constant value. (Math. 24) indicates that S(1) is proportional to C(1) in the case where C(1) is small, and that S(1) is saturated with respect to C(1) in the case where C(1) is large, gradually approaching a constant value. (Math. 25) indicates that S(2) is proportional to C(2) in the case where C(2) is small, and that S(2) is saturated with respect to C(2) in the case where C(2) is large, gradually approaching a constant value. Also, (Math. 27) indicates that S is proportional to C in the case where C is small, and that S is saturated with respect to C in the case where C is large, gradually approaching a constant value.

[0200] Thus, (Math. 24), (Math. 25), and (Math. 27) demonstrate the phenomenon in which, in capillary electrophoresis analysis, the signal intensity is proportional to the concentration of the analysis target DNA fragment contained in the sample when the concentration of the analysis target DNA fragment contained in the sample is low, and the signal intensity is saturated with respect to the concentration when the concentration of the analysis target DNA fragment contained in the sample is high. That is, the cause of the above phenomenon can be determined. This is an insight that has been obtained for the first time by introducing (Math. 14) and (Math. 15).

[0201] In addition, (Math. 9) and (Math. 17), (Math. 10) and (Math. 18), (Math. 11) and (Math. 19), (Math. 12) and (Math. 20), (Math. 13) and (Math. 21), (Math. 22) and (Math. 24), (Math. 23) and (Math. 25), and (Math. 26) and (Math. 27) coexist without contradiction, similarly to (Math. 8) and (Math. 16).

[0202] On the other hand, according to (Math. 8) and (Math. 9), since E, T, A, and μ are the same, the following equation holds.

[0203] [Equation 29]

[0204]

[0205] That is, the ratio of the number of molecules of the first DNA fragments to the number of molecules of the second DNA fragments injected into the capillary by the electric field injection is equal to the ratio of the concentration of the first DNA fragments to the concentration of the second DNA fragments in the sample. In addition, according to (Math. 8), (Math. 9), and (Math. 12), the following equation holds.

[0206] [Equation 30]

[0207]

[0208] [Equation 31]

[0209]

[0210] That is, the ratio of the number of molecules of the first DNA fragments or the second DNA fragments injected into the capillary by the electric field injection to the total number of molecules of the first DNA fragments and the second DNA fragments is consistent with the ratio of the concentration of the first DNA fragments or the second DNA fragments in the sample to the total concentration of the first DNA fragments and the second DNA fragments.

[0211] In addition, according to (Math. 10) and (Math. 11), E, T, A, and μ are the same, and thus the following equation holds.

[0212] [Equation 32]

[0213]

[0214] That is, the ratio of the amount of electric charge of the first DNA fragments to the amount of electric charge of the second DNA fragments injected into the capillary by the electric field injection is proportional to the ratio of the concentration of the first DNA fragments to the concentration of the second DNA fragments in the sample.

[0215] In addition, in the case where q = q(l) = q(2) is assumed, according to (Math. 10), (Math. 11), and (Math. 13), the following equation holds.

[0216] [Equation 33]

[0217]

[0218] [Equation 34]

[0219]

[0220] That is, the ratio of the amount of injected electric charge of the first DNA fragments or the second DNA fragments injected into the capillary by the electric field injection to the total amount of injected electric charge of the first DNA fragments and the second DNA fragments is consistent with the ratio of the concentration of the first DNA fragments or the second DNA fragments in the sample to the total concentration of the first DNA fragments and the second DNA fragments.

[0221] Also, according to (Math. 22) and (Math. 23), the following equation holds.

[0222] [Equation 35]

[0223]

[0224] That is, the ratio of the signal intensity of the first DNA fragment and the second DNA fragment to the total signal intensity of the first DNA fragment and the second DNA fragment injected into the capillary by the electric field injection is proportional to the ratio of the concentration of the first DNA fragment and the second DNA fragment in the sample.

[0225] Therefore, Figure 1A The capillary electrophoresis device of the present application can quantify the ratio of the concentration of the first DNA fragment to the concentration of the second DNA fragment in the sample based on the ratio of the signal intensity of the first DNA fragment to the signal intensity of the second DNA fragment.

[0226] In addition, particularly in the case where the concentration of the second DNA fragment in the sample is known, the concentration of the first DNA fragment in the sample can be quantified based on the ratio of the signal intensity of the first DNA fragment to the signal intensity of the second DNA fragment.

[0227] In addition, in the case where m = m(1) = m(2) is assumed, according to (Math. 22), (Math. 23), and (Math. 26), the following equation holds.

[0228] [Equation 36]

[0229]

[0230] [Equation 37]

[0231]

[0232] That is, the ratio of the signal intensity of the first DNA fragment or the second DNA fragment to the total signal intensity of the first DNA fragment and the second DNA fragment injected into the capillary by the electric field injection is identical to the ratio of the concentration of the first DNA fragment or the second DNA fragment to the total concentration of the first DNA fragment and the second DNA fragment in the sample.

[0233] It should be noted that (Formula 16), (Formula 17), and (Formula 20) do not contradict (Formula 29), (Formula 30), and (Formula 31), but rather coexist. Specifically, when the number of injected molecules of the first DNA fragment, the second DNA fragment, or the entire DNA fragment is proportional or disproportionate to the concentration of the first DNA fragment, the second DNA fragment, or the entire DNA fragment in the sample, the ratio of the number of injected molecules of the first DNA fragment to the second DNA fragment is consistent with the concentration ratio of the first DNA fragment to the second DNA fragment in the sample, and the ratio of the number of injected molecules of the first DNA fragment or the second DNA fragment relative to the entire DNA fragment is consistent with the concentration ratio of the first DNA fragment or the second DNA fragment relative to the entire DNA fragment in the sample.

[0234] Another point to note is that (Formula 18), (Formula 19), and (Formula 21) do not contradict (Formula 32), (Formula 33), and (Formula 34) but rather coexist. Specifically, when the amount of injected charge of the first DNA fragment, the second DNA fragment, or the entire DNA fragment is proportional or disproportionate to the concentration of the first DNA fragment, the second DNA fragment, or the entire DNA fragment in the sample, the ratio of the injected charge of the first DNA fragment to the second DNA fragment is proportional to the concentration ratio of the first DNA fragment to the second DNA fragment in the sample, and the ratio of the injected charge of the first DNA fragment or the second DNA fragment relative to the entire DNA fragment is consistent with the concentration ratio of the first DNA fragment or the second DNA fragment relative to the entire DNA fragment in the sample.

[0235] It should also be noted that (Formula 24), (Formula 25), and (Formula 27) do not conflict with (Formula 35), (Formula 36), and (Formula 37), but rather coexist. Specifically, when the signal intensities of the first DNA fragment, the second DNA fragment, or the entire DNA fragment are respectively proportional to the concentrations of the first DNA fragment, the second DNA fragment, or the entire DNA fragment in the sample, or when they are not proportional, the signal intensity ratio of the first DNA fragment to the second DNA fragment is proportional to the concentration ratio of the first DNA fragment to the second DNA fragment in the sample, and the signal intensity ratio of the first DNA fragment or the second DNA fragment relative to the entire DNA fragment is consistent with the concentration ratio of the first DNA fragment or the second DNA fragment relative to the entire DNA fragment in the sample.

[0236] If the second DNA fragment is used as an internal standard, (Math. 35) becomes a calibration curve of the same form as (Math. 3), and the influence of variations in the electric field strength E and the time T of the electric field injection can be avoided, and the first DNA fragment as the analysis target can be quantified with high precision. Therefore, as with (Math. 3) as the calibration curve of the conventional internal standard method, by using (Math. 35) as the calibration curve of the new internal standard method, high-precision quantification of the analysis target DNA fragment can be performed.

[0237] The conventional internal standard method can be applied only to cases where the signal strength is proportional to the concentration of the analysis target. In contrast, as shown in (Math. 24), (Math. 25), and (Math. 27), the new internal standard method can be applied not only to cases where the signal strength is proportional to the concentration of the analysis target, but also to any of cases where the signal strength is not proportional to the concentration of the analysis target, cases close to saturation, and cases of saturation.

[0238] [Specific example of measurement results]

[0239] In the capillary electrophoresis analysis, a specific example of a phenomenon in which the signal strength is proportional to the concentration of the analysis target DNA fragment included in the sample when the concentration of the analysis target DNA fragment included in the sample is low, and the signal strength is saturated with respect to the concentration when the concentration of the analysis target DNA fragment included in the sample is high, is shown.

[0240] STR-PCR for DNA-oriented examination was performed using the genome of a specific individual as a template, desalting was performed, and the solvent was set to formamide. Four kinds of samples in which the concentration of each of a plurality of DNA fragments as STR-PCR products was changed in the concentration range of 1-fold, 0.05-fold, 0.002-fold, and 0.0001-fold with respect to a reference concentration were prepared.

[0241] Using a capillary electrophoresis device of Figure 1A , a part of an electropherogram obtained by performing capillary electrophoresis analysis on samples with concentrations of 0.0001-fold, 0.002-fold, 0.05-fold, and 1-fold, respectively, using four capillaries is shown in (a), (b), (c), and (d) of Figure 3 .

[0242] Each peak on the electropherogram represents a signal of a DNA fragment as a STR-PCR product of each genetic locus. For example, a peak corresponding to a DNA fragment of a genetic locus of D8S1179 is shown in (a) of Figure 3In (a), (b), (c), and (d), the single peak observed around 4200 frames into the electrophoresis represents the signal of the DNA fragments that are the STR-PCR products of the D5S818 locus. As the concentration of the DNA fragments in the sample increases, the fluorescence intensity (representing signal intensity, the same applies hereafter) of each DNA fragment increases. Since the measured fluorescence intensities are adjusted to be equal when fluorescence of the same intensity is generated at the measurement points of each of the four capillaries, differences in the measured fluorescence intensities faithfully reflect differences in the fluorescence intensity emitted at the measurement points.

[0243] Figure 4 It will be Figure 3 The DNA fragments of the STR-PCR product of the gene locus D5S818 in the sample were used as the analysis object, and the fluorescence intensity of the peak of the gene locus D5S818 was plotted against the concentration of the DNA fragment contained in the sample to obtain a double logarithmic graph. Figure 3 Because the peak height, rather than the peak area, is considered equivalent in (a), (b), (c), and (d), the fluorescence intensity is represented by the peak height. The DNA fragment concentration on the horizontal axis represents the concentration of DNA fragments, i.e., STR-PCR products of the D5S818 locus, contained in the sample, but can also be considered to represent the overall concentration of DNA fragments contained in the sample.

[0244] When the concentration of the target DNA fragments contained in the sample is low, the fluorescence intensity of the target DNA fragments is proportional to the concentration of the DNA fragments contained in the sample. However, if the concentration of the target DNA fragments contained in the sample increases, the fluorescence intensity of the target DNA fragments deviates from the proportionality with respect to the concentration of the target DNA fragments contained in the sample and approaches saturation.

[0245] In (Formula 24), if y = S(1), x = C(1), a = m(1)·I·T / q(1), and b = (q(0)·μ(0)·C(0)+q(2)·μ·C(2)) / (q(1)·μ), then (Formula 28) is obtained. Furthermore, in (Formula 28), the fluorescence intensity of the target DNA fragment is S(1), and the concentration of the target DNA fragment contained in the sample is C(1).

[0246] Figure 4 The dotted line represents the relationship between S(1) and C(1) when a=3500 and b=0.17, that is, (Formula 28). Figure 4 The four points plotted, that is, the case where C(1) is low and the case where C(1) is high, are both good approximation curves. That is, as shown in (Formula 28), when C(1) is low, S(1) is proportional to C(1), but when C(1) becomes high, S(1) deviates from the proportionality with C(1), which proves that it is close to saturation.

[0247] On the other hand, Figure 4 The solid line indicates a case where S(l) = a / b - C(l) = 20000 - C(l), that is, a proportional relationship between S(l) and C(l), becomes a good approximation straight line with respect to the left 2 points of Figure 4 According to this result, it is known that S(l) and C(l) deviate from the proportional relationship to a non-proportional relationship at around 0.01 times of C(l). Thus, according to the conventional internal standard method, it is possible to perform quantification of a concentration range of C(l) of 0.0001 times to 0.01 times, that is, quantification of a concentration range of 2 bits of dynamic range. On the other hand, according to the new internal standard method, it is possible to perform quantification of a concentration range of C(l) of 0.0001 times to 1 times, that is, quantification of a concentration range of 4 bits of dynamic range.

[0248] Thus, by using the new internal standard method according to the present disclosure, it is possible to quantitatively determine DNA fragments contained in a sample in a wider concentration range than ever before with high accuracy.

[0249] Thus, by using the new internal standard method according to the present disclosure, it is possible to quantitatively determine DNA fragments contained in a sample in a wider concentration range than ever before with high accuracy.

[0250] The above analysis was performed focusing on the peak of the gene locus D5S818 in Figure 3 However, the same relationship is established for other peaks. That is, as the overall concentration of DNA fragments contained in a sample becomes higher, other peaks also deviate from the proportional relationship in synchronization with the peak of D5S818. In each electropherogram, although the fluorescence intensity of each peak is different, the reason for obtaining such a synchronized phenomenon is not saturation of the detector or self-quenching of the fluorescent body.

[0251] According to the above, the phenomenon observed in Figure 3 and Figure 4 is clearly explained and understood by the reason and theory examined in the above [principle].

[0252] Figure 5 is a double logarithmic graph in which the change in the fluorescence intensity of the analysis target DNA fragment is plotted against the change in the concentration of the analysis target DNA fragment contained in a sample for a sample different from Figure 3 and Figure 4 .

[0253] Five kinds of samples in which the concentration of the analysis target DNA fragment contained in a sample is changed in a concentration range of 100 times, 10 times, 1 times, 0.1 times, and 0.01 times with respect to a reference concentration were prepared. However, the reference concentration in Figure 5 is independent of the reference concentration in Figure 3 and Figure 4 .

[0254] Using a capillary electrophoresis device of the present disclosure, five samples were divided into two groups, and electrophoresis analysis was performed. As a result, as shown in FIG. 6, the fluorescence intensity of the DNA fragments contained in the samples was proportional to the concentration of the DNA fragments contained in the samples in the low concentration range, but deviated from the proportion in the high concentration range, and approached saturation. Figure 1A Using a capillary electrophoresis device of the present disclosure, five samples were divided into two groups, and electrophoresis analysis was performed. As a result, as shown in FIG. 6, the fluorescence intensity of the DNA fragments contained in the samples was proportional to the concentration of the DNA fragments contained in the samples in the low concentration range, but deviated from the proportion in the high concentration range, and approached saturation. Figure 4

[0255] As described above, when y = S(l), x = C(l), a = m(l) · I · T / q(l), and b = (q(0) · μ(0) · C(0) + q(2) · μ · C(2)) / (q(l) · μ) are set, (Math. 24) becomes (Math. 28). In (Math. 28), the fluorescence intensity of the DNA fragments of interest is set to S(l), and the concentration of the DNA fragments of interest contained in the sample is set to C(l).

[0256] Figure 5 The dotted line of FIG. 7 indicates the relationship between S(l) and C(l) in the case of a = 22000 and b = 10, that is, (Math. 28), plotted against the five points of FIG. 6, that is, both the low and high cases of C(l). That is, as indicated by (Math. 28), in the low case of C(l), S(l) is proportional to C(l), but if C(l) becomes high, S(l) deviates from the proportion to C(l), and approaches saturation. Figure 5 On the other hand, the solid line of FIG. 7 indicates the case where S(l) = a / b · C(l) = 2180 · C(l), that is, the proportional relationship between S(l) and C(l), plotted against the left three points of FIG. 6, that is, the low case of C(l). According to this result, S(l) and C(l) deviate from the proportional relationship to the non-proportional relationship at around 1 times C(l).

[0257] Figure 5 Figure 5 Accordingly, according to the conventional internal standard method, quantification in the concentration range of 0.01 times to 1 times C(l), that is, quantification in the concentration range of 2 bits of the dynamic range, is possible. In contrast, according to the new internal standard method, quantification in the concentration range of 0.01 times to 100 times C(l), that is, quantification in the concentration range of 4 bits of the dynamic range, is possible.

[0258] Thus, by using the new internal standard method according to the present disclosure, DNA fragments contained in a sample in a wider concentration range than before can be quantified with high accuracy. According to the above, in the new internal standard method according to the present disclosure, quantification in the concentration range of 0.01 times to 100 times C(l), that is, quantification in the concentration range of 4 bits of the dynamic range, is possible.

[0259] Thus, by using the new internal standard method according to the present disclosure, DNA fragments contained in a sample in a wider concentration range than before can be quantified with high accuracy. According to the above, in the new internal standard method according to the present disclosure, quantification in the concentration range of 0.01 times to 100 times C(l), that is, quantification in the concentration range of 4 bits of the dynamic range, is possible. Figure 5 ​​​The phenomena observed in the above [Principles] are clearly explained and understood through the causes and theories examined in the above [Principles].

[0260] [Example 1]

[0261] use Figure 1A The capillary electrophoresis apparatus shown below performs the following measurements. TM " mark indicates a trademark. Thermo Fisher Scientific's Hi-Di TM Formamide (hereinafter referred to as formamide) was used as a solvent to prepare two size standards: GeneScan TM 600LIZ TM Dye Size Standard (hereinafter, 600LIZ) and GeneScan TM 500ROX TM Dye Size Standard (hereinafter, 500ROX) was mixed with 4 samples in a specific ratio and used Figure 1A The analysis was performed using a capillary electrophoresis device. The four capillaries had an inner diameter of 50 μm, a total length of 47 cm, and an effective length of 36 cm. As the cathode side buffer and the anode side buffer, the Applied Biosystems of Thermo Fisher Scientific was used. TM 310 and 31xx Running Buffer, 10X was diluted 10 times with pure water before use. As the polymer solution, POP-4 from Thermo Fisher Scientific was used. TM Polymer, for3500 / SeqStudio TM Flex.

[0262] In addition, the concentration of 600LIZ contained in the four kinds of samples was changed to 1 / 2 times (0.5 times), 1 / 20 times (0.05 times), 1 / 200 times (0.005 times), and 1 / 2000 times (0.0005 times) relative to the reference concentration, and on the other hand, the concentration of 500ROX contained in the four kinds of samples was constant to 1 / 200 times (0.005 times) relative to the reference concentration. However, the reference concentration of 600LIZ is independent of the reference concentration of 500ROX. Here, 600LIZ and 500ROX respectively contain a variety of DNA fragments and change the overall concentration while maintaining their concentration ratio. The electric field injection of each sample is implemented by applying a voltage of 1.2kV for 9 seconds to the two ends of each capillary. Electrophoresis is implemented by applying a voltage of 8.5kV to the two ends of each capillary.

[0263] Figure 6(a) indicates an electropherogram of a sample in which the concentration of 600LIZ is 1 / 2000 times and the concentration of 500ROX is 1 / 200 times. Figure 6 (b) indicates an electropherogram of a sample in which the concentration of 600LIZ is 1 / 200 times and the concentration of 500ROX is 1 / 200 times. Figure 7 (a) indicates an electropherogram of a sample in which the concentration of 600LIZ is 1 / 20 times and the concentration of 500ROX is 1 / 200 times. Figure 7 (b) indicates an electropherogram of a sample in which the concentration of 600LIZ is 1 / 2 times and the concentration of 500ROX is 1 / 200 times. In any of the graphs, a solid line indicates the fluorescence intensity of 600LIZ and a dotted line indicates the fluorescence intensity of 500ROX. The horizontal axis indicates the electrophoresis time, the vertical axis on the left indicates the fluorescence intensity of 500ROX, and the vertical axis on the right indicates the fluorescence intensity of 600LIZ.

[0264] 600LIZ contains 36 kinds of single-stranded DNA fragments of 20, 40, 60, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280, 300, 314, 320, 340, 360, 380, 400, 414, 420, 440, 460, 480, 500, 514, 520, 540, 560, 580, and 600 bases in length, each labeled with a fluorescent body LIZ. Figure 6 and Figure 7 The electropherogram of (a) indicates the wave peaks of 15 kinds of DNA fragments of 20, 40, 60, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250 bases in length. Further, the wave peaks of DNA fragments of 40, 114, and 160 bases in length are indicated by arrows, and are denoted as LIZ40, LIZ114, and LIZ160.

[0265] 500ROX contains 16 kinds of single-stranded DNA fragments of 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500 bases in length, each labeled with a fluorescent body ROX. Figure 6 and Figure 7 The electropherogram of (a) indicates the wave peaks of 9 kinds of DNA fragments of 35, 50, 75, 100, 139, 150, 160, 200, 250 bases in length. Further, the wave peak of a DNA fragment of 160 bases in length is indicated by an arrow, and is denoted as ROX160.

[0266] The peaks of the DNA fragments of the same base length are observed at slightly different times as the peaks of LIZ160 and the peaks of ROX160, due to the difference in the mobility of the labeled fluorophores LIZ and ROX.

[0267] Figure 8 (a) is to be determined by Figure 6 and Figure 7 The double logarithmic graph of the fluorescence intensity of the peaks of LIZ160 and the peaks of ROX160 obtained from the 4 electropherograms of FIG. 9 is plotted against the concentration of 600LIZ contained in the sample. The horizontal axis can also be considered as the concentration of LIZ160 contained in the sample. Here, the width of each peak is considered to be approximately equal, and thus the height of each peak is taken as the fluorescence intensity. The black dots indicate the fluorescence intensity of LIZ160, and the white dots indicate the fluorescence intensity of ROX160.

[0268] When the concentration of 600LIZ contained in the sample is low, specifically, in the range of 1 / 2000 to 1 / 20 times the concentration of 600LIZ, the fluorescence intensity of LIZ160 is proportional to the concentration of 600LIZ contained in the sample, and can be approximated by a straight line with a slope of 1. On the other hand, when the concentration of 600LIZ contained in the sample is high, specifically, in the range of 1 / 20 to 1 / 2 times the concentration of 600LIZ, the fluorescence intensity of LIZ160 deviates from the proportion with respect to the concentration of 600LIZ contained in the sample, and approaches saturation. This phenomenon is the same as that observed in Figure 4 and Figure 5

[0269] Thus, the concentration range of the first component contained in the sample includes, in addition to the concentration range in which the signal intensity of the first component is proportional to the concentration of the first component, a concentration range in which, with respect to the concentration of the first component, the signal intensity of the first component is lower than the saturation signal intensity of the detector and deviates from the proportion to reach the saturation signal intensity. The capillary electrophoresis device of the present embodiment is also able to perform measurement of the concentration for such a sample.

[0270] On the other hand, when the concentration of 600LIZ contained in the sample is low, specifically, when the concentration of 600LIZ is in the range of 1 / 2000 to 1 / 200 times, the fluorescence intensity of ROX160 is constant with respect to the concentration of 600LIZ contained in the sample. This is easily understood since the concentration of ROX160 contained in the sample is constant. However, when the concentration of 600LIZ contained in the sample is high, specifically, when the concentration of 600LIZ is in the range of 1 / 200 to 1 / 2 times, the fluorescence intensity of ROX160 decreases with respect to the concentration of 600LIZ contained in the sample. Such a change is not easily understood since the concentration of ROX160 contained in the sample is constant. The above is a new phenomenon discovered in the present disclosure.​

[0271] Thus, the concentration range of the first component contained in the sample includes, in addition to the concentration range in which the signal intensity of the second component is constant with respect to the concentration of the first component, a concentration range in which the signal intensity of the second component decreases from the constant with respect to the concentration of the first component. The capillary electrophoresis apparatus of the present embodiment is also capable of measuring the concentration for such a sample.

[0272] The above phenomenon is explained by (Math. 24) and (Math. 25).

[0273] In (Math. 24) and (Math. 25), the first DNA fragments are regarded as all the DNA fragments contained in 600LIZ, and the second DNA fragments are regarded as all the DNA fragments contained in 500ROX. That is, S(l) is the sum of the fluorescence intensities of all the DNA fragments contained in 600LIZ, C(l) is the sum of the concentrations of all the DNA fragments contained in 600LIZ, q(l) is the average charge amount of all the DNA fragments contained in 600LIZ, S(2) is the sum of the fluorescence intensities of all the DNA fragments contained in 500ROX, C(2) is the sum of the concentrations of all the DNA fragments contained in 500ROX, and q(2) is the average charge amount of all the DNA fragments contained in 500ROX.

[0274] At this time, in (Math. 24), under the conditions that the concentration C(0) of the negative ions other than the DNA fragments is constant, and the concentration C(2) of 500ROX is constant, the fluorescence intensity S(l) of 600LIZ is proportional to the concentration C(l) of 600LIZ when the concentration C(l) of 600LIZ is low, and deviates from the proportion with respect to the concentration C(l) of 600LIZ to reach saturation when the concentration C(l) of 600LIZ is high. The fluorescence intensity of LIZ160 is proportional to the fluorescence intensity of 600LIZ, and thus both represent the same change. Therefore, Figure 8 The change in the fluorescence intensity of LIZ160 in the entire concentration range of LIZ160 of (a) of the above is explained by (Math. 24).

[0275] On the other hand, in (Math. 25), under the conditions that the concentration C(0) of the negative ions other than the DNA fragments is constant, and the concentration C(2) of 500ROX is constant, the fluorescence intensity S(2) of 500ROX is constant when the concentration C(l) of 600LIZ is low, and decreases with respect to the concentration C(l) of 600LIZ when the concentration C(l) of 600LIZ is high. The fluorescence intensity of ROX160 is proportional to the fluorescence intensity of 500ROX, and thus both represent the same change. Therefore, Figure 8 The change in the fluorescence intensity of ROX160 in the entire concentration range of LIZ160 of (a) of the above is explained by (Math. 25).

[0276] Further, the above is explained by (Math. 14) and (Math. 15). That is, under the condition that the current at the time of electric field injection is constant, when the injection charge amount Q(l) of the first DNA fragment increases by the increase of the concentration C(l) of the first DNA fragment, the injection charge amount Q(2) of the second DNA fragment decreases. This is achieved by the decrease of E on the left side of (Math. 15), that is, the electric field intensity in the sample near the sample injection end of the capillary at the time of electric field injection.

[0277] The above is the investigation focusing on the peak of LIZ160 and the peak of ROX160 in Figure 6 and Figure 7 , but the same investigation is also valid focusing on the peaks of DNA fragments other than them.

[0278] Figure 9 is a double logarithmic graph in which the fluorescence intensity of the peak of LIZ40 and LIZ114 is plotted against the concentration of 600LIZ contained in the sample, in addition to the fluorescence intensity of the peak of LIZ160 (the same data as (a) of Figure 8 . The horizontal axis can also be considered as the concentration of LIZ40, LIZ114, or LIZ160 contained in the sample.

[0279] When the concentration of 600LIZ contained in the sample is low, specifically, in the range where the concentration of 600LIZ is 1 / 2000 to 1 / 20 times, each fluorescence intensity is proportional to the concentration of 600LIZ contained in the sample, and can be approximated by a straight line with a slope of 1. On the other hand, when the concentration of 600LIZ contained in the sample is high, specifically, in the range where the concentration of 600LIZ is 1 / 20 to 1 / 2 times, each fluorescence intensity deviates from the proportion with respect to the concentration of 600LIZ contained in the sample, and approaches saturation. The above change in fluorescence intensity occurs synchronously for each DNA fragment. Of course, any change in fluorescence intensity can be approximated by (Math. 24).

[0280] In each electropherogram, although the fluorescence intensities of the peaks of LIZ40, LIZ114, and LIZ160 are different, the above change in fluorescence intensity occurs synchronously, which indicates that the cause is not the saturation of the detector or the self-quenching of the fluorophore, but is caused by the constant electric field injection amount found in the present disclosure. With respect to ROX, with respect to the peaks of DNA fragments other than ROX160, the same change in fluorescence intensity as that of ROX160 can also be obtained, and can be approximated by (Math. 25).

[0281] According to Figure 8(a) When the concentration of 600LIZ contained in the sample, where the fluorescence intensity of LIZ160 is proportional to the concentration of 600LIZ, is in the range of 1 / 2000 to 1 / 20, LIZ160 can be quantified with high precision using a conventional internal standard method using ROX160 as an internal standard. Alternatively, when the fluorescence intensity of ROX160 is constant relative to the concentration of 600LIZ contained in the sample, where the concentration of 600LIZ is in the range of 1 / 2000 to 1 / 200, LIZ160 can be quantified with high precision using a conventional internal standard method using ROX160 as an internal standard.

[0282] However, if the concentration of 600LIZ in the sample is halved, the internal standard method cannot be applied. Figure 8 In (a), the fluorescence intensity of the LIZ160 peak approaches saturation, and the fluorescence intensity of ROX160 decreases. Therefore, it is considered impossible to quantify LIZ160 using these ratios in the conventional concept.

[0283] In contrast, Figure 8 (b) is to Figure 8 In (a), the ratio of the fluorescence intensity of the LIZ160 peak to the fluorescence intensity of the ROX160 peak is plotted against the concentration of 600 LIZ contained in the sample. The horizontal axis can also be considered to be the concentration of LIZ160 contained in the sample.

[0284] according to Figure 8 (a) or the existing internal standard method is unimaginable. Figure 8 Over the entire concentration range from 1 / 2000 to 1 / 2 times the horizontal axis of (a), the ratio of the fluorescence intensity of the LIZ160 peak to the fluorescence intensity of the ROX160 peak is proportional to the concentration of 600 LIZ contained in the sample and can be approximated by a straight line with a slope of 1. In other words, the new internal standard method using ROX160 as the internal standard enables high-precision quantification of LIZ160 over a wider concentration range than before.

[0285] Figure 8 The result of (b) is explained by assuming that the first DNA fragment is LIZ160 and the second DNA fragment is ROX160 in (Equation 35). That is, the ratio of the fluorescence intensity of the LIZ160 peak to the fluorescence intensity of the ROX160 peak is proportional to the ratio of the LIZ160 concentration to the ROX160 concentration in the sample. Since the ROX160 concentration is constant, the ratio of the fluorescence intensity of the LIZ160 peak to the fluorescence intensity of the ROX160 peak is proportional to the LIZ160 concentration in the sample.

[0286] (Formula 35) does not include the parameters that can be changed, such as E, T, C(0), and so it can be quantified with high precision, similar to the conventional internal quantification method. Most importantly, (Formula 35) coexists with (Formula 24) and (Formula 25). That is, Figure 8 As shown in (a), regardless of whether the fluorescence intensity of the LIZ160 peak is proportional to or deviates from the proportion with respect to the concentration of LIZ160 contained in the sample, or regardless of whether the fluorescence intensity of the ROX160 peak is constant or decreases, the ratio of the fluorescence intensity of the LIZ160 peak to the fluorescence intensity of the ROX160 peak is always proportional to the concentration of LIZ160 contained in the sample.

[0287] Figure 10 Is with Figure 6-9 In the same experiment, the electric field injection time was increased from 9 seconds to 18 seconds. Figure 8 Corresponding experimental results. Figure 10 (a) Although in Figure 8 (a) was obtained by different electrophoresis analysis under different experimental conditions, but still shows the same Figure 8 The results show the same tendency as (a). However, the positions of the plotted points shift slightly up and down. Figure 10 (b) Although in Figure 8 (b) was obtained by different electrophoresis analysis under different experimental conditions, but shows the same Figure 8 The results are almost identical to those of (b), and the positional changes of the plotted points are also very small. Figure 10 The approximate straight line with slope 1 used in (b) is Figure 8 The above results indicate that the new internal standard method has high quantitative accuracy.

[0288] Figure 11 It will pass Figure 6 and Figure 7 A double logarithmic graph is created by plotting the ratio of the fluorescence intensity of the LIZ40 peak to the fluorescence intensity of the LIZ160 peak, and the fluorescence intensity of the LIZ114 peak, obtained from four electropherograms, against the concentration of 600 LIZ contained in the sample. The horizontal axis can also be considered to represent the concentration of LIZ40, LIZ114, or LIZ160 contained in the sample.

[0289] like Figure 6 、 Figure 7 and Figure 8 As shown in (a), the fluorescence intensity of each peak changes with the change of the concentration of 600LIZ contained in the sample, but Figure 11 The fluorescence intensity ratios shown remain constant. Figure 6 andFigure 7 The ratio of the fluorescence intensity of the peak of any two DNA fragments belonging to 600LIZ or 500ROX in each electropherogram remains constant.

[0290] This is illustrated by setting the first DNA fragment to LIZ40 or LIZ114 and the second DNA fragment to LIZ160 in (Math. 35). When the concentration of 600LIZ contained in the sample is changed, the concentration ratio of the plurality of DNA fragments contained in 600LIZ remains constant, and thus the right side remains constant, and the fluorescence intensity ratio of the left side remains constant. The above results confirm that the series of investigations disclosed in the above [Principle] are correct.

[0291] The present disclosure can be applied to any fragment analysis based on capillary electrophoresis. In each of the following examples, a part of a specific example thereof is shown.

[0292] [Example 2]

[0293] DNA typing based on short tandem repeat (STR) analysis is widely used for criminal investigations, identity confirmation in large-scale disasters, paternity testing, and the like, because of the high accuracy of personal identification. At present, various kits for STR analysis are sold. For example, in the PowerPlex (registered trademark) Fusion 6C System of Promega Corporation, human genomic DNA extracted from blood collected from a crime scene is used as a template, and 27 genetic loci of STR on the human genome are subjected to multiplex PCR using five fluorophores.

[0294] After 5 μl of PowerPlex Fusion 6C 5X Master Mix, 5 μl of PowerPlex Fusion 6C 5X Primer Pair Mix, and 25 μl of a pre-reaction solution containing extracted human genomic DNA were incubated at 96°C for 1 minute, heat cycles of 5 seconds at 96°C and 1 minute at 60°C were repeated 29 times, incubation at 60°C was performed for 10 minutes, and incubation at 4°C was performed. 25 μ of the post-reaction solution of the STR-PCR was subjected to ethanol precipitation, dissolved in 25 μl of pure water, and thus desalting was performed. 1 μl in 25 μl of the solution was mixed with 0.5 μl of a size standard labeled with a different fluorescent body from the above five fluorescent bodies, namely, WEN ILS 500 (hereinafter referred to as 500WEN) of Promega Corporation, and 9.5 μl of formamide, and thus 11 μl of a sample was obtained.

[0295] 500WEN contains 21 kinds of single-stranded DNA fragments of 60, 65, 80, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, and 500 bases in length, each labeled with a fluorescent body WEN. After heat denaturation at 95°C, the sample is subjected to rapid ice cooling, and then analyzed by capillary electrophoresis, whereby the DNA fragments of each base length labeled with any of the six fluorescent bodies contained in the sample are separated, and detected while identifying the labeled fluorescent bodies. By analyzing the electropherograms of the six fluorescent bodies respectively obtained, DNA typing is performed.

[0296] In DNA typing, it is important to accurately determine the base length of each DNA fragment in order to improve the accuracy of personal identification. However, in capillary electrophoresis, the migration speed of each DNA fragment varies in each capillary, each sample, and each analysis, and thus it is difficult to accurately determine the base length from the timing of the peak of each DNA fragment on the electropherogram. Therefore, as described above, a size standard of which the base length is known is mixed in the sample for analysis, and the timing of the peak of the DNA fragment of which the base length is known is referred to, whereby the base length of an arbitrary DNA fragment is accurately determined.

[0297] As described above, in the conventional DNA typing, the timing of the peak of the plurality of DNA fragments belonging to the size standard is flexibly used, but the fluorescence intensity of the peak is not flexibly used. However, the concentration of the size standard contained in the sample is known or fixed, and thus by referring to the fluorescence intensity of any of the peaks of the plurality of DNA fragments belonging to the size standard, the concentration of the DNA fragment contained in the sample can be quantified from the fluorescence intensity of any of the peaks of the plurality of DNA fragments contained in the sample as STR-PCR products. That is, not only the human genome as a template can be subjected to DNA typing, but also the plurality of DNA fragments contained in the sample as STR-PCR products can be individually quantified with high accuracy.

[0298] The high accuracy of this quantification is explained by setting the first DNA fragment to an arbitrary DNA fragment contained in the STR-PCR products and the second DNA fragment to an arbitrary DNA fragment contained in the size standard in (Mathematical expression 35). That is, the concentration of the first DNA fragment in the sample with respect to the concentration of the second DNA fragment in the sample can be obtained from the ratio of the fluorescence intensity of the peak of the first DNA fragment to the fluorescence intensity of the peak of the second DNA fragment.

[0299] In the case where the concentration of the size standard in the sample is constant, that is, the concentration of the second DNA fragment in the sample is constant, the concentration of the first DNA fragment in the sample can be obtained from the fluorescence intensity of the peak of the first DNA fragment relative to the fluorescence intensity of the peak of the second DNA fragment. In (Math. 35), because parameters capable of varying, E, T, C(0), and the like are not included, high-precision quantification can be performed as with the conventional internal quantification method. Such high-precision quantification is true in the case where the fluorescence intensity of the peak of the first DNA fragment is proportional to the concentration of the first DNA fragment contained in the sample and in the case where the proportion deviates, as shown in (Math. 24). That is, by the new internal standard method, the concentration of the first DNA fragment in a wider concentration range than in the past can be quantified with high precision.

[0300] Furthermore, by using the number of times of thermal cycles performed in the STR-PCR, the concentration of the human genome of the template contained in the solution before the STR-PCR can be quantified. The number of times of thermal cycles is set to N, the amplification efficiency of the STR-PCR is set to E(f), and the concentration of the human genome in the solution before the reaction of the STR-PCR is set to C(f). In the above example, N = 29. E(f) is E(f) = 1 under ideal conditions, but the actual value can be investigated in advance. In addition, an arbitrary STR-PCR product contained in the solution after the reaction of the STR-PCR is set to the first DNA fragment, and its concentration is set to B(l). Furthermore, the concentration of the first DNA fragment in the sample used for the electric field injection is set to C(l). Furthermore, the ratio of the concentration of the first DNA fragment in the sample used in the electric field injection C(l) to the concentration of the first DNA fragment in the solution after the reaction of the STR-PCR B(l), that is, the dilution rate of the solution after the reaction of the STR-PCR is set to D = C(l) / B(l). In the above example, D = 1 μl / 25 μl = 0.04. At this time, the following equation is established.

[0301] [Equation 38]

[0302] C1= D · C f · (1 + E f ) N (Equation 38)

[0303] In (Math. 35), when the second DNA fragment is set to an arbitrary DNA fragment contained in the size standard, C(l) can be obtained from (Math. 35). This is because S(l) and S(2) are obtained from the electropherogram, C(2) is known, and m(l) and m(2) can be investigated in advance. D, E(f), and N are known as described above, respectively. Therefore, according to (Equation 38), the concentration C(f) of the human genome contained in the solution before the reaction of the STR-PCR can be quantified with high precision.

[0304] [Example 3]

[0305] The SNaPshot (registered trademark) Multiplex system of Thermo Fisher Scientific is a kit for simultaneously typing SNPs (single nucleotide polymorphisms) at multiple places on a human genome using capillary electrophoresis. Template DNA amplified at multiple places on a human genome including SNPs is prepared in advance. For the template DNA, fluorescently unlabelled primers are hybridized at positions adjacent to each SNP, respectively, and single base extension reaction of each primer is performed using fluorescently labelled terminators. The fluorescently labelled terminators are ddATP, ddCTP, ddGTP, and ddTTP labelled with 4 different fluorophores, respectively. The base length of each primer varies depending on the corresponding SNP. Capillary electrophoresis analysis is performed on DNA fragments as a plurality of single base extension products, and electropherograms of each of the 4 fluorophores are obtained. On the electropherogram, the position of the corresponding SNP is determined from the electrophoresis time at which a peak is observed, that is, the base length of the corresponding DNA fragment. In addition, which one of A, C, G, T the SNP is determined from the fluorophore kind of the same peak. The above SNP typing can be performed simultaneously on SNPs at multiple sites.

[0306] In the above SNP typing, it is only necessary to determine whether each SNP is a homozygote (one SNP exists 100%) of any one of A, C, G, T, or a heterozygote (2 SNPs each exist 50%) of any 2 of A, C, G, T. However, generally, each SNP can be a base in which A, C, G, T are mixed at any ratio. For example, as cancer progresses, sometimes the wild type (hereinafter, WT) of an arbitrary SNP is any one of A, C, G, T, the mutant type (hereinafter, MT) is 3 of A, C, G, T other than the above 1, and the existence ratio of each is different. Conversely, by quantifying the existence ratio of the base kind of A, C, G, T in an arbitrary SNP, sometimes cancer can be diagnosed early, or the state of cancer can be grasped with high accuracy.

[0307] Suppose that M is set to an integer of 1 or more, the existence ratio of the base kind of A, C, G, T in the SNP at M, particularly the existence ratio of the base kind of the MT with respect to the base kind of the WT is quantified. For each of the SNPs at M, at most 4 kinds of single base extension products can be obtained. In this way, the reaction solution which maximally contains 4 x M kinds of DNA fragments is desalted by ethanol precipitation, dissolved in a desired amount of formamide, and used as a sample for field injection.

[0308] The electric field injection of each DNA fragment imparts a peak on the electropherogram of each of the four types of fluorescent bodies obtained by capillary electrophoresis analysis, and a maximum of 4 x M peaks are imparted. The SNP at M is assigned a SNP number i, which is set to i = 1, 2,..., M. The fluorescence intensity of the peak on the electropherogram of the single-base extension product of the base species A, C, G, T of the SNP number j, i.e., the DNA fragment, is set to S(ja), S(jc), S(jg), S(jt), the sensitivity coefficient of each is set to m(ja), m(jc), m(jg), m(jt), the average charge amount of each is set to q(ja), q(jc), q(jg), q(jt), and the concentration of each in the sample injected into the electric field injection capillary is set to C(ja), C(jc), C(jg), C(jt). The total injection charge amount Q of all the DNA fragments injected into the electric field injection capillary is, as with (Formula 13), the following formula.

[0309] [Formula 39]

[0310] Q = E - T - A -∑ i (q ia · μ · C ia + q ic · μ · C ic + q ig · μ · C ig + q it · μ · C it ) (Formula 39)

[0311] On the other hand, as with (Formula 15), the following formula.

[0312] [Formula 40]

[0313]

[0314] According to (Formula 39) and (Formula 40), as with (Formula 21), the following formula.

[0315] [Formula 41]

[0316]

[0317] q(0) · μ(0) · C(0) represents the injection charge amount of the negative ions other than the DNA fragments, and the Σ term represents the total injection charge amount of all the DNA fragments.

[0318] In Equation 41, when the Σ term is small compared to q(0) μ(0) C(0), Q is proportional to the Σ term, but when the Σ term becomes large compared to q(0) μ(0) C(0), Q deviates from the proportionality to the Σ term and reaches saturation. Thus, the total injection amount of all DNA fragments is proportional to the total concentration of all DNA fragments when the total concentration of all DNA fragments is low, but if the total concentration of all DNA fragments becomes high, it deviates from the proportionality and reaches saturation. Of course, the increase in the total concentration of all DNA fragments is caused by an increase in the concentration of an arbitrary DNA fragment. Moreover, if the total injection amount of all DNA fragments saturates, the injection amount of an arbitrary DNA fragment saturates or, on the contrary, decreases.

[0319] On the other hand, however, when arbitrary two DNA fragments among all DNA fragments are taken as the first DNA fragment and the second DNA fragment, Equations 8 to 11, 22, 23, 29, 32, and 35 are established. Equation 35 indicates that the fluorescence intensity ratio of the first DNA fragment and the second DNA fragment obtained by performing capillary electrophoresis analysis on the first DNA fragment and the second DNA fragment injected into the capillary by the electric field injection is proportional to the concentration ratio of the first DNA fragment and the second DNA fragment in the sample.

[0320] For example, when the single-base extension product, i.e., the DNA fragment, of the base species A, C, G, and T of SNP number j is focused on, Equations 22 and 23 are the following equations as well.

[0321] [Equation 42]

[0322] S ja = E · T · A · m ja · μ · C ja [Equation 42]

[0323] [Equation 43]

[0324] S jc = E · T · A · m jc · μ · C jc [Equation 43]

[0325] [Equation 44]

[0326] S jg = E · T · A · m jg · μ · C ig [Equation 44]

[0327] [Equation 45]

[0328] S jt = E · T · A · m jt · μ · Cjt (Formula 45)

[0329] Therefore, as an example, if the WT is A and the MT is C, G, or T, (Formula 35) becomes the following equation.

[0330] [Formula 46]

[0331]

[0332] [Formula 47]

[0333]

[0334] [Formula 48]

[0335]

[0336] That is, by the fluorescence intensities S(jc) / S(ja), S(jg) / S(ja), S(jt) / S(ja) of the peaks on the electropherogram, the concentration ratios C(jc) / C(ja), C(jg) / C(ja), C(jt) / C(ja) corresponding to the presence ratios of the three types of MTs with respect to the WT can be quantified with high precision. m(ja), m(jc), m(jg), and m(jt) are obtained in advance.

[0337] In the above, a case in which a maximum of 4 x M types of DNA fragments that are single base extension products are contained in the sample is assumed. In the following, a case in which, in addition to the maximum of 4 x M types of single base extension products, i.e., DNA fragments, one or more types of DNA fragments that are internal standards of which the concentration is known or fixed are contained in the sample is assumed. As the internal standard, a size standard that contains DNA fragments of various base lengths can be used. At this time, in (Formula 35), by taking any one of the single base extension products, i.e., DNA fragments, of the base types A, C, G, and T of the SNP number j as the first DNA fragment and any one of the DNA fragments of the internal standard as the second DNA fragment, the concentration of the first DNA fragment in the sample can be quantified with high precision.

[0338] Symbol Explanation

[0339] 1 capillary,

[0340] 2 sample injection end,

[0341] 3 sample elution end,

[0342] 4 negative electrode,

[0343] 5 positive electrode,

[0344] 6 negative electrode side buffer,

[0345] 7 buffer on the anode side,

[0346] 8 polymer solution,

[0347] 9 sample,

[0348] 10 pump block,

[0349] 11 valve,

[0350] 12 syringe,

[0351] 13 power supply,

[0352] 14 laser beam,

[0353] 15 laser light source,

[0354] 16 measurement point,

[0355] 17 detector,

[0356] 18 first DNA fragment (first component),

[0357] 19 second DNA fragment (second component),

[0358] 20 negative ions other than DNA fragments.

Claims

1. A capillary electrophoresis apparatus, performing the following processing: injecting a sample comprising a first component and a second component into a capillary; performing electrophoretic separation on the injected first component and the second component; as well as The luminescence from the first component and the luminescence from the second component induced by irradiating light onto the capillary are measured by a detector, thereby obtaining the signal intensity of the first component and the signal intensity of the second component, characterized in that: The concentration range of the first component contained in the sample includes, in addition to the concentration range in which the signal intensity of the first component is proportional to the concentration of the first component, a concentration range in which the signal intensity of the first component is lower than the saturation signal intensity of the detector relative to the concentration of the first component and deviates from the proportion to reach the saturation signal intensity. The capillary electrophoresis device quantifies the ratio of the concentration of the first component to the concentration of the second component in the sample based on the ratio of the signal intensity of the first component to the signal intensity of the second component.

2. A capillary electrophoresis apparatus for performing the following processes: injecting a sample comprising a first component and a second component into a capillary; performing electrophoretic separation on the injected first component and the second component; as well as The luminescence from the first component and the luminescence from the second component induced by irradiating light onto the capillary are measured by a detector, thereby obtaining the signal intensity of the first component and the signal intensity of the second component, characterized in that: The concentration range of the first component contained in the sample includes not only a concentration range in which the signal intensity of the second component is constant relative to the concentration of the first component, but also a concentration range in which the signal intensity of the second component deviates from the constant and decreases relative to the concentration of the first component. The capillary electrophoresis device quantifies the ratio of the concentration of the first component to the concentration of the second component in the sample based on the ratio of the signal intensity of the first component to the signal intensity of the second component.

3. The capillary electrophoresis device according to claim 1 or 2, characterized in that: the concentration of the second component in the sample is known, The concentration of the first component in the sample is quantified based on the ratio of the signal intensity of the first component to the signal intensity of the second component.

4. The capillary electrophoresis device according to any one of claims 1 to 3, characterized in that: The first component and the second component are both DNA fragments labeled with a fluorescent substance, and the base length of the first component is different from the base length of the second component. The luminescence is fluorescence, The signal intensity is the fluorescence intensity.

5. The capillary electrophoresis device according to claim 4, characterized in that The sample comprises size standards, The second component is the DNA fragment contained in the size standard.

6. The capillary electrophoresis device according to claim 4, characterized in that The sample comprises a PCR product, The first component is the DNA fragment contained in the PCR product.

7. The capillary electrophoresis device according to claim 4, characterized in that The sample contains a single base extension product, The first component is the first DNA fragment contained in the single-base extension product.

8. The capillary electrophoresis device according to claim 7, characterized in that: The second component is the second DNA fragment contained in the single-base extension product.

9. A capillary electrophoresis method comprising the following steps: injecting a sample comprising a first component and a second component into a capillary; performing electrophoretic separation on the injected first component and the second component; and The luminescence from the first component and the luminescence from the second component induced by irradiating light onto the capillary are measured by a detector, thereby obtaining the signal intensity of the first component and the signal intensity of the second component, characterized in that: The concentration range of the first component contained in the sample includes, in addition to the concentration range in which the signal intensity of the first component is proportional to the concentration of the first component, a concentration range in which the signal intensity of the first component is lower than the saturation signal intensity of the detector relative to the concentration of the first component and deviates from the proportion to reach the saturation signal intensity. The capillary electrophoresis method comprises the following steps: The ratio of the concentration of the first component to the concentration of the second component in the sample is quantified based on the ratio of the signal intensity of the first component to the signal intensity of the second component.

10. A capillary electrophoresis method comprising the following steps: injecting a sample comprising a first component and a second component into a capillary; performing electrophoretic separation on the injected first component and the second component; and The luminescence from the first component and the luminescence from the second component induced by irradiating light onto the capillary are measured by a detector, thereby obtaining the signal intensity of the first component and the signal intensity of the second component, characterized in that: The concentration range of the first component contained in the sample includes not only a concentration range in which the signal intensity of the second component is constant relative to the concentration of the first component, but also a concentration range in which the signal intensity of the second component deviates from the constant and decreases relative to the concentration of the first component. The capillary electrophoresis method comprises the following steps: The ratio of the concentration of the first component to the concentration of the second component in the sample is quantified based on the ratio of the signal intensity of the first component to the signal intensity of the second component.

11. The capillary electrophoresis method according to claim 9 or 10, characterized in that: the concentration of the second component in the sample is known, The concentration of the first component in the sample is quantified based on the ratio of the signal intensity of the first component to the signal intensity of the second component.

12. The capillary electrophoresis method according to any one of claims 9 to 11, characterized in that: The first component and the second component are both DNA fragments labeled with a fluorescent substance, and the base length of the first component is different from the base length of the second component. The luminescence is fluorescence, The signal intensity is the fluorescence intensity.

13. The capillary electrophoresis method according to claim 12, wherein: The sample comprises size standards, The second component is the DNA fragment contained in the size standard.

14. The capillary electrophoresis method according to claim 12, wherein: The sample comprises a PCR product, The first component is the DNA fragment contained in the PCR product.

15. The capillary electrophoresis method according to claim 12, wherein: The sample contains a single base extension product, The first component is the first DNA fragment contained in the single-base extension product.

16. The capillary electrophoresis method according to claim 15, wherein: The second component is the second DNA fragment contained in the single-base extension product.

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