Capillary electrophoresis device and capillary electrophoresis method

Abstract

Provided is a capillary electrophoresis device in which a sample that contains a first component and a second component is injected into a capillary, the injected first component and second component are subjected to electrophoretic separation, and emission of light from the first component and the second component induced by irradiating the capillary with light is measured by a detector, whereby the signal intensity of the first component and the signal intensity of the second component are acquired, wherein: 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 and reaches the saturation signal intensity when disproportional to the concentration of the first component; and the capillary electrophoresis device quantifies the ratio of the concentration of the first component to the concentration of the second component in the sample on the basis of the ratio of the signal intensity of the first component to the signal intensity of the second component.

Description

Capillary electrophoresis apparatus and capillary electrophoresis method

[0001] The present invention relates to a capillary electrophoresis apparatus and a capillary electrophoresis method.

[0002] Instrumental analysis in analytical chemistry is analysis using instruments that perform nuclear magnetic resonance spectroscopy, absorption spectroscopy, Raman spectroscopy, fluorescence spectroscopy, mass spectroscopy, chromatography, electrophoresis, and the like.

[0003] As shown in Non-Patent Documents 1 and 2, these instrumental analyses often use an external standard method (absolute calibration curve method) or an internal standard method (internal standard method) to quantify the unknown concentration of an analyte contained in a sample.

[0004] The external standard method is a method for quantifying unknown concentrations of an analyte in a sample by preparing a calibration curve, which shows the relationship between the analyte concentration and signal intensity, using standard samples containing various known concentrations of the analyte. Generally, it is assumed that the signal intensity is proportional to the analyte concentration, that is, the increase in signal intensity with respect to the analyte concentration is linear and the rate of increase in signal intensity with respect to the analyte concentration is constant.

[0005] However, the external standard method can work even when the increase in signal intensity with respect to analyte concentration is nonlinear, i.e., when the rate of increase in signal intensity with respect to analyte concentration decreases with increasing analyte concentration. However, the external standard method suffers from poor quantification accuracy due to the influence of sample matrix effects and variations in the amount of sample injected into the analytical instrument.

[0006] In contrast, the internal standard method reduces these effects and improves quantitative accuracy. This method involves using standard samples containing various known concentrations of the analyte and the internal standard to create a calibration curve in advance, which shows the relationship between the concentration ratio of the analyte to the internal standard and the signal intensity ratio of the analyte to the internal standard, and then quantifying the unknown concentration of the analyte in the sample.

[0007] Here, it is necessary to be able to measure the signal intensities of the internal standard and the analyte independently. As shown in Non-Patent Documents 1 and 2, in the internal standard method, the signal intensities of the analyte and the internal standard must be proportional to their concentrations, that is, the increase in the signal intensities of the analyte and the internal standard must be linear with respect to their concentrations.

[0008] On the other hand, the internal standard method does not work if the increase in signal intensity relative to the concentration of the analyte and the internal standard is nonlinear. In other words, it is not possible to improve quantitative accuracy by reducing the influence of the sample matrix effect or the influence of variations in the amount of sample injected into the analytical instrument. In fact, as pointed out in Non-Patent Document 2, it is known that quantitative accuracy is actually reduced.

[0009] The above will be explained more specifically in the case of quantifying the concentration C(tg) of a fluorescently labeled DNA fragment, which is the target of analysis contained in a sample, by capillary electrophoresis analysis using laser-induced fluorescence measurement. In this specification, the subscripts used for coefficients or variables used in mathematical formulas may be shown in parentheses. For example, when the subscript tg is added to the variable C, it may be expressed as C(tg), and C tg It is sometimes expressed as:

[0010] In FIG. 1 of Patent Document 1, a capillary electrophoresis apparatus is used that processes four capillary electrophoresis analyses in parallel, and electrophoresis analysis is performed using one of the capillaries. The sample may contain fluorescently labeled DNA fragments other than the fluorescently labeled DNA fragment to be analyzed. In addition, salts (ions) other than the DNA fragments contained in the sample are removed in advance as much as possible using ethanol precipitation or column purification.

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

[0012] Next, the injected DNA fragments are separated by base length as they move toward the sample elution end of the capillary by electrophoresis. During this process, the DNA fragments that pass the measurement point on the capillary by electrophoresis are irradiated with a laser beam, causing the fluorophores labeled on the DNA fragments to emit fluorescence.

[0013] The emitted fluorescence is sequentially measured by an image sensor, and the time series gives an electropherogram, on which peaks corresponding to the DNA fragments being analyzed are obtained.

[0014] The signal intensity S(tg) of the DNA fragment to be analyzed is generally expressed as the area of ​​the peak of the DNA fragment to be analyzed, but if the width of the peak can be considered constant, it can be expressed as the height of the peak of the DNA fragment to be analyzed. Since the signal intensity S(tg) of the DNA fragment to be analyzed is proportional to the amount of the DNA fragment to be analyzed injected into the capillary, the proportionality coefficient is K(tg), and where E is the effective electric field strength in the sample near the sample injection end of the capillary during electric field injection, and T is the time of electric field injection.

[0015] When E and T are fixed, Equation 1 shows that the signal intensity S(tg) of the analyte DNA fragment is proportional to the concentration C(tg) of the analyte DNA fragment in the sample, and is the calibration curve for the external standard method. Using Equation 1, the unknown concentration C(tg) of the analyte in the sample can be quantified from the measured signal intensity S(tg) of the analyte DNA fragment.

[0016] On the other hand, in the internal standard method, a sample is mixed with a DNA fragment serving as an internal standard of known concentration and then subjected to capillary electrophoresis analysis. The DNA fragment serving as the internal standard is also fluorescently labeled. On the electropherogram, the peaks of the DNA fragments to be analyzed and the peaks of the DNA fragments serving as the internal standard are observed independently. The signal intensity S(st) of the DNA fragments serving as the internal standard is proportional to the amount of the DNA fragments serving as the internal standard injected into the capillary, so that the proportionality coefficient is K(st), and the following is given:

[0017] Equation 2 shows 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. Since the electric field injection of the DNA fragment to be analyzed and the internal standard DNA fragment in the sample is carried out all at once, the electric field strength E and time T of the electric field injection in Equation 1 and Equation 2 are the same. Therefore, by taking the ratio of Equation 1 to Equation 2, Equation 3 shows that the signal intensity ratio S(tg) / S(st) of the DNA fragment to be analyzed relative to the internal standard is proportional to the concentration ratio C(tg) / C(st) of the DNA fragment to be analyzed relative to the internal standard in the sample, and is the calibration curve for the internal standard method.

[0018] Using Equation 3, the concentration ratio C(tg) / C(st) of the target DNA fragment to the internal standard in the sample can be determined from the signal intensity ratio S(tg) / S(st) of the target DNA fragment to the measured internal standard. Since the concentration C(st) of the internal standard DNA fragment in the sample is known, this is equivalent to determining the unknown concentration Ctg of the target DNA fragment in the sample.

[0019] In the external standard method using (Equation 1), for example, if the electric field strength E of the electric field injection varies by ±10%, as is clear from (Equation 1), this variation directly leads to a decrease in the quantitative accuracy of the concentration of the DNA fragment to be analyzed. In contrast, in the internal standard method using (Equation 3), since (Equation 3) does not include the electric field strength E of the electric field injection, this variation does not affect the quantitative accuracy of the concentration of the DNA fragment to be analyzed. This is because the influence of the variation in the electric field strength E in (Equation 1) and the influence of the variation in the electric field strength E in (Equation 2) are the same, and by taking the ratio of (Equation 1) to (Equation 2), these effects are canceled out. This is why the quantitative accuracy of the analyte can be improved using the internal standard method compared to the external standard method.

[0020] International Publication No. 2023 / 007567

[0021] Harvey, David. Modern analytical chemistry. Vol. 1. New York: McGraw-Hill, 2000.AK Hewavitharana (2009) Internal Standard-Friend or Foe?, Critical Reviews in Analytical Chemistry, 39:4, 272-275

[0022] If the signal intensity is not proportional to the analyte concentration, we consider why the external standard method may work but the internal standard method does not.

[0023] For example, following Non-Patent Document 2, if the signal intensity of the analyte is expressed as a quadratic function of the concentration, then, with L(tg) and K(tg) as coefficients, The constant term of the quadratic function is set to zero so that S(tg)=0 when C(tg)=0. Incidentally, when L(tg)=0, (Equation 4) becomes the same as (Equation 1).

[0024] In the external standard method, Equation 4 is the calibration curve. As in Equation 1, Equation 4 can be used to quantify the unknown concentration C(tg) of the analyte contained in the sample from the signal intensity S(tg) of the analyte DNA fragment being measured.

[0025] Similarly, if the signal intensity of the internal standard is expressed as a quadratic function of the concentration, then with L(st) and K(st) as coefficients, The constant term of the quadratic function is set to zero so that S(tg)=0 when C(tg)=0. Incidentally, when L(st)=0, (Equation 5) becomes the same as (Equation 2).

[0026] In this case, unlike Equation 3, taking the ratio of Equation 4 to Equation 5 does not simplify the equation, and the signal intensity ratio S(tg) / S(st) of the DNA fragment to be analyzed relative to the internal standard cannot be expressed as the concentration ratio C(tg) / C(st) of the DNA fragment to be analyzed relative to the internal standard in the sample. Furthermore, unlike Equation 3, the electric field strength E and time T of the electric field injection remain, making it impossible to cancel out their variations. Therefore, the quantitative accuracy of the analyte using the internal standard method is not improved compared to that using an external standard. In fact, the quantitative accuracy is lower than that of the external standard method using Equation 4. As described in Non-Patent Document 2, if the signal intensity of either the analyte or the internal standard is not proportional to its concentration, the quantitative accuracy of the internal standard method decreases.

[0027] The above discussion has focused on an example in which the signal intensities of the analyte and internal standard are expressed as quadratic functions of their respective concentrations, but the same applies to cases in which they are expressed as any function other than a linear function with a constant term of 0. As a result, if the signal intensity is not proportional to the analyte concentration, the internal standard method cannot be used to improve quantitative accuracy.

[0028] On the other hand, Patent Document 1 proposes improving the dynamic range of fluorescence measurement in a capillary electrophoresis device by optimizing the binning conditions according to the noise conditions of the image sensor. According to Patent Document 1, it is expected that it will be possible to quantify the concentration of DNA fragments in a sample over a wider concentration range than before.

[0029] However, when actually tested, it was found that when the concentration of the target DNA fragments in the sample was low, a signal intensity proportional to the concentration was obtained, but when the concentration of the target DNA fragments in the sample became high, the signal intensity saturated with respect to the concentration. Furthermore, because this saturated signal intensity was lower than the signal intensity at which the image sensor became saturated, it was found that the saturation of the image sensor was not the cause.

[0030] This phenomenon is a new issue that has become apparent with the use of capillary electrophoresis instruments with a wider dynamic range than conventional instruments. When the concentration of the target DNA fragments in a sample is low, the signal intensity obtained is proportional to the concentration of the target DNA fragments, making it possible to improve the quantitative accuracy of the target DNA fragments using the internal standard method. However, when the concentration of the target DNA fragments in a sample is high, the signal intensity obtained is not proportional to the concentration of the target DNA fragments, making it impossible to improve the quantitative accuracy of the target DNA fragments using the internal standard method.

[0031] The present invention has been made to solve these problems, and aims to provide a capillary electrophoresis apparatus and a capillary electrophoresis method that can improve the quantitative accuracy of an analyte using an internal standard method.

[0032] An example of a capillary electrophoresis apparatus according to the present invention comprises: injecting a sample containing a first component and a second component into a capillary; electrophoretically separating the injected first component and the second component; irradiating the capillary with light to induce luminescence from the first component and the second component with a detector, thereby obtaining the signal intensities of the first component and the second component; wherein 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 and deviates from the proportionality to the concentration of the first component, reaching the saturation signal intensity; and the capillary electrophoresis apparatus 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.

[0033] An example of a capillary electrophoresis apparatus according to the present invention comprises: injecting a sample containing a first component and a second component into a capillary; electrophoretically separating the injected first component and the second component; irradiating light onto the capillary to induced luminescence from the first component and the second component with a detector, thereby obtaining the signal intensities of the first component and the second component; wherein 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 second component is constant relative to the concentration of the first component, a concentration range in which the signal intensity of the second component decreases relative to the concentration of the first component; and the capillary electrophoresis apparatus 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.

[0034] An example of a capillary electrophoresis method according to the present invention comprises: injecting a sample containing a first component and a second component into a capillary; electrophoretically separating the injected first component and the second component; and measuring luminescence from the first component and the second component induced by irradiating light onto the capillary with a detector, thereby obtaining the signal intensities of the first component and the second component. The capillary electrophoresis method is 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 and deviates from the proportionality to the concentration of the first component, reaching the saturation signal intensity; and the capillary electrophoresis method comprises quantifying 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.

[0035] An example of a capillary electrophoresis method according to the present invention comprises: injecting a sample containing a first component and a second component into a capillary; electrophoretically separating the injected first component and the second component; and measuring, with a detector, luminescence from the first component and luminescence from the second component induced by irradiating light onto the capillary, thereby obtaining the signal intensities of the first component and the second component. The capillary electrophoresis method is characterized in that 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 second component is constant relative to the concentration of the first component, a concentration range in which the signal intensity of the second component decreases relative to the concentration of the first component, and the capillary electrophoresis method comprises quantifying 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.

[0036] By using the novel internal standard method discovered in the present invention, it becomes possible to quantify with high precision DNA fragments contained in a sample over a wider concentration range than ever before.

[0037] Problems, configurations, and effects other than those described above will become apparent from the following description of the embodiments.

[0038] Configuration of a capillary electrophoresis device according to an embodiment of the present inventionConfiguration of a capillary electrophoresis method according to an embodiment of the present inventionSchematic diagram of electric field injectionElectropherograms of samples with various concentrationsRelationship between sample concentration and fluorescence intensity (Part 1)Relationship between sample concentration and fluorescence intensity (Part 2)Electropherogram of a sample mixed with two types of size standards at different ratios (Part 1)Electropherogram of a sample mixed with two types of size standards at different ratios (Part 2)Relationship between sample concentration and fluorescence intensity and fluorescence intensity ratio of two types of DNA fragments (Part 1)Relationship between sample concentration and fluorescence intensity of three types of DNA fragmentsRelationship between sample concentration and fluorescence intensity and fluorescence intensity ratio of two types of DNA fragments (Part 2)Relationship between sample concentration and fluorescence intensity ratio of two types of DNA fragments

[0039] [Principle] In the capillary electrophoresis analysis described above, when the concentration of the DNA fragment to be analyzed in the sample is low, a signal intensity proportional to the concentration is obtained, but when the concentration of the DNA fragment to be analyzed in the sample is high, the signal intensity saturates with respect to the concentration. The cause of this phenomenon was considered.

[0040] If image sensor saturation is not the cause, another possible cause is self-quenching of the fluorophore at the measurement point. It is generally known that when the concentration of a fluorophore becomes very high, self-quenching of the fluorophore occurs, and the rate of increase in fluorescence intensity decreases depending on the concentration of the fluorophore, eventually reaching saturation. It is also known that if the concentration of the fluorophore increases further, the fluorescence intensity may begin to decrease.

[0041] To verify the presence or absence of self-quenching in the above-mentioned capillary electrophoresis analysis, samples containing multiple types of DNA fragments at different concentrations were analyzed using the above-mentioned capillary electrophoresis device. When increasing the concentration of the DNA fragments in the sample, the total concentration of the multiple DNA fragments was increased while maintaining the concentration ratio of the multiple DNA fragments in the sample.

[0042] The multiple DNA fragments were spatially separated by capillary electrophoresis and detected independently by laser-induced fluorescence measurement at the measurement point, each giving a distinct peak on the electropherogram.

[0043] DNA fragments with high concentrations in the sample gave peaks of high intensity, while DNA fragments with low concentrations in the sample gave peaks of low intensity. When the total concentration of the multiple DNA fragments to be analyzed in the sample was low, signal intensities proportional to the concentration of each DNA fragment were obtained. In contrast, when the total concentration of the multiple DNA fragments to be analyzed in the sample was high, signal intensities not proportional to the concentration of each DNA fragment were obtained. In other words, regardless of the intensity of the peaks on the electropherogram corresponding to the concentrations in the sample, a simultaneous change occurred from a state in which the signal intensity was proportional to the concentration of the DNA fragment in the sample to a state in which the signal intensity was not proportional to the concentration of the DNA fragment in the sample.

[0044] The degree of fluorophore self-quenching should depend on the fluorophore concentration at the measurement point on the capillary. However, the above change occurs independently of the fluorophore concentration at the measurement point on the capillary. From the above, it is clear that the cause of this phenomenon is not fluorophore self-quenching. Therefore, in the above capillary electrophoresis analysis, the concentrations of the DNA fragments and the fluorophores labeling the DNA fragments at the measurement point on the capillary do not reach concentrations that would cause self-quenching. Of course, this phenomenon does not occur due to saturation of the image sensor, so the cause of this phenomenon is not due to saturation of the image sensor. Therefore, in the above capillary electrophoresis analysis, the concentrations of the DNA fragments and the fluorophores labeling the DNA fragments at the measurement point on the capillary do not reach concentrations that would cause saturation of the image sensor.

[0045] Therefore, the cause of this phenomenon was independently elucidated through the study of the present invention as described below. This phenomenon was discovered for the first time through the study of the present invention, and its cause was also discovered for the first time through the study of the present invention. Furthermore, while allowing for this phenomenon, a unique new internal standard method was devised based on the discovered cause, enabling highly accurate quantitative analysis of the analyte.

[0046] [Embodiment 1] Fig. 1A is a diagram showing the configuration of a capillary electrophoresis device. Capillary electrophoresis devices are widely used as analytical devices for DNA sequencing and DNA fragment analysis. Four capillaries 1 are used, and each capillary 1 can analyze a different sample.

[0047] Figure 1B shows the configuration of capillary electrophoresis. One capillary electrophoresis analysis is performed by the capillary electrophoresis method comprising steps (1) to (8) in Figure 1B. Multiple capillary electrophoresis analyses can be performed by repeating steps (1) to (8).

[0048] (1) First, the sample injection ends 2 of the four capillaries 1 are immersed in the cathode buffer solution 6, and the sample elution ends 3 are connected to the anode buffer solution 7 via the polymer solution 8 inside the pump block 10.

[0049] (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 pushed down to pressurize the polymer solution 8 inside, and the polymer solution 8 is filled into the inside of each capillary 1 from the sample elution end 3 toward the sample injection end 2.

[0050] (3) Next, the valve 11 is opened, the sample injection ends 2 of the four capillaries 1 are immersed in different samples 9, and a constant voltage is applied between the negative electrode 4 and the positive electrode 5 by the power supply 13 for a certain period of time, thereby electrochemically injecting a portion of the different sample 9 (containing at least the first component and the second component) into each capillary 1 from the sample injection end 2. This causes the sample 9 containing the first component and the second component to be injected into the capillary.

[0051] (4) Then, the sample injection ends 2 of the four capillaries 1 are immersed in the cathode side buffer solution 6, and a high voltage is applied between the cathode 4 and the anode 5 by the power supply 13, thereby initiating capillary electrophoresis. The DNA fragments labeled with the fluorescent substance are electrophoresed from the sample injection end 2 toward the sample elution end 3. This allows the injected first and second components to be separated by electrophoresis.

[0052] (5) In parallel, a position of each capillary 1 electrophoresed a certain distance from the sample injection end 2 is set as a measurement point 16, and a laser beam 14 emitted from a laser light source 15 is irradiated simultaneously onto each measurement point 16. Here, the coating of each capillary 1 near the measurement point 16 is removed in advance, and each capillary 1 near the measurement point 16 is arranged on the same plane, and the laser beam 14 is focused and then introduced along the plane from the side of the above-mentioned arrangement plane.

[0053] (6) When the fluorescently labeled DNA fragments pass through the measurement points 16, they are excited by the laser beam 14 and emit fluorescence. In other words, the intensity of the fluorescence emitted from each of the four measurement points 16 changes from moment to moment as the DNA fragments move through the electrophoresis.

[0054] (7) Next, the fluorescence emitted from each measurement point 16 is measured by the detector 17. That is, the emitted fluorescence from the first component and the emitted fluorescence from the second component induced by irradiating the capillary 1 with a laser beam are measured by the detector 17, thereby obtaining the signal intensities of the first component and the second component. An electropherogram, which is time-series data of these signal intensities, is obtained and the sample 9 injected into each capillary 1 is analyzed. The detector 17 includes a spectrometer and an image sensor (not shown) and can simultaneously and independently measure the emitted fluorescence from the four measurement points 16. This makes it possible to distinguish between the emitted fluorescence of multiple types of fluorophores.

[0055] (8) Finally, the ratio of the concentration of the first component to the concentration of the second component in the sample is determined based on the ratio of the signal intensity of the first component to the signal intensity of the second component. This determination is possible based on the explanation of the above [Principle] and the method verified below.

[0056] Figure 2 shows a schematic diagram of the electric field injection of a sample 9 in capillary electrophoresis analysis. The sample injection end 2 of a capillary 1 filled with a polymer solution 8 is immersed in the liquid sample 9, with Figure 2(a) showing the state before electric field injection and Figure 2(b) showing the state after electric field injection. After Figure 2(b), the sample injection end 2 is immersed in a buffer solution on the cathode side, and electrophoresis begins.

[0057] The sample 9 contains a first DNA fragment 18 (first component), a second DNA fragment 19 (second component), which are negative ions, and negative ions 20 other than DNA fragments. The first DNA fragment 18 and the second DNA fragment 19 to be analyzed are both DNA fragments labeled with a fluorescent substance. The base length of the first DNA fragment 18 and the base length of the second DNA fragment 19 are different. The first DNA fragment 18 and the second DNA fragment 19 have different concentrations in the sample 9.

[0058] As in the specific example described below, sample 9 may contain a size standard, and second DNA fragment 19 may be a DNA fragment contained in the size standard. Alternatively, sample 9 may contain a PCR product, and first DNA fragment 18 may be a DNA fragment that is a PCR product or a DNA fragment derived from the PCR product. Alternatively, sample 9 may contain a single-base extension product, and first DNA fragment 18 may be a first DNA fragment that is a single-base extension product, and second DNA fragment 19 may be a second DNA fragment that is a single-base extension product.

[0059] In this example, two types of DNA fragments are mainly dealt with as the simplest example of multiple types of DNA fragments, but it goes without saying that a similar consideration can be made for any number of types of DNA fragments, three or more.

[0060] The cation and cathode electrodes are omitted from Figure 2. The solvent for Sample 9 is pure water or formamide. While negative ions (salts) other than DNA fragments are removed from Sample 9 in advance by ethanol precipitation or other methods, they cannot be reduced to zero.

[0061] In the state shown in Figure 2(a), electric field injection is performed by applying a voltage to both ends of the capillary 1, with the sample injection end 2 of the capillary 1 as the negative electrode and the sample elution end 3 as the positive electrode, so that the value of voltage x time is constant. For example, a constant voltage may be applied for a fixed period of time. This results in the state shown in Figure 2(b). As shown in Figure 2(b), the first DNA fragment 18, the second DNA fragment 19, which are negative ions in the sample 9, and some of the negative ions 20 other than the DNA fragments are injected into the capillary 1 from the sample injection end 2 of the capillary 1. The amount of each negative ion injected at this time is estimated as follows.

[0062] The current I that flows when a constant voltage V is applied between the cathode and anode electrodes is largely determined by the electrical resistance R of the capillary 1 filled with the polymer solution 8, and is expressed as I ≒ V / R. This is because the electrical resistance R(i) between the cathode 4 (Fig. 1A) and the sample injection end 2 of the capillary 1, and the electrical resistance R(o) between the sample elution end 3 (Fig. 1A) of the capillary and the anode 5 (Fig. 1A) are sufficiently small compared to R (R≫R(i), R(o)). In other words, the combined resistance R(i) + R + R(o) between the cathode 4 and anode 5 is approximately equal to R (R(i) + R + R(o) ≒ R).

[0063] For this reason, regardless of the composition of the sample, for example, whether the sample is pure water or a solution with a high ion concentration, the current I that flows when a constant voltage V is applied between the negative and positive electrodes remains almost constant. For this reason, "applying a constant voltage between the negative and positive electrodes" is sometimes expressed as "applying a constant voltage to both ends of the capillary."

[0064] Furthermore, the current I flowing between the negative electrode and the sample injection end of the capillary, the current I flowing through the capillary, and the current I flowing between the sample elution end of the capillary and the positive electrode are all equal due to the continuity of the current.

[0065] Here, the current I that flows between the cathode and the sample injection end of the capillary during field injection, i.e., the current I that flows through the sample, is carried by the negative ions injected into the capillary, ignoring positive ions for simplicity. Therefore, the total amount of negative ions injected into the capillary by field injection, which is achieved by applying a constant voltage to both ends of the capillary for a certain period of time, is constant regardless of the composition of the sample.

[0066] Let E be the effective electric field strength in the sample near the sample injection end of the capillary during electric field injection, T be the time for electric field injection, and A be the internal cross-sectional area of ​​the capillary. If the mobility of negative ions other than DNA fragments in the sample is μ(0) and their concentration is C(0), then the number of injected molecules of negative ions other than DNA fragments due to electric field injection, J(0), is: The mobility indicates the speed at which each negative ion moves per unit electric field strength.

[0067] When there are multiple types of negative ions other than DNA fragments, the average mobilities and concentrations of these ions are μ(0) and C(0). If the average charge per molecule of negative ions other than DNA fragments is q(0), the injected charge Q(0) of negative ions other than DNA fragments by electric field injection is is.

[0068] Furthermore, if the mobility of the first DNA fragment and the second DNA fragment in the sample is μ, the concentration of the first DNA fragment is C(1), and the concentration of the second DNA fragment is C(2), the number of injected molecules of the first DNA fragment J(1) and the number of injected molecules of the second DNA fragment J(2) by electric field injection are is.

[0069] The mobility of the first DNA fragment and the second DNA fragment is set to be equal because the mobility of DNA fragments in a solution without a molecular sieving effect, i.e., in a sample, is constant regardless of base length.

[0070] If the average charge amounts per molecule of the first DNA fragment and the second DNA fragment are q(1) and q(2), respectively, the charge amounts Q(1) and Q(2) injected into the first DNA fragment and the second DNA fragment by electric field injection are given by is.

[0071] If the total concentration of the first DNA fragment and the second DNA fragment in the sample is C = C(1) + C(2), the total number of molecules J injected by electric field injection of the first DNA fragment and the second DNA fragment is: is.

[0072] Here, if the average charge amount per molecule of the first DNA fragment and the second DNA fragment is equal and can be approximated as q = q(1) = q(2), the total charge amount Q injected into the first DNA fragment and the second DNA fragment by electric field injection is Alternatively, if the ratio of the concentrations C(1) and C(2) of the first and second DNA fragments to the total concentration C of the first and second DNA fragments in the sample is constant, then equation 13 can also be obtained by defining q = (q(1) C(1) + q(2) C(2)) / (C(1) + C(2)).

[0073] In (Equation 6) to (Equation 13), E, ​​T, and A are the same value. Since the total amount of injected charge of negative ions per unit time during electric field injection is equal to the current I, That is, is.

[0074] Since I is constant as described above, Equation 14 and Equation 15 mean that field injection is a competitive process among multiple types of negative ions, including DNA fragments, contained in the sample, and the injected charge of each negative ion is distributed according to the product of the charge, mobility, and concentration of each negative ion. To consider the contribution of positive ions to the current, simply multiply the right-hand side of Equation 14 by the contribution rate of negative ions to the current.

[0075] Using (Equation 15), (Equation 8) to (Equation 11) can be transformed as follows:

[0076] Furthermore, if q can be approximated as q = q(1) = q(2) as described above, then (Equation 12) and (Equation 13) can be transformed as follows:

[0077] Alternatively, if the ratio of the concentrations C(1) and C(2) of the first and second DNA fragments to the total concentration C of the first and second DNA fragments in the sample is constant, then (Equation 20) and (Equation 21) can also be obtained by defining q = (q(1) C(1) + q(2) C(2)) / (C(1) + C(2)).

[0078] On the other hand, the signal intensities S(1) and S(2) of the peaks of the first and second DNA fragments on the electropherogram obtained by capillary electrophoresis are given by (Equation 8) and (Equation 9), where m(1) and m(2) are the sensitivity coefficients, respectively. Using (Equation 15), The sensitivity coefficient includes the average number of labeled fluorophores per DNA fragment molecule, the excitation efficiency of the fluorophores, the quantum yield of the fluorophores, the light collection efficiency of the emitted fluorescence, the sensitivity of the image sensor, and so on.

[0079] Furthermore, when the sensitivity coefficients of the first DNA fragment and the second DNA fragment can be approximated as being equal to m(1) and m(2), respectively, assuming m = m(1) = m(2), the total signal intensity S of the peaks of the first DNA fragment and the second DNA fragment on the electropherogram obtained by capillary electrophoresis is given by (Equation 12): Alternatively, if the ratio of the concentrations C(1) and C(2) of the first and second DNA fragments to the total concentration C of the first and second DNA fragments in the sample is constant, then equation 26 can also be obtained by defining m as m = (m(1) C(1) + m(2) C(2)) / (C(1) + C(2)).

[0080] Using equation 15, we approximate q = q(1) = q(2), Alternatively, if the ratio of the concentrations C(1) and C(2) of the first and second DNA fragments to the total concentration C of the first and second DNA fragments in the sample is constant, then equation 27 can also be obtained by defining q = (q(1) C(1) + q(2) C(2)) / (C(1) + C2).

[0081] Equations 16 to 21, 24, 25, and 27 can be expressed as the following equations, where a and b are constants and y is a function of x.

[0082] For example, (Equation 16) becomes (Equation 28) when y = J(1), x = C(1), a = I T / q(1), b = (q(0) μ(0) C(0) + q(2) μ μ C(2)) / (q(1) μ). (Equation 28) shows that when x is small compared to b, the proportionality coefficient is a / b and y is proportional to x (y = a / b × x), but when x is large compared to b, y saturates with respect to x and asymptotically approaches a constant value a.

[0083] In other words, (Equation 16) shows that when C(1) is small compared to b = (q(0) μ(0) C(0) + q(2) μ C(2)) / (q(1) μ), J(1) is proportional to C(1), and when C(1) is large compared to b = (q(0) μ(0) C(0) + q(2) μ C(2)) / (q(1) μ), J(1) saturates with respect to C(1) and asymptotically approaches a constant value.

[0084] On the other hand, (Equation 8) shows that J(1) is proportional to C(1), so (Equation 8) and (Equation 16) appear to be contradictory. However, in fact, E included in (Equation 8) changes depending on C(1), so J(1) is not necessarily proportional to C(1) in (Equation 8). Therefore, (Equation 8) and (Equation 16) are not contradictory and are compatible.

[0085] Specifically, since I is constant in equation (15), if C(1) increases while C(0) and C(2) are constant, E will decrease. E is the effective electric field strength in the sample near the sample injection end of the capillary during electric field injection, and even if the voltage applied to both ends of the capillary is constant, i.e., the average electric field strength is constant, E can change depending on the composition of the sample.

[0086] Similarly, (Equation 17) shows that when C(2) is small, J(2) is proportional to C(2), and when C(2) is large, J(2) saturates with respect to C(2) and asymptotically approaches a constant value. (Equation 18) shows that when C(1) is small, Q(1) is proportional to C(1), and when C(1) is large, Q(1) saturates with respect to C(1) and asymptotically approaches a constant value. (Equation 19) shows that when C(2) is small, Q(2) is proportional to C(2), and when C(2) is large, Q(2) saturates with respect to C(2) and asymptotically approaches a constant value. (Equation 20) shows that when C is small, J is proportional to C, and when C is large, J saturates with respect to C and asymptotically approaches a constant value. (Equation 21) shows that when C is small, Q is proportional to C, and when C is large, Q saturates with respect to C and asymptotically approaches a constant value. (Equation 24) shows that when C(1) is small, S(1) is proportional to C(1), and when C(1) is large, S(1) saturates with respect to C(1) and asymptotically approaches a constant value. (Equation 25) shows that when C(2) is small, S(2) is proportional to C(2), and when C(2) is large, S(2) saturates with respect to C(2) and asymptotically approaches a constant value. And (Equation 27) shows that when C is small, S is proportional to C, and when C is large, S saturates with respect to C and asymptotically approaches a constant value.

[0087] Therefore, (Equation 24), (Equation 25), and (Equation 27) explain the phenomenon that in capillary electrophoresis analysis, when the concentration of the DNA fragment to be analyzed in the sample is low, a signal intensity proportional to the concentration is obtained, and when the concentration of the DNA fragment to be analyzed in the sample is high, the signal intensity saturates with respect to the concentration. In other words, the cause of the above phenomenon was identified. This knowledge was only obtained by introducing (Equation 14) and (Equation 15).

[0088] Furthermore, just as (Number 8) and (Number 16) are not contradictory, (Number 9) and (Number 17), (Number 10) and (Number 18), (Number 11) and (Number 19), (Number 12) and (Number 20), (Number 13) and (Number 21), (Number 22) and (Number 24), (Number 23) and (Number 25), and (Number 26) and (Number 27) are not contradictory and are compatible.

[0089] On the other hand, from (Equation 8) and (Equation 9), E, ​​T, A, and μ are common, so In other words, the ratio of the number of molecules of the first DNA fragment to the second DNA fragment injected into the capillary by electric field injection is equal to the concentration ratio of the first DNA fragment to the second DNA fragment in the sample. Furthermore, from (Equation 8), (Equation 9), and (Equation 12), That is, the ratio of the number of molecules of the first DNA fragment or the second DNA fragment to the total number of molecules of the first DNA fragment and the second DNA fragment injected into the capillary by electric field injection is equal 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.

[0090] Furthermore, from (Equation 10) and (Equation 11), since E, T, A, and μ are common, That is, the charge ratio between the first DNA fragment and the second DNA fragment injected into the capillary by electric field injection is proportional to the concentration ratio between the first DNA fragment and the second DNA fragment in the sample.

[0091] Also, if we can consider q = q(1) = q(2), then from (Equation 10), (Equation 11), and (Equation 13), That is, the ratio of the amount of charge injected into the capillary by electric field injection to the total amount of charge injected into the capillary by electric field injection of the first DNA fragment or the second DNA fragment is equal 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.

[0092] Furthermore, from (Equation 22) and (Equation 23), That is, the signal intensity ratio between the first DNA fragment and the second DNA fragment obtained by capillary electrophoresis analysis of the first DNA fragment and the second DNA fragment injected into the capillary by electric field injection is proportional to the concentration ratio of the first DNA fragment and the second DNA fragment in the sample.

[0093] Therefore, the capillary electrophoresis apparatus of Figure 1A can quantify the ratio of the concentration of the first DNA fragment to the concentration of the second DNA fragment in a sample based on the ratio of the signal intensity of the first DNA fragment to the signal intensity of the second DNA fragment.

[0094] In particular, when 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.

[0095] Also, if we can consider m = m(1) = m(2), then from (Equation 22), (Equation 23), and (Equation 26), That is, the ratio of the signal intensity of the first or second DNA fragment to the total signal intensity of the first and second DNA fragments injected into the capillary by electric field injection is equal to the ratio of the concentration of the first or second DNA fragment to the total concentration of the first and second DNA fragments in the sample.

[0096] What is noteworthy about the above is that (Equation 16), (Equation 17), and (Equation 20) are consistent and compatible with (Equation 29), (Equation 30), and (Equation 31). In other words, whether or not the number of injected molecules of the first DNA fragment, the second DNA fragment, or the entire DNA fragments is proportional to the concentration of the first DNA fragment, the second DNA fragment, or the entire DNA fragments in the sample, respectively, the ratio of the number of injected molecules of the first DNA fragment to the second DNA fragment corresponds to 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 to the entire DNA fragments corresponds to the concentration ratio of the first DNA fragment or the second DNA fragment to the entire DNA fragments in the sample.

[0097] Another thing to note is that (Equation 18), (Equation 19), and (Equation 21) are consistent and compatible with (Equation 32), (Equation 33), and (Equation 34). That is, whether or not the injected charge amount of the first DNA fragment, the second DNA fragment, or the entire DNA fragments is proportional to the concentration of the first DNA fragment, the second DNA fragment, or the entire DNA fragments in the sample, respectively, the ratio of the injected charge amount 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 amount of the first DNA fragment or the second DNA fragment to the entire DNA fragments coincides with the concentration ratio of the first DNA fragment or the second DNA fragment to the entire DNA fragments in the sample.

[0098] What is even more noteworthy is that (Equation 24), (Equation 25), and (Equation 27) are not contradictory and are compatible with (Equation 35), (Equation 36), and (Equation 37). That is, whether or not the signal intensities of the first DNA fragment, the second DNA fragment, or the entire DNA fragments are proportional to the concentrations of the first DNA fragment, the second DNA fragment, or the entire DNA fragments in the sample, respectively, 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 to the entire DNA fragments coincides with the concentration ratio of the first DNA fragment or the second DNA fragment to the entire DNA fragments in the sample.

[0099] If the second DNA fragment is used as the internal standard, then (Equation 35) will have the same form as (Equation 3), making it possible to avoid the effects of fluctuations in the electric field strength E and time T of electric field injection and to quantify the first DNA fragment, which is the target of analysis, with high accuracy. Therefore, by using (Equation 35) as the calibration curve for the new internal standard method, just like (Equation 3), which is the calibration curve for the conventional internal standard method, it is possible to quantify the target DNA fragment with high accuracy.

[0100] The conventional internal standard method was applicable only when the signal intensity was proportional to the analyte concentration, whereas the new internal standard method, as shown in Equations (24), (25), and (27), is applicable not only when the signal intensity is proportional to the analyte concentration, but also when it is not proportional, approaches saturation, or reaches saturation.

[0101] [Specific example of measurement results] In capillary electrophoresis analysis, when the concentration of the target DNA fragment contained in the sample is low, a signal intensity proportional to the concentration is obtained, and when the concentration of the target DNA fragment contained in the sample is high, the signal intensity saturates with respect to the concentration.

[0102] STR-PCR for DNA typing was performed using a specific individual's human genome as a template, and the mixture was desalted and the solvent was changed to formamide.Four types of samples were prepared, with the concentrations of the multiple DNA fragments produced by STR-PCR varied over a four-digit range: 1x, 0.05x, 0.002x, and 0.0001x the reference concentration.

[0103] Using the capillary electrophoresis apparatus shown in Figure 1A, samples with concentrations of 0.0001x, 0.002x, 0.05x, and 1x were electrophoretically analyzed using four capillaries. Parts of the electropherograms obtained are shown in Figure 3(a), (b), (c), and (d), respectively.

[0104] Each peak on the electropherogram represents the signal of a DNA fragment that is the STR-PCR product of each locus. For example, in Figures 3(a), (b), (c), and (d), the single peak observed around 4,200 frames of electrophoresis represents the signal of a DNA fragment that is the STR-PCR product of locus D5S818. Increasing the concentration of DNA fragments in the sample increases the fluorescence intensity (represented by signal intensity; the same applies below) of each DNA fragment. Because the four capillaries were adjusted so that the measured fluorescence intensities are equal when the same fluorescence intensity is generated at each measurement point, the differences in measured fluorescence intensity faithfully reflect the differences in the fluorescence intensity emitted at the measurement points.

[0105] Figure 4 is a log-log graph plotting the fluorescence intensity of the peak at locus D5S818 versus the concentration of the DNA fragment contained in the sample, using the DNA fragment that is the STR-PCR product of locus D5S818 in Figure 3 as the analysis target. Because the peak widths in Figures 3(a), (b), (c), and (d) are considered equivalent, the peak height, rather than the peak area, represents the fluorescence intensity. The DNA fragment concentration on the horizontal axis represents the concentration of the DNA fragment that is the STR-PCR product of locus D5S818 contained in the sample, but it can also be considered the total concentration of the DNA fragment contained in the sample.

[0106] When the concentration of the target DNA fragments in the sample is low, the fluorescence intensity of the target DNA fragments is proportional to the concentration of the DNA fragments in the sample. However, as the concentration of the target DNA fragments in the sample increases, the fluorescence intensity of the target DNA fragments becomes less proportional to the concentration of the target DNA fragments in the sample and approaches saturation.

[0107] If we let y = S(1), x = C(1), a = m(1) I T / q(1), and b = (q(0) μ(0) C(0) + q(2) μ μ C(2)) / (q(1) μ), then equation 24 becomes equation 28. In equation 28, the fluorescence intensity of the DNA fragment to be analyzed is S(1), and the concentration of the DNA fragment to be analyzed contained in the sample is C(1).

[0108] The dashed line in Figure 4 shows the relationship between S(1) and C(1) (Equation 28) when a = 3500 and b = 0.17, and is a good approximation curve for the four plots in Figure 4, that is, when C(1) is low or high. In other words, as shown in (Equation 28), when C(1) is low, S(1) is proportional to C(1), but as C(1) becomes high, S(1) deviates from the proportionality to C(1) and approaches saturation.

[0109] On the other hand, the solid line in Figure 4 shows the relationship when S(1) = a / b C(1) ≈ 20000 C(1), i.e., when S(1) is proportional to C(1), and is a good approximation line for the two plots on the left side of Figure 4, i.e., when C(1) is low. From this result, we can see that S(1) and C(1) deviate from a proportional relationship to a non-proportional relationship when C(1) is about 0.01 times the boundary.

[0110] Therefore, the conventional internal standard method allows quantification over a concentration range of 0.0001 to 0.01 times C(1), i.e., a dynamic range of two orders of magnitude.In contrast, the new internal standard method allows quantification over a concentration range of 0.0001 to 1 times C(1), i.e., a dynamic range of four orders of magnitude.

[0111] In this way, by using the novel internal standard method according to the present disclosure, it becomes possible to quantify with high accuracy DNA fragments contained in a sample over a wider concentration range than conventionally possible.

[0112] The above analysis focuses on the peak at locus D5S818 in Figure 3, but the same relationship holds true for the other peaks. That is, as the overall concentration of DNA fragments in the sample increases, the D5S818 peak deviates from the proportional relationship, and the other peaks also deviate from the proportional relationship in sync with this. Despite the different fluorescence intensities of the peaks in each electropherogram, the fact that such a synchronized phenomenon is observed indicates that the cause is not detector saturation or fluorophore self-quenching.

[0113] From the above, the phenomena observed in Figures 3 and 4 can be clearly explained and understood by the causes and theories discussed in the above [Principles].

[0114] FIG. 5 is a double logarithmic graph plotting the change in fluorescence intensity of the DNA fragment to be analyzed against the change in concentration of the DNA fragment to be analyzed contained in a sample different from those in FIGS.

[0115] Five types of samples were prepared in which the concentration of the target DNA fragment contained in the sample was varied over a four-digit concentration range: 100-fold, 10-fold, 1-fold, 0.1-fold, and 0.01-fold of the reference concentration. However, the reference concentrations in Figure 5 are unrelated to those in Figures 3 and 4.

[0116] Five types of samples were divided into two groups and each group was subjected to electrophoretic analysis using the capillary electrophoresis apparatus shown in Figure 1A. As in Figure 4, when the concentration of DNA fragments in the sample was low, the fluorescence intensity of the DNA fragments was proportional to the concentration of the DNA fragments in the sample, but as the concentration of the DNA fragments in the sample increased, the fluorescence intensity of the DNA fragments deviated from proportionality to the concentration of the DNA fragments in the sample and approached saturation.

[0117] Similarly to the above, (Equation 24) becomes (Equation 28) when y = S(1), x = C(1), a = m(1) I T / q(1), and b = (q(0) μ(0) C(0) + q(2) μ μ C(2)) / (q(1) μ). In (Equation 28), the fluorescence intensity of the DNA fragment to be analyzed is S(1), and the concentration of the DNA fragment to be analyzed contained in the sample is C(1).

[0118] The dashed line in Figure 5 shows the relationship between S(1) and C(1) (Equation 28) when a = 22000 and b = 10, and is a good approximation curve for the five plots in Figure 5, that is, when C(1) is low or high. In other words, as shown in (Equation 28), when C(1) is low, S(1) is proportional to C(1), but as C(1) becomes high, S(1) deviates from the proportionality to C(1) and approaches saturation.

[0119] On the other hand, the solid line in Figure 5 shows the relationship when S(1) = a / b C(1) ≈ 2180 C(1), i.e., when S(1) is proportional to C(1), and is a good approximation to the three plots on the left side of Figure 5, i.e., when C(1) is low. From this result, we can see that S(1) and C(1) deviate from a proportional relationship to a non-proportional relationship when C(1) is about 1.

[0120] Therefore, the conventional internal standard method allows quantification of C(1) in the concentration range of 0.01 to 1, i.e., a dynamic range of two orders of magnitude. In contrast, the new internal standard method allows quantification of C(1) in the concentration range of 0.01 to 100, i.e., a dynamic range of four orders of magnitude.

[0121] In this way, by using the novel internal standard method according to the present disclosure, it becomes possible to quantify with high accuracy DNA fragments contained in a sample over a wider concentration range than conventional methods. From the above, the phenomenon observed in Figure 5 can be clearly explained and understood based on the causes and theories discussed in the above [Principle].

[0122] [Example 1] The following measurements were carried out using the capillary electrophoresis apparatus shown in Figure 1A. TM " symbol represents a trademark. Hi-Di TM Formamide was used as a solvent to measure two size standards: GeneScan TM 600 LIZ TM Dye Size Standard (hereafter referred to as 600 LIZ), and GeneScan TM 500 ROX TM Four types of samples were prepared by mixing dye size standards (hereafter referred to as 500 ROX) at specific ratios and analyzed using the capillary electrophoresis system shown in Figure 1A. Each of the four capillaries had an inner diameter of 50 μm, a total length of 47 cm, and an effective length of 36 cm. Applied Biosystems (Thermo Fisher Scientific) buffer solutions were used as the cathode and anode buffer solutions. TM 310 and 31xx Running Buffer, 10X, were diluted 10 times with pure water and used. POP-4 (Thermo Fisher Scientific) was used as the polymer solution. TM Polymer for 3500 / SeqStudio TM Flex was used.

[0123] The concentrations of 600 LIZ in the four samples were varied to 1 / 2 (0.5x), 1 / 20 (0.05x), 1 / 200 (0.005x), and 1 / 2000 (0.0005x) of the reference concentration, respectively. The concentrations of 500 ROX in the four samples were kept constant at 1 / 200 (0.005x) of the reference concentration. However, the reference concentrations of 600 LIZ and 500 ROX were unrelated. While 600 LIZ and 500 ROX each contained multiple DNA fragments, their overall concentrations were varied while maintaining their concentration ratios. Field injection of each sample was performed by applying a voltage of 1.2 kV to both ends of each capillary for 9 seconds. Electrophoresis was performed by applying a voltage of 8.5 kV to both ends of each capillary.

[0124] Figure 6(a) shows an electropherogram of a sample with a 2000x reduction in 600 LIZ and a 200x reduction in 500 ROX. Figure 6(b) shows an electropherogram of a sample with a 200x reduction in 600 LIZ and a 200x reduction in 500 ROX. Figure 7(a) shows an electropherogram of a sample with a 20x reduction in 600 LIZ and a 200x reduction in 500 ROX. Figure 7(b) shows an electropherogram of a sample with a 20x reduction in 600 LIZ and a 200x reduction in 500 ROX. In both graphs, the solid line indicates the fluorescence intensity of 600 LIZ, and the dotted line indicates the fluorescence intensity of 500 ROX. The horizontal axis indicates electrophoresis time, the left vertical axis indicates the fluorescence intensity of 500 ROX, and the right vertical axis indicates the fluorescence intensity of 600 LIZ.

[0125] 600 LIZ contains 36 types of single-stranded DNA fragments with lengths 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, each labeled with the fluorophore LIZ. The electropherograms in Figures 6 and 7 show peaks for 15 types of DNA fragments, namely, 20, 40, 60, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, and 250 bases in length. Furthermore, the peaks for DNA fragments of 40, 114, and 160 bases in length are indicated by arrows and labeled LIZ 40, LIZ 114, and LIZ 160.

[0126] 500 ROX contains 16 single-stranded DNA fragments with lengths of 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500 bases, each labeled with the fluorescent ROX. The electropherograms in Figures 6 and 7 show peaks for nine of these DNA fragments: 35, 50, 75, 100, 139, 150, 160, 200, and 250 bases. The peak for the 160-base DNA fragment is indicated by an arrow and labeled ROX 160.

[0127] The peaks of DNA fragments of the same base length, such as the LIZ 160 peak and the ROX 160 peak, are observed at slightly different times due to the difference in mobility of the labeled fluorophores LIZ and ROX.

[0128] Figure 8(a) is a log-log graph plotting the fluorescence intensity of the LIZ 160 and ROX 160 peaks obtained from the four electropherograms in Figures 6 and 7 against the concentration of 600 LIZ contained in the sample. The horizontal axis can be considered to represent the concentration of LIZ 160 contained in the sample. Here, the width of each peak can be considered to be approximately equal, so the height of each peak was taken as the fluorescence intensity. The black circles represent the fluorescence intensity of LIZ 160, and the white circles represent the fluorescence intensity of ROX 160.

[0129] When the concentration of 600 LIZ in the sample is low, specifically in the range of 1 / 2000 to 1 / 20 times the concentration of 600 LIZ, the fluorescence intensity of LIZ 160 is proportional to the concentration of 600 LIZ in the sample and can be approximated by a straight line with a slope of 1. On the other hand, when the concentration of 600 LIZ in the sample is high, specifically in the range of 1 / 20 to 1 / 2 times the concentration of 600 LIZ, the fluorescence intensity of LIZ 160 deviates from proportionality to the concentration of 600 LIZ in the sample and approaches saturation. This phenomenon is the same as that observed in Figures 4 and 5.

[0130] Thus, the concentration range of the first component contained in the sample includes not only the concentration range in which the signal intensity of the first component is proportional to the concentration of the first component, but also the concentration range in which the signal intensity of the first component is lower than the saturation signal intensity of the detector and deviates from the proportionality to the concentration of the first component, reaching the saturation signal intensity. The capillary electrophoresis apparatus according to this embodiment can measure the concentration of such samples.

[0131] In contrast, the fluorescence intensity of ROX 160 remains constant relative to the concentration of 600 LIZ in the sample when the concentration of 600 LIZ in the sample is low, specifically when the concentration of 600 LIZ is in the range of 1 / 2000 to 1 / 200 times the original concentration. This is easily understood because the concentration of ROX 160 in the sample is constant. However, the fluorescence intensity of ROX 160 decreases relative to the concentration of 600 LIZ in the sample when the concentration of 600 LIZ in the sample is high, specifically when the concentration of 600 LIZ is in the range of 1 / 200 to 1 / 2 times the original concentration. This change is not easily understood because the concentration of ROX 160 in the sample is constant. The above is a new phenomenon discovered in the present disclosure.

[0132] Thus, the concentration range of the first component contained in the sample includes not only the concentration range in which the signal intensity of the second component is constant relative to the concentration of the first component, but also the 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 apparatus according to this embodiment can measure the concentration of such samples.

[0133] The above phenomenon is explained by (Equation 24) and (Equation 25).

[0134] In (Equation 24) and (Equation 25), the first DNA fragment is considered to be all DNA fragments contained in 600 LIZ, and the second DNA fragment is considered to be all DNA fragments contained in 500 ROX. That is, S(1) is the sum of the fluorescence intensities of all DNA fragments contained in 600 LIZ, C(1) is the sum of the concentrations of all DNA fragments contained in 600 LIZ, q(1) is the average charge of all DNA fragments contained in 600 LIZ, S(2) is the sum of the fluorescence intensities of all DNA fragments contained in 500 ROX, C(2) is the sum of the concentrations of all DNA fragments contained in 500 ROX, and q(2) is the average charge of all DNA fragments contained in 500 ROX.

[0135] In this case, under the conditions of constant concentration C(0) of anions other than DNA fragments and constant concentration C(2) of 500 ROX, the fluorescence intensity S(1) of 600 LIZ is proportional to the concentration C(1) of 600 LIZ when the concentration C(1) of 600 LIZ is low, but becomes non-proportional and saturates when the concentration C(1) of 600 LIZ is high. Because the fluorescence intensity of LIZ 160 is proportional to the fluorescence intensity of 600 LIZ, the two show similar changes. Therefore, the change in the fluorescence intensity of LIZ 160 over the entire concentration range of LIZ 160 in Figure 8(a) can be explained by Equation 24.

[0136] In contrast, in Equation 25, under the conditions of a constant concentration of anions other than DNA fragments, C(0), and a constant concentration of 500 ROX, C(2), the fluorescence intensity S(2) of 500 ROX is constant when the concentration C(1) of 600 LIZ is low, but decreases relative to the concentration C(1) of 600 LIZ when the concentration C(1) of 600 LIZ is high. Because the fluorescence intensity of ROX 160 is proportional to that of 500 ROX, the two show similar changes. Therefore, the change in the fluorescence intensity of ROX 160 over the entire concentration range of LIZ 160 in Figure 8(a) can be explained by Equation 25.

[0137] The above can be further explained by Equation 14 and Equation 15. That is, under the condition that the current during electric field injection is constant, if the injected charge amount Q(1) of the first DNA fragment increases due to an increase in the concentration C(1) of the first DNA fragment, the injected charge amount Q(2) of the second DNA fragment decreases. This is realized by a decrease in E on the left side of Equation 15, i.e., the electric field strength in the sample near the sample injection end of the capillary during electric field injection.

[0138] The above is a consideration focusing on the LIZ 160 peak and the ROX 160 peak in Figures 6 and 7, but the same consideration applies when focusing on peaks of DNA fragments other than these.

[0139] Figure 9 is a log-log graph plotting the peak fluorescence intensity of LIZ 160 (the same data as in Figure 8(a)) as well as the peak fluorescence intensity of LIZ 40 and LIZ 114, for example, against the concentration of 600 LIZ in the sample. The horizontal axis can be thought of as the concentration of LIZ 40, LIZ 114, or LIZ 160 in the sample.

[0140] When the concentration of 600 LIZ in the sample is low, specifically in the range of 1 / 2000 to 1 / 20 times, the fluorescence intensity is proportional to the concentration of 600 LIZ in the sample and can be approximated by a straight line with a slope of 1. On the other hand, when the concentration of 600 LIZ in the sample is high, specifically in the range of 1 / 20 to 1 / 2 times, the fluorescence intensity deviates from proportionality to the concentration of 600 LIZ in the sample and approaches saturation. The above changes in fluorescence intensity occur synchronously for each DNA fragment. Needless to say, all changes in fluorescence intensity can be approximated by (Equation 24).

[0141] Although the fluorescence intensities of the peaks of LIZ 40, LIZ 114, and LIZ 160 differ in each electropherogram, the above-mentioned changes in fluorescence intensity occur synchronously. This indicates that the cause is not detector saturation or fluorophore self-quenching, but rather the constant electric field injection amount discovered in this disclosure. The same changes in fluorescence intensity as ROX 160 are observed for the peaks of DNA fragments other than ROX 160, and can be approximated by (Equation 25).

[0142] As shown in Figure 8(a), when the concentration of 600 LIZ in the sample is in the range of 1 / 2000 to 1 / 20, the fluorescence intensity of LIZ 160 is proportional to the concentration of 600 LIZ contained in the sample, and it is possible to quantify LIZ 160 with high accuracy using the conventional internal standard method using ROX 160 as the internal standard. Alternatively, when the concentration of 600 LIZ in the sample is in the range of 1 / 2000 to 1 / 200, the fluorescence intensity of ROX 160 is constant, and it is possible to quantify LIZ 160 with high accuracy using the conventional internal standard method using ROX 160 as the internal standard.

[0143] However, outside these ranges, the conventional internal standard method cannot be applied. In particular, when the concentration of 600 LIZ contained in the sample is 1 / 2, as shown in Figure 8(a), the peak fluorescence intensity of LIZ 160 approaches saturation and the fluorescence intensity of ROX 160 decreases. Therefore, it would be impossible to quantify LIZ 160 based on these ratios using conventional methods.

[0144] In contrast, Figure 8(b) is a log-log graph plotting the ratio of the peak fluorescence intensity of LIZ 160 to the peak fluorescence intensity of ROX 160 in Figure 8(a) versus the concentration of 600 LIZ contained in the sample. The horizontal axis can be considered to be the concentration of LIZ 160 contained in the sample.

[0145] What would be unimaginable from Figure 8(a) or the conventional internal standard method is that, over the entire concentration range from 1 / 2000 to 1 / 2 times on the horizontal axis in Figure 8(a), the ratio of the fluorescence intensity of the LIZ 160 peak to that of the ROX 160 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, by using the new internal standard method that uses ROX 160 as an internal standard, it is possible to quantify LIZ 160 with high precision over a wider concentration range than before.

[0146] The results in Figure 8(b) can be explained by defining the first DNA fragment as LIZ 160 and the second DNA fragment as ROX 160 in Equation 35. That is, the ratio of the peak fluorescence intensity of LIZ 160 to the peak fluorescence intensity of ROX 160 is proportional to the ratio of the concentration of LIZ 160 to the concentration of ROX 160 contained in the sample. Here, since the concentration of ROX 160 is constant, the ratio of the peak fluorescence intensity of LIZ 160 to the peak fluorescence intensity of ROX 160 is proportional to the concentration of LIZ 160 contained in the sample.

[0147] Because Equation (35) does not include variable parameters such as E, T, and C(0), it enables highly accurate quantification, similar to conventional internal quantification methods. Most importantly, Equation (35) is compatible with Equations (24) and (25). That is, as shown in Figure 8(a), the ratio of the LIZ 160 peak fluorescence intensity to the ROX 160 peak fluorescence intensity is always proportional to the LIZ 160 concentration in the sample, regardless of whether the LIZ 160 peak fluorescence intensity is proportional or deviates from the proportionality, or whether the ROX 160 peak fluorescence intensity is constant or decreases.

[0148] Figure 10 shows the experimental results corresponding to Figure 8, but with the same experimental conditions and electrophoretic analysis performed, but with the field injection time increased from 9 seconds to 18 seconds. Figure 10(a) shows results similar to those in Figure 8(a), even though the experimental conditions and electrophoretic analysis were different. However, the positions of the plots fluctuate slightly. In contrast, Figure 10(b) shows results almost identical to those in Figure 8(b), even though the experimental conditions and electrophoretic analysis were different. The positional variation of the plots is very small. Furthermore, the approximation line with a slope of 1 used in Figure 10(b) is identical to the approximation line with a slope of 1 used in Figure 8(a). These results demonstrate the high quantitative accuracy of the new internal standard method.

[0149] Figure 11 is a log-log graph plotting the ratio of the peak fluorescence intensity of LIZ 40 and the ratio of the peak fluorescence intensity of LIZ 114 to the peak fluorescence intensity of LIZ 160 obtained in the four electropherograms of Figures 6 and 7 against the concentration of 600 LIZ contained in the sample. The horizontal axis can be considered to represent the concentration of LIZ 40, LIZ 114, or LIZ 160 contained in the sample.

[0150] As shown in Figures 6, 7, and 8(a), the fluorescence intensity of each peak changes with the concentration of 600 LIZ contained in the sample, but the fluorescence intensity ratio shown in Figure 11 remains constant. This result indicates that in each of the electropherograms in Figures 6 and 7, the ratio of the fluorescence intensities of the peaks of any two types of DNA fragments belonging to 600 LIZ or 500 ROX remains constant.

[0151] This can be explained by using Equation 35, where the first DNA fragment is LIZ 40 or LIZ 114 and the second DNA fragment is LIZ 160. When the concentration of 600 LIZ contained in the sample is changed, the concentration ratio of the multiple types of DNA fragments contained in 600 LIZ is kept constant, so the right-hand side remains constant and the fluorescence intensity ratio on the left-hand side remains constant. The above results support the correctness of the series of considerations disclosed in the above [Principle].

[0152] The present disclosure can be applied to any fragment analysis by capillary electrophoresis, and some specific examples are shown in the following examples.

[0153] Example 2: DNA typing using short tandem repeat (STR) analysis is widely used in criminal investigations, identity verification in large-scale disasters, paternity testing, and other applications due to its high accuracy in personal identification. Currently, various types of STR analysis reagent kits are commercially available. For example, Promega's PowerPlex® Fusion 6C System uses a human genome extracted from blood collected at a crime scene as a template to perform multiplex PCR of STRs at 27 loci on the human genome using five types of fluorophores.

[0154] 5 μl of PowerPlex Fusion 6C 5X Master Mix, 5 μl of PowerPlex Fusion 6C 5X Primer Pair Mix, and 25 μl of pre-reaction solution containing extracted human genome were incubated at 96°C for 1 minute, followed by 29 thermal cycles of 96°C for 5 seconds and 60°C for 1 minute, followed by a 10-minute incubation at 60°C, followed by a 4°C incubation. 25 μl of this STR-PCR post-reaction solution was ethanol precipitated and desalted by dissolving in 25 μl of pure water. 1 μl of this solution was mixed with 0.5 μl of Promega's WEN ILS 500 (hereafter referred to as 500 WEN), a size standard labeled with one fluorophore different from the five fluorophores listed above, and 9.5 μl of formamide to obtain an 11 μl sample.

[0155] The 500 WEN contains 21 single-stranded DNA fragments with lengths of 60, 65, 80, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, and 500 bases, each labeled with the fluorophore WEN. The sample is heat-denatured at 95°C, rapidly cooled on ice, and then subjected to capillary electrophoresis to separate DNA fragments of various lengths labeled with one of the six fluorophores contained in the sample, allowing for identification and detection of the labeled fluorophores. DNA typing is performed by analyzing the resulting electropherograms for each of the six fluorophores.

[0156] In DNA typing, accurately identifying the base length of each DNA fragment is important for improving the accuracy of personal identification. However, in capillary electrophoresis, the migration speed of each DNA fragment varies from capillary to capillary, sample to sample, and analysis to analysis, making it difficult to accurately identify the base length from the peak time of each DNA fragment on the electropherogram. Therefore, as described above, the sample is mixed with a size standard of known base length for analysis, and the base length of any DNA fragment can be accurately identified by referring to the peak time of the DNA fragment of known base length.

[0157] As mentioned above, conventional DNA typing utilizes the time at which the peaks of multiple DNA fragments belonging to a size standard are measured, but the fluorescence intensity of those peaks has not been utilized. However, because the concentration of the size standard contained in a sample is known or fixed, it is possible to quantify the concentration of a DNA fragment contained in the sample from the fluorescence intensity of the peak of one of the multiple DNA fragments belonging to the size standard by referencing the fluorescence intensity of that peak. In other words, not only can the DNA typing of the human genome template be performed, but each of the multiple DNA fragments contained in the sample that are STR-PCR products can also be quantified with high accuracy.

[0158] The high accuracy of this quantification can be explained by using Equation 35, where the first DNA fragment is any DNA fragment contained in the STR-PCR product and the second DNA fragment is any DNA fragment contained in the size standard. That is, the concentration of the first DNA fragment in a sample relative to the concentration of the second DNA fragment in a sample can be calculated from the ratio of the peak fluorescence intensity of the first DNA fragment to the peak fluorescence intensity of the second DNA fragment.

[0159] When the concentration of the size standard in a sample is constant, i.e., when the concentration of the second DNA fragment in the sample is constant, the concentration of the first DNA fragment in the sample can be determined from the peak fluorescence intensity of the first DNA fragment relative to the peak fluorescence intensity of the second DNA fragment. Because Equation 35 does not include variable parameters such as E, T, and C(0), it enables highly accurate quantification, similar to conventional internal quantification methods. As shown in Equation 24, this highly accurate quantification is valid both when the peak fluorescence intensity of the first DNA fragment is proportional to the concentration of the first DNA fragment in the sample and when it is not. In other words, the novel internal standard method makes it possible to accurately quantify the concentration of the first DNA fragment over a wider concentration range than conventional methods.

[0160] Furthermore, the concentration of the human genome template in the solution immediately before STR-PCR can be quantified by using the number of thermal cycles performed in STR-PCR. Let N be the number of thermal cycles, E(f) be the amplification efficiency of STR-PCR, and C(f) be the concentration of the human genome in the pre-STR-PCR solution. In the above example, N = 29. Under ideal conditions, E(f) = 1, but the actual value can be determined in advance. Let B(1) be the first DNA fragment, and its concentration be B(1). Let C(1) be the concentration of the first DNA fragment in the sample used for electric field injection. Let D = C(1) / B(1), be the ratio of C(1), the concentration of the first DNA fragment in the sample used for electric field injection, to B(1), the concentration of the first DNA fragment in the post-STR-PCR solution. This is the dilution ratio of the post-STR-PCR solution. In the above example, D = 1 μl / 25 μl = 0.04. holds true.

[0161] In (Equation 35), if the second DNA fragment is an arbitrary DNA fragment contained in the size standard, C(1) can be calculated from (Equation 35). This is because S(1) and S(2) can be obtained from the electropherogram, C(2) is known, and m(1) and m(2) can be determined in advance. D, E(f), and N are all known, as described above. Therefore, from (Equation 38), it is possible to quantify with high accuracy the concentration C(f) of human genome contained in the pre-reaction solution for STR-PCR.

[0162] Example 3: Thermo Fisher Scientific's SNaPshot® Multiplex system is a kit for simultaneously typing multiple SNPs (single nucleotide polymorphisms) in the human genome using capillary electrophoresis. Template DNA is prepared by amplifying a region containing multiple SNPs in the human genome. Non-fluorophore-labeled primers are hybridized to the template DNA at positions adjacent to each SNP, and a single-base extension reaction of each primer is performed using a fluorescently labeled terminator. The fluorescently labeled terminators are ddATP, ddCTP, ddGTP, and ddTTP, each labeled with four different fluorophores. The base length of each primer is varied depending on the corresponding SNP. DNA fragments representing the multiple single-base extension products are analyzed by capillary electrophoresis to obtain electropherograms for each of the four fluorophores. The location of the corresponding SNP is identified on the electropherogram based on the electrophoresis time at which a peak is observed, i.e., the base length of the corresponding DNA fragment. Furthermore, the type of fluorescent substance at the same peak is used to identify whether the SNP is A, C, G, or T. The above SNP typing can be performed simultaneously for SNPs at multiple locations.

[0163] In the above-mentioned SNP typing, it is sufficient to determine whether each SNP is a homozygote of one of A, C, G, or T (one SNP is present 100% of the time) or a heterozygote of two of A, C, G, and T (two SNPs are present 50% of the time). However, in general, each SNP may be a mixture of A, C, G, and T in any ratio. For example, as cancer progresses, the wild type (hereinafter referred to as WT) of a given SNP may be one of A, C, G, or T, and the mutant type (hereinafter referred to as MT) may be any of the three types of A, C, G, and T other than the one type mentioned above, with the abundance ratios of each type varying. Conversely, quantifying the abundance ratios of A, C, G, and T in a given SNP may enable early cancer diagnosis or accurate assessment of the state of cancer.

[0164] Assume that the abundance ratio of A, C, G, and T bases at M SNPs, where M is an integer of 1 or greater, is to be quantified, particularly the abundance ratio of MT bases relative to WT bases. For each of the M SNPs, up to four single-base extension products are obtained. The reaction solution containing up to 4 × M DNA fragments in total is desalted by ethanol precipitation and then eluted in a desired amount of formamide to prepare the sample for electroinjection.

[0165] Each DNA fragment injected into the field gives a peak on the electropherogram for each of the four types of fluorophores obtained by capillary electrophoresis analysis, giving a maximum of 4 × M peaks. SNP number i is assigned to each of the M SNPs, where i = 1, 2, ..., M. The fluorescence intensities of the peaks on the electropherogram of the DNA fragments that are single-base extension products of base types A, C, G, and T of SNP number j are S(ja), S(jc), S(jg), and S(jt), their respective sensitivity coefficients are m(ja), m(jc), m(jg), and m(jt), their respective average charge amounts are q(ja), q(jc), q(jg), and q(jt), and their respective concentrations in the sample injected into the capillary are C(ja), C(jc), C(jg), and C(jt). The total injected charge amount Q of all DNA fragments injected into the capillary is given by the following equation (equation 13): On the other hand, as in (Number 15), From (Equation 39) and (Equation 40), similarly to (Equation 21), q(0) μ(0) C(0) represent the injected charge of negative ions other than the DNA fragments, and the Σ term represents the total injected charge of all DNA fragments.

[0166] Equation 41 indicates that Q is proportional to the Σ term when the Σ term is small compared to q(0) μ(0) C(0), but when the Σ term becomes large compared to q(0) μ(0) C(0), the proportionality to the Σ term deviates from the Σ term and reaches saturation. Therefore, the total injected 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 as the total concentration of all DNA fragments increases, the proportionality deviates from the Σ term and reaches saturation. Needless to say, an increase in the total concentration of all DNA fragments is brought about by an increase in the concentration of any DNA fragment. And when the total injected amount of all DNA fragments reaches saturation, the injection amount of any DNA fragment will saturate or, conversely, decrease.

[0167] However, when any two types of DNA fragments among all the DNA fragments are designated as the first DNA fragment and the second DNA fragment, the following equations hold: (Equation 8) to (Equation 11), (Equation 22), (Equation 23), (Equation 29), (Equation 32), and (Equation 35). (Equation 35) indicates that the fluorescence intensity ratio of the first DNA fragment to the second DNA fragment obtained by capillary electrophoresis analysis of the first DNA fragment and the second DNA fragment injected into the capillary by electric field injection is proportional to the concentration ratio of the first DNA fragment to the second DNA fragment in the sample.

[0168] For example, when focusing on a DNA fragment that is a single base extension product of the base types A, C, G, and T of SNP number j, similarly to (Equation 22) and (Equation 23), is.

[0169] Therefore, for example, if WT is A and MT is C, G, T, then (Equation 35) becomes: In other words, the concentration ratios C(jc) / C(ja), C(jg) / C(ja), and C(jt) / C(ja), which correspond to the abundance ratios of the three types of MT relative to WT, can be quantified with high precision using the peak fluorescence intensities S(jc) / S(ja), S(jg) / S(ja), and S(jt) / S(ja) on the electropherogram. m(ja), m(jc), m(jg), and m(jt) are calculated in advance.

[0170] In the above, we have assumed that the sample contains up to 4 × M types of DNA fragments that are single-base extension products. Below, we will assume that the sample contains up to 4 × M types of DNA fragments that are single-base extension products, as well as one or more types of DNA fragments that serve as internal standards with known or fixed concentrations. A size standard containing DNA fragments of multiple base lengths may also be used as the internal standard. In this case, by using one of the DNA fragments that are single-base extension products of base types A, C, G, and T of SNP number j in (Equation 35) as the first DNA fragment and one of the internal standard DNA fragments as the second DNA fragment, the concentration of the first DNA fragment in the sample can be quantified with high accuracy.

[0171] 1 Capillary 2 Sample injection end 3 Sample elution end 4 Cathode 5 Anode 6 Cathode side buffer solution 7 Anode side buffer solution 8 Polymer solution 9 Sample 10 Pump block 11 Valve 12 Syringe 13 Power supply 14 Laser beam 15 Laser light source 16 Measurement point 17 Detector 18 First DNA fragment (first component) 19 Second DNA fragment (second component) 20 Negative ions other than DNA fragments

Claims

1. A sample containing a first component and a second component is analyzed, Injecting the sample into the capillary; separating the injected first and second components by electrophoresis; irradiating light onto the capillary to induce luminescence from the first component and luminescence from the second component, and measuring the luminescence with a detector, thereby obtaining the signal intensity of the first component and the signal intensity of the second component. In a capillary electrophoresis apparatus, 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 and deviates from the proportionality to the concentration of the first component, reaching 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. A capillary electrophoresis apparatus characterized by:

2. A sample containing a first component and a second component is analyzed, Injecting the sample into the capillary; separating the injected first and second components by electrophoresis; irradiating light onto the capillary to induce luminescence from the first component and luminescence from the second component, and measuring the luminescence with a detector, thereby obtaining the signal intensity of the first component and the signal intensity of the second component. In a capillary electrophoresis apparatus, the concentration range of the first component contained in the sample includes a concentration range in which the signal intensity of the second component is constant relative to the concentration of the first component, as well as a concentration range in which the signal intensity of the second component decreases relative to the concentration of the first component, and deviates from the constant; 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. A capillary electrophoresis apparatus characterized by:

3. 3. The capillary electrophoresis apparatus according to claim 1, the concentration of the second component in the sample is known; quantitating the concentration of the first component in the sample based on the ratio of the signal intensity of the first component to the signal intensity of the second component; A capillary electrophoresis apparatus characterized by:

4. 3. The capillary electrophoresis apparatus according to claim 1, 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 emission is fluorescence, The signal intensity is a fluorescence intensity. A capillary electrophoresis apparatus characterized by:

5. 5. The capillary electrophoresis apparatus according to claim 4, the sample includes a size standard; The second component is a DNA fragment contained in a size standard. A capillary electrophoresis apparatus characterized by:

6. 5. The capillary electrophoresis apparatus according to claim 4, the sample contains a PCR product; The first component is a DNA fragment contained in the PCR product. A capillary electrophoresis apparatus characterized by:

7. 5. The capillary electrophoresis apparatus according to claim 4, the sample contains a single-base extension product; the first component is a first DNA fragment contained in the single-base extension product; A capillary electrophoresis apparatus characterized by:

8. 8. The capillary electrophoresis apparatus according to claim 7, the second component is a second DNA fragment contained in the single-base extension product; A capillary electrophoresis apparatus characterized by:

9. Injecting a sample containing a first component and a second component into a capillary as an analysis target; Electrophoretically separating the injected first and second components; irradiating light onto the capillary to induced luminescence from the first component and luminescence from the second component are measured with a detector, thereby obtaining the signal intensity of the first component and the signal intensity of the second component; A capillary electrophoresis method comprising: 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 and deviates from the proportionality to the concentration of the first component, reaching the saturation signal intensity; The capillary electrophoresis method includes determining a ratio of the concentration of the first component to the 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. A capillary electrophoresis method characterized by:

10. Injecting a sample containing a first component and a second component into a capillary as an analysis target; Electrophoretically separating the injected first and second components; irradiating light onto the capillary to induced luminescence from the first component and luminescence from the second component are measured with a detector, thereby obtaining the signal intensity of the first component and the signal intensity of the second component; A capillary electrophoresis method comprising: the concentration range of the first component contained in the sample includes a concentration range in which the signal intensity of the second component is constant relative to the concentration of the first component, as well as a concentration range in which the signal intensity of the second component decreases relative to the concentration of the first component, and deviates from the constant; The capillary electrophoresis method includes determining a ratio of the concentration of the first component to the 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. A capillary electrophoresis method characterized by:

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

12. The capillary electrophoresis method according to claim 9 or 10, 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 emission is fluorescence, The signal intensity is a fluorescence intensity. A capillary electrophoresis method characterized by:

13. 13. The capillary electrophoresis method according to claim 12, the sample includes a size standard; The second component is a DNA fragment contained in a size standard. A capillary electrophoresis method characterized by:

14. 13. The capillary electrophoresis method according to claim 12, the sample contains a PCR product; The first component is a DNA fragment contained in the PCR product. A capillary electrophoresis method characterized by:

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

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

Images