CN120813826A

CN120813826A - Capillary electrophoresis device and capillary electrophoresis method

Info

Publication number: CN120813826A
Application number: CN202380094996.4A
Authority: CN
Inventors: 穴泽隆; 手塚沙也可; 安藤贵洋
Original assignee: Hitachi High Technologies Corp
Current assignee: Hitachi High Tech Corp
Priority date: 2023-07-03
Filing date: 2023-07-03
Publication date: 2025-10-17
Also published as: JPWO2025009018A1; DE112023005459T5; WO2025009018A1

Abstract

A capillary electrophoresis device injects a sample containing a first component and a second component into a capillary, performs electrophoretic separation on the injected first and second components, and uses a detector to measure luminescence from the first component and luminescence from the second component induced by irradiating light onto the capillary, thereby obtaining signal intensities of the first component and the second component. 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 relative to the concentration of the first component and deviates from the proportion to reach the saturation signal intensity. The capillary electrophoresis device quantifies the ratio of the concentration of the first component to the concentration of the second component in the sample based on the ratio of the signal intensity of the first component to the signal intensity of the second component.

Description

Capillary electrophoresis device and capillary electrophoresis method

Technical Field

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

Background

The analysis by an instrument in analytical chemistry is an analysis using an instrument that performs nuclear magnetic resonance spectroscopy, absorbance spectroscopy, raman spectroscopy, fluorescence spectroscopy, mass spectrometry, chromatography, electrophoresis analysis, or the like.

As shown in non-patent document 1 and non-patent document 2, in these instrument analyses, an external standard method (absolute calibration curve method) or an internal standard method (internal standard method) is often used to quantify the unknown concentration of an analysis target contained in a sample.

The external standard method is a method of quantifying an unknown concentration of an analyte contained in a sample by preparing a calibration curve in advance, which is a relationship between the concentration of the analyte and the signal intensity, using a standard sample containing the analyte of various known concentrations. In general, it is assumed that the signal intensity is proportional to the concentration of the analysis object, that is, the increase in the signal intensity with respect to the concentration of the analysis object is linear and the rate of increase in the signal intensity with respect to the concentration of the analysis object is constant.

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

On the other hand, the internal standard method is a method of reducing these influences and improving the quantitative accuracy. In this method, a calibration curve, which is a relationship between a concentration ratio of an analyte to an internal standard and a signal intensity ratio of the analyte to the internal standard, is prepared in advance using a standard sample including various analytes of known concentrations and internal standards of known concentrations, and the unknown concentration of the analyte contained in the sample is quantified.

Here, it is necessary to be able to measure the signal intensity of the internal standard and the analysis object separately. As shown in non-patent document 1 and non-patent document 2, in the internal standard method, it is necessary to scale the respective signal intensities with respect to the concentrations of the analysis object and the internal standard, that is, the increase in the respective signal intensities with respect to the concentrations of the analysis object and the internal standard is linear.

On the other hand, when the increase in the signal intensity with respect to the concentration of the analysis object and the internal standard is nonlinear, the internal standard method does not function. That is, the influence of the matrix effect of the sample and the influence of the variation in the injection amount of the sample into the analyzer cannot be reduced, and the quantitative accuracy cannot be improved. Furthermore, as indicated in non-patent document 2, it is known that the quantitative accuracy is rather lowered.

The case where the concentration C (tg) of the fluorescent-labeled DNA fragment as the analysis target contained in the sample is quantified by capillary electrophoresis analysis of laser-induced fluorescence measurement will be described in more detail. In the present specification, coefficients for mathematical formulas or subscripts for variables are sometimes indicated in brackets. For example, when a subscript tg is given to a variable C, C (tg) may be expressed as C _tg.

In fig. 1 of patent document 1, a capillary electrophoresis apparatus for performing electrophoresis analysis by parallel processing of 4 capillaries is used, and electrophoresis analysis is performed by using 1 capillary. The sample may contain a fluorescent-labeled DNA fragment other than the fluorescent-labeled DNA fragment to be analyzed. In addition, salts (ions) other than the DNA fragments contained in the sample are removed as much as possible in advance by ethanol precipitation and column purification.

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

Then, the injected DNA fragment is separated by the base length while moving to the sample elution end of the capillary by electrophoresis. At this time, the DNA fragment passing through the measurement point on the capillary by electrophoresis is irradiated with a laser beam, and fluorescence is emitted to the fluorescent substance labeled with the DNA fragment.

The emitted fluorescence is measured successively by the image sensor, the time series of which is given to the electropherogram. Peaks corresponding to the DNA fragments to be analyzed were obtained on the electropherogram.

The signal intensity S (tg) of the analysis target DNA fragment is generally represented by the area of the peak of the analysis target DNA fragment, but when the width of the peak is regarded as constant, it is represented by the height of the peak of the analysis target DNA fragment. 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, and therefore the proportionality coefficient is K (tg) as follows.

[ Number 1]

S _tg＝K_tg·E·T·C_tg (digital 1)

Here, E represents the effective electric field intensity in the sample near the sample injection end of the capillary at the time of electric field injection, and T represents the time of electric field injection.

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

On the other hand, in the internal standard method, a DNA fragment as an internal standard of a known concentration is mixed in a sample to perform capillary electrophoresis analysis. The internal standard DNA fragments are also fluorescently labeled. The peaks of the analysis target DNA fragments and the peaks of the internal standard DNA fragments were observed independently on the electropherogram. The signal intensity S (st) of the internal standard DNA fragment is proportional to the amount of the internal standard DNA fragment injected into the capillary, and therefore, the proportionality coefficient is set to K (st) as follows.

[ Number 2]

S _st＝K_st·E·T·C_st (number 2)

(Equation 2) shows that the signal intensity 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 analysis target DNA fragment and the internal standard DNA fragment in the sample is performed together, the electric field intensity E of the electric field injection of (expression 1) and (expression 2) is the same as the time T. Therefore, the following expression can be obtained by taking the ratio of (expression 1) to (expression 2).

[ Number 3]

The ratio S (tg)/S (st) of the signal intensity of the analysis target DNA fragment to the internal standard is proportional to the ratio C (tg)/C (st) of the concentration of the analysis target DNA fragment to the internal standard in the sample, and becomes a calibration curve of the internal standard method.

Using (expression 3), the concentration ratio C (tg)/C (st) of the analysis target DNA fragment in the sample with respect to the internal standard can be quantified from the measured signal intensity ratio S (tg)/S (st) of the analysis target DNA fragment with respect to the internal standard. This is synonymous with the quantification of the unknown concentration Ctg of the analysis target DNA fragment in the sample, since the concentration C (st) of the internal standard DNA fragment in the sample is known.

In the external standard method based on (expression 1), for example, if the electric field intensity E of the electric field injection varies by ±10%, it is known from (expression 1) that this variation directly causes a decrease in the accuracy of the quantification of the concentration of the analysis target DNA fragment. In contrast, in the internal standard method based on (expression 3), since the electric field intensity E of the electric field injection is not included in (expression 3), the deviation does not affect the accuracy of the quantification of the concentration of the analysis target DNA fragment. This is because the influence of the deviation of the electric field intensity E in (expression 1) is the same as the influence of the deviation of the electric field intensity E in (expression 2), and the influence thereof is eliminated by taking the ratio of (expression 1) to (expression 2). The above is why the accuracy of the quantification of the analysis target by the internal standard method can be improved as compared with the external standard method.

Prior art literature

Patent literature

Patent document 1 International publication No. 2023/007567

Non-patent literature

Non-patent document 1:Harvey,David.Modern analytical chemistry.Vol.1.New York:McGraw-Hill,2000.

Non-patent literature 2：A.K.Hewavitharana(2009)Internal Standard-Friend or Foe?,Critical Reviews in Analytical Chemistry,39:4,272-275

Disclosure of Invention

Problems to be solved by the invention

When the signal intensity is not proportional to the concentration of the analysis target, the reason why the external standard method can function but the internal standard method does not function is examined.

As an example, according to non-patent document 2, if the signal intensity of the analysis target is represented by a quadratic function of the concentration, the following expression is obtained using L (tg) and K (tg) as coefficients.

[ Number 4]

S _tg＝L_tg·(E·T·C_tg)²+K_tg·E·T·C_tg (number type 4)

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

In the external standard method, (equation 4) is a calibration curve. In the same manner as in the case of (expression 1), the unknown concentration C (tg) of the analysis target contained in the sample can be quantified from the measured signal intensity S (tg) of the analysis target DNA fragment using (expression 4).

On the other hand, similarly, if the signal intensity of the internal standard is represented by a quadratic function of the concentration, the coefficients L (st) and K (st) are represented by the following formulas.

[ Number 5]

S _st＝L_st·(E·T·C_st)²+·K_st·E·T·C_st (number 5)

The constant term of the quadratic function is set to zero so that S (tg) =0 when C (tg) =0. In the case where L (st) =0, (equation 5) is the same as (equation 2).

In this case, unlike (expression 3), even if the ratio of (expression 4) to (expression 5) is taken, the expression is not simplified, and the signal intensity ratio S (tg)/S (st) of the analysis target DNA fragment to the internal standard cannot be expressed by the concentration ratio C (tg)/C (st) of the analysis target DNA fragment to the internal standard in the sample. Further, unlike (equation 3), the influence of these variations cannot be eliminated due to the electric field strength E and the time T of the residual electric field injection. Therefore, the accuracy of the analysis target by the internal standard method is not improved as compared with the external standard. Furthermore, the quantitative accuracy is lower than that of the external standard method based on (equation 4). As described in non-patent document 2, if the signal intensity and the concentration of either the analysis target or the internal standard are not proportional, the accuracy of the quantification by the internal standard method is lowered.

In the above, the example in which the signal intensities of the analysis target and the internal standard are represented by the quadratic function of the respective concentrations was examined, but the same applies to the case in which the signal intensities are represented by any function other than the quadratic function in which the constant term is zero. As described above, in the case where the signal intensity is not proportional to the concentration of the analysis object, the quantitative accuracy cannot be improved by using the internal standard method.

On the other hand, patent document 1 proposes to improve the dynamic range of fluorescence measurement of a capillary electrophoresis device by optimizing the binning condition according to the noise condition of an image sensor. According to patent document 1, it is expected that the concentration of a DNA fragment in a sample can be quantified in comparison with the concentration of a DNA fragment in a wider range than before.

However, in actual attempts, when the concentration of the DNA fragment to be analyzed contained 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 contained in the sample becomes high, the signal intensity becomes saturated with respect to the concentration. In addition, since the signal intensity of the saturation is lower than that of the saturation of the image sensor, it is clear that the reason is not the saturation of the image sensor.

This phenomenon is a new problem that has been clarified by using a capillary electrophoresis device having a wider dynamic range than the conventional one. When the concentration of the analysis target DNA fragment contained in the sample is low, the signal intensity proportional to the concentration of the analysis target DNA fragment can be obtained, and therefore, the accuracy of the quantification of the analysis target can be improved by using the internal standard method. However, when the concentration of the analysis target DNA fragment contained in the sample is high, a signal intensity that is not proportional to the concentration of the analysis target DNA fragment is obtained, and therefore, the accuracy of the quantification of the analysis target cannot be improved by using the internal standard method.

The present invention has been made to solve the above problems, and an object of the present invention is to provide a capillary electrophoresis apparatus and a capillary electrophoresis method capable of improving the quantitative accuracy of an analysis target by using an internal standard method.

Means for solving the problems

In one example of the capillary electrophoresis device of the present invention,

The capillary electrophoresis device performs the following processing:

Injecting a sample comprising a first component and a second component into a capillary;

separating the injected first component and the injected second component electrophoretically, and

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

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

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

The capillary electrophoresis device performs the following processing:

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 reduced from the concentration of the first component by a constant deviation in addition to a concentration range in which the signal intensity of the second component is constant with respect to the concentration of the first component,

In one example of the capillary electrophoresis method of the present invention,

The capillary electrophoresis method comprises the following steps:

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

The capillary electrophoresis method comprises the following steps:

Quantifying a ratio of a concentration of the first component relative to a concentration of the second component in the sample based on the ratio of the signal intensity of the first component relative to the signal intensity of the second component.

The capillary electrophoresis method comprises the following steps:

Effects of the invention

By using the novel internal standard method found in the present invention, DNA fragments contained in a sample in a concentration range wider than the conventional one can be quantified with high accuracy.

Other problems, configurations and effects than those described above will be apparent from the following description of the embodiments.

Drawings

FIG. 1A is a schematic diagram showing a configuration of a capillary electrophoresis device according to an embodiment of the present invention.

Fig. 1B is a structure of a capillary electrophoresis method according to an embodiment of the present invention.

Fig. 2 is a schematic diagram of electric field injection.

FIG. 3 is an electrophoretogram of samples of various concentrations.

FIG. 4 is a graph showing the relationship between the concentration of a sample and the fluorescence intensity (1).

FIG. 5 is a graph showing the relationship between the concentration of a sample and the fluorescence intensity (2).

FIG. 6 is an electrophoretogram of a sample mixed with 2 size standards in varying ratios (1).

Fig. 7 is an electrophoretogram of a sample mixed with 2 size standards in varying ratios (2).

FIG. 8 is a graph showing the relationship between the concentration of a sample and the fluorescence intensities and the fluorescence intensity ratios of 2 DNA fragments (1).

FIG. 9 is a graph showing the relationship between the concentration of a sample and the fluorescence intensities of 3 DNA fragments.

FIG. 10 is a graph showing the relationship between the concentration of a sample and the fluorescence intensity and fluorescence intensity ratio of 2 DNA fragments (2).

FIG. 11 is a graph showing the relationship between the concentration of a sample and the fluorescence intensity ratio of 2 DNA fragments.

Detailed Description

Principle of

In the capillary electrophoresis analysis described above, the cause of the phenomenon that the signal intensity is proportional to the concentration when the concentration of the analysis target DNA fragment contained in the sample is low and the signal intensity is saturated with respect to the concentration when the concentration of the analysis target DNA fragment contained in the sample is high was examined.

If not the cause of saturation of the image sensor, another consideration is the self-extinction of the phosphor at the measurement point. In general, it is known that when the concentration of the fluorescent material becomes extremely high, self-extinction of the fluorescent material occurs, and the rate of increase in fluorescence intensity decreases according to the concentration of the fluorescent material, and the fluorescence intensity becomes saturated. In addition, it is also known that when the concentration of the fluorescent material is further increased, the fluorescence intensity is sometimes changed to decrease.

In order to verify the presence or absence of self-extinction in the capillary electrophoresis analysis, samples having different concentrations of each of a plurality of DNA fragments as an analysis target were analyzed by the capillary electrophoresis device. When the concentration of the DNA fragments in the sample is increased, the total concentration of the plurality of DNA fragments is increased while maintaining the concentration ratio of the plurality of DNA fragments in the sample.

The various DNA fragments are spatially separated by capillary electrophoresis, detected independently of each other by laser-induced fluorescence measurements at the measurement points, each assigned to a different peak on the electropherogram.

A high concentration of DNA fragments in the sample imparts a high intensity peak and a low concentration of DNA fragments in the sample imparts a low intensity peak. When the total concentration of the plurality of DNA fragments to be analyzed contained in the sample is low, a signal intensity proportional to the concentration of each DNA fragment is obtained. In contrast, when the total concentration of the plurality of DNA fragments to be analyzed contained in the sample is high, a signal intensity that is not proportional to the concentration of each DNA fragment is obtained. That is, a change from a state in which "the signal intensity is proportional to the concentration of the DNA fragment in the sample" to a state in which "the signal intensity is not proportional to the concentration of the DNA fragment in the sample" is simultaneously generated regardless of the magnitude of the intensity of the peak on the electropherogram corresponding to the concentration in the sample.

The degree of self-extinction of the phosphor should depend on the concentration of the phosphor at the measurement point on the capillary. However, the above-described variation is generated independently of the concentration of the phosphor at the measurement point on the capillary. From the above, the cause of this phenomenon is not self-extinction of the phosphor. Therefore, in the capillary electrophoresis analysis described above, the concentration of the DNA fragment at the measurement point of the capillary and the fluorescent substance labeled on the DNA fragment does not reach the concentration that causes self-extinction. Of course, this phenomenon does not occur due to saturation of the image sensor, and therefore the cause of this phenomenon is not saturation of the image sensor. Therefore, in the capillary electrophoresis analysis described above, the concentration of the DNA fragment at the measurement point of the capillary and the fluorescent substance labeled on the DNA fragment does not reach the concentration at which saturation of the image sensor occurs.

Therefore, as described below, the cause of this phenomenon was clarified solely by the study of the present invention. The phenomenon is first discovered in the study of the present invention, and the reason thereof is also first discovered in the study of the present invention. Furthermore, the present phenomenon is allowed, and based on the found cause, a new internal standard method alone is designed, so that high-precision quantitative analysis of the analysis target can be performed.

Embodiment 1

Fig. 1A is a structural view of a capillary electrophoresis device. Capillary electrophoresis devices are widely used as analysis devices for analyzing DNA sequences and DNA fragments. Using 4 capillaries 1, different samples can be analyzed in each capillary 1.

Fig. 1B is a structure of a capillary electrophoresis method. 1 capillary electrophoresis was performed by the capillary electrophoresis method including steps (1) to (8) in fig. 1B. By repeating the steps (1) to (8), a plurality of capillary electrophoresis analyses can be performed.

(1) First, the sample injection ends 2 of 4 capillaries 1 were immersed in the negative electrode side buffer solution 6, and the sample elution ends 3 were connected to the positive electrode side buffer solution 7 via the polymer solution 8 in the pump block 10.

(2) Next, the valve 11 of the pump block 10 is closed, and the piston of the syringe 12 connected to the pump block 10 is pressed, thereby pressurizing the polymer solution 8 inside, and filling the polymer solution 8 from the sample elution end 3 toward the sample injection end 2 into each capillary 1.

(3) Next, the valve 11 is opened, the sample injection ends 2 of the 4 capillaries 1 are immersed in the different samples 9, and a constant voltage is applied between the cathode electrode 4 and the anode electrode 5 for a constant time by the power supply 13, whereby a part of the different samples 9 (including at least the first component and the second component) is electric-field-injected from the sample injection ends 2 into the respective capillaries 1. Thereby, the sample 9 comprising the first component and the second component is injected into the capillary.

(4) Thereafter, the sample injection ends 2 of the 4 capillaries 1 were immersed in the cathode-side buffer solution 6, and a high voltage was applied between the cathode electrode 4 and the anode electrode 5 by the power supply 13, thereby starting capillary electrophoresis. The fluorescent-labeled DNA fragment was electrophoresed from the sample injection end 2 to the sample elution end 3. Thereby, the injected first component and second component are subjected to electrophoretic separation.

(5) In parallel, the position of each capillary 1 after electrophoresis at a constant distance from the sample injection end 2 is set as a measurement point 16, and the laser beam 14 oscillated from the laser light source 15 is irradiated to each measurement point 16 at once. Here, the capillaries 1 near the measurement point 16 are previously removed from the coating, the capillaries 1 near the measurement point 16 are aligned on the same plane, and the laser beam 14 is condensed and then introduced from the side of the alignment plane along the alignment plane.

(6) The DNA fragments labeled with the phosphor are then excited by the irradiation of the laser beam 14 while passing through the measurement point 16, and fluoresce. That is, the intensities of the fluorescent lights emitted from the 4 measurement points 16 each change from time to time with electrophoresis.

(7) Subsequently, the fluorescence emitted from each measurement point 16 is measured by the detector 17. That is, the detector 17 measures the luminescence fluorescence from the first component and the luminescence fluorescence from the second component induced by the irradiation of the laser beam onto the capillary 1, thereby obtaining the signal intensity of the first component and the signal intensity of the second component. An electrophoresis pattern, which is time-series data of these signal intensities, is acquired, and analysis of the sample 9 injected into each capillary 1 is performed. The detector 17 includes a spectroscope and an image sensor (not shown), and can simultaneously and independently spectroscopically measure luminescence fluorescence from the 4 measurement points 16. Therefore, the luminescence fluorescence of the plurality of phosphors can be recognized.

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

Fig. 2 schematically shows the electric field injection of a sample 9 in a capillary electrophoresis analysis. The sample injection end 2 of 1 capillary 1 filled with the polymer solution 8 is immersed in the solution-like sample 9, and fig. 2 (a) shows before electric field injection and fig. 2 (b) shows after electric field injection. After (b) of FIG. 2, the sample injection end 2 was immersed in the negative electrode side buffer solution, and electrophoresis was started.

The sample 9 contains negative ions 20 other than the first DNA fragment 18 (first component), the second DNA fragment 19 (second component), and the DNA fragment as negative ions. 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 is different from the base length of the second DNA fragment 19. The first DNA fragment 18 and the second DNA fragment 19 are different in concentration in the sample 9.

As will be described later in detail, the sample 9 may contain a size standard, and the second DNA fragment 19 may be a DNA fragment contained in the size standard. In addition, sample 9 may comprise a PCR product, and first DNA fragment 18 may be a DNA fragment that is a PCR product or a DNA fragment from a PCR product. In addition, sample 9 may contain a single base extension product, first DNA fragment 18 may be a first DNA fragment as a single base extension product, and second DNA fragment 19 may be a second DNA fragment as a single base extension product.

In this example, 2 types of DNA fragments are mainly treated as the simplest example of the plurality of types of DNA fragments, but it is needless to say that the same examination can be performed for any number of 3 or more types of DNA fragments.

The cation, the cathode, is omitted from fig. 2. The solvent of sample 9 was either pure water or formamide. Sample 9 was previously subjected to ethanol precipitation or the like to remove as much negative ions (salts) as possible other than the DNA fragment, but could not be set to zero.

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

The current I flowing when a constant voltage V is applied between the cathode and anode electrodes is approximately determined by the resistance R of the capillary 1 filled with the polymer solution 8, i≡v/R. This is because the resistance R (i) between the cathode electrode 4 (fig. 1A) and the sample injection end 2 of the capillary 1, and the resistance R (o) between the sample elution end 3 (fig. 1A) of the capillary and the anode electrode 5 (fig. 1A) are sufficiently small compared to R (R > > R (i), R (o)). That is, the combined resistance R (i) +r+r (o) between the cathode electrode 4 and the anode electrode 5 is approximately equal to R (i) +r+r (o) ≡r.

Therefore, the current I flowing when the constant voltage V is applied between the cathode electrode and the anode electrode hardly changes regardless of the composition of the sample, for example, regardless of whether the sample is pure water or a high ion concentration solution. Therefore, the expression "applying a constant voltage between the cathode electrode and the anode electrode" is sometimes expressed as "applying a constant voltage to both ends of the capillary".

The current I flowing between the negative electrode and the sample injection end of the capillary, the current I flowing in the capillary, and the current I flowing between the sample elution end of the capillary and the positive electrode are equal to each other in continuity of the current.

Here, the current I flowing between the cathode and the sample injection end of the capillary at the time of electric field injection, that is, the current I flowing in the sample is assumed by negative ions injected into the capillary if positive ions are omitted for simplicity. Therefore, the total amount of negative ions injected into the capillary by the electric field injection to which a constant voltage is applied for a constant time is constant regardless of the composition of the sample.

The effective electric field intensity in the sample near the sample injection end of the capillary at the time of electric field injection is set to E, the time of electric field injection is set to T, and the inner cross-sectional area of the capillary is set to a. When the mobility of the negative ions other than the DNA fragment in the sample is μ (0) and the concentration is C (0), the number of implanted molecules J (0) of the negative ions other than the DNA fragment implanted by the electric field is represented by the following formula.

[ Number 6]

J ₀＝E·T·A·μ₀·C₀ (digital type 6)

Mobility means the moving speed of each negative ion per unit electric field strength.

When negative ions other than a plurality of DNA fragments are present, the average of the mobility and concentration of the negative ions is represented by μ (0) and C (0). When the average charge amount per molecule of negative ions other than the DNA fragment is Q (0), the injection charge amount Q (0) of negative ions other than the DNA fragment injected by the electric field is represented by the following formula.

[ Number 7]

Q ₀＝q₀·J₀＝E·T·A·q₀·μ₀·C₀ (number 7)

The number of injection molecules J (1) of the first DNA fragment and the number of injection molecules J (2) of the second DNA fragment injected by the electric field are expressed as follows, assuming that 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).

[ Number 8]

J ₁＝E·T·A·μ·C₁ (number 8)

[ Number 9]

J ₂＝E·T·A·μ·C₂ (digital type 9)

Here, the mobility of the first DNA fragment and the second DNA fragment is made equal because the mobility of the DNA fragment in the solution without the molecular sieve effect, i.e., the sample is constant regardless of the base length.

When the average charge amounts per molecule of the first DNA fragment and the second DNA fragment are Q (1) and Q (2), the injection charge amounts Q (1) and Q (2) of the first DNA fragment and the second DNA fragment injected based on the electric field are the following formulas.

[ Number 10]

Q ₁＝q₁·J₁＝E·T·A·q₁·μ·C₁ (digital type 10)

[ Number 11]

Q ₂＝q₂·J₂＝E·T·A·q₂·μ·C₂ (digital type 11)

When the total concentration of the first DNA fragment and the second DNA fragment in the sample is c=c (1) +c (2), the total injection molecule number J of the first DNA fragment and the second DNA fragment injected by the electric field is represented by the following formula.

[ Number 12]

J=j ₁+J₂ = E.T.A mu.C (number 12)

Here, when the average charge amounts per molecule of the first DNA fragment and the second DNA fragment are equal and can be approximated as q=q (1) =q (2), the total injection charge amount Q of the first DNA fragment and the second DNA fragment injected by the electric field is represented by the following formula.

[ Number 13]

Q=q·j=e T.A. Q. Mu. C (number 13)

Alternatively, when the ratio of the concentrations C (1) and C (2) of the first DNA fragment and the second DNA fragment in the sample to the total concentration C of the first DNA fragment and the second DNA fragment is constant, the expression (formula 13) can be obtained by defining q= (q (1) ·c (1) +q (2) ·c (2))/(C (1) +c (2)).

In (expression 6) to (expression 13), E, T and a are the same value. Since the total amount of the injected charge of the negative ions per unit time at the time of electric field injection is equal to the current I, the following equation is adopted.

[ Number 14]

That is to say,

[ Number 15]

In (expression 14) and (expression 15), since I is constant as described above, electric field injection is a competition process between a plurality of negative ions including DNA fragments contained in a sample, meaning that the amount of injected electric charge of each negative ion is distributed according to the product of the amount of electric charge, mobility, and concentration of each negative ion. When the contribution of positive ions to the current is considered, the contribution ratio of negative ions to the current may be multiplied by the right side of (equation 14).

When (expression 15) is used, the following modifications can be made from (expression 8) to (expression 11).

[ Number 16]

[ Number 17]

[ Number 18]

[ Number 19]

In addition, in the case where q=q (1) =q (2) can be approximated as described above, (expression 12) and (expression 13) can be modified as follows.

[ Number 20]

[ Number 21]

Alternatively, when the ratio of the concentrations C (1) and C (2) of the first DNA fragment and the second DNA fragment in the sample to the total concentration C of the first DNA fragment and the second DNA fragment is constant, the (expression 20) and (expression 21) can be obtained by defining q= (q (1) ·c (1) +q (2) ·c (2))/(C (1) +c (2)).

On the other hand, the signal intensities S (1) and S (2) of the peaks of the first DNA fragment and the second DNA fragment on the electrophoresis pattern obtained by capillary electrophoresis are expressed by the following formulas based on the formulas (expression 8) and (expression 9) assuming that the sensitivity coefficients are m (1) and m (2).

[ Number 22]

S ₁＝m₁·J₁＝E·T·A·m₁·μ·C₁ (number 22)

[ Number 23]

S ₂＝m₂·J₂＝E·T·A·m₂·μ·C₂ (number 23)

When (formula 15) is used, the following formula is used.

[ Number 24]

[ Number 25]

In addition, the sensitivity coefficient includes the average number of labeled phosphors per molecule of the DNA fragment, excitation efficiency of the phosphors, quantum yield of the phosphors, light-collecting efficiency of luminescence fluorescence, sensitivity of the image sensor, and the like.

When the sensitivity coefficients of the first DNA fragment and the second DNA fragment can be approximated to be m (1) and m (2), the total signal intensity S of the peaks of the first DNA fragment and the second DNA fragment on the electrophoresis chart obtained by capillary electrophoresis is expressed by the following equation (equation 12) assuming that m=m (1) =m (2).

[ Number 26]

S=m·j=e T.A. m.mu.C (number 26)

Alternatively, when the ratio of the concentrations C (1) and C (2) of the first DNA fragment and the second DNA fragment in the sample to the total concentration C of the first DNA fragment and the second DNA fragment is constant, the expression (formula 26) can be obtained by defining m= (m (1) ·c (1) +m (2) ·c (2))/(C (1) +c (2)).

When the use (expression 15) is approximately q=q (1) =q (2), the following expression is obtained.

[ Number 27]

Alternatively, when the ratio of the concentrations C (1) and C (2) of the first DNA fragment and the second DNA fragment in the sample to the total concentration C of the first DNA fragment and the second DNA fragment is constant, the expression (formula 27) can be obtained by defining q= (q (1) ·c (1) +q (2) ·c (2))/(C (1) +c2).

The following expressions (16) to (21), (24), (25) and (27) are used, where a and b are constants and y is a function of x.

[ Number 28]

For example, when y=j (1), x=c (1), a=i·t/q (1), b= (q (0) ·μ (0) ·c (0) +q (2) ·μ·c (2))/(q (1) ·μ), the expression (16) is expressed as expression (28). (equation 28) where x is smaller than b, the scaling factor is a/b, y is proportional to x (y=a/b×x), and where x is larger than b, y is saturated with respect to x and gradually approaches a constant value a.

That is, (expression 16) indicates that J (1) is proportional to C (1) when C (1) is smaller than b= (q (0) ·μ (0) ·c (0) +q (2) ·μ·c (2))/(q (1) ·μ), and J (1) is saturated with respect to C (1) and gradually approaches a constant value when C (1) is larger than b= (q (0) ·μ (0) ·c (0) +q (2) ·μ·c (2))/(q (1) ·μ).

On the other hand, (expression 8) shows a proportional relationship between J (1) and C (1), and hence (expression 8) and (expression 16) seem to contradict. However, in reality, E contained in (formula 8) varies according to C (1), and thus J (1) is not necessarily proportional to C (1) in (formula 8). Therefore, (equation 8) and (equation 16) do not contradict each other and coexist.

Specifically, in (expression 15), I is constant, so when C (1) is raised in a state where C (0) and C (2) are constant, E is reduced. E is the effective electric field intensity in the sample near the sample injection end of the capillary at the time of electric field injection, and can vary depending on the composition of the sample even if the voltage applied to both ends of the capillary is made constant, that is, the average electric field intensity is made constant.

Similarly, (equation 17) indicates that J (2) is proportional to C (2) when C (2) is small, and J (2) is saturated with respect to C (2) when C (2) is large, and gradually approaches a constant value. (equation 18) indicates that Q (1) is proportional to C (1) when C (1) is small, and Q (1) is saturated with respect to C (1) when C (1) is large, gradually approaching a constant value. (equation 19) indicates that Q (2) is proportional to C (2) when C (2) is small, and Q (2) is saturated with respect to C (2) when C (2) is large, and gradually approaches a constant value. (equation 20) shows that J is proportional to C in the case where C is small, and J is saturated with respect to C in the case where C is large, gradually approaching a constant value. (equation 21) shows that Q is proportional to C when C is small, and Q is saturated with respect to C when C is large, and gradually approaches a constant value. (equation 24) indicates that S (1) is proportional to C (1) when C (1) is small, and that S (1) is saturated with respect to C (1) when C (1) is large, gradually approaching a constant value. (equation 25) indicates that S (2) is proportional to C (2) when C (2) is small, and that S (2) is saturated with respect to C (2) when C (2) is large, and gradually approaches a constant value. In addition, (equation 27) indicates that S is proportional to C when C is small, and S is saturated with respect to C when C is large, and gradually approaches a constant value.

Therefore, (expression 24), (expression 25) and (expression 27) illustrate the phenomenon that in capillary electrophoresis analysis, when the concentration of the analysis target DNA fragment contained in the sample is low, a signal intensity proportional to the concentration is obtained, and when the concentration of the analysis target DNA fragment contained in the sample is high, the signal intensity is saturated with respect to the concentration. That is, the cause of the above phenomenon can be determined. This is the first finding obtained by introducing (expression 14) and (expression 15).

In addition, as in (expression 8) and (expression 16), the contradictions are not present, and (expression 9) and (expression 17), (expression 10) and (expression 18), (expression 11) and (expression 19), (expression 12) and (expression 20), (expression 13) and (expression 21), (expression 22) and (expression 24), (expression 23) and (expression 25), and (expression 26) and (expression 27) are not present.

On the other hand, according to (expression 8) and (expression 9), since E, T, A and μ are the same, the following expression holds.

[ Number 29]

That is, 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 ratio of the concentrations of the first DNA fragment to the second DNA fragment in the sample. The following equations are established based on (expression 8), (expression 9), and (expression 12).

[ Number type 30]

[ Number 31]

That is, the ratio of the number of molecules of the first DNA fragment or the second DNA fragment injected into the capillary by electric field injection to the total number of molecules of the first DNA fragment and the second DNA fragment is identical to the ratio of the concentration of the first DNA fragment or the second DNA fragment in the sample to the total concentration of the first DNA fragment and the second DNA fragment.

Further, since E, T, A and μ are the same according to (expression 10) and (expression 11), the following expression holds.

[ Number 32]

That is, the ratio of the amount of charge of the first DNA fragment to the second DNA fragment injected into the capillary by electric field injection is proportional to the ratio of the concentrations of the first DNA fragment to the second DNA fragment in the sample.

When q=q (1) =q (2), the following equations are satisfied according to (expression 10), (expression 11), and (expression 13).

[ Number 33]

[ Number 34]

That is, the ratio of the amount of injected charge of the first DNA fragment or the second DNA fragment injected into the capillary by electric field injection to the total amount of injected charge of the first DNA fragment and the second DNA fragment is identical to the ratio of the concentration of the first DNA fragment or the second DNA fragment in the sample to the total concentration of the first DNA fragment and the second DNA fragment.

The following equations are established based on (expression 22) and (expression 23).

[ Number 35]

That is, the ratio of the signal intensities of the first and second DNA fragments obtained by subjecting the first and second DNA fragments injected into the capillary by electric field injection to capillary electrophoresis analysis is proportional to the ratio of the concentrations of the first and second DNA fragments in the sample.

Thus, the capillary electrophoresis device of FIG. 1A is capable of quantifying the ratio of the concentration of a first DNA fragment to the concentration of a 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.

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

When considered as m=m (1) =m (2), the following equations are given according to (equation 22), (equation 23), and (equation 26).

[ Number 36]

[ Number 37]

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

It should be noted that (expression 16), (expression 17), and (expression 20) are not contradictory to (expression 29), (expression 30), and (expression 31) and coexist. That is, when the number of injected molecules of the first DNA fragment, the second DNA fragment, or the whole DNA fragment is proportional to, or not proportional to, the concentration of the first DNA fragment, the second DNA fragment, or the whole DNA fragment in the sample, respectively, the ratio of the number of injected molecules of the first DNA fragment to the number of injected molecules of the second DNA fragment matches the ratio of the number of injected molecules of the first DNA fragment to the number of injected molecules of the second DNA fragment in the sample.

It should be noted that (expression 18), (expression 19), and (expression 21) are not contradictory to (expression 32), (expression 33), and (expression 34) and coexist. That is, in the case where the amount of injected charge of the first DNA fragment, the second DNA fragment, or the whole of the DNA fragment is proportional to the concentration of the first DNA fragment, the second DNA fragment, or the whole of the DNA fragment in the sample, respectively, and in the case where the amount of injected charge of the first DNA fragment and the second DNA fragment is not proportional to the concentration ratio of the first DNA fragment and the second DNA fragment in the sample, the amount of injected charge of the first DNA fragment or the second DNA fragment with respect to the whole of the DNA fragment is identical to the concentration ratio of the first DNA fragment or the second DNA fragment with respect to the whole of the DNA fragment in the sample.

It should be noted that (expression 24), (expression 25), and (expression 27) are not contradictory to (expression 35), (expression 36), and (expression 37) and coexist. That is, in the case where the signal intensity of the first DNA fragment, the second DNA fragment, or the whole of the DNA fragment is proportional to, or not proportional to, the concentration of the first DNA fragment, the second DNA fragment, or the whole of the DNA fragment 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 whole of the DNA fragment is identical to the concentration ratio of the first DNA fragment or the second DNA fragment to the whole of the DNA fragment in the sample.

If the second DNA fragment is used as the internal standard, the calibration curve of the same form as that of the expression (3) can be obtained by (expression 35), and the influence of the fluctuation of the electric field intensity E and the time T of the electric field injection can be avoided, so that the first DNA fragment to be analyzed can be accurately quantified. Therefore, similarly to the conventional calibration curve (expression 3) of the internal standard method, the analysis target DNA fragment can be precisely quantified by using the new calibration curve (expression 35) of the internal standard method.

The conventional internal standard method can be applied only to the case where the signal intensity is proportional to the concentration of the analysis object. In contrast, as shown in (expression 24), (expression 25) and (expression 27), the new internal standard method can be applied not only to the case where the signal intensity is proportional to the concentration of the analysis object but also to any of the case where the signal intensity is not proportional, the case where saturation is near, and the case where saturation is reached.

[ Concrete example of measurement results ]

In capillary electrophoresis analysis, a specific example of a phenomenon in which the signal intensity proportional to the concentration is obtained when the concentration of the analysis target DNA fragment contained in the sample is low, and the signal intensity is saturated with respect to the concentration when the concentration of the analysis target DNA fragment contained in the sample is high is shown.

STR-PCR for DNA-based examination was performed using the human genome of a specific person as a template, and desalting was performed with the solvent as formamide. 4 samples were prepared in which the concentrations of the various DNA fragments as STR-PCR products were varied from the standard concentration within a concentration range of 4-fold, 1-fold, 0.05-fold, 0.002-fold, and 0.0001-fold.

Parts of electrophoresis patterns obtained by subjecting samples having a concentration of 0.0001-fold, 0.002-fold, 0.05-fold, and 1-fold to electrophoresis analysis using 4 capillaries using the capillary electrophoresis apparatus of FIG. 1A are shown in FIGS. 3 (a), (b), (c), and (d), respectively.

Each peak on the electrophoretogram represents the signal of the DNA fragment as the STR-PCR product of each gene locus. For example, in FIGS. 3 (a), (b), (c) and (D), the single peak observed before and after the electrophoresis time 4200 frame represents the signal of the DNA fragment as the STR-PCR product of the gene locus D5S818, respectively. When the concentration of the DNA fragments contained in the sample is increased, the fluorescence intensity (signal intensity, hereinafter the same applies) of each DNA fragment increases. Since the fluorescence intensities measured when the fluorescence of the same fluorescence intensity is generated at the measurement points of the 4 capillaries are adjusted to be equal to each other, the difference in the measured fluorescence intensities faithfully reflects the difference in the fluorescence intensities emitted at the measurement points.

FIG. 4 is a bipartite graph obtained by plotting the fluorescence intensity of the peak of the gene locus D5S818 against the concentration of the DNA fragment contained in the sample, using the DNA fragment of the STR-PCR product of the gene locus D5S818 in FIG. 3 as an analysis object. Here, the width of the peak is considered to be equal in fig. 3 (a), (b), (c), and (d), and therefore, the peak height is taken as the fluorescence intensity instead of the peak area. The concentration of the DNA fragment on the horizontal axis is set to be the concentration of the DNA fragment which is the STR-PCR product of the gene locus D5S818 contained in the sample, but may be regarded as the whole concentration of the DNA fragment contained in the sample.

When the concentration of the analyte DNA fragment contained in the sample is low, the fluorescence intensity of the analyte DNA fragment is proportional to the concentration of the DNA fragment contained in the sample, but when the concentration of the analyte DNA fragment contained in the sample is high, the fluorescence intensity of the analyte DNA fragment is deviated from the concentration of the analyte DNA fragment contained in the sample, and the saturation is approached.

In expression 24, the expression "y=s (1), x=c (1), a=m (1) ·i·t/q (1), and b= (q (0) ·μ (0) ·c (0) +q (2) ·μ·c (2))/(q (1) ·μ) is expressed as expression 28. In expression 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).

The dashed line in fig. 4 shows that the relationship between S (1) and C (1) (equation 28) in the cases of a=3500 and b=0.17 is a good approximation curve for the case where C (1) is low and the case where C (1) is high, which is plotted against 4 points in fig. 4. That is, as shown in (equation 28), when C (1) is low, S (1) is proportional to C (1), but when C (1) becomes high, S (1) is deviated from C (1), and a near saturation is demonstrated.

On the other hand, the solid line in fig. 4 shows that when S (1) =a/b·c (1) ≡20000·c (1), that is, the relationship between S (1) and C (1) is proportional, a good approximate straight line is drawn with respect to the left 2 points in fig. 4, that is, when C (1) is low. From the results, it is clear that S (1) and C (1) are defined by C (1) being about 0.01 times, and are deviated from the proportional relationship to the non-proportional relationship.

Therefore, according to the conventional internal standard method, the concentration range of 0.0001 to 0.01 times of C (1), that is, the concentration range of 2 bits in the dynamic range can be quantified. In contrast, according to the new internal standard method, the concentration range of C (1) is 0.0001 to 1, that is, the concentration range of 4 bits in the dynamic range can be quantified.

Thus, by using the novel internal standard method according to the present disclosure, it is possible to accurately quantify a DNA fragment contained in a sample in a concentration range wider than before.

As described above, the peak of the gene locus D5S818 in fig. 3 was analyzed, but the same relationship is true for other peaks. That is, as the overall concentration of the DNA fragment contained in the sample increases, the other peaks deviate from the proportional relationship in synchronization with the proportional relationship of the peak deviation of D5S 818. In each of the electrophoresis patterns, although the fluorescence intensities of the peaks are different, the phenomenon of such synchronization is not due to saturation of the detector or self-extinction of the fluorescent material.

The phenomenon observed in fig. 3 and 4 is clearly described and understood by the reason and theory considered in the above-mentioned principle.

FIG. 5 is a bipartite graph showing a change in fluorescence intensity of an analysis target DNA fragment with respect to a change in concentration of the analysis target DNA fragment contained in a sample, for a sample different from that of FIGS. 3 and 4.

5 Samples were prepared in which the concentration of the DNA fragment to be analyzed contained in the sample was varied from the standard concentration by a factor of 100, 10, 1, 0.1, 0.01, and the concentration of the 4-position. The reference concentration in fig. 5 is independent of the reference concentrations in fig. 3 and 4.

The 5 samples were separated into 2 groups using the capillary electrophoresis apparatus of fig. 1A, and each was subjected to electrophoresis analysis. Similarly to fig. 4, when the concentration of the DNA fragment contained in the sample is low, the fluorescence intensity of the DNA fragment is proportional to the concentration of the DNA fragment contained in the sample, but when the concentration of the DNA fragment contained in the sample is high, the fluorescence intensity of the DNA fragment is deviated from the concentration of the DNA fragment contained in the sample by a proportion, and the saturation is approached.

Similarly to the above, when y=s (1), x=c (1), a=m (1) ·i·t/q (1), b= (q (0) ·μ (0) ·c (0) +q (2) ·μ·c (2))/(q (1) ·μ), the expression (24) becomes the expression (28). In expression 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).

The dashed line in fig. 5 shows that the relationship between S (1) and C (1) (expression 28) in the cases of a=22000 and b=10 is a good approximation curve for the case of C (1) being low and the case of C (1) being high, which are plotted at 5 points in fig. 5. That is, as shown in (equation 28), when C (1) is low, S (1) is proportional to C (1), but when C (1) becomes high, S (1) is deviated from C (1), and a near saturation is demonstrated.

On the other hand, the solid line in fig. 5 shows that when S (1) =a/b·c (1) ≡2180·c (1), that is, the relationship between S (1) and C (1) is proportional, a good approximate straight line is drawn with respect to the left 3 points in fig. 5, that is, when C (1) is low. From the results, it is clear that S (1) and C (1) are deviated from the proportional relationship to the non-proportional relationship by about 1 time as much as C (1).

Therefore, according to the conventional internal standard method, the concentration range of C (1) is 0.01 to 1 times, that is, the concentration range of 2 bits in the dynamic range can be quantified. In contrast, according to the new internal standard method, the concentration range of C (1) is 0.01 to 100 times, that is, the concentration range of 4 bits in the dynamic range can be quantified.

Thus, by using the novel internal standard method according to the present disclosure, it is possible to accurately quantify a DNA fragment contained in a sample in a concentration range wider than before. The phenomenon observed in fig. 5 is clearly explained and understood by the reason and theory considered in the above-mentioned [ principle ].

Example 1

The following measurements were performed using the capillary electrophoresis device shown in fig. 1A. Hereinafter, the symbol "^TM" represents a trademark. 4 kinds of samples obtained by mixing 2 kinds of size standards, geneScan ^TM 600LIZ^TM dye Size Standard (hereinafter, 600 LIZ) and GeneScan ^TM500ROX^TM dye Size Standard (hereinafter, 500 ROX), at a specific ratio were prepared using Hi-Di ^TM Formamide (hereinafter, formamide) from Thermo FISHER SCIENTIFIC as a solvent, and analyzed by using the capillary electrophoresis apparatus of FIG. 1A. The 4 capillaries have an inner diameter of 50 μm, a total length of 47cm and an effective length of 36cm. Applied Biosystems ^TM 310 and 31xx Running Buffer and 10X of Thermo FISHER SCIENTIFIC were diluted 10-fold with pure water as the negative electrode side buffer and the positive electrode side buffer. As the polymer solution, POP-4 ^TMPolymer、for3500/SeqStudio^TM Flex from Thermo FISHER SCIENTIFIC was used.

The concentration of 600LIZ contained in each of the 4 samples was varied 1/2 times (0.5 times), 1/20 times (0.05 times), 1/200 times (0.005 times), and 1/2000 times (0.0005 times) from the reference concentration, while the concentration of 500ROX contained in each of the 4 samples was constant 1/200 times (0.005 times) from the reference concentration. But the reference concentration of 600LIZ is independent of the reference concentration of 500 ROX. Here, 600LIZ and 500ROX each contain a plurality of DNA fragments, and the overall concentration is changed while maintaining their concentration ratio. The electric field injection of each sample was performed by applying a voltage of 1.2kV for 9 seconds to both ends of each capillary. Electrophoresis was performed by applying a voltage of 8.5kV across each capillary.

FIG. 6 (a) shows an electrophoresis pattern of a sample having a 600LIZ concentration of 1/2000 times and a 500ROX concentration of 1/200 times. FIG. 6 (b) shows an electrophoresis pattern of a sample having a 600LIZ concentration of 1/200 times and a 500ROX concentration of 1/200 times. FIG. 7 (a) shows an electrophoresis pattern of a sample having a 600LIZ concentration of 1/20 times and a 500ROX concentration of 1/200 times. FIG. 7 (b) shows an electrophoresis pattern of a sample having a concentration of 600LIZ of 1/2 times and a concentration of 500ROX of 1/200 times. In either graph, the solid line represents the fluorescence intensity of 600LIZ and the dashed line represents the fluorescence intensity of 500 ROX. The horizontal axis represents electrophoresis time, the left vertical axis represents fluorescence intensity of 500ROX, and the right vertical axis represents fluorescence intensity of 600 LIZ.

600LIZ contains 36 single-stranded DNA fragments of base lengths 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, labeled with a fluorescent LIZ, respectively. The electrophoresis patterns of FIGS. 6 and 7 show peaks of 15 DNA fragments of 20, 40, 60, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250 base lengths. Further, peaks of DNA fragments of 40, 114 and 160 bases are shown by arrows and are denoted as LIZ40, LIZ114 and LIZ160.

500ROX contains 16 single-stranded DNA fragments of base lengths 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490 and 500, respectively, labeled with a fluorescent ROX. The electrophoresis patterns of FIGS. 6 and 7 show peaks of 9 DNA fragments of 35, 50, 75, 100, 139, 150, 160, 200, 250 bases in length. Further, the peak of the 160-base-length DNA fragment is shown by an arrow, and is denoted as ROX160.

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

Fig. 8 (a) is a double-contrast plot obtained by plotting the fluorescence intensities of the peak of LIZ160 and the peak of ROX160 obtained from the 4 electrophoresis patterns of fig. 6 and 7 with respect to the concentration of 600LIZ contained in the sample. The horizontal axis may also be considered as the concentration of LIZ160 contained in the sample. Here, the width of each peak is regarded as being substantially equal, and therefore, the height of each peak is regarded as fluorescence intensity. The black dots indicate the fluorescence intensity of LIZ160 and the white dots indicate the fluorescence intensity of ROX 160.

When the concentration of 600LIZ contained in the sample is low, specifically, in the range of 1/2000 to 1/20 times the concentration of 600LIZ, the fluorescence intensity of LIZ160 is proportional to the concentration of 600LIZ contained in the sample, and can be approximated by a straight line having a slope of 1. On the other hand, when the concentration of 600LIZ contained in the sample is high, specifically, in the range of 1/20 to 1/2 times the concentration of 600LIZ, the ratio of the fluorescence intensity of the LIZ160 to the concentration of 600LIZ contained in the sample is deviated to approach saturation. This phenomenon is the same as that observed in fig. 4 and 5.

In this way, the concentration range of the first component contained in the sample includes a concentration range in which the signal intensity of the first component is lower than the saturation signal intensity of the detector and is deviated from the saturation signal intensity by a proportion so as to reach the saturation signal intensity, in addition to the concentration range in which the signal intensity of the first component is proportional to the concentration of the first component. The capillary electrophoresis device of the present embodiment can measure the concentration of such a sample.

In contrast, when the concentration of 600LIZ contained in the sample is low, specifically, when the concentration of 600LIZ is in the range of 1/2000 to 1/200, the fluorescence intensity of ROX160 is constant with respect to the concentration of 600LIZ contained in the sample. This is easily understood since the concentration of ROX160 contained in the sample is constant. However, when the concentration of 600LIZ contained in the sample is high, specifically, the concentration of 600LIZ is in the range of 1/200 to 1/2, the fluorescence intensity of ROX160 decreases relative to the concentration of 600LIZ contained in the sample. Such a change is not easily understood since the concentration of ROX160 contained in the sample is constant. The above is a novel phenomenon found in the present disclosure.

In this way, 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 reduced from the concentration of the first component by a constant deviation in addition to the concentration range in which the signal intensity of the second component is constant from the concentration of the first component. The capillary electrophoresis device of the present embodiment can measure the concentration of such a sample.

The above phenomenon is described by (expression 24) and (expression 25).

In (equation 24) and (equation 25), the first DNA fragment is regarded as all DNA fragments contained in 600LIZ, and the second DNA fragment is regarded as all DNA fragments contained in 500 ROX. That is, S (1) is the total of the fluorescence intensities of all the DNA fragments contained in 600LIZ, C (1) is the total of the concentrations of all the DNA fragments contained in 600LIZ, q (1) is the average charge amount of all the DNA fragments contained in 600LIZ, S (2) is the total of the fluorescence intensities of all the DNA fragments contained in 500ROX, C (2) is the total of the concentrations of all the DNA fragments contained in 500ROX, and q (2) is the average charge amount of all the DNA fragments contained in 500 ROX.

At this time, in the condition that the concentration C (0) of the negative ions other than the DNA fragment is constant and the concentration C (2) of 500ROX is constant, the fluorescence intensity S (1) of 600LIZ is proportional to the concentration C (1) of 600LIZ when the concentration C (1) of 600LIZ is low, and the ratio is deviated from the concentration C (1) of 600LIZ to saturate when the concentration C (1) of 600LIZ is high in (formula 24). The fluorescence intensity of LIZ160 is proportional to the fluorescence intensity of 600LIZ, so both represent the same change. Therefore, the change in fluorescence intensity of the LIZ160 in the full concentration range of the LIZ160 of fig. 8 (a) is described by (equation 24).

In contrast, in (expression 25), under the conditions that the concentration C (0) of negative ions other than DNA fragments is constant and the concentration C (2) of 500ROX is constant, the fluorescence intensity S (2) of 500ROX is constant when the concentration C (1) of 600LIZ is low, and is reduced relative to the concentration C (1) of 600LIZ when the concentration C (1) of 600LIZ is high. The fluorescence intensity of ROX160 is proportional to the fluorescence intensity of 500ROX, so both represent the same change. Therefore, the change in the fluorescence intensity of ROX160 in the full concentration range of LIZ160 of fig. 8 (a) is described by (equation 25).

Further, the above description is given by (expression 14) and (expression 15). That is, when the injection charge amount Q (1) of the first DNA fragment increases by an increase in the concentration C (1) of the first DNA fragment under the condition that the current at the time of electric field injection is constant, the injection charge amount Q (2) of the second DNA fragment decreases. This is achieved by the decrease in the electric field strength in the sample in the vicinity of the sample injection end of the capillary at the time of electric field injection, which is E on the left side of (equation 15).

The above is an examination focusing on the peak of the LIZ160 and the peak of the ROX160 in fig. 6 and 7, but the same examination is also true focusing on the peaks of DNA fragments other than these.

Fig. 9 is a double-log plot obtained by plotting the fluorescence intensities of the peaks of LIZ40 and LIZ114 against the concentration of 600LIZ contained in the sample, as an example, in addition to the fluorescence intensities of the peaks of LIZ160 (the same data as in fig. 8 (a)). The horizontal axis may also be considered as the concentration of LIZ40, LIZ114 or LIZ160 contained in the sample.

When the concentration of 600LIZ contained in the sample is low, specifically, in the range of 1/2000 to 1/20 times the concentration of 600LIZ, each fluorescence intensity is proportional to the concentration of 600LIZ contained in the sample, and can be approximated by a straight line having a slope of 1. On the other hand, when the concentration of 600LIZ contained in the sample is high, specifically, in the range of 1/20 to 1/2 times the concentration of 600LIZ, the ratio of the respective fluorescence intensities to the concentration of 600LIZ contained in the sample is deviated to be close to saturation. The above change in fluorescence intensity occurs synchronously for each DNA fragment. Of course, any change in fluorescence intensity can be approximated by (equation 24).

In each of the electrophoresis charts, although the fluorescence intensities of the peaks of LIZ40, LIZ114, and LIZ160 are different, the above-described changes in fluorescence intensities occur simultaneously, which means that the cause is not saturation of the detector or self-extinction of the phosphor, but is caused by a constant electric field injection amount found in the present disclosure. Regarding ROX, the peak of a DNA fragment other than ROX160 can also be approximated by (equation 25) by obtaining the same change in fluorescence intensity as ROX 160.

According to fig. 8 (a), if the concentration of 600LIZ, which is proportional to the concentration of 600LIZ contained in the sample, is in the range of 1/2000 to 1/20, the conventional internal standard method using ROX160 as the internal standard can be used to accurately quantify the LIZ 160. Alternatively, if the fluorescence intensity of the ROX160 is in the range of 1/2000 to 1/200 times the concentration of 600LIZ, which is a constant concentration of 600LIZ in the sample, the LIZ160 can be accurately quantified by using a conventional internal standard method using the ROX160 as an internal standard.

However, when the standard deviation is out of these ranges, the conventional internal standard method cannot be applied. In particular, when the concentration of 600LIZ contained in the sample is 1/2 times, the fluorescence intensity of the peak of LIZ160 is nearly saturated in fig. 8 (a), and the fluorescence intensity of ROX160 is reduced, so it is considered that quantification of LIZ160 by these ratios is impossible in the conventional concept.

In contrast, fig. 8 (b) is a double-logarithmic graph in which the ratio of the fluorescence intensity of the peak of LIZ160 to the fluorescence intensity of the peak of ROX160 in fig. 8 (a) is plotted against the concentration of 600LIZ contained in the sample. The horizontal axis may also be considered as the concentration of LIZ160 contained in the sample.

According to fig. 8 (a) or the conventional internal standard method, the ratio of the fluorescence intensity of the peak of LIZ160 to the fluorescence intensity of the peak of ROX160 is proportional to the concentration of 600LIZ contained in the sample, and can be approximated by a straight line having a slope of 1, for the entire concentration range of 1/2000 to 1/2 times the horizontal axis of fig. 8 (a). That is, by using a new internal standard method using ROX160 as an internal standard, LIZ160 can be quantified with higher accuracy over a wider concentration range than before.

The result of FIG. 8 (b) is illustrated by the case where the first DNA fragment is LIZ160 and the second DNA fragment is ROX160 in the expression (35). That is, the ratio of the fluorescence intensity of the peak of the LIZ160 to the fluorescence intensity of the peak of the ROX160 is proportional to the ratio of the concentration of the LIZ160 to the concentration of the ROX160 contained in the sample. Here, since the concentration of ROX160 is constant, the ratio of the fluorescence intensity of the peak of LIZ160 to the fluorescence intensity of the peak of ROX160 is proportional to the concentration of LIZ160 contained in the sample.

Since (equation 35) does not include parameters that can be changed such as E, T, C (0), the quantitative analysis can be performed with high accuracy in the same manner as in the conventional internal quantitative analysis. Most importantly, (equation 35) coexists with (equation 24) and (equation 25). That is, as shown in fig. 8 (a), the ratio of the fluorescence intensity of the peak of LIZ160 to the fluorescence intensity of the peak of ROX160 is always proportional to the concentration of LIZ160 contained in the sample, regardless of whether the fluorescence intensity of the peak of LIZ160 is proportional to the concentration of LIZ160 contained in the sample or is deviated from the concentration, or regardless of whether the fluorescence intensity of the peak of ROX160 is constant or reduced.

Fig. 10 shows experimental results corresponding to fig. 8 in the case of increasing the electric field injection time from 9 seconds to 18 seconds in the same experiment as fig. 6 to 9. Fig. 10 (a) shows the results of the same tendency as fig. 8 (a) although it was obtained by different electrophoresis analysis under different experimental conditions than fig. 8 (a). But the position of each plot point moves slightly up and down. In contrast, fig. 10 (b) shows a result almost completely equivalent to fig. 8 (b) even though it was obtained by different electrophoresis analysis under different experimental conditions from fig. 8 (b), and the variation in the position of each plot point is very small. Further, the approximate straight line of the slope 1 used in (b) of fig. 10 is the same as the approximate straight line of the slope 1 used in (a) of fig. 8. The above results indicate that the new internal standard method has high quantitative accuracy.

Fig. 11 is a double-contrast plot obtained by plotting the ratio of the fluorescence intensity of the peak of LIZ40 relative to the fluorescence intensity of the peak of LIZ160 and the fluorescence intensity of the peak of LIZ114 obtained from the 4 electrophoresis charts of fig. 6 and 7 against the concentration of 600LIZ contained in the sample. The horizontal axis may also be considered as the concentration of LIZ40, LIZ114 or LIZ160 contained in the sample.

As shown in fig. 6, 7 and 8 (a), the fluorescence intensity of each peak changes with a change in the concentration of 600LIZ contained in the sample, but the fluorescence intensity ratio shown in fig. 11 remains constant. This result shows that the ratio of fluorescence intensities of peaks of any 2DNA fragments belonging to 600LIZ or 500ROX is kept constant in each of the electrophoresis charts of FIGS. 6 and 7.

This is illustrated by setting the first DNA fragment to LIZ40 or LIZ114 and the second DNA fragment to LIZ160 in expression 35. When the concentration of 600LIZ contained in the sample was changed, the concentration ratio of the plurality of DNA fragments contained in 600LIZ was kept constant, and thus the right side was kept constant and the fluorescence intensity ratio on the left side was kept constant. The above results confirm that the series of observations disclosed in the above [ principle ] are correct.

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

Example 2

DNA-type inspection based on Short tandem repeat (Short TANDEM REPEAT, STR) analysis is widely used for crime search, identity confirmation in large-scale disasters, paternity test, etc. due to high accuracy of personal identification. Currently, various STR analysis kits are sold. For example, in PowerPlex (registered trademark) Fusion6C System from Promega corporation, multiplex PCR of STR at 27 loci on human genome was performed using 5 kinds of fluorescent materials with human genome extracted from blood collected from crime scene as a template.

After incubating 5 μl PowerPlex Fusion C5x Master Mix, 5 μl PowerPlex Fusion 6C5X Primer Pair Mix and 25 μl pre-reaction solution containing the extracted human genome at 96 ℃ for 1 minute, the thermal cycle at 96 ℃ for 5 seconds and at 60 ℃ for 1 minute was repeated 29 times, with incubation at 60 ℃ for 10 minutes, and incubation at 4 ℃. The STR-PCR reaction solution was subjected to ethanol precipitation at 25. Mu.m, and dissolved in purified water at 25. Mu.l, whereby desalting was performed. Mu.l of this solution (25. Mu.l) was mixed with 0.5. Mu.l of WEN ILS 500 (hereinafter referred to as 500 WEN) from Promega corporation, which was a size standard labeled with 1 type of phosphor different from the above 5 types of phosphors, and 9.5. Mu.l of formamide, to obtain 11. Mu.l of a sample.

500WEN comprises 21 single-stranded DNA fragments of base length 60, 65, 80, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500, each labeled with a fluorescent WEN. The sample was subjected to rapid ice-cooling after thermal denaturation at 95℃and then subjected to capillary electrophoresis analysis, whereby DNA fragments of various base lengths labeled with any one of 6 kinds of fluorescent materials contained in the sample were separated and detected while identifying the labeled fluorescent materials. DNA type examination was performed by analyzing the electrophoresis pattern of each of the 6 kinds of obtained fluorescent materials.

In DNA typing, it is important to accurately determine the base length of each DNA fragment to improve the accuracy of personal identification. However, in capillary electrophoresis, the electrophoresis speed of each DNA fragment varies for each capillary, each sample, and each analysis, and therefore it is difficult to accurately determine the base length from the timing of the peak of each DNA fragment on the electrophoretogram. Therefore, as described above, by mixing a size standard having a known base length with a sample and analyzing the mixture, the base length of an arbitrary DNA fragment can be accurately determined by referring to the time of the peak of the DNA fragment having a known base length.

As described above, in the conventional DNA type test, the time of measuring the peak of a plurality of DNA fragments belonging to the size standard is flexibly utilized, but the fluorescence intensity of the peak is not flexibly utilized. However, since the concentration of the size standard contained in the sample is known or fixed, by referring to the fluorescence intensity of any peak of the plurality of DNA fragments belonging to the size standard, the concentration of the DNA fragment contained in the sample can be quantified based on the fluorescence intensity of any peak of the plurality of DNA fragments contained in the sample as STR-PCR products. That is, it is possible to perform DNA typing of not only a human genome as a template but also a plurality of DNA fragments contained in a sample as STR-PCR products, respectively, with high accuracy.

In expression 35, the high accuracy of the quantification is demonstrated by setting the first DNA fragment to be an arbitrary DNA fragment contained in the STR-PCR product and setting the second DNA fragment to be an arbitrary DNA fragment contained in the size standard. That is, the concentration of the first DNA fragment in the sample can be obtained from the ratio of the fluorescence intensity of the peak of the first DNA fragment to the fluorescence intensity of the peak of the second DNA fragment.

When the concentration of the size standard in the sample, that is, the concentration of the second DNA fragment in the sample is constant, the concentration of the first DNA fragment in the sample can be obtained from the fluorescence intensity of the peak of the first DNA fragment relative to the fluorescence intensity of the peak of the second DNA fragment. Since the variable parameters such as E, T, C (0) are not included in the equation 35, the quantitative determination can be performed with high accuracy in the same manner as in the conventional internal quantitative determination method. As shown in (equation 24), such high-precision quantification is true both in the case where the fluorescence intensity of the peak of the first DNA fragment is proportional to the concentration of the first DNA fragment contained in the sample and in the case where the peak is deviated from the concentration. That is, the concentration of the first DNA fragment can be precisely quantified in a concentration range wider than the conventional concentration range by the new internal standard method.

Further, by using the number of thermal cycles performed in STR-PCR, the concentration of the human genome of the template contained in the solution before STR-PCR can be quantified. The number of thermal cycles was N, the amplification efficiency of STR-PCR was E (f), and the concentration of human genome in the solution before the reaction of STR-PCR was C (f). In the above example, n=29. E (f) is E (f) =1 under ideal conditions, but the actual value can be investigated in advance. In addition, the concentration of any STR-PCR product contained in the solution after the reaction of STR-PCR was set to B (1) as the first DNA fragment. Further, the concentration of the first DNA fragment in the sample for electric field injection was set to C (1). The ratio of the concentration C (1) of the first DNA fragment in the sample used for electric field injection to the concentration B (1) of the first DNA fragment in the post-reaction solution of STR-PCR, that is, the dilution ratio of the post-reaction solution of STR-PCR was set to d=c (1)/B (1). In the above example, d=1 μl/25 μl=0.04. At this time, the following expression holds.

[ Number 38]

C ₁＝D·C_f·(1+E_f)^N (number 38)

In expression 35, C (1) can be obtained from expression 35 when the second DNA fragment is an arbitrary DNA fragment contained in the size standard. This is because S (1) and S (2) are obtained from the electropherogram, C (2) is known, and m (1) and m (2) can be investigated in advance. D. E (f) and N are known as described above. Therefore, according to the expression 38, the concentration C (f) of the human genome contained in the pre-reaction solution of STR-PCR can be accurately quantified.

Example 3

The SNaPshot (registered trademark) Multiplex system of Thermo FISHER SCIENTIFIC is a kit for simultaneously typing SNPs (single nucleotide polymorphisms) at multiple locations on the human genome using capillary electrophoresis. Template DNA in which a plurality of SNP-containing regions on the human genome are amplified is prepared in advance. For the template DNA, the primers unlabeled with the fluorescent material were hybridized at positions adjacent to the SNPs, and a single-base extension reaction of each primer was performed using a fluorescent-labeled terminator. The fluorescent-labeled terminator is ddATP, ddCTP, ddGTP and ddTTP labeled with 4 different fluorophores, respectively. The base length of each primer varies depending on the corresponding SNP. The DNA fragments as a plurality of single base extension products were subjected to capillary electrophoresis analysis to obtain an electrophoresis pattern of each of 4 kinds of fluorescent materials. On the electropherogram, the position of the corresponding SNP is determined based on the electrophoresis time at which the peak is observed, i.e., the base length of the corresponding DNA fragment. In addition, which of the SNPs A, C, G, T is determined based on the type of phosphor at the same peak. The above SNP typing can be performed simultaneously for SNPs at a plurality of sites.

In the above SNP typing, it is sufficient if it is possible to determine whether each SNP is a homozygote of any one of A, C, G, T (100% of one SNP) or a heterozygote of any 2 of A, C, G, T (50% of each of 2 SNPs). However, in general, each SNP may be a base in which A, C, G, T are mixed at an arbitrary ratio. For example, with the progress of cancer, there are cases where the wild type (WT, hereinafter) of any SNP is any 1 of A, C, G, T, the mutant (MT, hereinafter) is 3 kinds other than the 1 kinds in A, C, G, T, and the respective existing ratios are different. In contrast, by quantifying the presence ratio of A, C, G, T base types in any SNP, it is possible to diagnose cancer early or grasp the cancer state with high accuracy.

Assuming that M is an integer of 1 or more, the presence ratio of the base type A, C, G, T in the SNP at M, in particular, the presence ratio of the base type of MT relative to the base type of WT is quantified. For each of the SNPs at M, a maximum of 4 single base extension products are available. Thus, the reaction solution containing a maximum of 4 XM DNA fragments was desalted by ethanol precipitation and eluted into a desired amount of formamide as a sample for electric field injection.

The electric field-injected DNA fragments were each provided with a peak on an electrophoresis pattern of each of 4 kinds of fluorescent materials obtained by capillary electrophoresis analysis, and a maximum of 4×M peaks were provided. SNPs at M were given SNP numbers i, i=1, 2,..and M. The fluorescence intensity of the peak on the electrophoresis pattern of the DNA fragment, which is the single-base extension product of the base type A, C, G, T of SNP number j, is defined as S (ja), S (jc), S (jg), S (jt), the sensitivity coefficients of the respective peaks are defined as m (ja), m (jc), m (jg), and m (jt), the average charge amounts of the respective peaks are defined as q (ja), q (jc), q (jg), and q (jt), and the concentrations of the electric field in the sample injected into the capillary are defined as C (ja), C (jc), C (jg), and C (jt). The total injection charge amount Q of all the DNA fragments of the electric field injection capillary is represented by the following formula, as in (formula 13).

[ Number 39]

Q＝E·T·A·∑_i(q_ia·μ·C_ia+q_ic·μ·C_ic+q_ig·μ·C_ig+q_it·μ·C_it) ( Number 39)

Meanwhile, the following expression is given as (expression 15).

[ Number 40]

The following equations are given according to (expression 39) and (expression 40), similarly to (expression 21).

[ Number 41]

Q (0) ·μ (0) ·c (0) represents the amount of injected negative ions other than the DNA fragment, and Σ represents the total amount of injected negative ions of all DNA fragments.

In (equation 41), Q is proportional to the Σ term when the Σ term is smaller than Q (0) ·μ (0) ·c (0), but Q is saturated with respect to the Σ term by a deviation ratio when the Σ term is larger than Q (0) ·μ (0) ·c (0)). Therefore, the total injection amount of all the DNA fragments is proportional to the total concentration of all the DNA fragments when the total concentration of all the DNA fragments is low, but when the total concentration of all the DNA fragments becomes high, the ratio is deviated to saturation. Of course, the increase in the total concentration of all the DNA fragments is caused by the increase in the concentration of any DNA fragment. If the total injection amount of all the DNA fragments is saturated, the injection amount of any DNA fragment is saturated or otherwise reduced.

However, when any 2 kinds of DNA fragments among all the DNA fragments are used as the first DNA fragment and the second DNA fragment, (expression 8) to (expression 11), (expression 22), (expression 23), (expression 29), (expression 32) and (expression 35) are established. (equation 35) shows that the ratio of fluorescence intensities of the first and second DNA fragments obtained by subjecting the first and second DNA fragments injected into the capillary by electric field injection to capillary electrophoresis analysis is proportional to the ratio of concentrations of the first and second DNA fragments in the sample.

For example, focusing on a DNA fragment which is a single base extension product of the base type A, C, G, T of SNP No. j, the following formula is given as in (expression 22) and (expression 23).

[ Number 42]

S _ja＝E·T·A·m_ja·μ·C_ja (number 42)

[ Number 43]

S _jc＝E·T·A·m_jc·μ·C_jc (number 43)

[ Number 44]

S _jg＝E·T·A·m_jg·μ·C_ig (number 44)

[ Number 45]

S _jt＝E·T·A·m_jt·μ·C_jt (number 45)

Therefore, for example, when WT is a and MT is C, G, T, the following expression is given (expression 35).

[ Number 46]

[ Number 47]

[ Number 48]

That is, the concentration ratios C (jc)/C (ja), C (jg)/C (ja), C (jt)/C (ja) corresponding to the presence ratios of 3 MTs to WT can be accurately quantified by the fluorescence intensities S (jc)/S (ja), S (jg)/S (ja), S (jt)/S (ja) of the peaks on the electrophoretogram. M (ja), m (jc), m (jg), and m (jt) are obtained in advance.

In the above, it is assumed that the sample contains a maximum of 4×m DNA fragments as single base extension products. In the following, it is assumed that 1 or more DNA fragments are contained in the sample as internal standards for known or fixed concentration in addition to DNA fragments which are single base extension products of the maximum total of 4 XM species. As the internal standard, a size standard containing DNA fragments of various base lengths can be used. In this case, in expression 35, the concentration of the first DNA fragment in the sample can be accurately determined by using any one of DNA fragments, which are single-base extension products of the base types A, C, G, T of SNP No. j, as the first DNA fragment and any one of DNA fragments of the internal standard as the second DNA fragment.

Symbol description

1 Capillary tube,

2 Sample injection end,

3 Sample dissolution end,

4 Cathode electrode,

5 Positive electrode,

6 Buffer solution at the cathode side,

7 Positive electrode side buffer solution,

8 Polymer solution,

9 Samples,

10 Pump blocks,

11 Valve,

12 Syringes,

13 Power source,

14 Laser beam,

15 Laser light source,

16 Measuring points,

17 Detector,

18 A first DNA fragment (first component),

19 A second DNA fragment (second component),

20 Negative ions other than DNA fragments.

Claims

1. A capillary electrophoresis apparatus, performing the following processing:

performing electrophoretic separation on the injected first component and the second component; and

The luminescence from the first component and the luminescence from the second component induced by irradiating light onto the capillary are measured by a detector, thereby obtaining the signal intensity of the first component and the signal intensity of the second component, characterized in that:

The concentration range of the first component contained in the sample includes, in addition to the concentration range in which the signal intensity of the first component is proportional to the concentration of the first component, a concentration range in which the signal intensity of the first component is lower than the saturation signal intensity of the detector relative to the concentration of the first component and deviates from the proportion to reach the saturation signal intensity.

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

2. A capillary electrophoresis apparatus for performing the following processes:

The concentration range of the first component contained in the sample includes not only a concentration range in which the signal intensity of the second component is constant relative to the concentration of the first component, but also a concentration range in which the signal intensity of the second component deviates from the constant and decreases relative to the concentration of the first component.

3. The capillary electrophoresis device according to claim 1 or 2, characterized in that:

the concentration of the second component in the sample is known,

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

4. The capillary electrophoresis device according to any one of claims 1 to 3, characterized in that:

The first component and the second component are both DNA fragments labeled with a fluorescent substance, and the base length of the first component is different from the base length of the second component.

The luminescence is fluorescence,

The signal intensity is the fluorescence intensity.

5. The capillary electrophoresis device according to claim 4, characterized in that

The sample contains size standards,

The second component is the DNA fragment contained in the size standard.

6. The capillary electrophoresis device according to claim 4, characterized in that

The sample comprises a PCR product,

The first component is the DNA fragment contained in the PCR product.

7. The capillary electrophoresis device according to claim 4, characterized in that

The sample contains a single base extension product,

The first component is the first DNA fragment contained in the single-base extension product.

8. The capillary electrophoresis device according to claim 7, characterized in that:

The second component is the second DNA fragment contained in the single-base extension product.

9. A capillary electrophoresis method comprising the following steps:

The capillary electrophoresis method comprises the following steps:

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

10. A capillary electrophoresis method comprising the following steps:

The capillary electrophoresis method comprises the following steps:

11. The capillary electrophoresis method according to claim 9 or 10, characterized in that:

the concentration of the second component in the sample is known,

12. The capillary electrophoresis method according to any one of claims 9 to 11, characterized in that:

The luminescence is fluorescence,

The signal intensity is the fluorescence intensity.

13. The capillary electrophoresis method according to claim 12, wherein:

The sample contains size standards,

The second component is the DNA fragment contained in the size standard.

14. The capillary electrophoresis method according to claim 12, wherein:

The sample comprises a PCR product,

The first component is the DNA fragment contained in the PCR product.

15. The capillary electrophoresis method according to claim 12, wherein the sample contains a single base extension product,

16. The capillary electrophoresis method according to claim 15, wherein: