METHOD FOR DETECTING A MUTANT GENE

Abstract

The present disclosure aims to provide a technique capable of accurately detecting a mutant gene and a mutation rate using capillary electrophoresis. A method for detecting a mutant gene according to the present disclosure classifies signal peaks included in a detected signal into a first group lower than a first threshold and a second group not lower than the first threshold, increases an injection voltage until the signal peak belonging to the first group becomes higher than or equal to the first threshold, and decreases the injection voltage after increasing the injection voltage until the signal peak belonging to the second group becomes lower than or equal to a second threshold higher than the first threshold.

Description

Technical area

[0001] The present disclosure relates to a method for detecting a mutant gene. State of the art

[0002] DNA analysis using electrophoresis includes fragment analysis and sequence analysis. Examples of fragment analysis include personal identification, microsatellite instability (MSI) analysis, and multiplex ligation-dependent probe amplification (MLPA). Methylation-specific MLPA (MS-MLPA) is also known as a method for detecting mutant genes using MLPA (Non-Patent Literature 1).

[0003] In MS-MLPA, two adjacent probes are used that can specifically bind (hybridize) to a target gene (a target region). A common sequence is bound to each probe, enabling PCR amplification with a universal primer. The probes are designed to provide different amplified fragment lengths. The two adjacent probes, which are hybridized to a target gene sequence, are then joined together by a ligase. After hybridization, separate tubes are used for copy number and methylation analysis, and simultaneously treated with a methylation-sensitive restriction enzyme Hha1 during the ligation reaction. Furthermore, a PCR reaction is performed. A probe in an unmethylated region is cleaved by the restriction enzyme and thus not amplified by the PCR.A probe in a methylated region, however, is not cleaved and thus amplified by PCR. Detected signals are obtained by electrophoresis of the resulting DNA fragment using a capillary electrophoresis device. Based on the difference between the peak positions of the detected signals, it is thus possible to identify an unmethylated cell (normal cell) and a methylated cell (cancer cell).

[0004] Patent Literature 1 describes DNA analysis using capillary electrophoresis. In the literature, when a detected signal obtained by electrophoresis is saturated (when the detected signal exceeds a recordable upper limit), a flag is issued, prompting a user to readjust an injection parameter (0165 of the literature). Furthermore, a signal-to-noise ratio of an optical signal is calculated using a median value of a signal peak and compared with noise estimated from a non-peak region (0166 of the literature). Literature listPatent literature

[0005] Patent Literature 1: US2020 / 0003728A1 Non-patent literature

[0006] Non-patent literature 1: https: / / www.falco-genetics.com / salsa / principle.html Summary of the inventionTechnical problem

[0007] Since the amount of a mutant gene is usually very small, a signal detected from a mutant gene is usually very weak, so the corresponding signal strength may fall below a detectable lower limit. In this case, an experimenter needs to increase an injection voltage and sample concentration and perform electrophoresis again. However, if the injection voltage and sample concentration are increased, the detected signal will simultaneously become saturated, and a mutation rate cannot be calculated. This is due to the fact that a signal level of a saturated peak cannot be identified, and therefore, the ratio of a signal peak level originating from a mutant type to a signal peak level originating from a wild type cannot be calculated.

[0008] In such a conventional technique as described in Patent Literature 1, all signal peaks must be higher than or equal to the detectable lower limit and unsaturated. If either condition is not met, the injection voltage and the like must be readjusted and electrophoresis must be performed again until both conditions are met. Therefore, from a DNA sample such as a mutant gene that causes a very weak signal peak and a normal signal peak at the same time, it is difficult to detect both signals simultaneously. This is due to the fact that either a peak lower than said lower limit or a saturated peak may occur.

[0009] The present disclosure has been made in view of the problems described above and accordingly aims to provide a technique capable of accurately detecting a mutant gene and a mutation rate using capillary electrophoresis. Solution to the problem

[0010] A method for detecting a mutant gene according to the present disclosure classifies signal peaks included in a detected signal into a first group lower than a first threshold and a second group not lower than the first threshold, increases an injection voltage until the signal peak belonging to the first group becomes higher than or equal to the first threshold, and decreases the injection voltage until the signal peak belonging to the second group becomes lower than or equal to a second threshold higher than the first threshold. Advantageous effects of the invention

[0011] According to the method for detecting a mutant gene according to the present disclosure, it is possible to accurately detect a mutant gene and a mutation rate using capillary electrophoresis. Other configurations, problems, advantages, and the like of the present disclosure will be clarified by the following description of the embodiments. Brief description of the drawings Fig. 1 is a configuration diagram of an electrophoresis system 1 according to a first embodiment. Fig. Figure 2 illustrates signal peaks indicating results obtained by measuring a nucleic acid sample containing a mutant gene by the electrophoresis system 1. Fig. 3 is an enlarged view of signal peaks of a probe group 202. Fig. Figure 4 illustrates results of measuring a DNA sample that was mixed with the sample in Fig. 2 is identical, by increasing an injection voltage, Fig. 5 illustrates a flowchart for explaining a general method for detecting a mutant gene as a comparative example. Fig. 6 illustrates a flowchart in a case where such a conventional technique as described in Patent Literature 1 is used for detecting a mutant gene as a comparative example. Fig. 7 is a flowchart for explaining a method for detecting a mutant gene according to the first embodiment. Description of embodiments<Erstes Ausführungsbeispiel: Systernkonfiguration>

[0012] Fig. 1 is a configuration diagram of an electrophoresis system 1 according to a first embodiment of the present disclosure. The electrophoresis system 1 includes an electrophoresis device 100 and a computing device 200 (computer). The electrophoresis device 100 is configured as a device that analyzes a component contained in a sample by electrophoresis of the sample using a capillary.

[0013] The electrophoresis device 100 includes a detection unit 116, a constant-temperature bath 118, a delivery unit 125, a high-voltage power source 104, a first ammeter 105, a second ammeter 112, the capillary 102, and a pumping mechanism 103. The detection unit 116 optically detects a sample. The constant-temperature bath 118 also maintains the capillary 102 at a constant temperature. The delivery unit 125 also delivers various containers to the capillary cathode ends. The high-voltage power source 104 applies a high voltage to the capillary 102. The first ammeter 105 also measures an electric current output by the high-voltage power source 104. The second ammeter 112 further measures an electric current flowing in an anode-side electrode 111. The pump mechanism 103 also injects a polymer into the capillary 102.

[0014] The capillary 102 is formed from a glass tube with an inner diameter of several tens to hundreds of micrometers and an outer diameter of several hundred micrometers, and has a surface coated with a polyimide to improve the strength of the capillary. However, a polyimide-coated film on a light irradiation section irradiated with laser light is removed, so that light emitted within the section easily leaks to the outside. The interior of the capillary 102 is also filled with a separation medium to provide a difference in migration speed during electrophoresis. Although a fluid separation medium and a non-fluid separation medium may be present, a fluid polymer is used as the separation medium in the first embodiment.

[0015] The detection unit 116 is also a region of a portion of the capillary 102. When excitation light is emitted from a light source 114 to the detection unit 116, fluorescence (hereinafter referred to as information light) with a sample-dependent wavelength is generated from the sample and emitted outside the capillary 102. This information light is then separated in a wavelength direction by a diffraction grating 132. An optical detector 115 further analyzes the sample by detecting the separated information light.

[0016] The capillary cathode ends 127 are fixed by hollow metal electrodes 126, and capillary tips protrude approximately 0.5 mm from the hollow electrodes 126. The hollow electrodes 126 arranged for each capillary are further integrally mounted on a load collector 129. Furthermore, all hollow electrodes 126 are electrically connected to the high-voltage power source 104 mounted on a main body of the device. The hollow electrodes 126 function as cathode electrodes when a voltage needs to be applied for electrophoresis, sample introduction, and the like.

[0017] Capillary ends (other ends) on the opposite side of the capillary cathode ends 127 are further bundled together by a capillary head 133. The capillary head 133 is capable of being pressure-tightly connected to a block 107. The high voltage output from the high-voltage power source 104 is further applied between the load collector 129 and the capillary head 133. A syringe 106 also fills the capillary from the other ends with a new polymer. More specifically, the polymer is refilled into the capillary after each measurement to improve the measurement performance.

[0018] The pump mechanism 103 further includes the syringe 106 and a mechanism system for pressurizing the syringe 106. The block 107 is a connecting element for connecting the syringe 106, the capillary 102, an anode buffer container 110, and a polymer container 109.

[0019] An optical detection unit that detects the information light from the sample further includes the light source 114, the optical detector 115 for detecting light emitted in the detection unit 116, and the diffraction grating 132. In order to detect the sample separated by electrophoresis and present in the capillary, the light source 114 irradiates the detection unit 116 of the capillary, the diffraction grating 132 separates the emitted light from the detection unit 116, and the optical detector 115 detects the separated information light.

[0020] The constant-temperature bath 118 is further covered with a thermal insulation material to maintain a constant temperature inside, and the temperature is controlled by a heating and cooling mechanism 120. A fan 119 also circulates and stirs the air in the constant-temperature bath 118 to maintain the temperature of the capillary 102 uniform and constant in position.

[0021] The conveyor unit 125 further contains up to three electric motors and a linear actuator and is movable along up to three axes in the vertical, horizontal, and depth directions. At least one or more containers can be placed on a table 130 of the conveyor unit 125. The table 130 is provided with an electric handle 131 for this purpose, and a user can grasp and release each container via the handle 131. Thus, a buffer container 121, a cleaning container 122, a waste liquid container 123, and a sample container 124 can be conveyed to the capillary cathode ends 127 as needed. An unused container can also be stored in a designated storage area in the device.

[0022] The calculation device 200 further acquires a result of detecting the information light from the optical detector 115, analyzes the detection result to generate a fluorescence intensity waveform, and performs processing such as calculating a base length of a substance to be measured. Details of the processing performed by the calculation device 200 will be described later. The calculation device 200 may be configured by a central processing unit (CPU), software executed by the CPU, and the like, but may also be configured by hardware such as a circuit device in which similar functions are implemented. <Erstes Ausführungsbeispiel: Probleme herkömmlicher Techniken>

[0023] Fig. Figure 2 illustrates signal peaks indicating results obtained by measuring a nucleic acid sample containing a mutant gene by the electrophoresis system 1. The purpose of the measurement is to calculate a mutation rate (methylation rate) of a DNA. In Fig. 2, 201 denotes a probe group that measures the methylation rate. 202 also denotes a probe group that is cleaved by a restriction enzyme. Furthermore, 203 denotes a reference probe group that is not cleaved by the restriction enzyme. It can be seen that the signal peaks of probe group 202 are significantly lower than those of probe group 203. This is due to the fact that the amount of the mutant gene is very small, and thus the detected signal level is also very low compared to a normal gene.

[0024] Fig. Figure 3 is an enlarged view of the signal peaks of probe group 202. Since a detected signal with a very low signal level has low reliability, it is common practice to exclude the detected signal from the analysis. For example, if a signal level (vertical axis) 300, which is Fig. 3, when an analyzable lower limit is set, 5 of the 16 probes included in probe group 202 to be detected fall below the analyzable lower limit. Therefore, it is difficult to accurately calculate a mutation rate of this DNA sample.

[0025] The analyzable lower limit for signal peak levels is determined based on whether the computing device 200 can acquire sufficiently reliable detected signal data. For example, if a detected signal below a certain signal level is known to have a large amount of noise and low reliability, the signal level is set as the analyzable lower limit. This reliability varies depending on the type (e.g., a product model number) of the electrophoresis device 100, thus making it possible to determine an analyzable lower limit for each type of the electrophoresis device 100.

[0026] Fig. Figure 4 illustrates results of measuring a DNA sample that was mixed with the sample in Fig. 2 is identical, by increasing an injection voltage. Here, it is assumed that if a signal peak is present below the analyzable lower limit, a signal level is increased such that the signal peak becomes higher than or equal to the analyzable lower limit. For example, the overall signal level can be increased by increasing the injection voltage to be applied to the capillary when performing electrophoresis. Fig. Figure 4 illustrates the corresponding results.

[0027] By increasing the injection voltage, the probe group 202 has higher signal peaks than those in Fig. 2 and Fig. 3. However, there is also a probe that shows a saturated signal peak of a normal gene (25000 is shown in the vertical axis in Fig. 4). Therefore, it is also difficult to accurately calculate the mutation rate in this case. This is due to the fact that signal levels (of saturated signal peaks) of some normal genes cannot be accurately measured.

[0028] In view of the above description, in a mutant gene detection method according to the present disclosure, after the injection voltage is increased to the extent that a signal peak of the mutant gene can be analyzed, the injection voltage is reduced to the extent that other signal peaks are not saturated. This achieves that the mutation rate can be accurately calculated. <Erste Ausführungsform: Verfahren zum Detektieren eines Mutantengens>

[0029] Fig. Figure 5 illustrates a flowchart for explaining a general method for detecting a mutant gene as a comparative example. In this method, electrophoresis is first performed on a DNA sample using a capillary sequencer (such as one Fig. 1), and a detected signal obtained as a result of electrophoresis is analyzed by software. If all the signal peaks to be measured are not higher than or equal to the analyzable lower limit, an error (analysis impossible) occurs because a signal level is insufficient. Conversely, if all the signal peaks to be measured are not lower than or equal to a saturation level, a similar error occurs. If both of these conditions are met, a ratio (mutation rate) of a signal peak of the mutant gene is calculated.

[0030] Fig. 6 illustrates a flowchart in a case where such a conventional technique as described in Patent Literature 1 is used to detect a mutant gene as a comparative example. When all the signal peaks to be measured are not higher than or equal to the analyzable lower limit, a sample injection voltage is increased to increase the signal peaks to a level higher than or equal to the lower limit. However, when a signal level of signal peaks of the normal genes is saturated, an error occurs. Conversely, when all the signal peaks to be measured are not lower than or equal to a saturation level, the sample injection voltage is decreased to decrease the signal peaks to a level lower than or equal to the saturation level.However, it may also occur that a signal peak of the mutant gene becomes lower than the analyzable lower limit, resulting in an error. Therefore, it is difficult to accurately calculate the mutation rate in conventional detection methods.

[0031] Fig. Fig. 7 is a flowchart for explaining a method for detecting a mutant gene according to the first embodiment. This flowchart can be executed by a manual operation by an experimenter or by the computing device 200 controlling the electrophoresis system 1. Here, it is assumed that the computing device 200 executes this flowchart of the following description, wherein each of the steps in Fig. 7 can be described as follows. (Fig. 7: Steps S701 to S703)

[0032] A user first sets a DNA sample (nucleic acid sample) as well as a necessary reagent, etc. (S701). The sample is then introduced into the electrophoresis system 1 (S702), and electrophoresis is performed (S703). (Fig. 7: Steps S704 to S706)

[0033] The calculation device 200 then analyzes a detected signal of a fragment obtained by electrophoresis (S704). If all detected signal peaks to be measured are higher than or equal to the analyzable lower limit, the process proceeds to S707 (S705: YES). However, if a detected signal peak is lower than the analyzable lower limit (S705: NO), the sample injection voltage (voltage to be applied to the capillary when electrophoresis is performed) of the electrophoresis system 1 is increased (S706). In this case, the amount of the increase can be determined in advance or based on the difference between the signal peak and the analyzable lower limit. At least until all signal peak levels of a signal peak group originating from a mutant type become higher than or equal to the analyzable lower limit, S706 must be continued.After S706, the process returns to S702 and electrophoresis is performed again using the increased injection voltage. (Fig. 7: Step S705: Supplement 1)

[0034] Among the peaks, a peak originating from the mutant type and a peak originating from a wild type are known in advance. Therefore, the information of the peak originating from either the mutant type or the wild type should be described in advance as attribute information, and each peak group originating from either the mutant type or the wild type can be identified by referring to the attribute data by the calculation device 200. The same applies to step S707. (Fig. 7: Step S705: Supplement 2)

[0035] When a signal peak is equal to the analyzable lower limit, the effect of noise is significant and the reliability of the signal is low. Since the reliability of the signal is generally determined by the type of the electrophoresis apparatus 100 (the electrophoresis system 1), the analyzable lower limit for signal peaks can be determined by each type of the electrophoresis apparatus 100. Therefore, the calculation device 200 can detect the type of the electrophoresis apparatus 100 and set an analyzable lower limit level according to the type. In other words, when a signal peak is lower than a certain lower limit threshold, and when the calculation device 200 cannot accurately identify a mutant gene corresponding to the signal peak, the lower limit threshold can be specifically set as the analyzable lower limit. (Fig. 7: Step S706: Addition)

[0036] In this step, after increasing the injection voltage, the previously used sample is reused (remeasured) to execute S702 and subsequent steps again. Therefore, steps S702 to S706 are executed again using the same sample, so it is possible to suppress a measurement error caused by a difference between samples or the like. The same applies to a case where the process returns from S708 to S702. (Fig. 7: Steps S707 and S708)

[0037] If all detected peaks to be measured are lower than or equal to the saturation level, the process proceeds to step S709 (S707: YES). If a detected peak higher than the saturation level is present (S707: NO), the sample injection voltage of the electrophoresis system 1 is reduced (S708). In this case, the amount of reduction can be determined in advance or based on the difference between the peak and the saturation level. Step S708 must be continued at least until all peak levels of a peak group originating from the wild type become lower than or equal to the saturation level. After step S708, however, the process returns to step S702, and electrophoresis is performed again using the reduced injection voltage. (Fig. 7: Steps S705 and S707: Supplement)

[0038] Only if "YES" is determined in each of these steps does the process proceed to step S709. In other words, the injection voltage is repeatedly adjusted so that the signal peaks fall within a range higher than or equal to the analyzable lower limit and lower than or equal to the saturation level by repeatedly performing steps S705 to S708 using the same sample. After this adjustment is completed, the signal peak originating from the wild type and the signal peak originating from the mutant type can be measured simultaneously by performing the next electrophoresis once. (Fig. 7: Step S707: Addition)

[0039] The saturation level used in step S707 can be determined based on the type of the electrophoresis apparatus 100, similar to step S705. That is, if there is an upper limit threshold value with which the electrophoresis apparatus 100 and the calculation device 200 can perform processing, the upper limit threshold value can be determined as the saturation level. For example, as described later, when the mutation rate is calculated using a ratio of signal peak levels, and the signal peak originating from the wild type reaches the saturation level, the mutation rate cannot be accurately calculated. This is due to the fact that the original signal peak level is higher than the saturation level. Therefore, in this case, an upper limit signal level that can be output from the electrophoresis apparatus 100 is used as the saturation level in this step. (Fig. 7: Step S709)

[0040] The calculation device 200 further calculates the mutation rate of the DNA sample using a fragment analysis result to identify a ratio between a normal gene and the mutated gene. Peak signal levels of normal genes and peak signal levels of mutated genes are each substantially equal. Therefore, it is possible to calculate the mutation rate based on a ratio between a peak signal level of the normal gene and a peak signal level of the mutated gene. <Erstes Ausführungsbeispiel: Schlussfolgerung>

[0041] The electrophoresis system 1 according to the first embodiment acquires in advance information indicating whether signal peaks obtained by performing capillary electrophoresis on the DNA sample are of the mutant type or the wild type, and classifies the signal peaks into derivation groups according to the information. For the mutant type derivation group, the injection voltage is increased so that all signal peaks become higher than or equal to the analyzable lower limit. For the wild type derivation group, on the other hand, the injection voltage is decreased so that all signal peaks become lower than or equal to the saturation level. Therefore, both the signal peaks originating from the mutant type and the signal peaks originating from the wild type can be measured by performing electrophoresis once. <Zweites Ausführungsbeispiel>

[0042] The first embodiment describes that the detected signal peaks obtained by electrophoresis are classified into two groups: the mutant gene group, which may be lower than the analyzable lower limit, and the normal gene group, which may be higher than the saturation level. However, the detected signal peaks may also be divided into three or more groups. For example, if the sample contains a fragment in which the signal intensity of A of the four bases ATGC of a gene is relatively higher than the signal intensity of TGC, a signal peak corresponding to the fragment may be classified into a third group. Meanwhile, a fragment in which a signal peak is relatively low may be classified into a fourth group.

[0043] Although this classification is not a mutant-type and wild-type classification, it can result in (a) a relatively high peak group higher than the saturation level of a wild-type peak, requiring the same processing as for the wild-type, and (b) a peak belonging to a relatively low peak group lower than the analyzable lower limit, requiring processing used for the mutant type. Therefore, in addition to the mutant-type and wild-type classification, peaks can also be classified based on whether the peaks are higher than the saturation level and lower than the analyzable lower limit. The classification of peaks described above can be used in addition to or instead of the mutant-type and wild-type classification.Therefore, the signal peaks can also be classified into three or more groups.

[0044] When groups are configured based on whether the signal peaks are higher than the saturation level and lower than the analyzable lower limit, a range in which a signal peak level of each of the groups lies is identified in advance, and the corresponding information is inserted into the attribute data to be used in step S705 (S707). That is, the information identifying whether the signal peaks lower than the analyzable lower limit and higher than the saturation level for each of the signal peaks is described in the attribute data. In step S705, it is also determined whether all the groups are higher than or equal to the analyzable lower limit. Furthermore, in step S707, it is determined whether all the groups are lower than or equal to the saturation level. Therefore, the flowchart of Fig.7 can also be used for this purpose as shown. <Modifikationen der vorliegenden Offenbarung>

[0045] The present disclosure is not limited to the embodiments described above and further includes various modifications. For example, the embodiments are described above in such detail only to explain the present disclosure in an easily understandable manner and are not necessarily limited to including all described configurations. In addition, some of the configurations described in a particular embodiment may also be replaced with a configuration described in the other embodiment. In addition, a configuration described in the other embodiment may be added to a configuration described in a particular embodiment. Furthermore, a configuration may also be added to, removed from, or replaced by some of the configurations described in each of the embodiments.

[0046] In the embodiments described above, step S706 is also performed to increase the detected signal peaks. If a similar effect can be achieved by other alternative means than increasing the injection voltage, these alternative means may also be used. For example, the amount (concentration) of the sample to be introduced into the capillary of the electrophoresis device 100 may also be increased. Furthermore, the amount of sample may also be increased in combination with increasing the injection voltage. Likewise, in step S708, the amount of sample to be introduced into the capillary of the electrophoresis device 100 may be reduced, or the amount of sample to be introduced into the capillary of the electrophoresis device 100 may be reduced in combination with reducing the injection voltage.

[0047] In the embodiments, the computing device 200 is described as the constituent component of the electrophoresis system 1, but the computing device 200 may also be configured as a constituent component of the electrophoresis device 100 to control each component of the electrophoresis device 100. List of reference symbols 1 electrophoresis system 100 electrophoresis device 200 Calculation device QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] US 2020 / 0003728A1

[0005] Cited non-patent literature

[0000] https: / / www.falco-genetics.com / salsa / principle.html

[0006]

Claims

[1] A mutant gene detection method for detecting a mutant gene in a nucleic acid sample containing a gene, the method comprising: a step of detecting a detected signal by measuring the nucleic acid sample using a capillary electrophoresis device; a step of acquiring attribute information describing information indicating whether signal peaks included in the detected signal are lower than a first threshold; a step of classifying the signal peaks included in the detected signal into a first group lower than the first threshold and a second group not lower than the first threshold according to the attribute information; a step of increasing a voltage applied to a capillary so that the capillary electrophoresis device performs electrophoresis on the nucleic acid sample until the signal peak belonging to the first group becomes higher than or equal to the first threshold value; and a step of decreasing the voltage until the signal peak belonging to the second group becomes lower than or equal to a second threshold value higher than the first threshold value. [2] The method for detecting a mutated gene according to claim 1, wherein the attribute information describes information indicating whether the signal peaks contained in the detected signal originate from a mutated type or from a wild type, in the step of classifying, the signal peaks contained in the detected signal are classified into the first group and the second group according to the attribute information, the first group is a group derived from the mutated type and the second group is a group derived from the wild type. [3] The method for detecting a mutated gene according to claim 1, wherein the capillary electrophoresis device contains a calculation device that processes the detected signal, the calculation device sets the first threshold based on a type of the capillary electrophoresis device, the first threshold is higher than or equal to a lower limit signal level with which the computing device is able to identify the mutated gene, and in the step of increasing the voltage, the voltage is increased until all signal peaks belonging to the first group become higher than or equal to the first threshold value. [4] The method for detecting a mutated gene according to claim 1, wherein the capillary electrophoresis device contains a calculation device that processes the detected signal, the calculation device sets the second threshold based on a type of the capillary electrophoresis device, the second threshold is lower than or equal to an upper limit signal level with which the computing device is able to analyze the detected signal, and in the step of decreasing the voltage, all signal peaks belonging to the second group become lower than or equal to the second threshold value. [5] The method for detecting a mutated gene according to claim 1, further comprising: after the step of increasing the voltage, a step of re-acquiring the detected signal by re-measuring the nucleic acid sample using the capillary electrophoresis device; and a step of re-executing the step of increasing the voltage for the re-acquired detected signal. [6] The method for detecting a mutated gene according to claim 1, further comprising: after the step of reducing the voltage, a step of re-acquiring the detected signal by re-measuring the nucleic acid sample using the capillary electrophoresis device; and a step of re-executing the step of decreasing the voltage for the re-acquired detected signal. [7] The method for detecting a mutated gene according to claim 1, further comprising: a step of calculating a mutation rate of the nucleic acid sample, wherein the method for detecting a mutated gene performs the step of calculating a mutation rate when the signal peak belonging to the first group is higher than or equal to the first threshold and the signal peak belonging to the second group is lower than or equal to the second threshold. [8] The method for detecting a mutated gene according to claim 7, wherein the method for detecting a mutated gene performs the step of increasing the voltage or the step of decreasing the voltage without performing the step of calculating the mutation rate when at least either the signal peak belonging to the first group is lower than the first threshold or the signal peak belonging to the second group is higher than the second threshold. [9] The method for detecting a mutated gene according to claim 1, wherein the attribute information describes information identifying whether the signal peaks are lower than the first threshold value for each of the signal peaks, and in the step of classifying the signal peaks, a signal peak included in the detected signal is classified into a third group different from the first group and the second group according to the attribute information, wherein the method for detecting a mutated gene further comprises: if the signal peak belonging to the third group is lower than the first threshold, a step of increasing the voltage until the signal peak belonging to the third group becomes higher than or equal to the first threshold; and if the signal peak belonging to the third group is higher than the second threshold, a step of decreasing the voltage until the signal peak belonging to the third group becomes lower than or equal to the second threshold. [10] The method for detecting a mutated gene according to claim 1, further comprising: a step of calculating a mutation rate of the sample based on a ratio of a signal level of the signal peak belonging to the first group and a signal level of the signal peak belonging to the second group. [11] The method for detecting a mutated gene according to claim 1, wherein in the step of increasing the voltage, an amount of the nucleic acid sample to be introduced into the capillary electrophoresis device is increased instead of or in combination with increasing the voltage, and in the step of reducing the voltage, the amount of nucleic acid sample to be introduced into the capillary electrophoresis device is reduced instead of or in combination with reducing the voltage. [12] The method for detecting a mutated gene according to claim 1, further comprising: a step of treating the nucleic acid sample using a multiplex ligation-dependent probe amplification, MLPA, method, wherein in the step of detecting the detected signal, the detected signal is detected by measuring the nucleic acid sample treated using the MLPA method.

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