Mutant gene detection method

Abstract

The purpose of the present disclosure is to provide a technique capable of detecting a mutant gene and a mutation rate with high accuracy using capillary electrophoresis. This mutant gene detection method classifies signal peaks of a detection signal into a first group that is less than a first threshold value and a second group other than the first group, and increases an injection voltage until the signal peaks belonging to the first group are equal to or greater than the first threshold value. The injection voltage is reduced until the signal peak belonging to the second group is equal to or less than a second threshold value greater than the first threshold value (referring to FIG. 7).

Description

Technical Field

[0001] The present disclosure relates to a method for detecting mutant genes. Background Art

[0002] Analysis of DNA using electrophoresis includes fragment analysis, sequence analysis, etc. Examples of fragment analysis include personal identification, MSI (MicroSatellite Instability) analysis, and MLPA (Multiplex Ligation-dependent Probe Amplification). As a method for detecting mutant genes using MLPA, there is MS-MLPA (Methylation-Specific MLPA) (Non-Patent Document 1).

[0003] In MS-MLPA, two adjacent probes that specifically bind (hybridize) to a target gene (region) are used. Universal sequences that enable PCR amplification using universal primers are attached to each probe. Each probe is designed to obtain different amplified fragment lengths. The two adjacent probes hybridized to the target gene sequence are ligated by a ligase to form a single strand. After hybridization, the tubes are divided into those for copy number analysis and methylation analysis, and are treated with the methylation-sensitive restriction enzyme Hha1 during the ligation reaction, and a PCR reaction is carried out. The probes in the unmethylated region are cut by the restriction enzyme, so no PCR amplification occurs. Since the probes in the methylated region are not cut, PCR amplification occurs. The obtained DNA fragments are electrophoresed using a capillary electrophoresis device, thereby obtaining detection signals. Based on the difference in the peak positions of the detection signals between them, unmethylated cells (normal cells) and methylated cells (cancer cells) can be identified.

[0004] Patent Document 1 below describes DNA analysis using capillary electrophoresis. In this document, when the detection signal obtained by electrophoresis saturates (when the detection signal exceeds the recordable upper limit value), a flag urging the user to adjust the injection parameters is output (0165 of this document). Also, the SN ratio of the optical signal is calculated using the median of the signal peaks and compared with the noise estimated from the non-peak region (0166 of this document).

[0005] Prior Art Documents

[0006] Patent Documents

[0007] Patent Document 1: US2020 / 0003728A1

[0008] Non-Patent Documents

[0009] Non - Patent Document 1: https: / www.falco - genetics.com / salsa / principle.html Summary of the Invention

[0010] Problems to be Solved by the Invention

[0011] Since the mutant gene is trace, the detection signal from the mutant gene is weak, and sometimes the signal intensity is lower than the detectable lower limit. At this time, the experimenter needs to increase the injection voltage and sample concentration to perform electrophoresis again. However, if the injection voltage and sample concentration are increased, the detection signal saturates and the mutation rate cannot be calculated. This is because since the signal level of the saturated peak cannot be determined, the ratio of the signal peak level from the mutant type to the signal peak level from the wild type cannot be calculated either.

[0012] In the prior art such as Patent Document 1, it is required that all signal peaks are above the detectable lower limit and not saturated. In the case where either condition is not met, the injection voltage is adjusted and electrophoresis is performed again until both conditions are satisfied. Thus, it is considered difficult to simultaneously detect a DNA sample in which a weak signal peak such as a mutant gene and a normal signal peak co - exist. This is because either a peak smaller than the lower limit or a saturated peak appears.

[0013] The present disclosure has been completed in view of the above - mentioned problems, and its object is to provide a technique capable of highly accurately detecting a mutant gene and a mutation rate using capillary electrophoresis.

[0014] Means for Solving the Problems

[0015] The mutant gene detection method of the present disclosure classifies the signal peaks of the detection signal into a first group smaller than a first threshold and a second group other than that, increases the injection voltage until the signal peaks belonging to the first group become above the first threshold, and decreases the injection voltage until the signal peaks belonging to the second group become below a second threshold larger than the first threshold.

[0016] Advantages of the Invention

[0017] According to the mutant gene detection method of the present disclosure, a mutant gene and a mutation rate can be highly accurately detected using capillary electrophoresis. Other structures, problems, advantages, etc. of the present disclosure will become clear from the following description of the embodiments. Brief Description of the Drawings

[0018] Figure 1 It is a structural diagram of the electrophoresis system 1 of Embodiment 1.

[0019] Figure 2is a signal peak representing the result of measuring a nucleic acid sample containing a mutant gene by the electrophoresis system 1.

[0020] Figure 3 is a graph that amplifies the signal peak of the probe set 202.

[0021] Figure 4 represents for Figure 2 the same DNA sample, the result of measurement with an increased injection voltage.

[0022] Figure 5 represents a flowchart illustrating a general mutant gene detection method as a comparative example.

[0023] Figure 6 represents a flowchart when using an existing technology such as Patent Document 1 in mutant gene detection as a comparative example.

[0024] Figure 7 is a flowchart explaining the mutant gene detection method in Embodiment 1. Detailed implementation mode

[0025] <Embodiment 1: System structure>

[0026] Figure 1 is a structural diagram of the electrophoresis system 1 of Embodiment 1 of the present disclosure. The electrophoresis system 1 is composed of an electrophoresis apparatus 100 and an arithmetic device 200 (computer). The electrophoresis apparatus 100 is a device that analyzes the components of a sample by electrophoresis of the sample using a capillary.

[0027] The electrophoresis apparatus 100 includes a detection unit 116, a thermostat 118, a conveyor 125, a high-voltage power supply 104, a first ammeter 105, a second ammeter 112, a capillary 102, and a pump mechanism 103. The detection unit 116 optically detects the sample. The thermostat 118 keeps the capillary 102 at a constant temperature. The conveyor 125 conveys various containers to the cathode end of the capillary. The high-voltage power supply 104 applies a high voltage to the capillary 102. The first ammeter 105 measures the current output by the high-voltage power supply 104. The second ammeter 112 measures the current flowing through the anode-side electrode 111. The pump mechanism 103 injects a polymer into the capillary 102.

[0028] The capillary 102 is made of a glass tube with an inner diameter of several tens to several hundreds of micrometers and an outer shape of several hundreds of micrometers. To improve the strength, the surface is coated with polyimide. However, the light irradiation portion irradiated with laser removes the polyimide coating film in such a way that the internal light emission easily leaks to the outside. The inside of the capillary 102 is filled with a separation medium for imparting a migration speed difference during electrophoresis. The separation medium includes a fluid medium and a non-fluid medium, but in the present Embodiment 1, a fluid polymer is used.

[0029] The detection unit 116 is a local area of the capillary 102. When the excitation light is irradiated from the light source 114 to the detection unit 116, fluorescence (hereinafter referred to as information light) having a wavelength dependent on the sample is generated from the sample and released to the outside of the capillary 102. The information light is spectroscopically separated in the wavelength direction by the diffraction grating 132. The optical detector 115 analyzes the sample by detecting the spectroscopically separated information light.

[0030] The cathode ends 127 of the capillary are respectively fixed by the metal hollow electrodes 126, and the front end of the capillary protrudes about 0.5 mm from the hollow electrode 126. All the hollow electrodes 126 equipped with the capillary are integrated and mounted on the loading head 129. All the hollow electrodes 126 are electrically connected to the high-voltage power supply 104 mounted on the apparatus main body, and when a voltage needs to be applied for electrophoresis, sample introduction, etc., the hollow electrode 126 acts as a cathode electrode.

[0031] The capillary end (the other end) on the side opposite to the cathode end 127 of the capillary is bundled together by the capillary head 133. The capillary head 133 can be connected to the block 107 in a pressure-resistant and sealed manner. The high voltage output by the high-voltage power supply 104 is applied between the loading head 129 and the capillary head 133. The syringe 106 fills the new polymer into the capillary from the other end. In order to improve the measurement performance, the polymer in the capillary is replaced at each measurement.

[0032] The pump mechanism 103 is composed of the syringe 106 and the mechanism system for pressurizing the syringe 106. The block 107 is a connecting member for connecting the syringe 106, the capillary 102, the anode buffer container 110, and the polymer container 109 respectively.

[0033] The optical detection unit for detecting the information light from the sample is composed of the light source 114, the optical detector 115 for detecting the light emission in the detection unit 116, and the diffraction grating 132. When detecting the sample in the capillary separated by electrophoresis, the detection unit 116 of the capillary is irradiated by the light source 114, the light emission from the detection unit 116 is spectroscopically separated by the diffraction grating 132, and the optical detector 115 detects the spectroscopically separated information light.

[0034] The thermostat 118 is covered with heat insulation material to keep the inside at a constant temperature, and the temperature is controlled by the heating and cooling mechanism 120. The fan 119 circulates and stirs the air in the thermostat 118 to keep the temperature of the capillary 102 spatially uniform and constant.

[0035] The conveyor 125 is equipped with a maximum of 3 electric motors and linear actuators and can move on up to 3 axes in the vertical, horizontal, and depth directions. At least one or more containers can be placed on the workbench 130 of the conveyor 125. An electric handle 131 is provided on the workbench 130, and the user can grasp and release each container via the handle 131. Thus, the buffer container 121, the cleaning container 122, the waste liquid container 123, and the sample container 124 can be transported to the capillary cathode end 127 as needed. Unwanted containers are stored at a predetermined storage location within the device.

[0036] The arithmetic unit 200 obtains the detection result of the information light from the optical detector 115, analyzes it to produce a fluorescence intensity waveform, and performs processes such as calculating the base length of the measurement target substance. Details of the processes performed by the arithmetic unit 200 will be described later. The arithmetic unit 200 can be composed of, for example, a central processing unit (CPU) and software executed by the CPU, or can be composed of hardware such as circuit devices that perform the same functions.

[0037] <Embodiment 1: Problems of the Prior Art>

[0038] Figure 2 It is a signal peak representing the result of measuring a nucleic acid sample containing a mutant gene by the electrophoresis system 1. The purpose of the measurement is to calculate the mutation rate (methylation rate) of DNA. Figure 2 In 201 is a probe set for measuring the methylation rate. 202 is a probe set cut by a restriction enzyme. 203 is a reference probe set not cut by a restriction enzyme. It can be seen that the signal peak of the probe set 202 is significantly smaller than that of the probe set 203. This is because the mutant gene is trace, and the detection signal level is very small compared to the normal gene.

[0039] Figure 3 It is a diagram amplifying the signal peak of the probe set 202. Since the reliability of detection signals with a very low signal level is low, they are generally not used as analysis targets. For example, when Figure 3 the signal level (vertical axis) 300 in is set as the resolvable lower limit, 5 out of the 16 detection target probes included in the probe set 202 are below the resolvable lower limit. Therefore, it is difficult to accurately calculate the mutation rate for this DNA sample.

[0040] The resolvable lower limit of the signal peak level can be determined based on whether the arithmetic unit 200 can obtain detection signal data with sufficient reliability. For example, if it is known that there is a lot of noise and low reliability in detection signals smaller than a certain signal level, then this signal level is used as the resolvable lower limit. This reliability varies depending on the type of electrophoresis device 100 (e.g., product model), so the resolvable lower limit can be determined according to the type of electrophoresis device 100.

[0041] Figure 4Shows the results of measuring by increasing the injection voltage for the same DNA sample as Figure 2 If there is a signal peak below the resolvable lower limit, it is considered that the signal level only needs to be increased so that the signal peak is above the resolvable lower limit. For example, by increasing the injection voltage applied to the capillary during electrophoresis, the signal level can be increased as a whole. Figure 4 Shows the results thereof.

[0042] By increasing the injection voltage, the probe set 202 shows a signal peak higher than Figures 2 to 3 . However, on the other hand, there are probes where the signal peak of the normal gene saturates (exceeds 25000 on the vertical axis in Figure 4 ). Therefore, in this case, it is also difficult to accurately calculate the mutation rate. This is because the signal level of a part of the normal gene (the gene with saturated signal peak) cannot be accurately measured.

[0043] In view of the above, the mutant gene detection method of the present disclosure, after increasing the injection voltage to a level where the signal peak of the mutant gene can be resolved, reduces the injection voltage to a level where other signal peaks are not saturated. Thus, it is considered that the mutation rate can be accurately calculated.

[0044] <Embodiment 1: Mutant gene detection method>

[0045] Figure 5 Shows a flowchart illustrating a general mutant gene detection method as a comparative example. In this method, first, electrophoresis is performed on a DNA sample using a capillary sequencer ( Figure 1 such as an electrophoresis system), and the detection signals obtained from the results are analyzed by software. If all the measured signal peaks are not above the resolvable lower limit, the signal level is insufficient, so it becomes an error (unresolvable). If all the measured signal peaks are not below the saturation level, it also becomes an error. If these conditions are satisfied simultaneously, the ratio (mutation rate) of the signal peaks of the mutant gene is calculated.

[0046] Figure 6 Shows a flowchart in the case of using the prior art such as Patent Document 1 in mutant gene detection as a comparative example. If all the measured signal peaks are not above the resolvable lower limit, the sample injection voltage is increased to lift the signal peak above the lower limit. However, as a result, if the signal level of one of the signal peaks of the normal gene saturates, it becomes an error. On the contrary, if all the measured signal peaks are not below the saturation level, the sample injection voltage is decreased to lower the signal peak below the saturation level. However, as a result, the signal peak of the mutant gene is less than the resolvable lower limit, becoming an error. Therefore, it is difficult for the conventional detection method to accurately calculate the mutation rate.

[0047] Figure 7This is a flowchart illustrating the mutant gene detection method in Embodiment 1. This flowchart can be implemented by an experimenter through manual operation or by controlling the electrophoresis system 1 with the arithmetic unit 200. Hereinafter, assuming that the arithmetic unit 200 implements this flowchart, each step of Figure 7 will be described.

[0048] ( Figure 7 : Steps S701 to S703)

[0049] The user adjusts the DNA sample (nucleic acid sample) and sets necessary reagents, etc. (S701). The sample is loaded into the electrophoresis system 1 (S702), and electrophoresis is performed (S703).

[0050] ( Figure 7 : Steps S704 to S706)

[0051] The arithmetic unit 200 analyzes the detection signals of the fragments obtained by electrophoresis (S704). If all the detection signal peaks of the measurement target are above the resolvable lower limit, it jumps to S707 (S705: Yes). When there are detection signal peaks less than the resolvable lower limit (S705: No), the sample injection voltage of the electrophoresis system 1 (the voltage applied to the capillary during electrophoresis) is increased (S706). The increase amount at this time can be determined in advance or appropriately determined according to the difference between the signal peak and the resolvable lower limit. It is necessary to perform S706 at least until all the signal peak groups derived from the mutant type become signal peak levels above the resolvable lower limit. After S706, return to S702 and perform electrophoresis again using the increased injection voltage.

[0052] ( Figure 7 : Supplementary One of Step S705)

[0053] Among each signal peak, the peaks derived from the mutant type and the peaks derived from the wild type are known in advance. Therefore, the information regarding which of the mutant type and the wild type each signal peak is derived from is described as attribute data in advance, and the arithmetic unit 200 refers to this attribute data, thereby being able to determine the signal peak groups derived from the mutant type and the wild type respectively. The same applies to Step S707.

[0054] ( Figure 7 : Supplementary Two of Step S705)

[0055] When the signal peak is at the lower limit of resolvability, the influence of noise is large and the reliability of the signal is low. Since the reliability of the signal is roughly determined according to the type of the electrophoresis apparatus 100 (electrophoresis system 1), the lower limit of resolvability of the signal peak may be determined according to the type of the electrophoresis apparatus 100. Therefore, the arithmetic unit 200 acquires the type of the electrophoresis apparatus 100 and sets the resolvability lower limit level corresponding to this type. In other words, when the signal peak is lower than a certain lower limit threshold, if the arithmetic unit 200 may not be able to accurately identify the mutant gene corresponding to this signal peak, this lower limit threshold may be determined as the resolvability lower limit.

[0056] ( Figure 7 : Step S706: Supplement)

[0057] After adding the injection voltage in this step, when S702 is implemented again later, the sample used last time is used (measured again). Therefore, since the same sample is used to implement S702 to S706, measurement errors caused by differences between samples and the like can be suppressed. The same applies when returning from S708 to S702.

[0058] ( Figure 7 : Steps S707 - S708)

[0059] If all the detected signal peaks to be measured are below the saturation level, it jumps to S709 (S707: Yes). In the case where there is a detected signal peak exceeding the saturation level (S707: No), the sample injection voltage of the electrophoresis system 1 is decreased (S708). The amount of decrease at this time can be determined in advance or appropriately determined according to the difference between the signal peak and the saturation level. It is necessary to implement S708 at least until all the signal peak groups derived from the wild type become signal peak levels below the saturation level. After S708, it returns to S702 and electrophoresis is implemented again using the decreased injection voltage.

[0060] ( Figure 7 : Steps S705, S707: Supplement)

[0061] When "Yes" holds in all these steps, it enters S709. In other words, by repeatedly implementing S705 to S708 using the same sample and repeatedly adjusting the injection voltage, the signal peak is converged within the range above the resolvability lower limit and below the saturation level. If this adjustment is completed, by implementing the next electrophoresis, the signal peaks derived from the wild type and the signal peaks derived from the mutant type can be measured simultaneously.

[0062] ( Figure 7 : Step S707: Supplement)

[0063] Regarding the saturation level in S707, it may be determined corresponding to the type of the electrophoresis apparatus 100 in the same manner as in S705. That is, when there is an upper limit threshold that can be processed by the electrophoresis apparatus 100 and the arithmetic unit 200, this upper limit threshold may be determined as the saturation level. For example, as described later, in the case of calculating the mutation rate using the ratio of the signal peak levels, if the signal peak from the wild type reaches the saturation level, the mutation rate cannot be accurately calculated. This is because the original signal peak level is greater than the saturation level. Therefore, in this case, the upper limit signal level that the electrophoresis apparatus 100 can output is used as the saturation level in this step.

[0064] ( Figure 7 : Step S709)

[0065] The arithmetic unit 200 uses the result of fragment analysis to determine the ratio between the normal gene and the mutant gene, and thereby calculates the mutation rate of the DNA sample. The signal peak levels of each normal gene are substantially the same, and the signal peak levels of the mutant genes are substantially the same. Therefore, the mutation rate can be calculated based on the ratio between the signal peak level of the normal gene and the signal peak level of the mutant gene.

[0066] <Embodiment 1: Summary>

[0067] The electrophoresis system 1 of this Embodiment 1 pre-obtains information on whether the signal peak obtained by performing capillary electrophoresis on a DNA sample is from the mutant type or the wild type, and divides the signal peaks into respective source groups according to this information. For the mutant type source group, the injection voltage is increased so that all signal peaks are above the resolvable lower limit. For the wild type source group, the injection voltage is decreased so that all signal peaks are below the saturation level. Thereby, it is possible to measure the signal peaks from the mutant type and the signal peaks from the wild type by one electrophoresis.

[0068] <Embodiment 2>

[0069] In Embodiment 1, it was described that the detected signal peaks obtained by electrophoresis are divided into two parts: a mutant gene group that may be lower than the resolvable lower limit and a normal gene group that may exceed the saturation level. The detected signal peaks can also be divided into three or more groups. For example, in the case where a sample contains a fragment in which the signal intensity of A among the four bases ATGC of a gene is relatively higher than the signal intensity of TGC, the signal peak corresponding to this fragment may be divided into a third group. Conversely, fragments with relatively low signal peaks may be divided into a fourth group.

[0070] Such a division is not the division between mutant type and wild type. However, (a) a relatively high signal peak group may exceed the saturation level in the same way as the wild type signal peak, so the same treatment as the wild type is required, and (b) a signal peak group below the resolvable lower limit in the relatively low signal peak group requires the same treatment as the mutant type. Therefore, in addition to the distinction between mutant type and wild type, it is also possible to distinguish signal peaks based on whether the signal peaks exceed the saturation level / are less than the resolvable lower limit. Such a division based on signal peaks can be used on the basis of or in place of the division between mutant type / wild type. Therefore, signal peaks can be divided into three or more groups.

[0071] When forming groups based on whether signal peaks exceed the saturation level / are less than the resolvable lower limit, it is determined in advance which range the signal peak levels of each group are in, and this information is described in the attribute data used in S705 (S707). That is, information capable of determining whether there are signal peaks less than the resolvable lower limit / exceeding the saturation level according to signal peaks is described in the attribute data. It is only necessary to determine in S705 whether all groups are above the resolvable lower limit and in S707 whether all groups are below the saturation level, so it can be used directly Figure 7 of the flowchart.

[0072] <Regarding the modification example of the present disclosure>

[0073] The present disclosure is not limited to the above-described embodiments and includes various modification examples. For example, the above-described embodiments are embodiments described in detail for the purpose of easily understanding the present disclosure and are not necessarily limited to having all the structures described. In addition, a part of the structure of a certain embodiment can be replaced with the structure of other embodiments, and in addition, the structure of other embodiments can be added to the structure of a certain embodiment. In addition, regarding a part of the structure of each embodiment, addition, deletion, and replacement of other structures can be performed.

[0074] In the above embodiments, S706 is used to increase the detected signal peaks. Therefore, as long as the same effect can be achieved by a substitute means other than increasing the injection voltage, that substitute means can also be used. For example, it is only necessary to increase the amount (concentration) of the sample introduced into the capillary of the electrophoresis apparatus 100. Increasing the injection voltage and increasing the sample amount can also be used in combination. Similarly, in S708, the amount of the sample introduced into the capillary of the electrophoresis apparatus 100 can be reduced, or reducing the injection voltage can also be used in combination.

[0075] In the above embodiments, the arithmetic device 200 has been described as a component of the electrophoresis system 1. However, as a component of the electrophoresis apparatus 100, the arithmetic device 200 can also be configured to control each part of the electrophoresis apparatus 100.

[0076] Symbol Explanation

[0077] 1: Electrophoresis system

[0078] 100: Electrophoresis device

[0079] 200: Computing device.

Claims

1. A method for detecting a mutant gene, which detects a mutant gene in a nucleic acid sample containing the gene, characterized in that, The method for detecting a mutant gene has the following steps: Obtaining a detection signal by measuring the nucleic acid sample using a capillary electrophoresis device; Obtaining attribute information describing information indicating whether a signal peak included in the detection signal is less than a first threshold; Classifying, according to the attribute information, the signal peaks included in the detection signal into a first group having signal peaks less than the first threshold and a second group other than the first group; Increasing the voltage applied to the capillary by the capillary electrophoresis device for performing electrophoresis on the nucleic acid sample until the signal peaks belonging to the first group become equal to or greater than the first threshold; And Reducing the voltage until the signal peaks belonging to the second group become equal to or less than a second threshold greater than the first threshold.

2. The method for detecting a mutant gene according to claim 1, characterized in that, The attribute information describes information indicating whether a signal peak included in the detection signal is a signal peak derived from a mutant type or a signal peak derived from a wild type, In the classifying step, the signal peaks included in the detection signal are classified into the first group and the second group according to the attribute information, The first group is a group derived from a mutant type, and the second group is a group derived from a wild type.

3. The method for detecting a mutant gene according to claim 1, characterized in that, The capillary electrophoresis device includes an arithmetic device that processes the detection signal, The arithmetic device sets the first threshold based on the type of the capillary electrophoresis device, The first threshold is equal to or greater than a lower limit signal level at which the arithmetic device can identify the mutant gene, In the step of increasing the voltage, the voltage is increased until all the signal peaks belonging to the first group become equal to or greater than the first threshold.

4. The method for detecting a mutant gene according to claim 1, characterized in that, The capillary electrophoresis device includes an arithmetic device that processes the detection signal, The arithmetic device sets the second threshold based on the type of the capillary electrophoresis device, The second threshold is equal to or less than an upper limit signal level at which the arithmetic device can analyze the detection signal, In the step of reducing the voltage, the voltage is reduced until all the signal peaks belonging to the second group become equal to or less than the second threshold.

5. The method for detecting a mutant gene according to claim 1, characterized in that, The method for detecting a mutant gene further has the following steps: After the step of increasing the voltage, measuring the nucleic acid sample again using the capillary electrophoresis device to thereby obtain the detection signal again; and Repeating the step of increasing the voltage for the detection signal obtained again.

6. The method for detecting a mutant gene according to claim 1, characterized in that, The method for detecting a mutant gene further has the following steps: After the step of reducing the voltage, measuring the nucleic acid sample again using the capillary electrophoresis device to thereby obtain the detection signal again; and Repeating the step of reducing the voltage for the detection signal obtained again.

7. The method for detecting a mutant gene according to claim 1, characterized in that, The method for detecting a mutant gene further has a step of calculating a mutation rate of the nucleic acid sample, The method for detecting a mutant gene performs the step of calculating the mutation rate when the signal peaks belonging to the first group are equal to or greater than the first threshold and the signal peaks belonging to the second group are equal to or less than the second threshold.

8. The method for detecting a mutant gene according to claim 7, characterized in that, When at least one of the following conditions is met: the signal peak belonging to the first group is less than the first threshold, or the signal peak belonging to the second group exceeds the second threshold, the step of calculating the mutation rate is not performed, and instead, the step of increasing the voltage or the step of decreasing the voltage is performed.

9. The mutant gene detection method according to claim 1, wherein The attribute information describes information that can determine whether the signal peak is less than the first threshold based on the signal peak. In the step of classifying the signal peaks, according to the attribute information, the signal peaks of the detection signal are classified into a third group that is different from both the first group and the second group. The mutant gene detection method further includes the following steps: When the signal peak belonging to the third group is less than the first threshold, increase the voltage until the signal peak belonging to the third group becomes equal to or greater than the first threshold. And When the signal peak belonging to the third group exceeds the second threshold, decrease the voltage until the signal peak belonging to the third group becomes equal to or less than the second threshold.

10. The mutant gene detection method according to claim 1, wherein The mutant gene detection method further includes the following step: calculating the mutation rate of the sample based on the ratio between the signal level of the signal peak belonging to the first group and the signal level of the signal peak belonging to the second group.

11. The mutant gene detection method according to claim 1, wherein In the step of increasing the voltage, instead of increasing the voltage or in combination with increasing the voltage, increase the amount of the nucleic acid sample introduced into the capillary electrophoresis device. In the step of decreasing the voltage, instead of decreasing the voltage or in combination with decreasing the voltage, decrease the amount of the nucleic acid sample introduced into the capillary electrophoresis device.

12. The mutant gene detection method according to claim 1, wherein The mutant gene detection method further includes a step of processing the nucleic acid sample using the MLPA method, where the MLPA method is the multiplex ligation-dependent probe amplification method. In the step of obtaining the detection signal, the detection signal is obtained by measuring the nucleic acid sample processed using the MLPA method.

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