Method for detecting mutated genes

Abstract

The purpose of the present disclosure is to provide a technology that makes it possible to use capillary electrophoresis to accurately detect a mutant gene and a mutation rate. This mutant gene detection method involves sorting the signal peaks of a detection signal into a first group that is below a first threshold value and a second group that is not, increasing an injection voltage until the signal peaks of the first group are at least the first threshold value, and then reducing the injection voltage until the signal peaks of the second group are no higher than a second threshold value that is greater than the first threshold value.

Description

Technical Field

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

Background Art

[0002] Analyses of DNA using electrophoresis include fragment analysis and sequence analysis. 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. A common sequence that enables PCR amplification by a universal primer is attached to each probe. Each probe is designed so that different amplified fragment lengths are obtained. Two adjacent probes hybridized to the target gene sequence are ligated by ligase to form a single strand. After hybridization, the tubes are separated for copy number analysis and methylation analysis, and simultaneously treated with the methylation-sensitive restriction enzyme Hha1 during the ligation reaction, followed by PCR reaction. Probes in non-methylated regions are cleaved by the restriction enzyme and thus not PCR amplified. Probes in methylated regions are not cleaved and thus are PCR amplified. The obtained DNA fragments are electrophoresed using a capillary electrophoresis apparatus to obtain detection signals. Based on the difference in the peak positions of these detection signals, non-methylated cells (normal cells) and methylated cells (cancer cells) can be discriminated.

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

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

[0006] [Non-Patent Document 1] https: / / www.falco-genetics.com / salsa / principle.html [Overview of the project] [Problems that the invention aims to solve]

[0007] Since the amount of mutated genes is minute, the detection signal from them is weak, and the signal intensity may fall below the lower limit of detection. In this case, the experimenter needs to increase the injection voltage or sample concentration and repeat the electrophoresis. However, if the injection voltage or sample concentration is increased, the detection signal becomes saturated, and it becomes impossible to calculate the mutation rate. This is because the signal level of the saturated peak cannot be identified, and therefore it is impossible to calculate the ratio of the signal peak level from the mutant to the signal peak level from the wild type.

[0008] In conventional techniques such as those described in Patent Document 1, it is necessary that all signal peaks are above the detectable lower limit and are not saturated. If either condition is not met, the injection voltage and other parameters are adjusted and the electrophoresis is repeated until both conditions are met. In such cases, it is considered difficult to simultaneously detect weak signal peaks, such as those of mutant genes, and normal signal peaks in a DNA sample, because either a peak smaller than the lower limit or a saturated peak will occur.

[0009] This disclosure has been made in view of the above-mentioned issues and aims to provide a technology that can accurately detect mutant genes and mutation rates using capillary electrophoresis. [Means for solving the problem]

[0010] The mutant gene detection method according to this disclosure classifies the signal peaks of the detection signal into a first group of peaks below a first threshold and a second group of peaks other than the first threshold, increases the injection voltage until the signal peaks belonging to the first group are equal to or greater than the first threshold, and decreases the injection voltage until the signal peaks belonging to the second group are equal to or less than a second threshold greater than the first threshold. [Effects of the Invention]

[0011] The mutant gene detection method described herein allows for the accurate detection of mutant genes and mutation rates using capillary electrophoresis. Other features, challenges, and advantages of this disclosure will become clear from the following description of embodiments. [Brief explanation of the drawing]

[0012] [Figure 1] This is a diagram showing the configuration of the electrophoresis system 1 according to Embodiment 1. [Figure 2] This signal peak shows the result of measuring nucleic acid samples containing mutated genes using electrophoresis system 1. [Figure 3] This is a magnified view of the signal peaks of probe group 202. [Figure 4] The results of measurements using the same DNA sample as in Figure 2, but with an increased injection voltage, are shown. [Figure 5] As a comparative example, a flowchart illustrating a common method for detecting mutated genes is shown. [Figure 6] As a comparative example, a flowchart is shown illustrating the use of a conventional technique, such as that described in Patent Document 1, in the detection of mutated genes. [Figure 7] This is a flowchart illustrating the method for detecting mutated genes in Embodiment 1. [Modes for carrying out the invention]

[0013] <Embodiment 1: System Configuration> Figure 1 is a diagram showing the configuration of an electrophoresis system 1 according to Embodiment 1 of the present disclosure. The electrophoresis system 1 consists of an electrophoresis apparatus 100 and a computing device 200 (computer). The electrophoresis apparatus 100 is a device that analyzes the components of a sample by performing electrophoresis on the sample using a capillary.

[0014] The electrophoresis apparatus 100 comprises a detection unit 116, a constant temperature bath 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 constant temperature bath 118 maintains 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 to the anode side electrode 111. The pump mechanism 103 injects polymer into the capillary 102.

[0015] The capillary 102 is composed of a glass tube with an inner diameter of several tens to several hundreds of microns and an outer shape of several hundreds of microns, and its surface is coated with polyimide to improve strength. However, the polyimide coating is removed from the light irradiation part where the laser light is irradiated so that the internal light emission is likely to leak to the outside. The inside of the capillary 102 is filled with a separation medium for providing a difference in migration speed during electrophoresis. There are fluid and non-fluid separation media, but in the first embodiment, a fluid polymer is used.

[0016] The detection unit 116 is a part of the capillary 102. When excitation light is irradiated from the light source 114 to the detection unit 116, fluorescence having a wavelength dependent on the sample (hereinafter referred to as information light) is generated from the sample and emitted outside 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.

[0017] The capillary cathode ends 127 are each fixed through a metal hollow electrode 126, and the capillary tip protrudes about 0.5 mm from the hollow electrode 126. All the hollow electrodes 126 equipped for each capillary are integrated and mounted on the load header 129. All the hollow electrodes 126 are connected to the high-voltage power supply 104 mounted on the apparatus main body, and when it is necessary to apply a voltage for electrophoresis, sample introduction, etc., the hollow electrode 126 operates as a cathode electrode.

[0018] The capillary end (the other end) on the side opposite to the capillary cathode end 127 is bundled together by the capillary head 133. The capillary head 133 can be connected to the block 107 in a pressure-resistant and confidential manner. The high voltage output from the high-voltage power supply 104 is applied between the load header 129 and the capillary head 133. The syringe 106 fills the capillary with a new polymer from the other end. The polymer replacement in the capillary is carried out for each measurement to improve the measurement performance.

[0019] The pump mechanism 103 consists of a syringe 106 and a mechanism for pressurizing the syringe 106. 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.

[0020] The optical detection unit for detecting information light from a sample consists of a light source 114, an optical detector 115 for detecting light emission within the detection unit 116, and a diffraction grating 132. When detecting a sample in a capillary separated by electrophoresis, the light source 114 irradiates the detection unit 116 of the capillary, the diffraction grating 132 spectrally analyzes the light emission from the detection unit 116, and the optical detector 115 detects the spectrally analyzed information light.

[0021] The constant temperature bath 118 is covered with insulating material to maintain a constant internal temperature, and its temperature is controlled by the heating and cooling mechanism 120. The fan 119 circulates and agitates the air inside the constant temperature bath 118, keeping the temperature of the capillary 102 uniform and constant in terms of location.

[0022] The conveyor 125 is equipped with up to three electric motors and linear actuators, and is capable of moving in up to three axes: up and down, left and right, and depth. The stage 130 of the conveyor 125 can hold at least one container. The stage 130 is equipped with an electric grip 131, which the user can use to grasp and release each container. This allows the buffer container 121, washing container 122, waste liquid container 123, and sample container 124 to be transported to the capillary cathode 127 as needed. Unnecessary containers are stored in designated storage areas within the device.

[0023] The computing unit 200 acquires the detection result of information light from the optical detector 115, analyzes it to create a fluorescence intensity waveform, and performs processing such as calculating the base length of the substance to be measured. Details of the processing performed by the computing unit 200 will be described later. The computing unit 200 can be configured by, for example, a Central Processing Unit (CPU) and software executed by the CPU, or by hardware such as a circuit device that implements similar functions.

[0024] <Embodiment 1: Problems of the prior art> Figure 2 shows the signal peaks obtained by measuring nucleic acid samples containing mutated genes using electrophoresis system 1. The purpose of the measurement is to calculate the mutation rate (methylation rate) of DNA. Of the probes in Figure 2, 201 is the probe group used to measure the methylation rate. 202 is the probe group that is cleaved by restriction enzymes. 203 is the reference probe group that is not cleaved by restriction enzymes. It can be seen that the signal peak of probe group 202 is significantly smaller than that of probe group 203. This is because the amount of mutated genes is minute, and the detection signal level is very small compared to that of normal genes.

[0025] Figure 3 shows a magnified view of the signal peaks of probe group 202. Detection signals with very low signal levels are generally unreliable and should be excluded from analysis. For example, when the signal level (vertical axis) of 300 in Figure 3 is set as the lower limit of analysis, 5 of the 16 detection probes included in probe group 202 fall below this lower limit. Therefore, it is difficult to accurately calculate the mutation rate from this DNA sample.

[0026] The lower limit of the analyzable signal peak level can be determined by whether the computing unit 200 can acquire detection signal data with sufficient reliability. For example, if it is known that detection signals below a certain signal level are noisy and unreliable, then that signal level can be set as the lower limit of the analyzable level. Since this reliability varies depending on the type of electrophoresis apparatus 100 (e.g., product model number), the lower limit of the analyzable level can be set for each type of electrophoresis apparatus 100.

[0027] Figure 4 shows the results of measurements using the same DNA sample as in Figure 2, but with an increased injection voltage. If there are signal peaks below the analyzable lower limit, it is reasonable to increase the signal level so that those signal peaks are above the analyzable lower limit. For example, increasing the injection voltage applied to the capillary during electrophoresis can raise the overall signal level. Figure 4 shows the results.

[0028] By increasing the injection voltage, probe group 202 shows higher signal peaks than those in Figures 2 and 3. However, on the other hand, some probes exhibit saturated signal peaks for normal genes (exceeding 25,000 on the vertical axis in Figure 4). Therefore, even in this case, it is difficult to accurately calculate the mutation rate because the signal levels of some of the normal genes (those with saturated signal peaks) cannot be accurately measured.

[0029] In view of the above, the mutant gene detection method described herein involves increasing the injection voltage to a level sufficient to analyze the signal peak of the mutant gene, and then decreasing the injection voltage to a level that does not saturate other signal peaks. This is expected to enable accurate calculation of the mutation rate.

[0030] <Embodiment 1: Method for detecting mutant genes> Figure 5 shows a flowchart illustrating a general mutant gene detection method as a comparative example. In this method, electrophoresis is first performed on a DNA sample using a capillary sequencer (an electrophoresis system like the one in Figure 1), and the resulting detection signals are analyzed by software. If all the target signal peaks are not above the analyzable lower limit, the signal level is insufficient, resulting in an error (analysis impossible). Similarly, if all the target signal peaks are not below the saturation level, an error occurs. If both of these conditions are met, the ratio of signal peaks of mutant genes (mutation rate) is calculated.

[0031] Figure 6 shows a flowchart illustrating the use of a conventional technique, such as that described in Patent Document 1, in mutant gene detection, as a comparative example. If all target signal peaks are not above the analyzable lower limit, the sample injection voltage is increased to push the signal peaks above the lower limit. However, this results in an error if the signal level of any of the normal gene signal peaks becomes saturated. Conversely, if all target signal peaks are not below the saturation level, the sample injection voltage is decreased to lower the signal peaks below the saturation level. However, this results in an error if the signal peak of the mutant gene falls below the analyzable lower limit. Therefore, it is difficult to accurately calculate the mutation rate with conventional detection methods.

[0032] Figure 7 is a flowchart illustrating the mutant gene detection method in this embodiment 1. This flowchart may be performed manually by the experimenter, or it may be performed by the computing device 200 controlling the electrophoresis system 1. In the following description, each step in Figure 7 will be explained assuming that the computing device 200 performs this flowchart.

[0033] (Figure 7: Steps S701-S703) The user prepares the DNA sample (nucleic acid sample) and sets up the necessary reagents (S701). The sample is placed into the electrophoresis system 1 (S702) and electrophoresis is performed (S703).

[0034] (Figure 7: Steps S704-S706) The computing unit 200 analyzes the detection signals of the fragments obtained by electrophoresis (S704). If all the detection signal peaks being measured are above the analyzable lower limit, the process skips to S707 (S705: YES). If there are detection signal peaks below the analyzable lower limit (S705: NO), the sample injection voltage of the electrophoresis system 1 (the voltage applied to the capillary when performing electrophoresis) is increased (S706). The amount of increase at this time may be predetermined, or it may be determined as appropriate according to the difference between the signal peak and the analyzable lower limit. At a minimum, S706 must be performed until all signal peak groups derived from the mutant are above the analyzable lower limit. After S706, the process returns to S702 and electrophoresis is repeated using the increased injection voltage.

[0035] (Figure 7: Step S705: Supplement 1) It is known in advance which of the signal peaks originate from the mutant and which from the wild type. Therefore, information on whether each signal peak originates from the mutant or the wild type is described in advance as attribute data, and the arithmetic unit 200 can identify the signal peak groups originating from the mutant and wild types by referring to this attribute data. The same applies to S707.

[0036] (Figure 7: Step S705: Supplement 2) When the signal peak is at the lower limit of analysis, the influence of noise is significant and the reliability of the signal is low. The reliability of the signal is generally determined by the type of electrophoresis apparatus 100 (electrophoresis system 1), so the lower limit of analysis of the signal peak should be determined for each type of electrophoresis apparatus 100. Therefore, the computing unit 200 should acquire the type of electrophoresis apparatus 100 and set the corresponding lower limit of analysis level. In other words, if the signal peak falls below a certain lower threshold, and there is a possibility that the computing unit 200 may not be able to accurately identify the mutant gene corresponding to that signal peak, then that lower threshold should be set as the lower limit of analysis.

[0037] (Figure 7: Step S706: Supplement) In this step, after increasing the injection voltage, when repeating steps S702 and beyond, the previously used sample is reused (re-measured). Therefore, steps S702 to S706 are performed using the same sample, which helps to suppress measurement errors due to differences between samples. The same applies when returning from S708 to S702.

[0038] (Figure 7: Steps S707-S708) If all detection signal peaks being measured are below the saturation level, skip to S709 (S707: YES). If there are detection signal peaks above the saturation level (S707: NO), reduce the sample injection voltage of electrophoresis system 1 (S708). The amount of reduction at this time may be predetermined or may be determined as appropriate according to the difference between the signal peak and the saturation level. At a minimum, S708 must be performed until all signal peak groups originating from the wild type are below the saturation level. After S708, return to S702 and repeat electrophoresis using the reduced injection voltage.

[0039] (Figure 7: Steps S705, S707: Supplementary Information) Only when all of these steps are met with a 'YES' will the process proceed to S709. In other words, by repeatedly performing S705 to S708 using the same sample, the injection voltage is repeatedly adjusted so that the signal peak falls within the range above the analyzable lower limit but below the saturation level. Once this adjustment is complete, the signal peaks from the wild type and the mutant can be measured simultaneously by performing the next electrophoresis.

[0040] (Figure 7: Step S707: Supplement) The saturation level in S707 should be determined in accordance with the type of electrophoresis apparatus 100, similar to S705. That is, if there is an upper threshold that the electrophoresis apparatus 100 and the calculation unit 200 can process, that upper threshold should be set as the saturation level. For example, as will be described later, when calculating the mutation rate using the ratio of signal peak levels, if the signal peak derived from the wild type reaches the saturation level, the mutation rate cannot be calculated accurately. 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 will be used as the saturation level in this step.

[0041] (Figure 7: Step S709) The computing device 200 calculates the mutation rate of a DNA sample by determining the ratio between normal and mutant genes using the results of fragment analysis. The signal peak levels of each normal gene are approximately the same, and the signal peak levels of mutant genes are also approximately the same. Therefore, the mutation rate can be calculated from the ratio between the signal peak levels of normal genes and mutant genes.

[0042] <Embodiment 1: Summary> The electrophoresis system 1 according to this embodiment 1 acquires information in advance on whether the signal peaks obtained by capillary electrophoresis on a DNA sample originate from a mutant or a wild-type DNA, and classifies the signal peaks into their respective origin groups according to this information. For the mutant-derived group, the injection voltage is increased so that all signal peaks are above the analyzable lower limit. For the wild-type-derived group, the injection voltage is decreased so that all signal peaks are below the saturation level. As a result, both mutant-derived and wild-type-derived signal peaks can be measured in a single electrophoresis.

[0043] <Embodiment 2> In Embodiment 1, the detection signal peaks obtained by electrophoresis were divided into two groups: a group of mutant genes that may be below the analyzable lower limit and a group of normal genes that may exceed the saturation level. The detection signal peaks can also be divided into three or more groups. For example, if a sample contains a fragment in which the signal intensity of A among the four bases A, T, C, and G of the gene is relatively higher than the signal intensity of T, C, and G, the signal peak corresponding to that fragment may be classified as a third group. Conversely, fragments with relatively low signal peaks may be classified as a fourth group.

[0044] While this classification is not a distinction between mutant and wild-type, (a) relatively high signal peak groups may exceed the saturation level, similar to wild-type signal peaks, and therefore require the same processing as wild-type; and (b) relatively low signal peak groups that fall below the analyzable lower limit require the same processing as mutants. Therefore, in addition to the mutant and wild-type distinction, it is also possible to classify signal peaks based on whether they exceed the saturation level or fall below the analyzable lower limit. Such classification based on signal peaks can be used in addition to, or as an alternative to, the mutant / wild-type distinction. Thus, it is possible to classify signal peaks into three or more groups.

[0045] When grouping based on whether the signal peak is above the saturation level or below the analyzable lower limit, the range of the signal peak level for each group should be identified in advance, and this information should be included in the attribute data used in S705 (S707). In other words, information that identifies whether or not there are signal peaks below the analyzable lower limit or above the saturation level for each signal peak should be included in the attribute data. In S705, it is necessary to determine whether or not all groups are above the analyzable lower limit, and in S707, it is necessary to determine whether or not all groups are below the saturation level, so the flowchart in Figure 7 can be used as is.

[0046] <Regarding variations of this disclosure> This disclosure is not limited to the embodiments described above, but includes various modifications. For example, the embodiments described above are described in detail for the purpose of explaining this disclosure clearly, and are not necessarily limited to those having all the configurations described. Furthermore, it is possible to replace parts of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add configurations from other embodiments to the configuration of one embodiment. In addition, it is possible to add, delete, or replace parts of the configuration of each embodiment with other configurations.

[0047] In the embodiments described above, S706 is for increasing the detection signal peak, so if an alternative means other than increasing the injection voltage can achieve a similar effect, that alternative means may be used. For example, the amount (concentration) of sample introduced into the capillary of the electrophoresis apparatus 100 can be increased. Increasing the injection voltage and increasing the sample amount can also be done in combination. Similarly, in S708, the amount of sample introduced into the capillary of the electrophoresis apparatus 100 can be decreased, or the injection voltage can be decreased in combination with this.

[0048] In the embodiments described above, the arithmetic unit 200 was described as a component of the electrophoresis system 1, but the arithmetic unit 200 may also be configured as a component of the electrophoresis apparatus 100 to control each part of the electrophoresis apparatus 100. [Explanation of Symbols]

[0049] 1: Electrophoresis system 100: Electrophoresis apparatus 200: Arithmetic device

Claims

1. A method for detecting mutant genes in nucleic acid samples containing genes, A step of obtaining a detection signal by measuring the nucleic acid sample using a capillary electrophoresis apparatus. A step of acquiring attribute data that describes information indicating whether or not the signal peak of the detection signal is less than a first threshold, A step of classifying the signal peaks of the detection signal into a first group of peaks that are below the first threshold and a second group of peaks that are not, according to the attribute data. The step of increasing the voltage applied by the capillary electrophoresis apparatus to the capillary in order to perform electrophoresis on the nucleic acid sample until the signal peaks belonging to the first group are equal to or greater than the first threshold, A step of reducing the voltage until the signal peaks belonging to the second group become less than or equal to a second threshold greater than the first threshold, It has, The attribute data describes information that allows for the identification of whether or not the signal peak is less than the first threshold for each signal peak. In the step of classifying the signal peaks, the signal peaks of the detected signal are classified into a third group that is different from both the first group and the second group, according to the attribute data. The aforementioned method for detecting mutated genes further, If the signal peak belonging to the third group is less than the first threshold, the step of increasing the voltage until it is equal to or greater than the first threshold, If the signal peak belonging to the third group exceeds the second threshold, the step is to reduce the voltage until it falls below the second threshold. has A method for detecting mutant genes, characterized by the features described above.

2. The attribute data describes information indicating whether the signal peak in the detection signal originates from a mutant or a wild type. In the classification step, the signal peaks of the detection signal are classified into the first group and the second group according to the attribute data. The first group is derived from mutants, and the second group is derived from wild types. The method for detecting a mutant gene according to claim 1, characterized in that it is a feature of the present invention.

3. The capillary electrophoresis apparatus includes a computing device for processing the detection signal, The calculation device sets the first threshold based on the type of capillary electrophoresis apparatus, The first threshold is greater than or equal to the lower signal level at which the computing device can identify the mutated gene. In the step of increasing the voltage, the voltage is increased until all of the signal peaks belonging to the first group are equal to or greater than the first threshold. The method for detecting a mutant gene according to claim 1, characterized in that it is a feature of the present invention.

4. The capillary electrophoresis apparatus includes a computing device for processing the detection signal, The calculation device sets the second threshold based on the type of capillary electrophoresis apparatus, The second threshold is below the upper limit signal level at which the arithmetic unit can analyze the detection signal. In the step of reducing the voltage, the voltage is reduced until all of the signal peaks belonging to the second group are below the second threshold. The method for detecting a mutant gene according to claim 1, characterized in that it is a feature of the present invention.

5. The aforementioned method for detecting mutated genes further, After the step of increasing the voltage, the detection signal is reacquired by re-measuring the nucleic acid sample using the capillary electrophoresis apparatus. With respect to the reacquired detection signal, the step of increasing the voltage is repeated. A method for detecting a mutant gene according to claim 1, characterized by having the following features.

6. The aforementioned method for detecting mutated genes further, After the step of reducing the voltage, the detection signal is reacquired by re-measuring the nucleic acid sample using the capillary electrophoresis apparatus. With respect to the reacquired detection signal, the step of reducing the voltage is repeated. A method for detecting a mutant gene according to claim 1, characterized by having the following features.

7. The mutated gene detection method further includes the step of calculating the mutation rate of the nucleic acid sample. The mutant gene detection method performs the step of calculating the mutation rate when the signal peaks belonging to the first group are above the first threshold and the signal peaks belonging to the second group are below the second threshold. The method for detecting a mutant gene according to claim 1, characterized in that it is a feature of the present invention.

8. The mutant gene detection method, if at least one of the following is true—that the signal peak belonging to the first group is below the first threshold, or that the signal peak belonging to the second group is above the second threshold—is performed without performing the step of calculating the mutation rate, by performing the step of increasing the voltage or the step of decreasing the voltage. The method for detecting a mutant gene according to claim 7, characterized in that it is as described in the present invention.

9. The mutant gene detection method further includes the step of 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. The method for detecting a mutant gene according to claim 1, characterized in that it is a feature of the present invention.

10. In the step of increasing the voltage, instead of increasing the voltage, or in combination therewith, the amount of nucleic acid sample introduced into the capillary electrophoresis apparatus is increased. In the step of reducing the voltage, instead of reducing the voltage, or in combination therewith, the amount of nucleic acid sample introduced into the capillary electrophoresis apparatus is reduced. The method for detecting a mutant gene according to claim 1, characterized in that it is a feature of the present invention.

11. The aforementioned mutant gene detection method further comprises the step of processing the nucleic acid sample using the MLPA (Multiplex Ligation-dependent Probe Amplification) method, In the step of acquiring the detection signal, the detection signal is acquired by measuring the nucleic acid sample processed using the MLPA method. The method for detecting a mutant gene according to claim 1, characterized in that it is a feature of the present invention.

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