Point mutation rate detection method

Abstract

Provided is a point mutation rate detection method capable of conducting quantitative examination. The point mutation rate detection method according to the present invention is used for multiplex ligation-dependent probe amplification (MLPA) measurement, the method involving a measurement step for measuring, among strength SMT of a mutation-derived signal, which is a fluorescent signal emitted from a mutation site of a sample, and strength SWT of a wild-derived signal, which is a fluorescent signal emitted from a site other than the mutation site of the sample, at least the SMT using an electrophoresis device, and a rate calculation step for calculating the rate of the SMT to a strength reference value greater than the SMT, wherein the upper limit of the measurement dynamic range for a fluorescent signal of the electrophoresis device is equal to or greater than a predetermined value.

Description

Point mutation rate detection method

[0001] The present invention relates to a method for detecting a point mutation rate.

[0002] It has become clear that of the approximately 20,000 genes, only a few hundred are oncogenes or tumor suppressor genes. Gene mutations that cause cancer in humans are still being discovered, but it is predicted that the number will ultimately remain at around a few hundred. In fact, the OncoGuide NCC Oncopanel for next-generation sequencers, jointly developed by the National Cancer Center Research Institute and Sysmex, which was covered by insurance in 2019, contains 124 genes. Furthermore, the Foundation-one CDx, developed by Foundation Medicine in the United States, contains 324 genes. These developments demonstrate the advancement of cancer diagnosis by detecting the behavior of a limited number of genes, a few hundred in number. Among these genes, point mutations are particularly important. This is because cancer develops and progresses through the accumulation of random point mutations.

[0003] However, these tests are inevitably expensive because they use next-generation sequencing, a method for decoding genes in massive parallel sequences. In fact, the cost of the two cancer gene panel tests mentioned above is 560,000 yen, a significant burden for the average patient. Therefore, there is a need for a technology that can measure a limited number of genes, approximately 100, inexpensively, quickly, and accurately. With current qPCR, the number of genes that can be measured in a single tube is limited to the use of multiple fluorescent dyes, with a maximum of five colors, or five genes. Next-generation sequencers, on the other hand, are expensive. On the other hand, capillary sequencers perform electrophoresis after PCR, allowing them to separate and detect PCR products according to molecular weight. Therefore, information on the "length of DNA molecules" that conventional qPCR lacks can be obtained.

[0004] The characteristics of capillary sequencers are utilized in short tandem repeat analysis and multiplex ligation-dependent probe amplification (MLPA) for human personal identification. Both of these techniques extract and add molecular length information from the amplified products after PCR amplification using capillary electrophoresis, thereby enabling determination and diagnosis that cannot be achieved by simple PCR alone. In human personal identification, "separation by DNA molecule length" is achieved by focusing on repetitive sequences called short tandem repeats, which are unique to genomic DNA molecules, while MLPA actively and artificially achieves "separation by DNA molecule length" from the outside by adding PCR probes of different lengths from the outside. In other words, the former uses the intrinsic characteristics of biological samples, while the latter involves external molecular design by humans, which is a contrast.

[0005] The MLPA method, developed by Shoten in the Netherlands, involves performing PCR with varying probe lengths and then analyzing the resulting products using capillary electrophoresis. Patent Document 1 describes the basic technology of the MLPA method. Furthermore, MRC Holland, a company founded by Shoten, sells MLPA kits. The MLPA method can detect copy number changes (deletions and duplications), DNA methylation, gene expression, and point mutations, which are essential for cancer diagnosis. Specific details regarding the detection of point mutations using the MLPA method are described in Non-Patent Document 1. Non-Patent Document 2 provides a product description (instruction manual) for a commercially available MLPA kit for myeloproliferative tumors, capable of detecting point mutations in eight genes, including JAK2, with a high sensitivity of 1-5%. The kit cautions that this kit cannot be used for quantitative measurement of point mutations and should only be used for qualitative measurement. The term "qualitative" means that the signal is compared with a control called binning DNA provided with the kit, and if the signal is greater than that of the binning DNA, it is determined that "mutation is present," and if the signal is smaller, it is determined that "mutation is not present."

[0006] Furthermore, to detect the presence or absence of a mutation, it is necessary to change the length of the stuffer sequence contained in the left LPO (Left Probe Oligonucleotide) of the two probes hybridizing to the target sequence in the MLPA method. This is because LPO plays a role in detecting the state of the mutation, and is necessary to convert this state of the mutation into molecular length information during electrophoresis. A kit for detecting two types of single-base substitutions with different sequences for a single point mutation is also commercially available, and this kit is described in Non-Patent Document 3. Specifically, the wild type for the IDH2 gene is guanine. In contrast, a 151-base-long probe is assigned to the adenine point mutation, and a 145-base-long probe is assigned to the thymine point mutation. In other words, it can be seen that the length of the LPO has been changed in the commercially available kit.

[0007] On the other hand, the kits described in Non-Patent Documents 2 and 3 do not include probes for confirming wild type status. Point mutations generally occur in a portion of a cell population. The ratio of cells with point mutations (mutant type) to cells with wild type, which is a normal state without point mutations, (MT / WT) is important information for cancer diagnosis. However, conventional MLPA kits do not have probes for wild type and therefore cannot confirm MT / WT. This is because the signal detected with 1% point mutations is approximately 3,000 [ADU]. 1% point mutations means a state in which 1% mutant type and 99% wild type are mixed. In this case, when detecting a signal from the wild type probe, the signal from the wild type will be approximately 300,000 [ADU]. However, this signal amount exceeds the saturation upper limit of 32,767 [ADU] that can be detected by conventional capillary sequencers, making it impossible to measure. This is the reason why probes for measuring wild type are not arranged in the MLPA method for detecting point mutations. In other words, the reason why wild type probes cannot be arranged is due to the narrow dynamic range of the device.

[0008] Meanwhile, Non-Patent Document 4 reports a technology that can detect the MT / WT ratio, which is the ratio of mutant type to wild type, down to 0.01% by expanding the dynamic range of a conventional capillary sequencer. The mutation detection limit for fragment analysis using conventional capillary sequencers is said to be 1-5%. The reason for this is that the dynamic range of conventional capillary sequencers is narrower than three orders of magnitude. Non-Patent Document 4 reports a technology that expands the dynamic range from three or more orders of magnitude to four orders of magnitude. In this patent, the capillary electrophoresis analysis technology with a dynamic range expanded to three or more orders of magnitude is called HiDy.

[0009] WO 2001 / 61033

[0010] Multiplex ligation-dependent probe amplification (MLPA) in tumor diagnostics and prognostics, Diagn Mol Pathol. 2012 Dec;21(4): P.189-206. doi: 10.1097 / PDM.0b013e3182595516.Product Description SALSA MLPA Probemix P520-A2 MPN mix 2 (Product description version A2-03; Issued 13 July 2022), [Search date 2022.09.08], Internet <URL:https: / / www.mrcholland.com / products / 32322 / Product%20description%20P520-A2%20MPN%20mix%202-v03.pdf> Product description ME012-A1 MGMT-IDH1-IDH2 (Product Description version A1-03; Issued 03 August 2021), [Retrieved 2022.09.08], Internet <URL:https: / / www.mrcholland.com / products / 30043 / Product%20description%20ME012-A1%20MGMT-IDH1-IDH2-v03.pdf> Highly sensitive mutation quantification by highdynamic-range capillary-array electrophoresis (HiDy CE), Lab Chip, 2020, 20, P.1083-1091

[0011] The MLPA method, one of the most common applications of capillary electrophoresis, allows for multiplexing of 40 probes. However, point mutation measurement using conventional MLPA methods is determined by comparing the signal intensity from the gene in the target sample with that in the control sample provided with the kit. Therefore, while it was possible to determine the presence or absence of Mutation Type (MT), it was not possible to measure the ratio. In other words, point mutation measurement using conventional MLPA methods could perform qualitative testing, but not quantitative testing.

[0012] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for detecting a point mutation ratio that allows quantitative testing.

[0013] The method for detecting a point mutation ratio according to the present invention, which has solved the above-mentioned problems, is used in Multiplex Ligation-dependent Probe Amplification (MLPA) measurement, and involves measuring the intensity S of a mutation-derived signal, which is a fluorescent signal emitted from a mutated portion of a sample, using an electrophoresis device. ＭＴ and the intensity S of the wild-derived signal, which is a fluorescent signal emitted from a portion of the sample other than the mutated portion. ＷＴ Among these, at least the S ＭＴ a measuring step of measuring the S ＭＴ The S for a reference value of strength higher than ＭＴ and a ratio calculation step of calculating the ratio of the fluorescent signal obtained by the electrophoresis device, wherein the upper limit of the dynamic range of measurement for the fluorescent signal is equal to or greater than a predetermined value.

[0014] The present invention can provide a method for detecting a point mutation rate that can perform quantitative testing. Problems, configurations, and advantages other than those described above will become apparent from the following description of the embodiments.

[0015] FIG. 1 is an explanatory diagram of an analytical reaction for explaining an example of a method for detecting point mutations in a DNA base sequence. FIG. 2 is an explanatory diagram of an analytical reaction of a first embodiment of the present invention. FIG. 3 is an explanatory diagram of an analytical reaction of a second embodiment of the present invention. FIG. 4 is an explanatory diagram of an analytical reaction of a third embodiment of the present invention. FIG. 5 is an explanatory diagram of an analytical reaction of a fifth embodiment of the present invention. FIG. 6 is an explanatory diagram of an analytical reaction of a sixth embodiment of the present invention. FIG. 7 is an explanatory diagram of an analytical reaction of a seventh embodiment of the present invention. FIG. 8 is a diagram showing analytical reaction analysis results in an eighth embodiment of the present invention.

[0016] Hereinafter, an embodiment of the present invention will be described with reference to FIGS. 1 to 9. First, an example of a method for detecting point mutations in a DNA base sequence will be described with reference to FIG. 1. FIG. 1 is an explanatory diagram of an analytical reaction illustrating an example of a method for detecting point mutations in a DNA base sequence. The method shown in FIG. 1 is a technique for multiplex detection of multiple fragments for multiple point mutations by capillary electrophoresis. This method is generally known as MLPA (Multiplex Ligation-dependent Probe Amplification).

[0017] The method shown in Figure 1 relates to a method for detecting point mutations in DNA base sequences. As shown in Figure 1 (before hybridization), at this stage, DNA target sequences 112 and 113, the state of which point mutations are to be investigated, LPO (Left Probe Oligonucleotide), and RPO (Right Probe Oligonucleotide), are not hybridized. DNA target sequences 112 and 113 are composed of molecules of four types of nucleotides: guanine, adenine, cytosine, and thymine. DNA target sequence 112 has a point mutation of one base pair, guanine 103. On the other hand, DNA target sequence 113 is a wild type having a normal gene sequence and has a normal base, cytosine 114, which is a regular sequence. The difference between DNA target sequence 112 and DNA target sequence 113 is only one base difference between point mutation guanine 103 and normal base cytosine 114, and surrounding sequences 101 and 102 located upstream and downstream of point mutation guanine 103 are identical sequences.

[0018] As shown in the pre-hybridization state of FIG. 1 , LPO has a base cytosine 106 that matches the point mutation guanine 103 in the DNA target sequence 112. LPO has a sequence 104 complementary to sequence 101 and stuffer sequences 107 and 111 for adjusting the length of the probe. Note that stuffer sequence 107 does not hybridize with the DNA target sequences 112 and 113. In this method, after hybridization of these sequences, a ligation reaction is carried out using a ligase enzyme to link LPO and RPO, which are then amplified by PCR. LPO has a primer sequence 109 complementary to a PCR primer for amplification by PCR.

[0019] 1, the RPO has a sequence 105 that hybridizes complementarily to the sequence 102. Furthermore, the RPO has a primer sequence 110 that is complementary to a PCR primer for amplification by PCR.

[0020] In a typical MLPA method, a stuffer sequence 111 for adjusting the length of the PCR product is inserted between the sequence 105 and the primer sequence 110 in the RPO. However, in order to detect point mutations, it is desirable to insert a stuffer sequence 107 into the LPO, as shown in FIG. 1. This is because it is the LPO that actually detects point mutations. In order to convert this point mutation information into base length, it is absolutely necessary to place the stuffer sequence 107 in the LPO. In other words, even if the RPO contains the stuffer sequence 107, it is difficult to detect point mutations as base length.

[0021] Next, as shown in the hybridization ligation diagram of FIG. 1, LPO (Left Probe Oligonucleotide) and RPO (Right Probe Oligonucleotide) are hybridized to the DNA target sequences 112 and 113 that are the targets of hybridization.

[0022] For example, as shown in the hybridization / ligation diagram of Figure 1, 50-100 ng of DNA target sequences 112 and 113 are heated in 5 µL of 10 mM Tris buffer (pH 8.0) at 98°C for 5 minutes, cooled to room temperature, and LPO and RPO are added. After incubation at 60°C for 18 hours, the DNA target sequence 112 hybridizes with the LPO and RPO. Furthermore, the DNA target sequence 113 hybridizes with the LPO and RPO.

[0023] Here, cytosine 106 at the 3' end of LPO is complementary to the point mutation guanine 103, and therefore hybridizes. After hybridization, cytosine 106 at the 3' end of LPO and the 5' end of sequence 105 in RPO are adjacent, allowing a ligase enzyme to link the two. That is, in the mutant type, LPO and RPO can form a single DNA strand. On the other hand, in the wild type, DNA target sequence 113 contains the normal base cytosine 114, and therefore cannot form a complementary strand with cytosine 106 at the 3' end of LPO. Therefore, a ligase enzyme cannot link LPO and RPO. That is, in the wild type, LPO and RPO cannot form a single DNA strand.

[0024] Next, the PCR step shown in FIG. 1 is performed. In the PCR step, the DNA target sequence 112 and the single-stranded DNA strand formed by the ligation of LPO and RPO are dissociated at 94°C. The DNA target sequence 113 is also dissociated with the LPO and RPO at 94°C. After dissociation, primers added to the solution hybridize to the primer sequences 109 and 110 of the dissociated single-stranded DNA, and the desired DNA fragment can be exponentially amplified by repeated heat cycles. It is important to note that only mutant-type DNA fragments in which LPO and RPO are linked by hybridization and ligation in FIG. 1 are PCR-amplified, while wild-type DNA fragments are not amplified. In other words, if the DNA target sequence 113 contains the normal base cytosine 114, the LPO and RPO are not linked and therefore not amplified, resulting in no PCR product being generated.

[0025] The above-described reaction details one specific point mutation on the genome. This reaction can also be performed in parallel (i.e., multiplexed) to detect multiple point mutations present on the genome. In fact, the MLPA reaction can simultaneously detect 40 or more probes for Copy Number Variation and methylation detection. Therefore, the MLPA reaction can also be applied to point mutation detection, allowing simultaneous detection of 40 or more probes.

[0026] Next, as shown in the capillary electrophoresis (CE) diagram in Figure 1, after the PCR reaction, the resulting 40 PCR product DNA fragments are electrophoresed using a capillary sequencer. The base lengths of the resulting PCR products are determined by varying the lengths of the LPO stuffer sequences 107 and 111 applied to each point mutation, so the base lengths of the PCR products 115, 116, and 117 can be varied to any desired length. In the example illustrated in Figure 1, the base length to be varied was 15 bases. The difference in the base length of the stuffer sequence is not limited to 15 bases, but can be varied from 1 to 100 bases.

[0027] In the example illustrated in FIG. 1 , the target DNA sequence 112 having a mutation of Mutant Type is amplified, while the target DNA sequence 113 of Wild Type is not amplified. Therefore, PCR products 115, 116, and 117 amplified from the multiple target DNA sequences 112 having mutations are developed in the capillary. The molecular lengths of the PCR products 115, 116, and 117 are different because their stuffer sequences are different. Therefore, they are separated by capillary electrophoresis, and signals corresponding to different times are detected. This is represented by an electropherogram 120.

[0028] 1 alone can confirm the presence or absence of mutation, but cannot perform quantitative testing. In this embodiment, in order to perform quantitative testing, the intensity S of the mutation-derived signal, which is a fluorescent signal emitted from the mutated portion of the sample, is ＭＴ S for a reference value of strength higher than ＭＴ Calculate the ratio.

[0029] One example of this is a point mutation ratio detection method having a measurement step and a ratio calculation step. Here, the measurement step uses an electrophoresis device to measure the intensity S of a mutation-derived signal, which is a fluorescent signal emitted from a mutated portion of a sample. ＭＴ and the intensity S of the wild-derived signal, which is the fluorescent signal emitted from the part of the sample other than the mutated part. ＷＴ Among them, at least S ＭＴ This measurement step can be performed by capillary electrophoresis as described with reference to Fig. 1 or by an embodiment as described with reference to Fig. 2 and subsequent figures.

[0030] The ratio calculation step is performed by the S ＭＴ The S for a reference value of strength higher than ＭＴ The reference value is, for example, a step of calculating the ratio of the intensity S ＷＴ The reference value is a very high signal intensity, so the upper limit of the dynamic range of the measurement of the fluorescent signal of the electrophoresis device is set to a predetermined value or more. In this way, the point mutation ratio detection method can detect the intensity S of the mutation-derived signal. ＭＴThe relative ratio can be calculated, allowing quantitative testing.

[0031] The maximum saturation value of the mutant type is the intensity of the fluorescent signal obtained when 100% of the cells are derived from the mutant type (derived from the mutation). The maximum saturation value may be measured in advance using cultured cells obtained by cloning living cells of the mutant type that have undergone the corresponding point mutation. In other words, the maximum saturation value (reference value) may be measured in advance independently of the measurement step described above.

[0032] The upper limit of the dynamic range can be, for example, 200,000 [ADU] or more, 400,000 [ADU] or more, 600,000 [ADU] or more, 800,000 [ADU] or more, or 1,000,000 [ADU] or more, but is not limited to these. Therefore, the dynamic range can be, for example, 0 to 200,000 [ADU], 0 to 400,000 [ADU], 0 to 600,000 [ADU], 0 to 800,000 [ADU], or 0 to 1,000,000 [ADU], but is not limited to these. The dynamic range can also be, for example, 0 to 1,500,000 [ADU]. When the upper limit or dynamic range is set in this way, the S ＭＴ Even if a reference value of intensity higher than the reference value is measured, the intensity is unlikely to saturate, so more accurate quantitative testing can be performed.

[0033] In the point mutation rate detection method according to this embodiment, examples of the above-mentioned quantitative testing include calculating the ratio of mutant type to wild type, and calculating the ratio of mutant type to the saturation maximum value. To calculate the ratio of mutant type to wild type, PCR amplification may be performed on a DNA fragment containing the wild type DNA target sequence 113. Below, several examples of the point mutation rate detection method according to this embodiment that employs these techniques will be described.

[0034] 2 is an explanatory diagram of an analytical reaction in a first embodiment of the present invention. In this embodiment, a technique is described that enables the amount of mutations and wild type to be measured and the ratio of mutant type to wild type to be quantified when up to three types of mutations occur relative to the wild type at one point mutation site.

[0035] As shown in FIG. 2 , sample tube 201 contains DNA target sequences 222, 223, and 224 that have a point mutation at a specific location in the genome. In this example, DNA target sequences 222, 223, and 224 contain a mutation, and DNA target sequence 225 is a wild-type sequence. The point mutations in the respective target sequences are cytosine, guanine, thymine, and adenine. As described with reference to FIG. 1 , four types of LPOs 202, 203, 204, and 205 that can form complementary strands for each of these four different target sequences are coexistent. The bases at the 5′ ends of LPOs 202, 203, 204, and 205 are guanine, cytosine, adenine, and thymine, respectively. Furthermore, the stuffer sequences of these LPOs are such that the stuffer sequence of LPO 202 is the shortest, and the stuffer sequence of LPO 205 is the longest. The difference between the respective stuffer sequences is 15 bp, and the difference between the maximum and minimum base lengths is 45 bp. The reason why 15 bp is suitable for the spacing between the linked probes is that if the difference is smaller than 15 bp, the peaks will be close to each other during electrophoresis, making it difficult to separate the two. In particular, when detecting the ratio MT / WT of the mutant type to the wild type of 1% or 0.1%, it is desirable that the distance between the two be 15 bp or more. However, the difference between the stuffer sequences is not limited to 15 bp. The ratio MT / WT of the mutant type to the wild type can be determined by measuring the intensity S of the mutation-derived signal, which is a fluorescent signal emitted from the mutated portion of the sample, using an electrophoresis device. ＭＴ and the intensity S of the wild-derived signal, which is the fluorescent signal emitted from the part of the sample other than the mutated part. ＷＴ From the above, ＭＴ and the above S ＷＴ Ratio S ＭＴ / S ＷＴThis can be suitably calculated by calculating

[0036] Furthermore, since the same RPO206 may be used for detecting a single point mutation in this reaction, the PCR reaction can be performed using one type of RPO206. Since the ligation reaction proceeds only when the 3' end of LPO is complementary to the point mutation sequence in the target sequence, the information on the "point mutation" can be converted into information on the "length" of the base length of the stuffer sequence.

[0037] It is worth noting here that the MLPA method used for conventional point mutation detection only measures mutations in mutations, not wild-type mutations. The MLPA method used for conventional point mutation detection is based on the signal from a control sample that is said to contain 1% of the mutation amount included in the kit. The signal amount of the control sample for a specific point mutation is compared with the signal amount from the sample to be measured, and if the signal amount from the sample is greater than the signal amount from the control sample, it is judged as "mutation present," and if it is smaller, it is judged as "absence of mutation." In addition, the manual clearly states that the MLPA method used for conventional point mutation detection can only state the presence or absence of mutation, and cannot quantify MT / WT (MRC Holland Product Description SALSA® MLPA® Probemix P520-A2 MPN mix 2).

[0038] The primary reason for the inability to calculate MT / WT is that the kit does not include a WT LPO probe. The reason the kit cannot include a WT LPO probe is that the measurement of the WT LPO probe reaches saturation with conventional capillary sequencers, making accurate measurements impossible. In other words, the dynamic range of conventional capillary sequencers is insufficient. Specifically, while the maximum signal measurement value (saturation measurement value) of conventional capillary sequencers is 32,767 [ADU], the signal from the WT significantly exceeds this maximum measurement value. In other words, substantial saturation occurs, making it impossible to accurately obtain the WT signal value. This makes it difficult to measure MT / WT. More specifically, since the signal from 1% MT is approximately 2,000 [ADU], the signal from 100% WT is expected to be approximately 200,000 [ADU]. However, the saturation signal intensity of current capillary electrophoresis is 32,767 [ADU], so 100% WT cannot be measured.

[0039] In contrast, capillary electrophoresis measurement methods with a high dynamic range reported in recent papers have been reported to be capable of expanding the dynamic range. More specifically, the dynamic range, which was previously less than three orders of magnitude, has been expanded to three or even four orders of magnitude. Therefore, by using this technology, signals derived from wild types can be detected without saturation. Therefore, it is possible to calculate the MT / WT ratio. In this example, the capillary electrophoresis analysis technology with a dynamic range expanded to three or more orders of magnitude is called HiDy.

[0040] There are other advantages to being able to measure wild type. Another reason why conventional kits cannot quantify mutant type is the variability in the amount of sample introduced into the capillary during injection. Conventional methods measure only mutant type for a single point mutation, so the signal value includes variability in the amount of sample introduced during injection. However, if both mutant type and wild type can be injected into the same capillary during the same injection, the MT / WT ratio can be calculated to offset and cancel the injection variability. In this example, the wild type is measured and the MT / WT ratio is calculated, enabling more accurate and quantitative measurements. Furthermore, in this example, even in samples in which the point mutation status is unknown, signals for both the mutant type and the wild type can be detected simultaneously in a single electrophoresis, thereby achieving the effect of quantitatively, simply, quickly, and inexpensively detecting the contamination rate of abnormal cells such as cancer cells.

[0041] In electrophoresis, peaks 212, 213, 214, and 215 can be confirmed on an electropherogram 250. Each peak corresponds to a DNA target sequence 222, 223, 224, and 225. MT / WT can be calculated by dividing the signal values ​​of peaks 212, 213, and 214 derived from mutations by the signal value of peak 215, which corresponds to the wild type.

[0042] While this example demonstrates the ability to identify and quantify four different bases for a single point mutation, this does not impose any limitations on the number of multiplexes for point mutations. As shown in Figure 1, the number of point mutations to be measured can be multiplexed. Generally, fragment analysis using a capillary sequencer involves separation in the range of 100 to 500 bp. If a stuffer sequence is designed with a base spacing of 15 bp, the number of measurable mutations is (500 - 100) bp ÷ 15 bp, meaning that approximately 25 mutations can be measured and analyzed in a single electrophoresis run. Furthermore, if the PCR primer sequences in the LPO and RPO can be amplified with, for example, six different color primer sets, multicolor analysis becomes possible. This allows for simultaneous measurement of 25 mutations x 6 colors = 150 mutations in a single electrophoresis run.

[0043] Next, a second embodiment of the present invention will be described with reference to FIG. 3. FIG. 3 is an explanatory diagram of the analytical reaction of the second embodiment of the present invention. This embodiment differs from the first embodiment in the following respects. In the first embodiment, there were three types of point mutations in the DNA target sequence, whereas in the second embodiment, there are two types of point mutations. Specifically, DNA target sequences 322 and 325 contain adenine, whereas DNA target sequence 323 contains a guanine point mutation and DNA target sequence 324 contains a thymine point mutation. Note that DNA target sequences 325 and 322 are wild type, and the other DNA target sequences 324 and 323 are mutant type.

[0044] In the second example, as in the first example, four types of LPOs 302, 303, 304, and 305 and one type of RPO 306 are added to a single sample tube 301 containing a mixture of DNA target sequences. While LPO and RPO hybridize to the DNA target sequence, the G at the 3' end of LPO 302 is not complementary to the point mutation A in DNA target sequence 322, preventing complete hybridization. Therefore, the ligation reaction following hybridization does not allow the 3' end of LPO 302 to be ligated to the 5' end of RPO 306. Therefore, PCR amplification derived from DNA target sequence 322 does not proceed in the PCR reaction in the next step.

[0045] Therefore, in fragment analysis by capillary electrophoresis, the detected peaks are peaks 313, 314, and 315, which correspond to DNA target sequences 323, 324, and 325, as shown in electropherogram 350. Peak 312, which corresponds to DNA target sequence 322, is not detected. By comparing these peak values, it is possible to determine not only the presence or absence of mutations, but also to calculate the MT / WT ratio for each mutation. Therefore, quantitative measurements can be performed.

[0046] Although this example describes the detection of a single point mutation, as can be easily understood, this method enables multiplex detection of multiple point mutations, and it is possible to measure mutations at 40 or more locations simultaneously in a single electrophoresis run.

[0047] Next, a third embodiment of the present invention will be described with reference to FIG. 4. FIG. 4 is an explanatory diagram of an analytical reaction in the third embodiment of the present invention. This embodiment differs from the first embodiment in the following respects. In the first embodiment, there were three types of point mutations in the DNA target sequence, whereas in the third embodiment, there is only one type of point mutation. Specifically, DNA target sequences 422, 423, and 425 contain adenine, whereas DNA target sequence 424 contains a thymine point mutation. Note that DNA target sequences 422, 423, and 425 are wild type, having adenine at the corresponding mutation site, while the other DNA target sequence 424 is mutant type, having a thymine point mutation.

[0048] In the third example, similar to the first example, four types of LPOs 402, 403, 404, and 405 and one type of RPO 406 are added to a single sample tube 401 with a mixture of DNA target sequences. LPO and RPO hybridize to the DNA target sequence, but complete hybridization is not possible because the G at the 3' end of LPO 402 is not complementary to the point mutation A in DNA target sequence 422. Furthermore, the C at the 3' end of LPO 403 is not complementary to the point mutation A in DNA target sequence 423, so complete hybridization is not possible.

[0049] Therefore, the 3' end of LPO and the 5' end of RPO cannot be ligated in the ligation reaction after hybridization in the DNA target sequences 422 and 423. Therefore, in the PCR reaction of the next step, PCR amplification derived from the DNA target sequences 422 and 423 does not proceed.

[0050] Therefore, in fragment analysis by capillary electrophoresis, the detected peaks are peaks 414 and 415, which correspond to DNA target sequences 424 and 425, as shown in electropherogram 450. Peaks 412 and 413, which correspond to DNA target sequences 422 and 423, are not detected. By comparing these peak values, it is possible to determine not only the presence or absence of mutations, but also to calculate the MT / WT ratio for each mutation. Therefore, quantitative measurements can be performed.

[0051] Although this example describes the detection of a single point mutation, as can be easily understood, this method enables multiplex detection of multiple point mutations, and it is possible to measure mutations at 40 or more locations simultaneously in a single electrophoresis run.

[0052] Next, a fourth embodiment of the present invention will be described with reference to FIG. 5. FIG. 5 is an explanatory diagram of the analytical reaction of the fourth embodiment of the present invention. Unlike the first to fourth embodiments, in which ligation and hybridization reactions are performed in a single sample tube, this embodiment differs in that the initial DNA 501 is divided into four parts and added in equal amounts to four different sample tubes 511, 512, 513, and 514. Another difference is that different LPOs 531, 532, 533, and 534 are added to each of these parts. The 3' ends of the illustrated LPOs 531, 532, 533, and 534 are guanine, cytosine, adenine, and thymine, respectively. Furthermore, the same RPOs 541, 542, 543, and 544 are added to each tube. Note that, for simplicity, this embodiment illustrates one point mutation; however, in actuality, there are as many sets of LPOs and RPOs as there are point mutations in the reagents. In other words, the sample tube 514 contains as many LPO534 fragments for the corresponding wild types as there are wild types. In addition, the 3' end of LPO534 is not always thymine, but LPO534 is designed based on the sequence information of the wild type in each point mutation. In other words, when this method is applied, the peak of the wild type-derived DNA fragment in one point mutation becomes longer than the peaks derived from the other three DNA fragments (the intensity of the wild type-derived signal S ＷＴ (The peak of the wild type-derived DNA fragment elutes the slowest compared to the other three peaks.)

[0053] On the other hand, point mutation information other than the wild type is assigned to the remaining three LPOs 531, 532, and 533. The sequence information for these point mutations need only be different and exclusive. Alternatively, a useful method is to fix the 3' ends of LPOs 531, 532, 533, and 534 added to sample tubes 511, 512, 513, and 514 to guanine, cytosine, adenine, and thymine, respectively, without considering the wild type state. It is useful to perform these fixation states on multiple point mutation groups.

[0054] By adjusting the length of the stuffer sequence, the base length of each probe can be designed. Therefore, the base length of the PCR product identified by electrophoresis can be easily sized, and it is possible to distinguish which peak is the wild type and which peak is the mutant type. The signals of the point mutation and the wild type are calculated from the assigned peaks, and MT / WT are quantified (the intensity of the mutation-derived signal S ＭＴ and the strength of the wild-derived signal S ＷＴ Ratio S ＭＴ / S ＷＴ This makes it possible to quantify the MT / WT ratio. In this example, for each point mutation, LPO534, which corresponds to the wild type, has the longest stuffer sequence. This avoids the effect of sloping, in which signal intensity decreases with increasing base length. In other words, by grouping electrophoresis for each point mutation, for example, if the differential base length of the stuffer sequence is 15 bp, the difference in base length within the point mutation can be reduced to 15 bp x (4 bases - 1) = 45 bp. This enables more accurate calculation of MT / WT. Furthermore, to make the detection of low-frequency point mutations even slightly more advantageous, it is effective to shorten the PCR fragments associated with the mutations and run them earlier.

[0055] The advantage of this embodiment over the first to third embodiments is that by dividing LPO according to the base type at the 3' end, competitive hybridization to the target DNA sequence that may occur between LPO probes can be avoided, thereby suppressing competition between LPO probes and improving the reliability of the MT / WT numerical value.

[0056] The 3' ends of LPOs 531, 532, 533, and 534 contain guanine, cytosine, adenine, and thymine, respectively, and thus hybridize with the cytosine, guanine, thymine, and adenine of the DNA target sequences 521, 522, 523, and 524 dispensed into each sample tube. Furthermore, the DNA target sequences 521, 522, 523, and 524 hybridize with RPOs 541, 542, 543, and 544. Ligation is performed separately for sample tubes 511, 512, 513, and 514. Then, sample tubes 511, 512, 513, and 514 are combined into a single sample tube and PCR is performed. After PCR, each fragment can be separated by molecular weight size within a single capillary by capillary electrophoresis. Specifically, the electropherogram 551 shown in Figure 5 can be obtained. As shown in the electropherogram 551, MT / WT can be calculated for each of Mutations 1, 2, 3, . . . N.

[0057] In this example, fluorescent primers are used to fluorescently label the PCR products during PCR. These fluorescent primers are not limited to fluorescent dyes of one color; labeling can also be performed with fluorescent dyes of different wavelengths for each sample tube. It is also possible to design probe groups within the LPO and RPO so that each point mutation can be labeled with a different fluorescent dye.

[0058] Next, a fifth embodiment of the present invention will be described with reference to FIG. 6 . FIG. 6 is an explanatory diagram of an analytical reaction in the fifth embodiment of the present invention. Compared to the fourth embodiment, this embodiment is effective in reducing the number of divided sample tubes and reducing the cost required per electrophoresis run. Point mutations do not necessarily occur in the three types of bases other than the wild type; in most cases, they can be limited to two or fewer types. In particular, cancer is highly diverse, and different point mutations occur in organ-specific forms, such as lung cancer, breast cancer, and pancreatic cancer. Therefore, limiting the number of point mutations to be detected to two or fewer types is extremely useful in increasing the number of multiplexes detectable from a single capillary in a single electrophoresis run.

[0059] In this example, the initial DNA 601 is divided into three parts and added in equal amounts to three different sample tubes 612, 613, and 614. Different LPOs 632, 633, and 634 are added to each of these. The 3' ends of the illustrated LPOs 632, 633, and 634 are cytosine, adenine, and thymine, respectively. The same RPOs 642, 643, and 644 are added to each tube. While this example illustrates one point mutation for simplicity, the actual reagents contain the same number of LPO and RPO sets as the number of point mutations. In other words, the sample tube 614 contains the same number of corresponding wild type LPOs 634 as the number of wild types. Furthermore, the 3' end of LPO 634 is not always thymine; LPO 634 is designed based on the sequence information of the wild type for each point mutation. In other words, when this method is applied, the peak of the wild type-derived DNA fragment in one point mutation becomes longer than the peaks of the other three DNA fragments (the intensity of the wild type-derived signal S ＷＴ (The peak of the wild type DNA fragment is the fragment with the slowest elution time compared to the other three peaks.)

[0060] Meanwhile, the remaining two LPOs 632 and 633 are assigned point mutation information other than the wild type. The information needs only to be different and exclusive. In sample tubes 612, 613, and 614, the LPOs and RPOs hybridize with DNA target sequences 622, 623, and 624, respectively. The LPOs and RPOs are linked by a ligation reaction and amplified by PCR. The base length of each probe can be designed by adjusting the length of the stuffer sequence.

[0061] Therefore, the base length of the PCR product identified by electrophoresis can be easily determined, and it is possible to distinguish which peak is the wild type and which peak is the mutant type. Specifically, an electropherogram 651 shown in FIG. 6 can be obtained. As shown in the electropherogram 651, MT / WT can be calculated for each mutation 1, 2, 3, ... N. In other words, the signals of the point mutation and wild type are calculated from the assigned peaks, and MT / WT is quantified (the intensity S of the mutation-derived signal). ＭＴ and the strength of the wild-derived signal S ＷＴ Ratio S ＭＴ / S ＷＴ (quantification). In this example, for each point mutation, LPO634, which corresponds to the wild type, has the longest stuffer sequence. This makes it possible to avoid the effect of sloping, in which signal intensity decreases with increasing base length. In other words, by grouping electrophoresis for each point mutation, for example, if the differential base length of the stuffer sequence is 15 bp, the difference in base length within the point mutation can be reduced to 15 bp x (3 bases - 1) = 30 bp. This makes it possible to calculate MT / WT more accurately in this example. Furthermore, in order to make the detection of low-frequency point mutations even slightly more advantageous, it is effective to shorten the PCR fragments related to the mutations and run them earlier.

[0062] Next, a sixth embodiment of the present invention will be described with reference to FIG. 7. FIG. 7 is an explanatory diagram of an analytical reaction in the sixth embodiment of the present invention. In this embodiment, the number of tubes to be divided is limited to two. One sample tube 713 is assigned to a reaction of a representative point mutation to be detected, and the other sample tube 714 is assigned to a reaction of the wild type. The advantage of this method is that by limiting the number of sample tubes to be divided to two, the reagent costs and labor required for the reaction are reduced. Note that base information regarding the point mutations of LPO733 and 734 can be obtained from an existing database. In addition, since competitive hybridization between wild type LPO and mutant type LPO can be avoided, the ratio MT / WT of wild type and mutant type molecules present in the sample (the intensity of the mutation-derived signal S) can be calculated. ＭＴ and the strength of the wild-derived signal S ＷＴ Ratio S ＭＴ / S ＷＴ ) can be detected more accurately.

[0063] In this example, the initial DNA 701 is divided into two and added in equal amounts to two different sample tubes 713 and 714. Different LPOs 733 and 734 are added to each tube. The 3' ends of the illustrated LPOs 733 and 734 are adenine and thymine, respectively. Furthermore, identical RPOs 743 and 744 are added to each tube for the point mutation. While one point mutation is illustrated in this example for simplicity, LPO and RPO are reagents for a probe group containing multiple point mutations. Furthermore, not all of the 3' ends of LPO 734 are thymine. A probe group having a sequence complementary to the point mutation group corresponding to the wild type is selected for the 3' end of LPO 734. Therefore, wild type bases reflecting the information of each point mutation are placed at the 3' ends of multiple LPOs used to detect the wild type. These base sequences can be either adenine, guanine, cytosine or thymine.

[0064] By adjusting the length of the stuffer sequence, the base length of each probe can be designed. Therefore, the base length of the PCR product identified by electrophoresis can be easily sized, and it is possible to distinguish which peak is the wild type and which peak is the mutant type. Specifically, an electropherogram 751 shown in FIG. 7 can be obtained. As shown in the electropherogram 751, MT / WT can be calculated for each mutation 1, 2, 3, ... N. In other words, the signals of the point mutation and wild type are calculated from the assigned peaks, and MT / WT is quantified (the intensity of the mutation-derived signal S ＭＴ and the strength of the wild-derived signal S ＷＴ Ratio S ＭＴ / S ＷＴ (quantification). In this example, for each point mutation, it is considered that LPO734, which corresponds to the wild type, has a longer stuffer sequence than LPO733. In other words, a shorter stuffer sequence is assigned to the mutant type. The reason for this is that the abundance ratio of the mutant type relative to the wild type is generally low. In particular, the amount of the mutant type is small in micromutations. On the other hand, it is known that in capillary electrophoresis, the longer the length of the DNA fragment, the lower the amount introduced into the capillary. Therefore, in order to better detect even the slightest micromutations, a technique of arranging a short stuffer sequence for point mutations is useful.

[0065] However, if an excessive amount of wild type is present, it may cause background noise in the electrophoretic range of molecular weights smaller than that of the wild type. In this case, it is useful to conversely place a stuffer sequence shorter in LPO734, which corresponds to the wild type, than in LPO733. Furthermore, it is useful to group electrophoresis data by each point mutation to more directly compare the wild type and mutant type.

[0066] If the difference in base length of the stuffer sequence is 15 bp, the difference in base length within the point mutation can be limited to 15 bp x (2 bases - 1) = 15 bp. This allows for more accurate MT / WT calculations in this example. This example also increases the number of point mutation genes that can be detected in a single electrophoresis run. This example shortens the base length of low-frequency point mutations and wild type genes to 15 bp, which has the effect of almost negligible sloping effects.

[0067] Next, a seventh embodiment of the present invention will be described with reference to Fig. 8. Fig. 8 is an explanatory diagram of an analytical reaction in the seventh embodiment of the present invention. In this embodiment, a measurement method for detecting only mutant types without using wild types will be described.

[0068] 10 to 100 μg of sample DNA 801 is added to one sample tube 813. LPO 833 and RPO 843, which hybridize to a DNA target sequence 832 with a point mutation in DNA 801, are added. Note that the illustration shows the behavior for one point mutation sequence, but in an actual reaction, multiple point mutation sequences exist in DNA 801. The explanation here is merely given using one point mutation sequence as an example.

[0069] The 3' end of the illustrated LPO833 is an adenine. RPO843 is also added. A point mutation sequence, point mutation thymine 823, exists in the DNA target sequence 832. Point mutation thymine 823 forms a complementary strand with the adenine at the 3' end of LPO833. Therefore, the gap between LPO833 and RPO843 can be ligated with a ligase enzyme, and LPO833 and RPO843 form a single DNA strand.

[0070] Although one point mutation is illustrated in this example for simplicity, LPO and RPO are probe group reagents containing multiple point mutations. Therefore, the 3' end of LPO833 is not always adenine for every point mutation site. Depending on the actual point mutation, one of the four bases, adenine, guanine, cytosine, or thymine, is linked to the 3' end of each LPO833.

[0071] By adjusting the length of the stuffer sequence, the base length of each probe can be designed. Therefore, the base length of the PCR product identified by electrophoresis can be easily determined, and an electropherogram 851 shown in Figure 8 can be obtained. As shown in the electropherogram 851, multiple peaks 852, 853, 854, and 855 can be obtained for each mutation 1, 2, 3, ..., N.

[0072] Graph 860 shown in FIG. 8 shows the normalized signal intensity of a mutation when the cell ratio of the input wild type to mutant type is changed for a certain point mutation. The normalized signal intensity is calculated by dividing the signal amount of the mutant type at different cell ratios by the signal amount of the mutant type when the mutant type ratio is 100%. The proportion of the mutant type in the cell and the signal amount from the mutant type are in a proportional relationship. For this reason, if the signal intensity when the mutant type is 100% is measured in advance independently of the measurement by electrophoresis described above, the signal intensity of the mutation-derived signal S ＭＴ By comparing the signal intensity derived from a point mutation measured in a certain experiment with the saturated maximum value of the signal intensity, the content (proportion) of mutant cells in the sample used can be estimated. In other words, quantitative testing can be performed in this example as well.

[0073] This technique cannot be achieved with conventional capillary sequencers. The reason is that if the cell ratio is 100% mutant type, the peak signal will saturate within the conventional dynamic range, making accurate measurement impossible. More specifically, the signal detected with 1% point mutations is approximately 3,000 [ADU], so with 100% point mutations, it will be approximately 300,000 [ADU]. The upper saturation limit detectable with conventional capillary sequencers is 32,767 [ADU], resulting in saturation. Therefore, to perform quantitative point mutation measurement using LPO and RPO for mutant types, it is recommended to use a capillary sequencer with a high dynamic range. Examples of a high dynamic range include, but are not limited to, an upper limit of 200,000 [ADU] or more, as described above, and also, for example, 0 to 200,000 [ADU].

[0074] Next, an eighth embodiment of the present invention will be described with reference to FIG. 9 . FIG. 9 is a diagram showing analytical reaction analysis results in the eighth embodiment of the present invention. In graph 901 shown in FIG. 9 , the horizontal axis of the graph indicates the ratio of the amount of mutant type to the wild type for a certain point mutation, where the amount of wild type is fixed at, for example, 100 μg. The vertical axis of the graph indicates the signal intensity of the mutant type peak relative to the MT / WT cell mixture ratio.

[0075] In graph 901, the triangles in the figure indicate that in measurements performed with a conventional capillary sequencer (CE), the signal saturates when the MT / WT cell ratio is between 10% and 100%. In other words, with a conventional capillary sequencer, the signal saturates when 100 μg of mutant type DNA is reacted, making accurate measurements impossible. On the other hand, as indicated by the circles in the figure, with a HiDy capillary sequencer (HiDy CE) with a high dynamic range, it can be confirmed that the signal from the mutant type increases proportionally even when the MT / WT cell ratio increases from 10% to 100%. Therefore, to detect mutation ratios over a wide range from 0.1% to 100%, it is recommended to use a HiDy capillary sequencer with a high dynamic range.

[0076] Next, graph 902 shown in FIG. 9 will be described. In graph 902, the horizontal axis of the graph also shows the ratio of the amount of mutant type to the amount of wild type for a certain point mutation, where the amount of wild type is fixed at, for example, 100 μg. The vertical axis of the graph is normalized by dividing the signal intensity of the mutant type peak relative to the MT / WT mixture ratio of cells by the signal value of the mutant type at 100% MT / WT.

[0077] Graph 902 shows the measurement results when only the mutant type was targeted, without using the LPO and RPO probes for the wild type, as described in the seventh example. Therefore, because there is no signal derived from the wild type, signal normalization is performed solely by division within the mutant type. Note that the seventh example has the problem that the signal values ​​of the 100% MT / WT mutant type used for normalization and the signal values ​​of the mutant type with a different MT / WT cell mixture ratio are electrophoresed separately, making it impossible to correct for variations in sample injection during electrophoresis.

[0078] 9, the mutant type and wild type were paired for one point mutation, and LPO and RPO probes were placed on the DNA target sequence, and measurements were performed. The difference from graph 902 is that signals from the mutant type and wild type can be measured simultaneously during each electrophoresis run.

[0079] The vertical axis of the graph in graph 903 can be calculated for each electrophoresis by dividing the signal derived from the mutant type by the signal derived from the wild type. Therefore, there is an advantage that the variation in sample injection during electrophoresis can be corrected for each electrophoresis. This directly leads to improvement in measurement accuracy. In graph 902, the linearity is R ２ = 0.9849, whereas in graph 903 the linearity is R ２ = 1, indicating that simultaneous measurement of the mutant type and wild type is more accurate. In particular, as the MT / WT ratio decreases from 1% to 0.1%, the normalized signal values ​​in graph 902 deviate from the ideal linear approximation line, whereas graph 903 shows a better fit. This indicates that simultaneous measurement of signals from the mutant type and wild type, especially at low MT / WT, allows for more sensitive and reliable measurements. Therefore, it can be said that it is preferable to measure the mutant type and wild type in pairs when detecting mutations using a capillary sequencer with a high dynamic range.

[0080] The mutation ratio detection method according to the present invention has been described in detail above through examples (embodiments), but the present invention is not limited to the above examples and includes various modifications. For example, the above examples have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those including all of the described configurations. Furthermore, it is possible to replace part of the configuration of one example with the configuration of another example, or to add the configuration of another example to the configuration of one example. Furthermore, it is possible to add, delete, or replace part of the configuration of each example with other configurations.

[0081] 101, 102 Sequence 103 Point mutation guanine 104, 105 Sequence 106 Cytosine 107 Stuffer sequence 109, 110 Primer sequence 111 Stuffer sequence 112, 113 DNA target sequence 114 Normal base cytosine 115-117 PCR product 120 Electropherogram 201 Sample tube 202-205 LPO 206 RPO 212-215 Peak 222-225 DNA target sequence 250 Electropherogram 301 Sample tube 302-305 LPO 306 RPO 312-315 Peak 322-325 DNA target sequence 350 Electropherogram 401 Sample tube 402-405 LPO 406 RPO 412-415 Peak 422-425 DNA target sequence 350 Electropherogram 501 DNA 511-514 Sample tube 521-524 DNA target sequence 531-534 LPO 541-544 RPO 551 Electropherogram 601 DNA 612-614 Sample tube 622-624 DNA target sequence 632-634 LPO 642-644 RPO 651 Electropherogram 701 DNA 713, 714 Sample tube 733, 734 LPO 743, 744 RPO 751 Electropherogram 801 DNA 813 Sample tube 823 Point mutation thymine 832 DNA target sequence 833 LPO 843 RPO 851 Electropherogram 852-855 Peak 860 Graph 901-903 Graph

Claims

1. used in multiplex ligation-dependent probe amplification (MLPA) measurement, Using an electrophoresis apparatus, the intensity S of a mutation-derived signal, which is a fluorescence signal emitted from a mutated portion of a sample ＭＴ and the intensity S of a wild-derived signal, which is a fluorescence signal emitted from a portion other than the mutated portion of the sample ＷＴ Among them, at least the S ＭＴ measurement step of measuring the said S ＭＴ the ratio of the said S to a reference value of higher strength ＭＴ a ratio calculation step of calculating the ratio having the upper limit of the dynamic range of the measurement for the fluorescence signal of the electrophoresis apparatus is a predetermined value or more, The reference value is the saturation maximum value of the S ＭＴ measured in advance independently of the measurement process, and The ratio calculation step calculates the ratio of S obtained in the measurement step from the saturation maximum value ＭＴ to A method for detecting a point mutation ratio, characterized in that.

2. used in multiplex ligation-dependent probe amplification (MLPA) measurement, Using an electrophoresis apparatus, the intensity S of a mutation-derived signal, which is a fluorescence signal emitted from a mutated portion of a sample ＭＴ and the intensity S of a wild-derived signal, which is a fluorescence signal emitted from a portion other than the mutated portion of the sample ＷＴ a measuring step of measuring the said S ＭＴ a ratio of the said S to a reference value of higher strength ＭＴ a ratio calculation step of calculating the ratio having the upper limit of the dynamic range of the measurement for the fluorescence signal of the electrophoresis apparatus is a predetermined value or more, wherein the reference value is the S ＷＴ and The ratio calculation step calculates the ratio S ＭＴ and the S ＷＴ to obtain the ratio S ＭＴ and the S ＷＴ of S ＭＴ / S ＷＴ from the S In the MLPA measurement, the sample is divided into three or two parts, and in one of them, the S ＷＴ is generated independently and selectively, and in the remaining two or one, the S ＭＴ is generated independently and selectively A method for detecting a point mutation ratio, characterized in that.

3. (Deleted)

4. In Claim 1 or Claim 2, the upper limit of the dynamic range is 200,000 [ADU] or more, 400,000 [ADU] or more, 600,000 [ADU] or more, 800,000 [ADU] or more, or 1,000,000 [ADU] or more A method for detecting a point mutation ratio, characterized in that.

5. In Claim 1 or Claim 2, the dynamic range is 0 to 200,000 [ADU], 0 to 400,000 [ADU], 0 to 600,000 [ADU], 0 to 800,000 [ADU], or 0 to 1,000,000 [ADU] A method for detecting a point mutation ratio, characterized in that.