Point variation ratio detection method

Abstract

Provided is a point variation ratio detection method capable of performing a quantitative test. The point variation ratio detection method according to the present invention is used for multiple ligation probe amplification (MLPA) measurement, and comprises: a measurement step for measuring a point variation ratio by using an electrophoresis device; measuring at least an intensity SMT derived from a variation signal, which is a fluorescence signal emitted from a variation portion of the sample, and an intensity SWT derived from a wild signal, which is a fluorescence signal emitted from a portion other than the variation portion of the sample; and a ratio calculation step for calculating the ratio of the SMT to a reference value having a higher intensity than the SMT, the upper limit of the dynamic range of the measurement of the fluorescence signal by the electrophoresis device being equal to or greater than a predetermined value.

Description

Technical Field

[0001] The invention relates to a point variation ratio detection method. Background Art

[0002] It is known that among the approximately 20,000 genes, there are only a few hundred oncogenes and cancer suppressor genes. Gene mutations that cause cancer in humans are being discovered, but their number is expected to remain at around a few hundred. In fact, the number of genes carried on the OncoGuide NCC OncoPanel for next-generation sequencers jointly developed by the National Cancer Center of the National Research and Development Corporation and Sysmex, which was included in the insurance in 2019, is 124. In addition, the number of genes in Foundation-one CDx developed by Foundation Medicine in the United States is 324. From these conditions, it can be seen that cancer diagnosis by detecting the behavior of hundreds of limited genes is advancing. Point mutations are particularly important among these genes. The reason is that cancer develops and develops due to the accumulation of random point mutations.

[0003] On the other hand, since these tests use a massively parallel method of reading genes, such as next-generation sequencers, the cost is high. In fact, the cost of the above two cancer gene panel tests is 560,000 yen, which is a heavy burden for ordinary patients.

[0004] Therefore, a technology that can measure about 100 limited genes cheaply, quickly and accurately is required. In the current qPCR, the number of genes that can be measured in one pass through the tube is limited to the multicolorization of fluorescent pigments, and the maximum is 5 colors, that is, 5 genes. On the other hand, the cost of the next generation sequencer is high. On the other hand, the capillary sequencer can separate and detect PCR products according to molecular weight by performing electrophoresis after PCR. Therefore, it is possible to obtain information on the "length of the DNA molecule" that the previous qPCR did not have.

[0005] The characteristics of the capillary sequencer are utilized in personal identification by short tandem repeat analysis and multiplex ligation-dependent probe amplification (MLPA). These technologies are to extract and attach information on the molecular length to the amplification product by capillary electrophoresis after PCR amplification, thereby implementing judgments and diagnoses that cannot be achieved by simple PCR alone. In personal identification, "separation based on DNA molecule length" is performed by focusing on the repetitive arrangement of short tandem repeats that are unique to genomic DNA molecules. On the other hand, MLPA artificially implements "separation based on DNA molecule length" from the outside by actively attaching PCR probes of different lengths from the outside. It can be said that the former uses the inherent characteristics of biological samples, which is in contrast to the latter that artificially designs molecules from the outside.

[0006] The MLPA method is a technology developed by Shouten in the Netherlands. It is a method of performing PCR by changing the length of the probe and developing its product by capillary electrophoresis. The basic technology of the MLPA method is described in Patent Document 1. In addition, companies such as MRC Holland established by Shouten are selling reagents that have been converted into MLPA kits. The MLPA method can detect copy number changes (deletions, duplications), DNA methylation, gene expression, and point mutations, which are the most important for cancer diagnosis. Specific details about the detection of point mutations by the MLPA method are described in Non-Patent Document 1. In addition, Non-Patent Document 2 is a product description (instruction manual) of a commercially available MLPA kit that can detect point mutations in 8 genes such as JAK2, which are the main cause of myeloproliferative tumors, with a high sensitivity of 1% to 5%. Among them, regarding point mutations, this kit cannot be used for quantitative measurement, but only for qualitative measurement and attention. Qualitative means that if the signal is larger than the combined DNA compared to the control such as the combined DNA attached to the kit, the judgment of "there is a mutation" is implemented, and if the signal is small, the judgment of "no mutation" is implemented.

[0007] In addition, in order to detect the presence or absence of mutations, it is necessary to change the length of the stuffing sequence contained in the left LPO (Left Probe Oligonucleotide) of the two probes hybridized to the target sequence in the MLPA method. This is because LPO plays the role of detecting the state of mutation, and it is necessary to convert the state of the mutation into information on the molecular length in electrophoresis.

[0008] Here, a kit for detecting two single base substitutions with different sequences for one point mutation is also on sale, and this kit is described in non-patent document 3. Specifically, regarding the gene IDH2, the wild type is guanine. In contrast, a probe with a length of 151 bases is assigned to the point mutation adenine, and a probe with a length of 145 bases is assigned to the point mutation thymine. That is, it can be seen that the length of LPO has changed in the commercially available kit.

[0009] On the other hand, the kits shown in these non-patent documents 2 and 3 do not include probes for confirming the state of the wild type. Usually, point mutations occur in a part of the cell cluster. Usually, the ratio MT / WT of cells (mutant type) that have undergone point mutations relative to wild-type cells that maintain a normal state without point mutations becomes important information for cancer diagnosis. However, the existing MLTA kit does not have a probe for the wild type, so MT / WT cannot be confirmed. The reason is that the signal detected in 1% of the point mutation is about 3000 [ADU]. 1% of the point mutation refers to a state in which 1% of the mutant type and 99% of the wild type are mixed. In this case, when the signal is detected from the wild-type probe, the signal from the wild type is about 300000 [ADU]. However, the signal amount exceeds the saturation upper limit value of 32767 [ADU] that can be detected by the previous capillary sequencer, and the measurement cannot be performed. This is the reason why there is no probe for measuring the wild type in the MLPA method for detecting point mutations. In other words, the reason why the wild-type probe cannot be configured is due to the narrow dynamic range of the device.

[0010] On the other hand, non-patent document 4 reports a technology that can detect the ratio of mutant to wild type, i.e., MT / WT, to 0.01% by expanding the dynamic range of the previous capillary sequencer. The mutation detection limit of fragment analysis in the previous capillary sequencer was set at 1% to 5%. The reason is that the dynamic range of the previous capillary sequencer was narrower than 3 digits. Non-patent document 4 reports a technology that expands the dynamic range to more than 3 digits to 4 digits. In this patent, the capillary electrophoresis analysis technology that expands the dynamic range to more than 3 digits is called HiDy.

[0011] Prior art literature

[0012] Patent Literature

[0013] Patent Document 1: International Publication No. 2001 / 61033

[0014] Non-patent literature

[0015] Non-patent document 1: 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.

[0016] Non-patent document 2: Product Description SALSA MLPA Probemix P520-A2 MPN mix 2 (Product description version A2-03; Issued 13July 2022), [Retrieved on 2022.09.08], Internet

[0017] <URL:https: / / www.mrcholland.com / products / 32322 / Product%20description%20P520-A2%20MPN%20mix%202-v03.pdf>

[0018] Non-patent document 3: Product description ME012-A1 MGMT-IDH1-IDH2 (Product Description version A1-03; Issued 03August 2021), [retrieved on 2022.09.08], Internet

[0019] <URL:https: / / www.mrcholland.com / products / 30043 / Product%20description%20M E012-A1%20MGMT-IDH1-IDH2-v03.pdf>

[0020] Non-patent literature 4: Highly sensitive mutation quantification by highdynamic-range capillary-array electrophoresis (HiDy CE), Lab Chip, 2020, 20, P.1083-1091 Summary of the invention

[0021] Problems to be solved by the invention

[0022] The MLPA method, one of the representative applications of capillary electrophoresis, can realize multiplexing of 40 probes. However, in the conventional point variation measurement by the MLPA method, the comparison of the signal intensity from the gene of the control sample attached to the kit and the target sample is used to determine the presence or absence of the mutant form (MT), but the ratio cannot be measured. That is, in the conventional point variation measurement by the MLPA method, qualitative inspection can be performed, but quantitative inspection cannot be performed.

[0023] The present invention has been made in view of the above-mentioned situation. An object of the present invention is to provide a point variation ratio detection method capable of quantitative inspection.

[0024] Means for solving problems

[0025] The point variation ratio detection method of the present invention that solves the above-mentioned problems is used for multiplex ligation probe amplification (MLPA) measurement, and comprises: a measurement step, using an electrophoresis device, measuring the intensity S of the fluorescent signal emitted from the variation part of the sample, that is, the variation-derived signal MT and the intensity S of the fluorescent signal emitted from the part other than the variant part of the sample, i.e., the wild-type signal. WT At least one of the above S MT ; Ratio calculation step, calculate the above S MT Relative to the strength ratio above S MT When the ratio of the reference value is high, the upper limit of the dynamic range of the electrophoresis device for measuring the fluorescent signal is equal to or greater than a predetermined value.

[0026] Effects of the Invention

[0027] The present invention can provide a point variation ratio detection method capable of performing quantitative inspection.

[0028] The following description of the embodiments will make other problems, structures, and effects clear. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is an analytical reaction explanatory diagram for explaining an example of a method for detecting a point mutation in a DNA base sequence.

[0030] Figure 2 It is a diagram for explaining the analytical reaction of the first embodiment of the present invention.

[0031] Figure 3 It is a diagram for explaining the analytical reaction of the second embodiment of the present invention.

[0032] Figure 4 It is a diagram for explaining the analytical reaction of the third embodiment of the present invention.

[0033] Figure 5It is a diagram for explaining the analytical reaction according to the fourth embodiment of the present invention.

[0034] Figure 6 It is a diagram for explaining the analytical reaction of the fifth embodiment of the present invention.

[0035] Figure 7 It is a diagram for explaining the analytical reaction of the sixth embodiment of the present invention.

[0036] Figure 8 It is an explanatory diagram of the analytical reaction of the seventh embodiment of the present invention.

[0037] Fig. 9 The results of analytical reaction analysis according to the eighth embodiment of the present invention are shown. DETAILED DESCRIPTION

[0038] Below, use Figures 1 to 9 Embodiments of the present invention are described.

[0039] First, use Figure 1 An example of a method for detecting a point mutation in a DNA base sequence is described. Figure 1 This is an analytical reaction explanatory diagram for explaining an example of a method for detecting a point mutation in a DNA base sequence.

[0040] Figure 1 The method shown is a technique for multiplex detection of multiple fragments by capillary electrophoresis for multiple point mutations. This method is generally called MLPA (Multiplex Ligation-dependent Probe Amplification).

[0041] Figure 1 The method shown relates to a method for detecting point variations in a DNA base sequence.

[0042] like Figure 1As shown before hybridization, at this stage, the DNA target sequences 112, 113, LPO (Left Probe Oligonucleotide) and RPO (Right Probe Oligonucleotide) whose states of point mutations are to be investigated are in an unhybridized state. The DNA target sequences 112, 113 are composed of molecules of four nucleotides, namely guanine, adenine, cytosine, and thymine. The DNA target sequence 112 has a point mutation guanine 103 of 1 base pair as a point mutation. On the other hand, the DNA target sequence 113 is a wild type (Wild Type) having a normal gene sequence, and has a normal base cytosine 114 as a regular sequence. The difference between the DNA target sequence 112 and the DNA target sequence 113 is only the difference of one base between the point mutation guanine 103 and the normal base cytosine 114, and the sequences 101, 102 located upstream and downstream of the point mutation guanine 103 are the same sequence.

[0043] Here, as mentioned above Figure 1 As shown before hybridization, LPO has a base cytosine 106 that matches the point variation guanine 103 in the DNA target sequence 112. LPO has a sequence 104 complementary to the sequence 101 and stuffer sequences 107 and 111 for adjusting the probe length. In addition, the stuffer sequence 107 does not hybridize with the DNA target sequences 112 and 113. In addition, in this method, after hybridization of these sequences, a ligation reaction is performed using a ligase to connect LPO and RPO, and then amplification is performed by PCR. LPO has a primer sequence 109 complementary to the PCR primer used for amplification by this PCR.

[0044] In addition, as mentioned above Figure 1 As shown before hybridization, RPO has a sequence 105 that hybridizes complementarily to the sequence 102. In addition, RPO has a primer sequence 110 that is complementary to the PCR primer amplified by PCR as described above.

[0045] In addition, in the conventional MLPA method, a stuffer sequence 111 for adjusting the length of the PCR product is inserted between the sequence 105 in the RPO and the primer sequence 110. However, in order to detect point mutations, such as Figure 1 As shown, it is preferred to insert a stuffing sequence 107 in LPO. The reason is that it is LPO that actually detects the point variation. In order to convert the information of the point variation into base length, the stuffing sequence 107 must be configured in LPO anyway. In other words, even if there is a stuffing sequence 107 in RPO, it is difficult to detect the point variation as base length.

[0046] Then, if Figure 1As shown in the hybridization connection, LPO (Left Probe Oligonucleotide) and RPO (Right Probe Oligonucleotide) are hybridized with the DNA target sequences 112 and 113 as the hybridization targets.

[0047] For example, Figure 1 As shown in the hybridization connection, 5 μL of 10 mM Tris buffer (pH 8.0) containing 50 to 100 ng of DNA target sequences 112 and 113 was heated at 98°C for 5 minutes, cooled to room temperature, and LPO and RPO were added. By culturing at 60°C for 18 hours, DNA target sequences 112, LPO and RPO hybridized. In addition, DNA target sequences 113, LPO and RPO hybridized.

[0048] Here, the cytosine 106 at the 3' end of LPO is complementary to the point variant guanine 103, so they hybridize. After hybridization, the cytosine 106 at the 3' end of LPO is adjacent to the 5' end of the sequence 105 in RPO, so the ligase can connect the two. That is, in the mutant type (Mutant Type), LPO and RPO can be made into one DNA chain. On the other hand, in the case of the wild type (Wild Type), the DNA object sequence 113 has a normal base cytosine 114, so it cannot form a complementary chain with the cytosine 106 at the 3' end of LPO. Therefore, the ligase cannot connect LPO and RPO. That is, in the wild type, LPO and RPO cannot form one DNA chain.

[0049] Next, enter Figure 1 In the PCR step, the DNA target sequence 112 is separated from the DNA strand that is linked to LPO and RPO by the ligation reaction to form a single strand by a temperature of 94° C. In addition, the DNA target sequence 113 is separated from LPO and RPO by a temperature of 94° C.

[0050] Then, the primers added to the solution after dissociation are hybridized with the primer sequences 109 and 110 of the dissociated single-stranded DNA, and the thermal cycle is repeated, thereby amplifying the predetermined DNA fragment in an exponential function. Figure 1 In the hybridization connection, only the mutant DNA fragment formed by connecting LPO and RPO is amplified by PCR, and the wild-type DNA fragment is not amplified. In other words, when the DNA target sequence 113 has the normal base cytosine 114, LPO and RPO are not connected, and therefore are not amplified, resulting in no PCR product.

[0051] In the above reaction, a specific point variation on the genome is described in detail. In addition, the reaction can detect multiple point variations on the genome in parallel (= multiplexing). In fact, in the MLPA reaction, more than 40 probes can be detected at the same time in the detection of Copy Number Valiation and methylation. Therefore, the MLPA reaction can also be used for point variation detection, and more than 40 probes can be detected at the same time.

[0052] Then, if Figure 1 As shown in the capillary electrophoresis (CE), after the PCR reaction, the 40 kinds of DNA fragments generated as PCR products were electrophoresed using a capillary sequencer. As for the base length of the generated PCR products, since the length of the filling sequence 107 and 111 of the LPO applicable to each point mutation is changed, the base length of the PCR products 115, 116, and 117 can be changed to any length. Figure 1 In the example described in , the length of the bases to be changed is set to 15 bases. In addition, the difference in the length of the bases of the stuffing sequence is not limited to 15 bases, and can be changed by 1 to 100 bases.

[0053] In addition, Figure 1 In the example described in , a mutant DNA target sequence 112 is amplified, and a wild-type DNA target sequence 113 is not amplified. Therefore, PCR products 115, 116, and 117 amplified from multiple mutant DNA target sequences 112 are developed in the capillary. Since the filling sequences are different, the molecular lengths of PCR products 115, 116, and 117 are also different. Therefore, they are separated in capillary electrophoresis, and signals corresponding to different times are detected. This is shown in the electrophoretogram 120.

[0054] Only in Figure 1 In the example described in , although the presence or absence of mutation can be confirmed, quantitative inspection cannot be performed. In this embodiment, in order to perform quantitative inspection, the intensity S of the fluorescent signal emitted from the mutant part of the sample, that is, the signal derived from the mutation, is calculated. MT Relative to S MT The ratio of the high strength to the baseline value.

[0055] As an example of a specific implementation thereof, there is a point variation ratio detection method having a measurement step and a ratio calculation step.

[0056] Here, in the measurement step, the intensity S of the fluorescence signal emitted from the mutant part of the sample, that is, the mutant signal, is measured using an electrophoresis device. MT and the intensity S of the wild-type signal, which is the fluorescence signal emitted from the part other than the variant part of the sample. WTAt least S MT The measurement process can be done by referring to Figure 1 Capillary electrophoresis, reference Figure 2 This is done by referring to the embodiments described in the accompanying drawings and the subsequent drawings.

[0057] In addition, in the ratio calculation step, the above S MT Relative to the strength ratio above S MT As the above-mentioned reference value, for example, the intensity S of the above-mentioned wild signal can be cited. WT , the saturation maximum value of the mutant type, etc. In addition, since the signal intensity of the above-mentioned reference value is very high, the upper limit of the dynamic range of the electrophoresis device for measuring the fluorescent signal is set to be greater than a predetermined value.

[0058] In this way, the point variation ratio detection method can calculate the strength S of the variation signal MT The relative ratio of the two components can therefore be examined quantitatively.

[0059] In addition, the saturation maximum value of the mutant type is the intensity of the fluorescent signal obtained when the ratio of cells is 100% derived from the mutant type (derived from mutation). The above-mentioned saturation maximum value can be measured in advance using cultured cells cloned from mutant biological cells in which the corresponding point mutation has occurred. That is, the above-mentioned saturation maximum value (reference value) can be measured in advance independently of the above-mentioned measurement step.

[0060] The upper limit of the dynamic range may be set to, 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 thereto.

[0061] Therefore, the dynamic range can be set to, 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 thereto. The dynamic range can also be set to, for example, 0 to 1,500,000 [ADU], etc.

[0062] If the upper limit of the dynamic range is set in this way, the dynamic range will be MT The intensity is still difficult to saturate even if the baseline value is reached, so quantitative inspection can be performed more accurately.

[0063] In the point variation ratio detection method of the present embodiment, as an example of a method for performing the above-mentioned quantitative inspection, calculation of the ratio of mutant type to wild type and calculation of the ratio of mutant type to saturation maximum value can be cited. In order to calculate the ratio of mutant type to wild type, PCR amplification can be performed on a DNA fragment containing the wild-type DNA target sequence 113.

[0064] Hereinafter, the point variation ratio detection method of the present embodiment using these methods will be described with reference to several examples.

[0065] Figure 2 This is an illustration of the analysis reaction of the first embodiment of the present invention. In this embodiment, a technique is described in which, when a point mutation occurs at a location and a maximum of three mutations occur relative to the wild type, the amount of these mutations and the wild type can be measured to quantify the ratio of the mutant type to the wild type.

[0066] like Figure 2 As shown, the sample tube 201 contains DNA target sequences 222, 223, and 224, and the DNA target sequences 222, 223, and 224 have a point mutation at a specific location in the genome. In addition, in this embodiment, the DNA target sequences 222, 223, and 224 contain mutations, and the DNA target sequence 225 is a wild-type sequence. The point mutations in each target sequence are cytosine, guanine, thymine, and adenine. As shown in FIG. Figure 1 As described, four types of LPO202, 203, 204, and 205 that can form complementary chains for each of these four different target sequences coexist. The bases at the 5' ends of LPO202, 203, 204, and 205 are guanine, cytosine, adenine, and thymine, respectively. In addition, among the filling sequences of these LPOs, the filling sequence of LPO202 is the shortest, and the filling sequence of LPO205 is the longest. The difference between each filling sequence is 15bp, and the difference between the maximum and minimum base lengths is 45bp. The reason why the interval between the connected probes is preferably 15bp is that if the difference is less than 15bp, the peaks during electrophoresis are close, and it becomes difficult to separate the two. In particular, when detecting the ratio MT / WT of 1% or 0.1% mutant type relative to wild type, it is preferred that the distance between the two is greater than 15bp. However, the difference in the filling sequence is not limited to 15bp. The ratio of mutant type to wild type (MT / WT) can be determined by using an electrophoresis device based on the intensity S of the fluorescent signal emitted from the mutant part of the sample, that is, the signal derived from the mutation. MT and the intensity S of the wild-type signal, which is the fluorescence signal emitted from the part other than the variant part of the sample. WT , calculate the above S MT With the above S WT The ratio S MT / S WTto calculate appropriately.

[0067] In addition, in this reaction, all RPO206 used for detecting a point mutation at one location can be the same, so the PCR reaction can be performed using a single RPO206. The ligation reaction is performed only when the 3' end of the LPO is complementary to the point mutation sequence in the target sequence, so the information of the "point mutation" can be converted into the information of the "length" of the base length of the filler sequence.

[0068] It should be noted here that the MLTA method used in previous point variation detection only measures mutation variation, but does not measure wild-type variation. The MLPA method used in existing point variation detection uses the signal from the control sample that is included in the kit and is made to contain 1% variation as a benchmark. The signal amount of the control sample of a specific point variation is compared with the signal amount from the sample to be measured. When the signal amount from the sample is greater than the signal amount of the control sample, it is judged as "there is variation", and when the signal amount from the sample is less than the signal amount of the control sample, it is judged as "there is no variation". In addition, it is clearly stated in the manual that the MLPA method used in previous point variation detection can only mention the presence or absence of variation, and cannot perform MT / WT quantification (MRC Holland Product Description SALSA (registered trademark) MLPA (registered trademark) Probemix P520-A2 MPN mix2).

[0069] The reason why MT / WT cannot be calculated is that the kit does not contain an LPO probe for WT. The reason why the LPO probe for WT cannot be included in the kit is that the measurement of the LPO probe for WT in the previous capillary sequencer reaches a saturation value, and accurate measurement cannot be performed. In other words, this is because the dynamic range of the previous capillary sequencer is insufficient. In detail, the maximum measurement value (saturation measurement value) of the signal amount of the previous capillary sequencer is 32767 [ADU], and the signal from WT greatly exceeds this measurement maximum value. In other words, since substantial saturation occurs, the signal value of WT cannot be accurately obtained. Therefore, it is difficult to measure MT / WT. More specifically, the signal of 1% MT is about 2000 [ADU], so it is assumed that the signal of 100% WT is about 200000 [ADU]. However, the saturation signal intensity of the current capillary electrophoresis is 32767 [ADU], so 100% WT cannot be measured.

[0070] In contrast, the capillary electrophoresis measurement method under the high dynamic range reported in the paper in recent years is reported to be able to expand the dynamic range. More specifically, the dynamic range that was previously less than 3 digits is expanded to more than 3 digits to 4 digits. Therefore, if this technology is used, the signal from the wild type can be detected without saturation. Therefore, the ratio of MT / WT can be calculated. In this embodiment, the capillary electrophoresis analysis technology that expands the dynamic range to more than 3 digits is called HiDy.

[0071] In addition, other advantages can also be cited when the wild type can be measured. Another reason why the mutant type cannot be quantitatively measured in the previous kit is that the sample introduction amount to the capillary during injection produces a deviation. In the existing method, only the mutant type is measured for a point mutation, so the signal value includes the deviation of the sample introduction amount during injection. However, if both the mutant type and the wild type can be injected into the same capillary during the same injection, the deviation of the injection can be offset and eliminated by calculating MT / WT. In the present embodiment, the wild type is measured and MT / WT is calculated, so a more accurate measurement can be performed, and a quantitative measurement can be performed. In addition, in the present embodiment, even in a sample with an unclear point mutation state, by simultaneously detecting the signals of the mutant type and the wild type in one electrophoresis, the effect of being able to quantitatively, simply, quickly and cheaply detect the mixing rate of abnormal cells such as cancer cells can be obtained.

[0072] In electrophoresis, peaks 212, 213, 214, and 215 can be confirmed on the electrophoretogram 250. Each peak corresponds to a DNA target sequence 222, 223, 224, and 225. MT / WT can be calculated by dividing the signal value from the peaks 212, 213, and 214 corresponding to the mutant by the signal value of the peak 215 corresponding to the wild type.

[0073] In addition, in this example, it was shown that four different bases can be identified and quantified for one point variation, but this does not impose a limitation on the multiplicity associated with the point variation. Figure 1 As shown, the number of point mutations measured can be multiplexed. Generally, in the fragment analysis using a capillary sequencer, separation is performed in the range of 100 to 500 bp. If the base interval is set to 15 bp to design the filling sequence, the number of mutations that can be measured is (500-100) bp ÷ 15 bp, and about 25 mutations can be measured and analyzed by one electrophoresis. Moreover, if the PCR primer sequences in LPO and RPO can be amplified using, for example, 6 different primer sets, it can be multicolored. Thus, 25 mutations × 6 colors = 150 mutations can be measured at once by one electrophoresis.

[0074] Next, refer to Figure 3 A second embodiment of the present invention will be described. Figure 3This is an analytical reaction explanation diagram of the second embodiment of the present invention. This embodiment is different from the first embodiment in the following aspects. In the first embodiment, there are three types of point mutations in the DNA target sequence, while in the second embodiment, there are two types of point mutations. Specifically, the DNA target sequences 322 and 325 are adenine, while in the DNA target sequence 323 there is a point mutation of guanine, and in the DNA target sequence 324 there is a point mutation of thymine. In addition, the DNA target sequences 325 and 322 are wild-type, and the other DNA target sequences 324 and 323 are mutants.

[0075] In the second embodiment, similarly to the first embodiment, four types of LPO 302, 303, 304, 305 and one type of RPO 306 are added to a sample tube 301 in a state where the target DNA sequence is mixed. LPO and RPO hybridize with the target DNA sequence respectively, but the G at the 3' end of LPO 302 is not complementary to the point mutation A of the target DNA sequence 322, and thus cannot hybridize at all. Therefore, in the ligation reaction after hybridization, the 3' end of LPO 302 cannot be ligated to the 5' end of RPO 306. Therefore, in the PCR reaction of the next step, PCR amplification from the target DNA sequence 322 does not proceed.

[0076] Therefore, in the fragment analysis in capillary electrophoresis, as shown in the electropherogram 350, the peaks detected are peaks 313, 314, and 315 corresponding to the DNA target sequences 323, 324, and 325. The peak 312 at the position corresponding to the DNA target sequence 322 is not detected. By comparing these peaks, not only can the presence or absence of mutations be detected, but also MT / WT can be calculated for each mutation. Therefore, quantitative measurement can be performed.

[0077] In this example, the detection of a single point mutation was described. However, as can be easily inferred, the present method can perform multiplex detection of multiple point mutations and can collectively measure 40 or more mutations by a single electrophoresis.

[0078] Next, refer to Figure 4 A third embodiment of the present invention will be described. Figure 4: is an analytical reaction explanation diagram of the third embodiment of the present invention. This embodiment is different from the first embodiment in the following aspects. In the first embodiment, there are three types of point mutations in the DNA target sequence, while in the third embodiment, there is one type of point mutation. Specifically, in the DNA target sequences 422, 423, and 425, there is adenine, while in the DNA target sequence 424, there is a point mutation of thymine. In addition, the DNA target sequences 422, 423, and 425 are wild-type having adenine at the mutation corresponding position, and the other DNA target sequence 424 is a mutant having a point mutation of thymine.

[0079] In the third embodiment, similarly to the first embodiment, four types of LPO 402, 403, 404, 405 and one type of RPO 406 are added to a sample tube 401 in a state where the target DNA sequence is mixed. LPO and RPO hybridize with the target DNA sequence respectively, but the G at the 3' end of LPO 402 is not complementary to the point mutation A of the target DNA sequence 422, and thus cannot hybridize at all. In addition, the C at the 3' end of LPO 403 is not complementary to the point mutation A of the target DNA sequence 423, and thus cannot hybridize at all.

[0080] Therefore, the 3' end of LPO and the 5' end of RPO cannot be connected in the ligation reaction after hybridization of the DNA target sequences 422 and 423. Therefore, in the PCR reaction of the next step, PCR amplification from the DNA target sequences 422 and 423 does not proceed.

[0081] Therefore, in the fragment analysis in capillary electrophoresis, as shown in the electropherogram 450, the peaks detected are peaks 414 and 415 corresponding to the DNA target sequences 424 and 425. The peaks 412 and 413 at the positions corresponding to the DNA target sequences 422 and 423 are not detected. By comparing these peaks, not only the presence or absence of mutations can be detected, but also MT / WT can be calculated for each mutation. Therefore, quantitative measurement can be performed.

[0082] In this example, the detection of a single point mutation was described. However, as can be easily inferred, the present method can perform multiplex detection of multiple point mutations and can collectively measure 40 or more mutations by a single electrophoresis.

[0083] Next, refer to Figure 5 A fourth embodiment of the present invention will be described. Figure 5: This is an explanatory diagram of the analysis reaction of the fourth embodiment of the present invention. The difference of this embodiment is that the first to fourth embodiments perform the reaction related to ligation and hybridization in one sample tube, whereas the initial DNA 501 is divided into four parts and the equal parts are added to four different sample tubes 511, 512, 513, and 514. In addition, it is also different in that different LPOs 531, 532, 533, and 534 are added to each of these sample tubes. The 3' ends of the LPOs 531, 532, 533, and 534 shown in the figure are guanine, cytosine, adenine, and thymine, respectively. In addition, the same RPOs 541, 542, 543, and 544 are added to each tube. In addition, in this embodiment, one point variation is shown in the figure for simplicity, but in actual reagents, there are the number of LPOs and RPOs corresponding to the number of point variations. In other words, there are LPOs 534 for wild type corresponding to the number of wild type in the sample tube 514. 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, if this method is applied, the peak of the DNA fragment derived from the wild type in one point mutation is longer than the peaks derived from the other three DNA fragments (the intensity of the signal derived from the wild type is S WT In addition, regarding the elution time, the peak derived from the wild-type DNA fragment eluted the slowest compared to the other three peaks.

[0084] On the other hand, point mutation information other than the wild type is assigned to the remaining three LPOs 531, 532, and 533. Sequence information related to these point mutations may be different from each other and be exclusive.

[0085] Alternatively, regardless of the wild-type state, a method is also useful to fix the 3' ends of LPO531, 532, 533, 534 added to sample tubes 511, 512, 513, 514 to guanine, cytosine, adenine, and thymine, respectively. It is useful to implement these fixed states for multiple point mutation groups.

[0086] By adjusting the length of the stuffing sequence, the base length of each probe can be designed. Therefore, the base length of the PCR product identified by electrophoresis can be easily shaped, and it can be determined which peak is the wild type and which peak is the mutant type. The signals of the point mutation and wild type are calculated from the assigned peaks, and MT / WT can be quantified (the intensity S of the signal derived from the mutation is calculated). MT and the strength S of the wild signal WT The ratio S MT / S WTquantification). In addition, in the case of the present embodiment, among the various point mutations, LPO534 that is consistent with the wild type has the longest stuffing sequence. Thus, the influence of slow changes such as the signal intensity decreases with longer base lengths can be avoided. In other words, by grouping the electrophoresis according to each point mutation, for example, when the difference base length of the stuffing sequence is 15bp, the difference in base length within the point mutation can be suppressed to 15Bp×(4 bases-1)=45bp. Thus, a more accurate calculation of MT / WT can be performed. In addition, in order to facilitate the detection of low-frequency point mutations as much as possible, the method of making the PCR fragment associated with the mutation shorter and pre-ejaculation is effective.

[0087] In addition, the advantage of this embodiment over the first to third embodiments is that by dividing the LPO according to the base type at the 3' end, competitive hybridization between LPO probes to the DNA target sequence can be avoided. Thus, competition between LPO probes can be suppressed and the reliability of the MT / WT value can be improved.

[0088] The 3' ends of LPO531, 532, 533, and 534 have guanine, cytosine, adenine, and thymine, respectively, and therefore hybridize with the cytosine, guanine, thymine, and adenine of the DNA target sequences 521, 522, 523, and 524 respectively injected into the sample tubes. In addition, the DNA target sequences 521, 522, 523, and 524 hybridize with RPO541, 542, 543, and 544. The sample tubes 511, 512, 513, and 514 are connected separately. Thereafter, the sample tubes 511, 512, 513, and 514 are combined into one sample tube to perform PCR. After PCR, they are provided to capillary electrophoresis, thereby being able to separate the fragments according to the molecular weight within one capillary. Specifically, it is possible to obtain Figure 5 Shown is electropherogram 551. As shown in electropherogram 551, MT / WT can be calculated for each mutation 1, 2, 3...N.

[0089] In addition, in this embodiment, fluorescent primers are used to fluorescently label the PCR products during PCR. The fluorescent primers are not limited to a single color of fluorescent dye, and each sample tube can be labeled with a fluorescent dye of a different wavelength. In addition, the probe sets in LPO and RPO can be designed so that each point mutation can be labeled with a different fluorescent dye.

[0090] Next, refer to Figure 6 A fifth embodiment of the present invention will be described. Figure 6This is an illustration of the analytical reaction of the fifth embodiment of the present invention. Compared with the fourth embodiment, this embodiment is effective in reducing the number of divided sample tubes and reducing the cost required for each electrophoresis. Point mutations do not necessarily occur in the three bases other than the wild type, and in most cases can be limited to two or less. In particular, cancer is diverse, and point mutations with different organ specificities such as lung cancer, breast cancer, and pancreatic cancer occur. Therefore, it is very useful to increase the number of repetitions that can be detected from one capillary in one electrophoresis by specifically limiting the number of point mutations to be detected to two or less.

[0091] In this embodiment, the initial DNA 601 is divided into three parts, and equal amounts are added to three different sample tubes 612, 613, and 614. Different LPOs 632, 633, and 634 are added to them respectively. The 3' ends of the LPOs 632, 633, and 634 shown in the figure are cytosine, adenine, and thymine, respectively. In addition, the same RPOs 642, 643, and 644 are added to each tube. In addition, in this embodiment, for the sake of simplicity, one point variation is shown in the figure, but the actual reagent has the number of groups of LPOs and RPOs corresponding to the number of point variations. In other words, the number of LPOs 634 for wild type corresponding to the number of wild type exists in the sample tube 614. In addition, the 3' end of LPO 634 is not always thymine, but LPO 634 is designed based on the sequence information of the wild type in each point variation. In other words, if this method is applied, the peak of the DNA fragment derived from the wild type in one point mutation is longer than the peaks derived from the other three DNA fragments (the intensity S of the signal derived from the wild type is greater than 1 / 2). WT In addition, regarding the elution time, the peak derived from the wild-type DNA fragment is the slowest elution fragment compared to the other three peaks.

[0092] On the other hand, for the remaining two LPOs 632 and 633, point mutation information other than the wild type is assigned. These information are different from each other as long as they are exclusive. The LPOs and RPOs in the sample tubes 612, 613, and 614 are hybridized with the DNA target sequences 622, 623, and 624, respectively. LPO and RPO are connected by a ligation reaction and amplified by PCR. In addition, by adjusting the length of the stuffing sequence, the base length of each probe can be designed.

[0093] Thus, the base length of the PCR product identified by electrophoresis can be easily shaped, and it is possible to distinguish which peak is the wild type and which peak is the mutant type. Figure 6 As shown in the electropherogram 651, MT / WT can be calculated for each mutation 1, 2, 3, ... N. That is, the signals of the point mutation and the wild type can be calculated from the assigned peaks, and MT / WT can be quantified (the intensity S derived from the mutation signal is calculated).MT and the strength S of the wild signal WT The ratio S MT / S WT quantification). In addition, in the case of the present embodiment, among the various point mutations, LPO634 that is consistent with the wild type has the longest filler sequence. Thus, the present embodiment can avoid the influence of slow changes such that the longer the base length, the lower the signal intensity. In other words, by grouping the electrophoresis according to each point mutation, for example, when the difference base length of the filler sequence is 15bp, the difference in base length within the point mutation can be suppressed to 15Bp×(3 bases-1)=30bp. Thus, the present embodiment can perform more accurate calculations of MT / WT. In addition, in order to facilitate detection related to low-frequency point mutations as much as possible, the method of making the PCR fragment associated with the mutation shorter and pre-ejaculation is effective.

[0094] Next, refer to Figure 7 A sixth embodiment of the present invention will be described. Figure 7 This is an illustration of the analytical reaction of the sixth embodiment of the present invention. This embodiment limits the number of divided tubes to two. One sample tube 713 is allocated to the reaction of the representative point mutation to be detected, and the other sample tube 714 is allocated to the reaction of the wild type. The advantage of this method is that by reducing the number of divided sample tubes to two, the reagent cost and man-hours required for the reaction are reduced. In addition, the base information related to the point mutation of LPO733 and 734 can be obtained from the existing database. In addition, since competitive hybridization between wild-type LPO and mutant LPO can be avoided, the ratio MT / WT (derived from the intensity S of the mutation signal) of wild-type and mutant molecules present in the sample can be detected more accurately. MT and the strength S of the wild signal WT The ratio S MT / S WT ).

[0095] In this embodiment, the initial DNA 701 is divided into two parts, and equal amounts are added to two different sample tubes 713 and 714. Different LPOs 733 and 734 are added to them respectively. The 3' ends of the illustrated LPOs 733 and 734 are adenine and thymine, respectively. In addition, the same RPOs 743 and 744 are added to the point mutation in each tube. In addition, in this embodiment, one point mutation is illustrated for simplicity, but LPO and RPO are reagents for probe sets containing multiple point mutations. In addition, the 3' ends contained in LPO 734 are not all thymine. A probe set having a sequence complementary to the point mutation group equivalent to the wild type is selected at the 3' end of LPO 734. Therefore, the wild-type bases reflecting the information of each point mutation are configured at the 3' ends of multiple LPOs used to detect the wild type. These base sequences can be any one of adenine, guanine, cytosine, and thymine.

[0096] By adjusting the length of the stuffing sequence, the base length of each probe can be designed. Thus, the base length of the PCR product identified by electrophoresis can be easily shaped, and it can be determined which peak is the wild type and which peak is the mutant type. Specifically, Figure 7 As shown in the electropherogram 751, MT / WT can be calculated for each mutation 1, 2, 3 ... N. That is, the signals of the point mutation and the wild type can be calculated from the assigned peaks, and MT / WT can be quantified (the intensity S derived from the mutation signal is calculated). MT and the strength S of the wild signal WT The ratio S MT / S WT quantification). In addition, in the case of this embodiment, it is considered that in each point mutation, LPO734, which is consistent with the wild type, has a longer filler sequence than LPO733. That is, a shorter filler sequence is assigned to the mutant. The reason is that the existence ratio of the mutant relative to the wild type is usually small. In particular, in minor mutations, the amount of the mutant is small. 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 detect trace minor mutations as well as possible, a method of configuring a short filler sequence for point mutations is useful.

[0097] However, when there is an excess of wild-type, background noise may be generated in the migration range of a molecular weight smaller than that of the wild-type. In this case, it is useful to configure a stuffer sequence in which LPO734 is shorter than LPO733, which is consistent with the wild-type.

[0098] Additionally, it is useful to more directly compare wild-type to mutant forms by grouping the electrophoresis by individual point variants.

[0099] When the difference base length of the filling sequence is set to 15bp, the difference in base length within the point mutation can be suppressed to 15Bp×(2 bases-1)=15bp. As a result, this embodiment can perform more accurate MT / WT calculations. In addition, this embodiment can increase the number of point mutation genes that can be detected in one electrophoresis. This embodiment shortens the base length of low-frequency point mutations and wild types to 15bp, which has the effect of almost ignoring the impact of slow changes.

[0100] Next, refer to Figure 8 A seventh embodiment of the present invention will be described. Figure 8 This is a diagram for explaining the analytical reaction of the seventh embodiment of the present invention. In this embodiment, a measurement method for detecting only mutant types without using wild types is described.

[0101] 10 to 100 μg of DNA 801 as a sample is added to a sample tube 813. LPO 833 and RPO 843 are added to hybridize with a DNA target sequence 832 having a point mutation in DNA 801. In addition, the behavior for one point mutation sequence is shown in the figure here, but in the actual reaction, multiple point mutation sequences exist in DNA 801. Here, only one point mutation sequence is used as an example for explanation.

[0102] The 3' end of LPO833 shown in the figure is adenine. In addition, RPO843 is added. The point variation thymine 823 as a point variation sequence exists in the DNA target sequence 832. The point variation thymine 823 forms a complementary chain with the adenine at the 3' end of LPO833. Therefore, the gap between LPO833 and RPO843 can be connected by ligase, and LPO833 and RPO843 form a DNA chain.

[0103] In addition, in this embodiment, a single point mutation is shown for simplicity, but LPO and RPO are reagents of a probe set including multiple point mutations. Therefore, for any point mutation site, the 3' end of LPO833 is not always all adenine, but any one of the four bases of adenine, guanine, cytosine, and thymine is connected to the 3' end of each LPO833 according to the actual point mutation.

[0104] By adjusting the length of the stuffing sequence, the base length of each probe can be designed. Therefore, the base length of the PCR product identified by electrophoresis can be easily shaped, and the Figure 8 The electrophoretogram 851 is shown. As shown in the electrophoretogram 851, multiple peaks 852, 853, 854, 855 can be obtained for each mutation 1, 2, 3...N.

[0105] in addition, Figure 8The graph 860 shown shows the normalized signal intensity of the mutant when the ratio of the wild-type and mutant cells used as input is changed for a certain point mutation. The normalized signal intensity is calculated by dividing the signal amount of the mutant at different cell ratios by the signal amount of the mutant when the ratio of the mutant is set to 100%. The ratio of the mutant in the cell is proportional to the signal amount from the mutant. Therefore, if the signal intensity when the mutant is 100% is pre-determined independently from the above-mentioned measurement by electrophoresis (the intensity S of the signal derived from the mutant is measured), the normalized signal intensity is calculated. MT saturation maximum value), then by comparing it, the content rate (ratio) of mutant cells in the sample used can be estimated based on the signal intensity derived from point mutations measured in a certain experiment. That is, quantitative testing can also be performed in this embodiment.

[0106] In addition, this method cannot be implemented by conventional capillary sequencers. The reason is that when the ratio of cells is 100% derived from mutants, the peak signal causes saturation within the conventional dynamic range, and accurate measurement cannot be performed. More specifically, the signal detected in a 1% point variation is about 3000 [ADU], so it is about 300,000 [ADU] in a 100% point variation. Since the saturation upper limit that conventional capillary sequencers can detect is 32767 [ADU], saturation occurs. Therefore, in order to perform quantitative point variation measurement for mutants by LPO and RPO, a capillary sequencer with a high dynamic range can be used. As a high dynamic range, for example, as described above, the upper limit is 200,000 [ADU] or more, and in addition, for example, it is 0 to 200,000 [ADU], but it is not limited to this.

[0107] Next, refer to Fig. 9 An eighth embodiment of the present invention will be described. Fig. 9 The results of analytical reaction analysis in the eighth embodiment of the present invention are shown.

[0108] exist Fig. 9 In the graph 901 shown, the horizontal axis of the graph represents the ratio of the mutant type relative to the wild type when the amount of the wild type is fixed at, for example, 100 μg for a certain point mutation. In addition, the vertical axis of the graph represents the signal intensity of the mutant type peak relative to the MT / WT mixing ratio of the cell.

[0109] In the graph 901, it can be seen from the △ in the figure that in the measurement performed using the conventional capillary sequencer (CE), the signal is saturated when the cell ratio MT / WT is 10% to 100%. That is, in the conventional capillary sequencer, if 100 μg of DNA derived from the mutant is reacted, the signal is saturated and accurate measurement cannot be performed. On the other hand, as shown by ○ in the figure, in the HiDy capillary sequencer (HiDy CE) with a high dynamic range, even if the cell ratio MT / WT increases from 10% to 100%, it can be confirmed that the signal from the mutant increases proportionally. Therefore, in order to detect the ratio of mutations in a wide range of 0.1% to 100%, a HiDy capillary sequencer with a high dynamic range can be used.

[0110] Next, Fig. 9 The graph 902 shown in FIG. 902 also shows the ratio of the mutant type relative to the wild type when the amount of the wild type is changed under the condition that the amount of the wild type is fixed at, for example, 100 μg for a certain point mutation. In addition, the vertical axis of the graph is a value normalized by dividing the signal intensity of the peak of the mutant type relative to the MT / WT mixing ratio of the cell by the signal value of the mutant type at MT / WT 100%.

[0111] As described in the seventh embodiment, graph 902 is the measurement result when only the mutant type is used as the object and the LPO and RPO probes related to the wild type are not used. Therefore, since there is no signal derived from the wild type, the signal normalization is only implemented by the division operation within the mutant type. In addition, in the seventh embodiment, the signal value of the mutant type with MT / WT 100% and the signal value of the mutant type with the MT / WT mixing ratio of the cell changed are different electrophoresis, so there is a problem that the deviation of the sample injection during electrophoresis cannot be corrected.

[0112] On the other hand, Fig. 9 In the diagram 903 shown, a mutant and a wild type are paired for one point variation, and LPO and RPO probes are arranged in the DNA target sequence and measured. The difference from the diagram 902 is that the signals from the mutant and the wild type can be measured simultaneously during each electrophoresis.

[0113] Regarding the vertical axis of the graph in graph 903, it is possible to calculate for each electrophoresis by dividing the signal derived from the mutant by the signal derived from the wild type. Therefore, there is an advantage that the deviation of sample injection during electrophoresis can be corrected for each electrophoresis. This is directly related to the improvement of 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 mutant and wild type is more accurate. In particular, as the MT / WT ratio decreases from 1% to 0.1%, the standardized signal value deviates from the ideal linear approximation straight line in graph 902, whereas a better fit is shown in graph 903. This indicates that, especially at low MT / WT, measurements can be made with higher sensitivity and reliability by simultaneously measuring signals from mutant and wild type. Therefore, in variant detection using a capillary sequencer with a high dynamic range, it can be said that it is preferable to measure mutant and wild type in pairs.

[0114] The above has described the variation ratio detection method of the present invention in detail through the embodiments (implementation methods), but the present invention is not limited to the above embodiments, and includes various modified examples. For example, the above embodiments are examples described in detail in order to easily understand the present invention, and are not limited to all the structures described. In addition, a part of the structure of an embodiment can be replaced with the structure of other embodiments, and the structure of other embodiments can be added to the structure of a certain embodiment. In addition, for a part of the structure of each embodiment, other structures can be added, deleted, or replaced.

[0115] Description of Reference Numerals

[0116] 101, 102 sequences

[0117] 103 guanine mutations

[0118] 104, 105 sequences

[0119] 106 Cytosine

[0120] 107 Filling sequence

[0121] 109, 110 primer sequences

[0122] 111 Filling Sequence

[0123] 112, 113DNA object sequence

[0124] 114 Normal base cytosine

[0125] 115~117PCR product

[0126] 120 electrophoresis images

[0127] 201 sample tube

[0128] 202~205LPO

[0129] 206RPO

[0130] 212~215 peak

[0131] 222~225DNA target sequence

[0132] 250 electropherogram

[0133] 301 sample tube

[0134] 302~305LPO

[0135] 306RPO

[0136] 312~315 peak

[0137] 322~325DNA target sequence

[0138] 350 electrophoresis

[0139] 401 sample tube

[0140] 402~405LPO

[0141] 406RPO

[0142] 412~415 peak

[0143] 422~425DNA target sequence

[0144] 350 electrophoresis

[0145] 501DNA

[0146] 511~514 sample tube

[0147] 521~524DNA target sequence

[0148] 531~534LPO

[0149] 541~544RPO

[0150] 551 electrophoresis

[0151] 601DNA

[0152] 612~614 sample tube

[0153] 622~624DNA target sequence

[0154] 632~634LPO

[0155] 642~644RPO

[0156] 651 electrophoresis

[0157] 701DNA

[0158] 713, 714 sample tube

[0159] 733, 734LPO

[0160] 743, 744RPO

[0161] 751 electrophoresis

[0162] 801DNA

[0163] 813 sample tube

[0164] 823 thymine mutations

[0165] 832DNA object sequence

[0166] 833LPO

[0167] 843RPO

[0168] 851 electrophoresis

[0169] 852~855 peak

[0170] 860 Charts

[0171] Charts 901 to 903.

Claims

1. A point variation ratio detection method for multiplex ligation probe amplification (MLPA) measurement, characterized in that: The point variation ratio detection method has the following features: In the measurement process, an electrophoresis device is used to measure the intensity S of the fluorescent signal emitted from the mutant part of the sample, that is, the signal derived from the mutant. MT and the intensity S of the fluorescent signal emitted from the part other than the variant part of the sample, that is, the intensity S derived from the wild-type signal WT At least the S MT ; The ratio calculation step is to calculate the S MT Relative to the intensity ratio S MT The ratio of high baseline values, An upper limit of a dynamic range of the electrophoresis device for measuring a fluorescent signal is equal to or greater than a predetermined value.

2. The point variation ratio detection method according to claim 1, characterized in that: In the measurement step, the S MT and the S WT , The reference value is the S WT , In the ratio calculation step, based on the S obtained in the measurement step MT and the S WT , calculate the S MT With the S WT The ratio S MT / S WT .

3. The point variation ratio detection method according to claim 1, characterized in that: The reference value is the S measured in advance independently of the measurement process. MT The saturation maximum value of In the ratio calculation step, the S obtained in the measurement step is calculated according to the saturation maximum value. MT ratio.

4. The point variation ratio detection method according to claim 1, characterized in that: 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.

5. The point variation ratio detection method according to claim 1, characterized in that: 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].

6. The point variation ratio detection method according to claim 2, characterized in that: In the MLPA measurement, the samples are split to generate the S independently and selectively. MT and the S WT .

Images