Method for reducing non-specific byproducts in ligation reaction using ligase

Abstract

To provide a method for reducing nonspecific by-products in a ligation reaction using a ligase.SOLUTION: The present invention provides a method for reducing a non-specific by-product in a ligation reaction using a ligase, the method including a step of heating a reaction solution containing a reaction product and a by-product in the presence of a first washing buffer after the ligation reaction, and a step of washing the reaction product with a second washing buffer after the heating step.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention broadly relates to a method for reducing non-specific by-products in a ligation reaction using a ligase. [Background technology]

[0002] One method for detecting a gene having a mutation is to use a ligase, which can distinguish mismatched base pairs near the junction. For example, a nucleic acid sample containing the mutant sequence and two adjacent probes complementary to the mutant sequence can be treated with the ligase to detect the presence or absence of a mutation. Specifically, the two probes hybridized to the mutant sequence do not form mismatched base pairs with the mutant sequence and are therefore joined by the ligase, whereas the two probes hybridized to the wild-type sequence form mismatched base pairs with the wild-type sequence and are therefore only joined by the ligase at a low frequency. Therefore, when a nucleic acid sample contains a mutant sequence, a reaction product containing the two probes joined by the ligase is obtained at a higher frequency, and the presence or absence of a mutation can be determined by analyzing the reaction product. Methods for detecting gene mutations using ligase include, for example, the ligase detection reaction (LDR method) (Non-Patent Document 1) and the ligase chain reaction (LCR method) (Non-Patent Document 2).

[0003] When analyzing the reaction products, it is necessary to reduce by-products such as probes not ligated by the ligase, nucleic acid samples, and complexes thereof, in order to reduce unnecessary background and noise. To reduce by-products, washing is generally performed using a high-pH buffer or a buffer containing a denaturant. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Masahiko Hashimoto et al. Polymerase chain reaction / ligase detection reaction / hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations. Biosensors and Bioelectronics. 2006, 21(10), 1915-1923. [Non-patent document 2] Yueying Sun et al. Real-time fluorescence ligase chain reaction for sensitive detection of single nucleotide polymorphism based on fluorescence resonance energy transfer. Biosensors and Bioelectronics. 2015, 74, 705-710. Summary of the Invention [Problem to be solved by the invention]

[0005] However, it has been found that these conventional methods are unable to sufficiently reduce by-products and the like from the reaction products. The problem to be solved by the present invention is to provide a novel method for reducing non-specific by-products and the like in a ligation reaction. [Means for solving the problem]

[0006] As a result of extensive research, the inventors have found that by heating the reaction solution containing the reaction product and by-products in the presence of a first wash buffer after the ligation reaction, and then washing the reaction product with a second wash buffer after heating, it is possible to reduce by-products more effectively than with conventional methods.

[0007] That is, the present application includes the following inventions. [1] A method for reducing non-specific by-products in a ligation reaction using a ligase, comprising: After the ligation reaction, heating the reaction solution containing the reaction product and by-product in the presence of a first washing buffer; After the heating step, washing the reaction product with a second wash buffer; A method comprising: [2] The method according to [1], further comprising the step of washing the reaction product with a wash buffer before the heating step. [3] The method according to [1] or [2], wherein the washing buffer has a pH of 8.5 or higher. [4] The method according to any one of [1] to [3], wherein the washing buffer contains a nonionic surfactant. [5] The method according to any one of [1] to [4], wherein the step of washing the reaction product after the heating step is carried out at a temperature of less than 80°C. [6] The method according to any one of [1] to [5], wherein a ligation reaction is carried out in the detection of a mutated target sequence. [7] The method according to [6], wherein the mutation is a single nucleotide polymorphism. [8] The method according to [6] or [7], wherein in the reaction product, the capture probe and the detection probe bound to the solid phase are annealed to the target sequence. [9] The method according to [8], wherein the solid phase is magnetic beads.

[10] The method according to [8] or [9], wherein when the target sequence is mutated, the capture probe and the detection probe are ligated in the reaction product.

[11] The method according to any one of [6] to

[10] , wherein background in mutation detection is reduced by reducing non-specific by-products. [Effects of the Invention]

[0008] According to the present invention, non-specific by-products in a ligation reaction can be reduced. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows a schematic diagram of the ligation reaction and the washing step after the ligation reaction. [Figure 2] The results of detecting the FAM fluorescent signal labeled on the detection probe after washing by a standard method are shown. [Figure 3] The results of investigating the contribution of each component to the detection of nonspecific FAM fluorescence signals are shown. [Figure 4] The results of detecting FAM fluorescent signals when washing was performed at different heat denaturation temperatures are shown. [Figure 5] The results of detecting FAM fluorescent signals when washed with buffers containing each DNA denaturant are shown. [Figure 6] The results of detecting FAM fluorescent signals when washing was performed using the replacement method or the dilution method are shown. [Figure 7] The results of detecting FAM fluorescent signals when washing was performed using a combination of the displacement method and buffers containing each DNA denaturant are shown. [Figure 8] 1 shows the results of detecting FAM fluorescent signals when washing was performed at different washing temperatures and surfactant concentrations in the displacement method. [Figure 9] The figure shows the results of detecting FAM fluorescent signals when samples subjected to ligation reactions with and without ligase were washed by the substitution method with PBST or 10 mM CAPS buffer (pH 11) containing 0.05% Tween-20 as the washing buffer. [Figure 10] This shows the results of detecting FAM fluorescent signals when washing was performed under different conditions (with or without heat denaturation) and on different washing scales in the displacement method. [Figure 11]This shows the results of detecting FAM fluorescent signals when washing was performed for different numbers of times in the displacement method. [Figure 12] In the displacement method, washing was performed twice with different pH values ​​of the washing buffer, and the results of detecting FAM fluorescent signals are shown. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described, but the scope of the present invention should not be interpreted as being limited to the following embodiment.

[0011] In a first embodiment, there is provided a method for reducing non-specific by-products in a ligation reaction using a ligase, the method comprising the steps of: after the ligation reaction, heating a reaction solution containing the reaction product and the by-product in the presence of a first wash buffer; and, after the heating step, washing the reaction product with a second wash buffer.

[0012] The method of this embodiment makes it possible to reduce unnecessary background and noise caused by non-specific by-products in the analysis of reaction products.

[0013] As used herein, "ligase" refers to an enzyme that catalyzes the formation of a phosphodiester bond between nucleic acids. Examples of "ligase" include DNA ligase.

[0014] As used herein, "ligation" refers to a reaction in which nucleic acids are joined together by forming a phosphodiester bond, and can be used synonymously with conjugation.

[0015] The reaction conditions for the ligation reaction in this embodiment, such as the reaction time and treatment temperature, can be appropriately determined by those skilled in the art.

[0016] The ligation reaction in this embodiment may be performed in any experiment involving the joining of nucleic acids, but is preferably performed in the detection of a mutated target sequence. The ligation reaction in the detection of a mutated target sequence is a ligation reaction that uses a ligase that can distinguish mismatched base pairs near the junction. Specifically, the mutated target sequence can be detected by the following reaction. For example, when a nucleic acid sample containing two adjacent probes complementary to a mutated target sequence and the mutated target sequence is treated with a ligase capable of discriminating between mismatched base pairs, the two probes hybridized to the mutated target sequence are joined by the ligase because they do not form mismatched base pairs with the mutated target sequence. On the other hand, when a sample containing two adjacent probes complementary to the mutated target sequence and a nucleic acid sample containing a wild-type sequence is treated with a ligase capable of discriminating between mismatched base pairs, the two probes hybridized to the wild-type sequence are joined by the ligase only at a low frequency because they form mismatched base pairs with the wild-type sequence. Therefore, when a nucleic acid sample contains a mutated target sequence, a product containing two probes joined by the ligase is obtained at a higher frequency, and the presence or absence of a mutation can be determined by analyzing the product. In other words, the mutated target sequence can be detected.

[0017] As used herein, the term "mutation" refers to one or more bases in which a base substitution, deletion, or insertion has occurred, or to the state in which the base substitution or insertion has occurred. "Mutation" can be used interchangeably with "polymorphism." The position of the base where the base substitution, deletion, or insertion occurs, the number of bases substituted or deleted, and the number of bases inserted vary depending on the target sequence being analyzed. The type of mutation is not particularly limited, but includes missense mutation, nonsense mutation, frameshift mutation, silent mutation, and the like. In this embodiment, the mutation may be a polymorphism, and is preferably a single nucleotide polymorphism.

[0018] Furthermore, in this embodiment, the mutated target sequence detected by the ligation reaction may be in any form, for example, it may be part of genomic DNA, a cell sample, a tissue sample, etc., or it may be an amplification product obtained by amplifying a region containing the target sequence by PCR or the like, and it may be a single-stranded sequence or a double-stranded sequence.

[0019] When the ligation reaction of this embodiment is performed to detect a mutated target sequence, the ligation reaction is preferably performed in a reaction solution containing the target sequence to be detected for mutation, two adjacent probes complementary to the mutated target sequence, and a ligase capable of discriminating between mismatched base pairs. If a mutated target sequence is present in the reaction solution, the two probes are ligated by the ligase. Thus, the reaction solution after the ligation reaction contains two ligated probes, and these two ligated probes correspond to the reaction product of this embodiment. Note that the two ligated probes are annealed to the mutated target sequence.

[0020] The reaction solution after the ligation reaction contains not only the reaction product but also nonspecific by-products. For example, nonspecific by-products include (i) a product formed by nonspecific association between the target sequence and two unligated probes, (ii) a product formed by nonspecific association between the target sequence and one of the two probes, and (iii) a product formed by nonspecific association between the two probes. As used herein, "nonspecific association" refers to a state in which nucleic acids are not complementary bound to each other but are nonspecifically hybridized.

[0021] Furthermore, when the ligation reaction in this embodiment is performed to detect a mutated target sequence, and the ligation reaction solution does not contain the mutated target sequence but only the wild-type target sequence, the two probes complementary to and adjacent to the mutated target sequence will not be ligated. Therefore, the reaction solution after the ligation reaction does not contain a reaction product, but may contain nonspecific by-products. Such nonspecific by-products include, for example, (i) a product formed by nonspecific association between the wild-type target sequence and two unligated probes, (ii) a product formed by nonspecific association between the wild-type target sequence and one of the two probes, and (iii) a product formed by nonspecific association between the two probes.

[0022] Non-specific by-products can cause unnecessary background or noise in the analysis of reaction products, which can hinder the analysis of reaction products. Prior to the analysis of reaction products, such non-specific by-products must be reduced. Furthermore, since the two probes, either ligated or unligated, are annealed to the target sequence, it is preferable to dissociate these double strands before analyzing the reaction products.

[0023] After the ligation reaction, a reaction solution containing the reaction product and by-products is heated in the presence of a first washing buffer. The method of this embodiment includes a step of heating the reaction solution containing the reaction product and by-product in the presence of a first wash buffer after the ligation reaction. That is, the first wash buffer is added to the reaction solution after the ligation reaction, and then the heating step is performed. Heating denatures the nucleic acids, which dissociates the double-stranded chain containing the reaction product and can also dissociate the nucleic acids that form nonspecific by-products. Dissociating the nucleic acids makes it easier to recover the desired reaction product.

[0024] The reaction conditions in the heating step, such as the heating temperature and heating time, can be appropriately determined by those skilled in the art, and may be any conditions as long as the nucleic acids dissociate from each other. The heating temperature may be any temperature as long as it is higher than the Tm value of the nucleic acids so that the nucleic acids dissociate from each other, but heating at about 65 to 100°C for about 1 minute to 1 hour is preferred, and for example, the reaction solution may be heated at about 65 to 75°C for 3 to 10 minutes. In one embodiment, the method of this embodiment comprises the step of heating the reaction mixture in the presence of a first wash buffer to about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75°C for 3, 4, 5, 6, 7, 8, 9, or 10 minutes.

[0025] The amount (volume) of the washing buffer in the heating step can be determined appropriately by those skilled in the art, but the ratio of the amount (volume) of the ligation reaction solution to the amount (volume) of the washing buffer in the heating step is preferably 1:50 to 200 (ligation reaction solution:washing buffer), and more preferably 1:80 to 110 (ligation reaction solution:washing buffer).

[0026] Furthermore, after the heating step, it is preferable to rapidly cool the reaction solution. The method for rapid cooling can be determined appropriately by those skilled in the art, but it is preferable to quickly cool the reaction solution on ice for about 5 seconds to about 30 minutes after the heating step.

[0027] By performing the heating step in the presence of the first wash buffer, the rate at which nucleic acids that form nonspecific by-products dissociate from each other can be increased compared to when the reaction solution is heated directly without adding the wash buffer.

[0028] The first wash buffer used in the heating step may be any wash buffer known to those skilled in the art, such as Tris-HCl, sodium acetate, ammonium acetate, GnSCN (guanidine thiocyanate) buffer, TE buffer, TAE buffer, TBE buffer, PBS buffer, TBS buffer, SSC buffer, or Good's buffer such as TAPS buffer, CHES buffer, or CPAS buffer, but is preferably a wash buffer adjusted to pH 8.5 or higher, for example, a wash buffer adjusted to pH 8.5 to 12. In one embodiment, the method comprises heating the reaction in the presence of a first wash buffer at pH 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12. The wash buffer may contain any additives as appropriate depending on the purpose, such as a surfactant or a nucleic acid denaturant, and preferably contains a surfactant. The surfactant may be any surfactant known to those skilled in the art, but is preferably a nonionic surfactant, such as Tween®-20, Tween®-80, Triton® X-100, Triton® X-114, Brij®-35, Brij®-58, or SDS (sodium dodecyl sulfate). Examples of nucleic acid denaturants include formamide, formaldehyde, dimethyl sulfoxide, and urea.

[0029] In one embodiment, the method of this embodiment comprises the step of heating the reaction solution in the presence of a wash buffer comprising CPAS buffer at pH 10, 10.5, 11, 11.5, or 12 supplemented with Tween (registered trademark)-20.

[0030] After the heating step, it is preferable to recover the reaction product from the reaction solution. When the reaction solution is rapidly cooled after the heating step, the recovery step is carried out after the rapid cooling step. The method for recovering the reaction product may be carried out by any method known to those skilled in the art, such as extraction using a solid phase, extraction with an organic solvent, extraction with a silica membrane, extraction with an ion exchange column, etc., but extraction using a solid phase is preferable.

[0031] In the present embodiment, when the ligation reaction is carried out to detect a mutated target sequence, and the two ligated probes complementary to the mutated target sequence as the reaction product are extracted using a solid phase after the heating step, it is preferred that one of the two probes (referred to as a complementary probe in this specification) is bound to a solid phase. Here, the solid phase may be any carrier, such as polymer beads, silica gel beads, graphite carbon beads, etc., but is preferably a magnetic bead containing a magnetic molecule. Alternatively, a solid phase having high temperature resistance may be used. Here, in this specification, a solid phase having high temperature resistance refers to a solid phase having the property of not denaturing even in an environment of, for example, 80°C or higher. When a solid phase having high temperature resistance, such as magnetic beads having high temperature resistance, is used, the heating temperature in the heating step is preferably 80°C or higher, and may be, for example, about 90 to 99°C. In one embodiment, the method of this embodiment comprises the step of heating the reaction mixture in the presence of a first wash buffer to about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99°C for 3, 4, 5, 6, 7, 8, 9, or 10 minutes.

[0032] When the solid phase is magnetic beads, the ligated reaction product is recovered using a magnet. For example, if a container containing a reaction solution containing the reaction product is placed on a magnetic stand with a magnet near the bottom, the reaction product will accumulate at the bottom of the container, and the reaction solution containing the reaction product can be recovered by discarding the supernatant. The recovered reaction solution may contain nonspecific by-products along with the reaction product. A washing step using a second wash buffer is performed after the reaction solution recovery step.

[0033] By including a step of heating the reaction solution in the presence of a first wash buffer, the reaction products can be recovered with higher selectivity than when the reaction solution is heated directly without adding a wash buffer. Conventionally, the products recovered using a magnet include not only the reaction products formed by ligation of the capture probe and the other probe (detection probe), but also a particularly large amount of products formed by nonspecific association of the capture probe, detection probe, and target sequence (the nonspecific by-products (i) above). However, the method of this embodiment can significantly reduce the nonspecific by-products (i) above recovered together with the reaction products. In other words, the capture probe, detection probe, and target sequence that form the nonspecific by-products are dissociated, and the detection probe and target sequence that are not ligated to the capture probe are not recovered by the magnet but are discarded as the supernatant.

[0034] washing the reaction product with a washing buffer prior to the heating step; This embodiment preferably includes a step of washing the reaction product with a wash buffer before the heating step.

[0035] The wash buffer may be the same as or different from the first wash buffer used in the heating step, but like the first wash buffer, it is preferably a buffer adjusted to pH 8.5 or higher, and more preferably a buffer containing a nonionic surfactant.

[0036] The reaction conditions such as temperature in the washing step before the heating step can be appropriately determined by those skilled in the art, but washing is preferably carried out at less than 80°C, for example, at about 15 to 79°C. In one embodiment, the washing step before the heating step is carried out at about 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50, 45 to 55, 50 to 60, 55 to 65, 60 to 70, 65 to 70, or 70 to 79°C. More preferably, the washing step before the heating step is carried out at room temperature, which may be, for example, 10 to 30°C. In one embodiment, the washing step of the heating step is carried out at about 10 to 20, 15 to 25, or 20 to 30°C.

[0037] The procedure for the washing step can also be determined appropriately by those skilled in the art, but for example, washing can be carried out by adding a washing buffer to the reaction solution and suspending it. The amount (volume) of the washing buffer in the washing step before the heating step can be determined appropriately by those skilled in the art, but is preferably 200 μL or less, more preferably 50 μL or less. The ratio of the amount (volume) of the ligation reaction solution to the amount (volume) of the washing buffer in the heating step is preferably 1:2-20 (ligation reaction solution:washing buffer), more preferably 1:4-10 (ligation reaction solution:washing buffer).

[0038] When a washing step is included before the heating step, the amount (volume) of the washing buffer in the heating step is preferably 200 μL or less, more preferably 50 μL or less.

[0039] After washing, the reaction product is preferably recovered. If the reaction product is bound to magnetic beads, the reaction product is recovered using a magnet. The reaction solution containing the recovered reaction product also contains non-specific by-products. After the reaction solution is recovered, it is subjected to a heating step.

[0040] After the heating step, washing the reaction product with a second wash buffer. The method of this embodiment includes, after the heating step, a step of washing the reaction product with a second wash buffer.

[0041] The second washing buffer may be the same as or different from the first washing buffer used in the heating step or the washing buffer used in the washing step before the heating step. However, like the first washing buffer and the washing buffer used before the heating step, the second washing buffer is preferably a buffer adjusted to a pH of 8.5 or higher, and more preferably a buffer containing a nonionic surfactant.

[0042] The reaction conditions in the washing step, such as temperature, can be appropriately determined by those skilled in the art, but washing is preferably carried out at less than 80°C, for example, at about 15 to 79°C. In one embodiment, the washing step of the heating step is carried out at about 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50, 45 to 55, 50 to 60, 55 to 65, 60 to 70, 65 to 70, or 70 to 79°C. More preferably, the washing step before the heating step is carried out at room temperature, which may be, for example, 10 to 30°C. In one embodiment, the washing step of the heating step is carried out at about 10 to 20, 15 to 25, or 20 to 30°C.

[0043] The procedure for the washing step can also be determined appropriately by those skilled in the art, but for example, washing can be performed by adding a second washing buffer to the reaction solution containing the reaction product recovered in the heating step and suspending it. If nonspecific by-products are also recovered together with the reaction product, the second washing buffer is added to the reaction solution containing the reaction product and the nonspecific by-products.

[0044] The amount (volume) of the second wash buffer used for washing can be appropriately determined by those skilled in the art, but is preferably 200 μL or less, more preferably 50 μL or less.

[0045] After washing, the reaction products are preferably recovered. If the reaction products are bound to magnetic beads, they are recovered using a magnet.

[0046] The step of washing the reaction product with the second washing buffer may be carried out any number of times, but is preferably carried out 1, 2, 3, 4, or 5 times, and more preferably 1 or 2 times.

[0047] When the method of this embodiment includes a step of washing the reaction product with a wash buffer before the heating step, it may be a displacement method. When the method of this embodiment does not include a step of washing the reaction product with a wash buffer before the heating step, it may be a dilution method. The method of this embodiment is preferably a displacement method.

[0048] The method of this embodiment can reduce nonspecific by-products more than conventional methods. When the ligation reaction in the method of this embodiment is performed to detect a mutated target sequence and two ligated probes are obtained as reaction products, the nonspecific by-products formed by the nonspecific association of the mutated target sequence with the two unligated probes are reduced more. In other words, the method of this embodiment can separate the nucleic acids forming the nonspecific by-products more than conventional methods, making it possible to recover the reaction products with higher selectivity. By reducing non-specific by-products and recovering reaction products with higher selectivity, noise or background in the analysis of reaction products, preferably in the detection of mutations, is reduced.

[0049] The method for analyzing the reaction products in the reaction solution after the reaction can be appropriately selected by those skilled in the art depending on the purpose of the analysis. For example, analysis methods include analysis of the reaction products by microarray analysis, flow cytometry analysis, etc., and analysis of the amplified products of the reaction products by quantitative PCR, etc., but it is preferable to analyze the reaction products by flow cytometry analysis. Reaction products labeled by any method, such as with a fluorescent substance or a specific sequence, may also be analyzed. In the embodiment, when the ligation reaction is carried out to detect a mutated target sequence and two ligated probes are obtained as the reaction product, the reaction product to be analyzed is preferably not annealed to the target sequence, but may be annealed to the target sequence. The reaction product annealed to the target sequence may show the same results in analysis as the reaction product not annealed to the target sequence. The method of this embodiment can be applied to various analyses of reaction products.

[0050] The reduction of non-specific reaction products by the method of this embodiment can be evaluated by a method known to those skilled in the art. An example of an evaluation method for the case where the ligation reaction in the method of this embodiment is carried out for the detection of a mutated target sequence, and the reaction product is fluorescently labeled and analyzed by flow cytometry is shown below. First, a reaction solution containing a mutated target sequence and two adjacent probes complementary to the mutated target sequence, but without ligase, is prepared, along with an identical reaction solution containing ligase. Next, after reaction under ligation conditions, the reaction products are subjected to flow cytometry, and the amount of nonspecific reaction products (fluorescence intensity) is compared to evaluate the reduction of nonspecific reaction products. In the ligase-free reaction solution, no ligation reaction occurs, and no ligated probes are obtained as reaction products. Therefore, little or no fluorescence should be detected in the flow cytometry analysis. However, if nonspecific byproducts are not removed, fluorescence equivalent to that of the ligase-containing reaction product is detected. Therefore, if the fluorescence intensity detected in the flow cytometry analysis of the ligase-free reaction solution is lower than that detected in the flow cytometry analysis of the ligase-containing reaction solution, the method of this embodiment can be evaluated as indicating a reduction of nonspecific reaction products. Even when the fluorescence intensity detected by flow cytometry analysis of a reaction solution not containing ligase is close to 0, it can be evaluated that the nonspecific reaction products have been reduced by the method of this embodiment.

[0051] The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples. [Example]

[0052] Test Example 1 In Test Example 1, the cleaning effect of the conventional method was examined. <Immobilization of azide-modified DNA onto alkyne beads> 5.0 μL of alkyne beads (Tamagawa Seiki, Cat. No. TAS8848N1161) and 1 μM azide-modified capture oligo (oligo of SEQ ID NO: 1) were reacted in a click reaction solution (0.2 M NaCl (Promega, Cat. No. V4221), 1.38 mM THPTA (Sigma, Cat. No. 752342-500MG), 0.4 mM copper sulfate (Nacalai Tesque, Cat. No. 09605-04), 2 mM sodium ascorbate (Nacalai Tesque, Cat. No. 11692-52), total reaction volume: 500 μL) with stirring at room temperature for 2 hours. 5 μL of Tween-20 (Nacalai Tesque, Cat. No. 28353-14) was added to a final concentration of 0.1%, mixed, and the beads were collected using a magnetic stand. The supernatant was discarded, and the beads were resuspended in 500 μL of PBS containing 0.05% Tween-20, pH 7.4 (137 mM sodium chloride (Nacalai Tesque, Cat. No. 31333-45), 2.7 mM potassium chloride (Nacalai Tesque, Cat. No. 28538-75), 10 mM disodium hydrogen phosphate (Nacalai Tesque, Cat. No. 31738-55), 1.8 mM potassium dihydrogen phosphate (Nacalai Tesque, Cat. No. 28736-75), hereafter referred to as PBST). The beads were again collected using a magnetic stand. This procedure was repeated twice, and the beads were resuspended in 50 μL of distilled water (Thermo Fisher Scientific, Cat. No. 10977-023) and stored in a refrigerator until use.

[0053] <Ligase reaction of DNA-immobilized alkyne beads and labeled oligos via cross-linked oligos> Two microliters of a 10% alkyne bead suspension, 2 μM FAM-labeled detection probe (oligonucleotide of SEQ ID NO: 2), 2 μM bridge nucleic acid (oligonucleotide of SEQ ID NO: 3), and 0.2 μL HiFi Taq DNA Ligase (New England Biolabs, Cat. No. M0647S) were reacted in 1X HiFi Taq Ligase Buffer (included in M0647S, total reaction volume: 10 μL, reaction vessel: general-purpose PCR tube) (Figure 1). The reaction was performed at 70°C for 5 minutes, followed by 50°C for 60 minutes and rapid cooling. In some cases, a negative control group was also tested without ligase or bridge oligo as a negative control. After washing under the conditions specified for each test, the magnetic beads were separated using a flow cytometer (Beckton Dickinson, Fortessa X-20) and the FAM fluorescent signal of the detection probe conjugated to the magnetic beads was detected.

[0054] [Table 1]

[0055] After the ligation reaction, the reaction mixture was heated at 70°C for 5 minutes, then rapidly cooled on ice. The mixture was then suspended in a 1.5mL microtube containing 1mL of ice-cold PBST as a wash buffer, and the beads were collected using a magnetic stand. The supernatant was then collected and resuspended in 1mL of ice-cold PBST, and the beads were collected using a magnetic stand. This procedure was repeated once more, and the beads were resuspended in PBS. The magnetic beads were then fractionated using a flow cytometer, and the FAM fluorescent signal labeled on the detection probe was detected.

[0056] <Test example result 1> When only the detection probe was added to magnetic beads with immobilized capture probes, no fluorescent signal was detected, regardless of the presence or absence of ligase (Figure 2, sample numbers 1 and 3). However, when bridging nucleic acid was added, a fluorescent signal was detected at the same level as under ligase reaction conditions, even though no ligase was added (Figure 2, sample numbers 2 and 4). This suggests that the detection probe is nonspecifically bound to the magnetic beads via the bridging nucleic acid, or that the ternary complex of the capture probe, bridging nucleic acid, and detection probe on the magnetic beads was not separated during the washing process.

[0057] Test Example 2 In Test Example 2, the identity of the substance that could not be separated by conventional methods was investigated. As a negative control, beads on which no capture probe was immobilized ("unimmobilized" beads) were used for the ligase reaction. The other procedures were the same as in Test Example 1.

[0058] <Test example result 2> When solid-phase beads without capture probe were used, the FAM fluorescence value did not increase even when bridging nucleic acid and detection probe were added (Figure 3, sample numbers 2 and 5). Nor did the FAM fluorescence value increase when only the capture probe and detection probe were added (Figure 3, sample numbers 3 and 6). A strong fluorescent signal was observed when the capture probe, bridging nucleic acid, and detection probe were all present (Figure 3, sample numbers 1 and 4). Although it was expected that no fluorescent signal would be observed in sample number 1, a strong fluorescent signal was observed. These results suggest that the ternary complex of the capture probe, bridging nucleic acid, and detection probe on the magnetic beads was not separated during the washing process.

[0059] Test Example 3 In Test Example 3, the effect of the denaturation temperature on the separation of the ternary complex of the capture probe, the bridging nucleic acid, and the detection probe was examined. The Tm value of the ternary complex of the capture probe, bridging nucleic acid, and detection probe was approximately 64°C, and it was thought that sufficient dissociation would occur at a thermal denaturation temperature of 70°C before washing. However, to increase the dissociation efficiency, the denaturation temperature was increased to 70°C, 80°C, 90°C, and 95°C, and the procedure of Test Example 1 was performed. Only capture probe-immobilized beads and detection probe were added to all samples; no ligase was added. In order to generate a ternary complex of the capture probe, bridging nucleic acid, and detection probe, which was to be removed by washing, no ligase was added, i.e., the situation in which the ternary complex was generated in the ligation reaction was reproduced, and it was examined whether the ternary complex could be separated by washing.

[0060] <Test example result 3> Under thermal denaturation conditions of 70°C, the FAM fluorescence intensity increased significantly with the addition of crosslinked nucleic acid alone compared to the condition without crosslinked nucleic acid (Figure 4, sample number 5), and the tripartite complex was not eliminated even when the denaturation temperature was raised to 95°C (Figure 4, sample numbers 1 to 4).

[0061] Test Example 4 In Test Example 4, the effect of a denaturant on the separation of a ternary complex of a capture probe, a bridging nucleic acid, and a detection probe was examined. Known methods for disrupting DNA double-stranded structures include high temperature treatment, high pH, ​​and denaturing agents such as formamide and urea. Therefore, instead of PBST, we used the following wash buffers: 10 mM CAPS, pH 11 (Dojindo Chemical Industries, Cat. 347-00482), PBS containing 20% ​​formamide (Fujifilm Wako Pure Chemical Industries, Cat. 066-02301), PBS containing 7 M urea (Nacalai Tesque, Cat. 35940-65), and PBS containing 3 M urea, each supplemented with 0.05% Tween-20. Each buffer was ice-cooled. Only capture probe-immobilized beads and detection probes were added to all samples; no ligase was added.

[0062] <Test example result 4> No reduction in nonspecific FAM fluorescence signals was observed under any of the buffer conditions. Because the nucleic acid concentration in the reaction solution after thermal denaturation was high and the effects of thermal denaturation, pH denaturation, and denaturants were thought to be limited, the reaction solution was diluted before thermal denaturation (Example 1).

[0063] Example 1-1: Substitution method To the product (10 μL) from which the ligase reaction had been completed without the addition of ligase, 90 μL of PBST was added, the beads were accumulated using a magnetic stand, the supernatant was discarded, and 10 μL of PBST was added. The heat treatment and subsequent procedures were similar to those in Test Example 1. All buffers used were ice-cooled.

[0064] Example 1-2: Dilution method To the product (10 μL) from which the ligase reaction had been completed without the addition of ligase, 1.0 mL of PBST was added, and the mixture was heat-treated at 70°C and then rapidly cooled. The beads were accumulated using a magnetic stand, the supernatant was discarded, and the mixture was washed three times with PBST according to the procedure in Test Example 1. All buffers used were ice-cooled.

[0065] As in Test Example 3, in Examples 1-1 and 1-2, ligase was not added in order to generate a ternary complex of the capture probe, bridging nucleic acid, and detection probe, which was intended to be removed by washing. That is, the situation in which the ternary complex was generated in the ligation reaction was reproduced to examine whether the ternary complex could be separated by washing. In Examples 2 and 3, ligase was not added for the same purpose.

[0066] <Example Result 1> Nonspecific fluorescent signals were dramatically improved by buffer replacement or buffer dilution before heat denaturation (Figure 6, sample numbers 1 and 2). Further investigation was performed using a replacement method that was more effective at reducing background signals and allowed heat denaturation to be performed using a general-purpose thermal cycler.

[0067] Example 2 In Example 2, the washing buffer used in the washing step was examined. The procedure for the replacement method in Example 1-1 was followed, except that the washing buffers used were PBST, 10 mM CAPS, pH 11, each containing 0.05% Tween-20, 20% formamide-containing PBS, 7 M urea-containing PBS, and 3 M urea-containing PBS. All buffers were ice-cooled.

[0068] <Example Result 2> The combination of DNA denaturants (formamide, urea) did not improve washing efficiency (Figure 7, sample numbers 2 to 4), but the background was significantly reduced by using a high-pH buffer (Figure 7, sample number 1). Going forward, we plan to use a high-pH buffer, 10 mM CAPS buffer (pH 11). Washing was performed under ice-cooled conditions to maintain the detachment of crosslinked nucleic acids, but sufficient washing effectiveness was achieved, so we decided to examine the washing temperature conditions. We also planned to evaluate whether increasing the surfactant concentration could improve the washing effectiveness of PBST.

[0069] Example 3 In Example 3, the temperature conditions of the washing operation and the washing effect of a washing buffer containing a surfactant were examined. The procedure for the replacement method in Example 1-1 was followed, except that the washing buffers used were 10 mM CAPS buffer containing 0.05% Tween-20, pH 11, and 10 mM CAPS buffer containing 0.1% Tween-20, pH 11. The washing procedure was carried out at room temperature or heated to 70°C.

[0070] <Example Result 3> By setting the washing temperature to 70°C, the background signal was sufficiently reduced even with PBST washing (Figure 8, Sample No. 3 vs. Sample No. 6). However, maintaining the buffer, reaction solution, and magnetic stand at 70°C during washing is cumbersome and poses a risk of burns. On the other hand, when 10 mM CAPS buffer was used, sufficient cleaning effect was maintained even at room temperature, and a surfactant concentration of 0.05% was sufficient (Figure 8, Sample No. 5). Based on these findings, we decided to perform washing at room temperature with 10 mM CAPS buffer containing 0.05% Tween-20, pH 11. Since a washing method that sufficiently reduced nonspecific FAM fluorescent signals was established, we decided to perform a follow-up test of Test Example 1 (Example 4).

[0071] Example 4 In Example 4, the washing effect under the room temperature washing conditions of Example 3 was examined when ligase was actually added. The test was carried out according to the operating procedure under room temperature washing conditions in Example 3. The reaction conditions were a combination of the presence or absence of cross-linked nucleic acid and the presence or absence of added ligase, and the washing buffer was PBST or 10 mM CAPS buffer containing 0.05% Tween-20, pH 11.

[0072] <Example Result 4> Although the improved washing process significantly reduced the background even with PBST, the histogram for the positive control (Figure 9, sample number 5) was somewhat broad, and the background (Figure 9, sample numbers 6 to 8) also had a tail that increased the FAM fluorescent signal, and the S / N ratio was poorer than with CAPS buffer (Figure 9, sample numbers 1 and 2).When CAPS buffer was used, the histograms for both the positive control (Figure 9, sample number 1) and the negative control (Figure 9, sample numbers 2 to 4) were sharp, and the distance between the two tails (the distance between the orange lines in the figure) was wide. While FAM fluorescent signals were detected when ligase was added (Figure 9, sample numbers 1 and 5), nonspecific FAM fluorescent signals were significantly reduced when ligase was not added (Figure 9, sample numbers 3 and 7). Therefore, the effectiveness of the improved washing method was confirmed in the actual detection system. Therefore, we decided to investigate reducing the buffer volume during washing to further improve operability.

[0073] Example 5 In Example 5, the scale of the wash buffer under the room temperature wash conditions of Example 4 was examined. The test was performed according to the operating procedure for room temperature washing conditions in Example 4. However, the amount of buffer used during washing was reduced from 1 mL to 200 μL or 50 μL. The washing buffer used was 10 mM CAPS buffer containing 0.05% Tween-20, pH 11. To further confirm the importance of heat denaturation treatment, washing without heat denaturation treatment was also examined.

[0074] <Example Result 5> A sufficient washing effect was obtained even when the buffer volume used during washing was reduced (Figure 10, sample numbers 1 and 2). Without heat treatment, the FAM fluorescent signal increased depending on the buffer volume, reaffirming the necessity of a heat treatment step before washing. Since the buffer volume during washing could be reduced to 50 μL, transferring from PCR tubes to 1.5 mL microtubes during washing was no longer necessary, improving operability. Next, we decided to investigate reducing the number of washes.

[0075] Example 6 In Example 6, the number of washing cycles under the room temperature washing conditions of Example 5 was examined. The test was performed according to the operating procedure of Example 5, under the conditions of room temperature washing and 50 μL of buffer volume during washing. The amount of buffer volume during washing was reduced from 1 mL to 200 μL or 50 μL. The number of washes was reduced from the standard four times to three or two times.

[0076] <Example Result 6> It was confirmed that sufficient washing effect could be obtained even when the number of washings was reduced to three or two times (Fig. 11, sample numbers 1 and 2). Finally, we decided to evaluate the pH dependency of the washing buffer.

[0077] Example 7 In Example 7, the influence of the pH value of the washing buffer on the washing effect under the conditions of Example 6 was examined. The test was performed according to the procedure of Example 6, with two washes, except that the wash buffers used were 10 mM CAPS buffer, pH 11, 10.5, or 10.0, each containing 0.05% Tween-20; 100 mM CHES (Dojindo Chemical Industries, Ltd., Cat. No. 342-04692) buffer, pH 9.5 or 9.0; 10 mM Tris (Nacalai Tesque, Ltd., Cat. No. 35434-05) buffer, pH 8.5; and PBS, pH 7.4.

[0078] <Example Result 7> Even when the pH was increased from 8.5 to 11.0, sufficient cleaning effect was maintained (Fig. 12, sample numbers 1 to 6).

Claims

1. A method for reducing non-specific by-products in a ligation reaction using a ligase, comprising: After the ligation reaction, heating the reaction solution containing the reaction product and by-product in the presence of a first washing buffer; After the heating step, washing the reaction product with a second wash buffer; A method comprising:

2. The method of claim 1 , further comprising the step of washing the reaction product with a wash buffer prior to the heating step.

3. The method according to claim 1 or 2, wherein the wash buffer has a pH of 8.5 or higher.

4. The method of claim 1 or 2, wherein the wash buffer comprises a non-ionic detergent.

5. 3. The method of claim 1 or 2, wherein the step of washing the reaction product after the heating step is carried out at a temperature of less than 80°C.

6. The method of claim 1 or 2, wherein a ligation reaction is carried out in the detection of the mutated target sequence.

7. The method of claim 6 , wherein the mutation is a single nucleotide polymorphism.

8. 7. The method of claim 6, wherein in the reaction product, the capture probe and the detection probe bound to the solid phase are annealed to the target sequence.

9. The method of claim 8, wherein the solid phase is a magnetic bead.

10. 9. The method of claim 8, wherein the capture probe and the detection probe are ligated in the reaction product when the target sequence is mutated.

11. The method of claim 6, wherein the reduction of non-specific by-products reduces background in mutation detection.

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