Primers, DNA detection methods, and DNA detection kits

Abstract

The present invention relates to: a primer for a nucleic acid amplification reaction, in particular, an asymmetric nucleic acid amplification reaction; a DNA detection method; and a DNA detection kit. Specifically, the present invention relates to: a primer for introducing a mutation into a subject nucleic acid and amplifying the nucleic acid, the primer comprising, in the sequence thereof, a mutation for introducing a mutation in the subject nucleic acid that results in, at the temperature at which a probe and a nucleic acid amplified from the subject nucleic acid are bonded, an increased ratio of the single-strand formation bases in the probe bonding region of the nucleic acid amplified from the subject nucleic acid; and a DNA detection method and a DNA detection kit which use the primer.

Description

Technical Field

[0001] The present invention relates to primers, a DNA detection method, and a DNA detection kit in nucleic acid amplification reactions, particularly asymmetric nucleic acid amplification reactions.

Background Art

[0002] For genetic testing, there are techniques such as polymerase chain reaction (PCR), real-time PCR, and digital PCR. In real-time PCR and digital PCR, which enable more accurate quantification than PCR, DNA is detected using intercalators or fluorescently labeled probes. Among the DNA detection methods using fluorescently labeled probes, two types, hydrolysis probes and molecular beacons, are commonly used. Different from hydrolysis probes, in which a fluorescent dye is released from the probe and emits fluorescence when the probe is decomposed using the nuclease activity of DNA polymerase, molecular beacons form a stem-loop in the free state and bind to the detection target DNA at the loop portion. Molecular beacons have the characteristic of not being decomposed during PCR, and not only can the amplification be determined by the fluorescence intensity after PCR, but also melting curve analysis can be performed. [[ID=Z13]]

[0003] Here, in DNA detection using molecular beacons, in order to increase the binding amount between the molecular beacon and the amplified detection target DNA and increase the fluorescence intensity, a method has been reported (Non-Patent Document 1) in which the concentration of the forward primer and the reverse primer is made asymmetric to asymmetrically amplify the detection target DNA so that a strand complementary to the molecular beacon is amplified excessively.

[0004] The present inventors have developed a technique for applying an asymmetric nucleic acid amplification reaction using a molecular beacon to digital PCR and identifying the genotype of a target gene with high sensitivity and high multiplexing by melting curve analysis after amplification (Patent Document 1, Non-Patent Document 2).

[0005] An example of a digital PCR detection method is shown below. First, DNA polymerase, primers, and a fluorescently labeled probe necessary for PCR are added to the limitingly diluted sample to prepare the PCR reaction mixture. The PCR reaction mixture is divided into small compartments such as wells or droplets. At this time, each compartment should either contain one molecule of the target gene or not.

[0006] Next, the target gene within each microcompartment is amplified by PCR. After PCR, the fluorescence intensity of each microcompartment is measured, and the number of microcompartments with fluorescence intensity exceeding a threshold is counted to quantify the target gene.

[0007] When an asymmetric nucleic acid amplification reaction using molecular beacons is applied, after PCR, melting curve analysis of the target gene amplified within a microcompartment and the molecular beacon can be performed, allowing for the observation and differentiation of different melting temperatures (Tm) for each genotype of the target gene. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2018-108063 [Non-patent literature]

[0009] [Non-Patent Document 1] BMC Microbiol., 13, pp295, 2013 [Non-Patent Document 2] Anal. Chem.,92,pp11705-11713,2020 [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] However, the inventors were the first to recognize that in asymmetric nucleic acid amplification reactions, one strand of the excessively amplified DNA forms a secondary structure within the molecule. Molecular beacons designed for the single-stranded region bind to the amplified target DNA, while molecular beacons designed for the double-stranded region experience a decrease in binding and fluorescence intensity. In particular, in PCR performed in a micro-compartment, such as digital PCR, rather than in a tube, such as real-time PCR, a decrease in fluorescence intensity can lead to detection failure due to insufficient fluorescence intensity, or make it difficult to calculate the melting temperature in melting curve analysis due to small changes in fluorescence intensity, thus reducing measurement sensitivity and accuracy.

[0011] Therefore, the object of the present invention is to provide a novel primer, DNA detection method, and DNA detection kit that, in an asymmetric nucleic acid amplification reaction, reduce the proportion of molecular beacon binding regions on one strand of the excessively amplified DNA that form a double-stranded structure and increase the proportion that form a single-stranded structure, thereby suppressing the decrease in the binding ratio between molecular beacons and amplified target DNA, and enabling accurate and highly sensitive detection of target genes and genotype determination. [Means for solving the problem]

[0012] The inventors of this invention have revealed that in asymmetric nucleic acid amplification reactions, even when amplified with the same primer set, the fluorescence intensity differs significantly depending on the molecular beacon binding region, and that molecular beacons designed in regions where one of the amplified strands forms a double-stranded structure within the molecule have low fluorescence intensity. Therefore, they discovered that by intentionally introducing mutations different from the target gene sequence into the primers used in the asymmetric nucleic acid amplification reaction and performing the asymmetric nucleic acid amplification reaction, the proportion of molecular beacon binding regions forming a double-stranded structure decreases, increasing fluorescence intensity, thus completing the present invention.

[0013] One embodiment of the present invention is, A step of amplifying the nucleic acid to be tested in the presence of a primer pair including a forward primer and a reverse primer and a probe, A step of measuring the binding of nucleic acids amplified by the forward primer and the reverse primer to the probe. A method for detecting target nucleic acids, including The method wherein either or both of the forward primer or the reverse primer include in their sequence a mutation for introducing a mutation into the amplified nucleic acid such that, at the temperature at which the probe is bound to the amplified nucleic acid, the proportion of single-stranded bases in the probe-binding region of the amplified nucleic acid is increased.

[0014] Another embodiment of the present invention is a primer for introducing a mutation into a test nucleic acid and amplifying it, wherein the primer includes in its sequence a mutation for introducing a mutation into the test nucleic acid such that, at the temperature at which the nucleic acid amplified from the test nucleic acid is bound to the probe, the proportion of single-stranded bases in the probe binding region of the nucleic acid amplified from the test nucleic acid is increased.

[0015] Another embodiment of the present invention is a kit for detecting a target nucleic acid, A primer pair including a forward primer and a reverse primer, A probe that binds to nucleic acids amplified using forward primers and reverse primers. Includes, The kit is wherein either or both of the forward primers or reverse primers contain mutations in their sequences to introduce mutations into the nucleic acid to be amplified such that, at the temperature at which the probe is bound to the nucleic acid to be amplified, the proportion of single-stranded bases in the probe-binding region of the nucleic acid to be amplified is increased. [Effects of the Invention]

[0016] The present invention provides novel primers, a DNA detection method, and a DNA detection kit that can more accurately and sensitively detect or quantify a target gene in an asymmetric nucleic acid amplification reaction. Therefore, the present invention is useful in fields such as basic research, testing, and drug discovery for gene detection, genotype discrimination, and the like.

Brief Description of the Drawings

[0017] [Figure 1] It is a schematic diagram showing an example of a method for measuring the melting temperature of DNA using a fluorescently labeled probe. [Figure 2] It is a schematic diagram showing an example of the behavior of a fluorescently labeled probe in a symmetric nucleic acid amplification reaction and an asymmetric nucleic acid amplification reaction. [Figure 3] It is a schematic diagram showing the secondary structure of amplified target DNA and the binding of a fluorescently labeled probe when performing an asymmetric nucleic acid amplification reaction using a primer for introducing a mutation in one embodiment of the present invention. [Figure 4] It is a schematic diagram showing the secondary structure of amplified target DNA and the binding of a fluorescently labeled probe when performing an asymmetric nucleic acid amplification reaction using a primer for introducing a tagged mutation in one embodiment of the present invention. [Figure 5] It is a diagram showing an example of a melting curve when using a mutation-introducing primer in one embodiment of the present invention. [Figure 6] It is a flowchart showing one embodiment of a DNA detection method for performing an asymmetric nucleic acid amplification reaction using a mutation-introducing primer. [Figure 7A] In the examples of the present invention, it is a diagram showing the positional relationship between the secondary structure of amplified single-stranded DNA of the detection target and the binding region of a fluorescently labeled probe corresponding to the wild type. [Figure 7B] In the examples of the present invention, it is a diagram showing the positional relationship between the secondary structure of amplified single-stranded DNA of the detection target and the binding region of a fluorescently labeled probe corresponding to the mutant type. [Figure 7C] In the examples of the present invention, it is a diagram showing the measurement results (differential curve of the melting curve) when not using a mutation-introducing primer. [Figure 8A]This figure shows the secondary structure of the amplified single-stranded DNA to be detected, the positional relationship of the binding region of the fluorescently labeled probe corresponding to the wild type, and the location where mutations are planned to be introduced by the primer. [Figure 8B] This figure shows the positional relationship between the secondary structure of the amplified single-stranded DNA to be detected and the binding region of the fluorescently labeled probe corresponding to the mutant type, as well as the location of the mutation introduced by the primer, in an embodiment of the present invention. [Figure 9A] This figure shows the secondary structure of the amplified single-stranded DNA to be detected, the positional relationship of the binding region of the fluorescently labeled probe corresponding to the wild type, and the location where mutations are planned to be introduced by the primer. [Figure 9B] This figure shows the positional relationship between the secondary structure of the amplified single-stranded DNA to be detected and the binding region of the fluorescently labeled probe corresponding to the mutant type, as well as the location of the mutation introduced by the primer, in an embodiment of the present invention. [Modes for carrying out the invention]

[0018] The object, features, advantages, and ideas related thereto of the present invention will be apparent to those skilled in the art from the description herein. Those skilled in the art will be able to easily reproduce the present invention from the description herein. The embodiments and specific examples of the invention described below are examples of preferred embodiments of the present invention and are provided for illustrative or explanatory purposes only; the present invention is not limited thereto. It will be apparent to those skilled in the art that various modifications and modifications can be made based on the description herein without departing from the intent and scope of the present invention as disclosed herein.

[0019] (1) Primer In one embodiment, the present invention provides a primer for introducing a mutation into a test nucleic acid and amplifying it, wherein the primer contains in its sequence a mutation to introduce a mutation into the test nucleic acid such that, at a temperature at which the nucleic acid amplified from the test nucleic acid is bound to the probe, the proportion of single-stranded bases in the probe binding region of the nucleic acid amplified from the test nucleic acid is increased. Such a primer may be used as a reagent for introducing a mutation into a test nucleic acid and amplifying it.

[0020] In the present invention, the test nucleic acid is not particularly limited as long as it contains or may contain the target nucleic acid to be detected, and includes, for example, messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, genomic DNA, and their fragments, DNA-RNA hybrid nucleic acids, etc., and may be single-stranded or double-stranded. If the target nucleic acid to be detected is RNA such as mRNA, the test nucleic acid may be DNA (such as cDNA) obtained by reverse transcription of that RNA.

[0021] The primer may be a forward primer or a reverse primer, and both primers may contain the mutation. Preferably, the primer is a reverse primer.

[0022] The primer contains a mutation to amplify the test nucleic acid. This mutation is introduced to increase the proportion of single-stranded bases in the probe binding region of the amplified nucleic acid (the ratio of single-stranded bases to double-stranded bases) at the temperature at which the probe is bound to the amplified nucleic acid. While the amplified nucleic acid has a single-stranded structure, it may form a double-stranded structure internally due to the presence of complementary bases. The structure of the amplified nucleic acid depends on its sequence composition, and in this art, programs (e.g., OligoAnalyzer) are used to predict such structures. TMTool (IDT), RNAfold (University of Vienna) are conventionally used. Therefore, such programs are used to predict the structure of the nucleic acid to be amplified and to determine the percentage of single-stranded bases in the probe-binding region of the amplified nucleic acid. If the percentage of single-stranded bases is low, for example less than 50%, mutations are introduced into the nucleic acid to be amplified so that the percentage of single-stranded bases increases, for example to at least 50%, preferably 60% or more.

[0023] Aside from the mutations mentioned above, primers can be designed using conventional primer design methods and can be designed with lengths, sequence compositions, and melting temperatures suitable for specific binding to target nucleic acids.

[0024] The primers may further contain a tag sequence to increase the proportion of single-stranded bases as described above. Such a tag sequence should have a length and composition that does not affect the detection of the target nucleic acid. The tag sequence may be included in either the forward primer or the reverse primer, or both may further contain the tag sequence. Nucleic acids amplified using such primers will contain the tag sequence.

[0025] One embodiment disclosed herein is a primer for asymmetric nucleic acid amplification reactions, in which a mutation is introduced such that, at the temperature at which a fluorescently labeled probe such as a molecular beacon is bound, the proportion of double-stranded structures and the proportion of single-stranded structures in the fluorescently labeled probe-binding region of the DNA to be detected is reduced. This primer will be described in detail below with reference to the schematic diagrams in Figures 1 to 5.

[0026] Figure 1 is a schematic diagram showing the behavior of a fluorescently labeled probe when the temperature of the reaction solution is changed. Examples of fluorescently labeled probes include molecular beacons and Taqman probes, but here we will explain Figure 1 using molecular beacons as an example.

[0027] Molecular beacons are composed of oligonucleotides and have a sequence complementary to the sequence between the primer pair used in nucleic acid amplification reactions to amplify the target gene. Furthermore, molecular beacons have complementary sequences at both ends; one end has a fluorescent dye 103, and the other end has a quencher (quencher) 104. In the nucleic acid amplification reaction, initially, molecular beacons 102 exist as free molecules, as shown in Figure 1B. At this time, molecular beacons 102 form a stem-loop, and since the fluorescent dye 103 and quencher 104 are in close proximity, no fluorescence is emitted. In the initial denaturation step, when the sample solution is heated, it adopts a highly flexible structure as shown in Figure 1C, but since the fluorescent dye and quencher dye are never separated, the fluorescence remains quenched. In the annealing step, when the temperature is lowered to approximately room temperature, the loop portion of molecular beacon 102 anneals to the DNA 101 amplified in the sample solution, as shown in Figure 1A. As a result, the fluorescent dye 103 and the quencher 104 are constantly separated, causing the fluorescently labeled probe 102 to emit strong fluorescence. In the next extension step, the molecular beacon 102 is released, and the situation returns to that shown in Figure 1B, and the fluorescence is quenched. In the next denaturation step, the situation returns to that shown in Figure 1C, and the fluorescence remains quenched. In the nucleic acid amplification reaction, this process is repeated, so the fluorescence intensity can be measured at some point during heating or cooling. The same method can be used to measure the fluorescence intensity after the nucleic acid amplification reaction is complete. After the nucleic acid amplification reaction is complete, heating or cooling may be performed solely to measure the fluorescence intensity.

[0028] When using a DNA intercalator instead of a combination of fluorescent dye and quencher, the DNA intercalator intercalates between the double helix strands of the sample DNA when the sample DNA is double-stranded, causing fluorescence. However, when the sample DNA is single-stranded, the DNA intercalator is released, and the fluorescence is quenched. Therefore, in nucleic acid amplification reactions, similar to molecular beacon 102, the fluorescence intensity can be measured at some stage, either during heating or cooling. Alternatively, the fluorescence intensity may be measured after the nucleic acid amplification reaction is complete, solely for the purpose of heating or cooling.

[0029] In the molecular beacon 102 used here, the combination of fluorescent dye 103 and quencher 104 is not particularly limited as long as it is a combination commonly used in real-time PCR. For example, examples of fluorescent dye 103 include FAM, VIC, ROX, Cy3, and Cy5, and examples of quencher 104 include TAMRA, BHQ1, BHQ2, and BHQ3. All of these are commonly used and are commercially available.

[0030] When two different target genes with distinct sequences are used, molecular beacon 102 sequences are prepared that specifically bind to each target gene, and by attaching different fluorescent dyes, the two target genes can be distinguished and detected in a single reaction system.

[0031] The DNA intercalator is not particularly limited as long as it increases fluorescence intensity upon binding to double-stranded DNA and can be used for detecting double-stranded DNA. Specifically, SYBR® Green I, SYBR Gold, PicoGreen®, SYTO® Blue, SYTO Green, SYTO Orange, SYTO Red, POPO®-1, BOBO®-1, YOYO®-1, TOTO®-1, JOJO®-1, POPO-3, LOLO®-1, BOBO-3, YOYO-3, TOTO-3, PO-Pro®-1, YO-Pro®-1, TO-Pro®-1, JO-Pro®-1, PO-Pro-3, YO-Pro-3, TO-Pro-3, TO-Pro-5, ethidium bromide, etc., are applicable and all are commercially available. If the DNA intercalator is heat-resistant, it can be added to the reaction solution before the nucleic acid amplification reaction is performed.

[0032] Figure 2 is a schematic diagram illustrating the behavior of a fluorescently labeled probe in symmetric and asymmetric nucleic acid amplification reactions. As shown in Figure 2A, when the concentrations of forward primer 201 and reverse primer 202 are the same, both complementary strands of the target DNA 204 are amplified equally, resulting in the generation of many double-stranded molecules 205 and a small amount of single-stranded molecules 206 with the fluorescently labeled probe 203 bound to them. As shown in Figure 2B, when the concentrations of forward primer 201 and reverse primer 202 are asymmetric (in Figure 2B, the amount of reverse primer 202 is increased), one strand 207 of the target DNA 204, which is the complementary strand of the molecular beacon, is excessively amplified, resulting in the generation of many single-stranded molecules 206 with the fluorescently labeled probe 203 bound to them. Performing an asymmetric nucleic acid amplification reaction in this way increases the amount of the fluorescently labeled probe bound to the amplified target DNA, and thus increases the fluorescence intensity.

[0033] Figure 3 is a schematic diagram showing the binding of a fluorescently labeled probe to the secondary structure of the amplified target DNA when an asymmetric nucleic acid amplification reaction is performed using a primer that introduces a mutation. As shown in Figure 3A, a forward primer 301, a mutation-introduced reverse primer 302 with mutation 309 introduced, and a fluorescently labeled probe 303 are prepared for the target DNA 304. At this time, the concentration of the reverse primer 302 is excessive compared to the concentration of the forward primer 301, and the asymmetric nucleic acid amplification reaction is performed. Figure 3B is a schematic diagram showing the binding of the fluorescently labeled probe 303 to the secondary structure 305 of the single-stranded target DNA amplified by the asymmetric nucleic acid amplification reaction using a reverse primer that does not introduce mutation 309. (Conventional method) Since mutation 309 is not introduced, position 306 on the secondary structure 305 of the amplified DNA remains the original base of the target DNA. In the secondary structure 305 of the amplified single-stranded target DNA, the binding region of the fluorescently labeled probe 303 forms a double-stranded structure, and the amount of fluorescently labeled probe 303 that binds is reduced. On the other hand, Figure 3C is a schematic diagram showing the binding of the fluorescently labeled probe 303 to the secondary structure 307 of the single-stranded target DNA amplified by an asymmetric nucleic acid amplification reaction using the mutagenesis reverse primer 302 (invention). Due to the mutation 308 introduced by the mutagenesis reverse primer 302, the secondary structure 307 of the amplified single-stranded target DNA forms a different secondary structure from the secondary structure 305 of the amplified single-stranded target DNA in Figure 3B. Therefore, the proportion of the binding region of the fluorescently labeled probe 303 that forms a double-stranded structure decreases in Figure 3C compared to Figure 3B, and the proportion that forms a single-stranded structure increases, resulting in an increase in the amount of fluorescently labeled probe 303 that binds.

[0034] Figure 4 is a schematic diagram showing the binding of the fluorescently labeled probe to the secondary structure of the amplified target DNA when an asymmetric nucleic acid amplification reaction is performed using a primer that introduces a tagged mutation. As shown in Figure 4A, a forward primer 401, a mutation-introducing reverse primer 402 with a tag sequence 405 added and mutation 411 introduced, and a fluorescently labeled probe 403 are prepared for the target DNA 404. At this time, the concentration of the reverse primer 402 is excessive compared to the concentration of the forward primer 401, and the asymmetric nucleic acid amplification reaction is performed. Figure 4B is a schematic diagram showing the binding of the fluorescently labeled probe 403 to the secondary structure 406 of the single-stranded target DNA amplified by the asymmetric nucleic acid amplification reaction using a reverse primer (not shown) that does not have a tag sequence and does not introduce mutation 411 (similar to Figure 3B). Since mutation 411 is not introduced, position 407 on the secondary structure 406 of the amplified DNA remains the original base of the target DNA. In the secondary structure 406 of the amplified single-stranded target DNA, the binding region of the fluorescently labeled probe 403 forms a double-stranded structure, resulting in a decrease in the amount of fluorescently labeled probe 403 that binds. On the other hand, Figure 4C is a schematic diagram showing the binding of the fluorescently labeled probe 403 to the secondary structure 408 of the single-stranded target DNA amplified by an asymmetric nucleic acid amplification reaction using a tagged mutagenesis reverse primer 402. Due to the mutation 409 introduced by the tagged mutagenesis reverse primer 402 and the addition of a tag sequence 410, the secondary structure 408 of the amplified single-stranded target DNA forms a different secondary structure from the secondary structure 406 of the amplified single-stranded target DNA in Figure 4B. Therefore, the proportion of the binding region of the fluorescently labeled probe 403 that forms a double-stranded structure decreases in Figure 4C compared to Figure 4B, and the proportion that forms a single-stranded structure increases, resulting in an increase in the amount of fluorescently labeled probe 403 that binds.

[0035] Figure 5 shows examples of melting curves for asymmetric nucleic acid amplification reactions using and without mutagenesis primers. When asymmetric nucleic acid amplification reactions are performed using mutagenesis primers, measuring the change in fluorescence intensity of the solution with respect to temperature results in a melting curve with a large change in fluorescence intensity, as shown in Figure 5A. The differential curve shows a high peak, as shown in Figure 5B, and the temperature at which this peak is the melting temperature (Tm) of 501. However, when asymmetric nucleic acid amplification reactions are performed without mutagenesis primers, measuring the change in fluorescence intensity of the solution with respect to temperature results in a decrease in fluorescence intensity compared to Figure 5A, as shown in Figure 5C. Therefore, the differential curve shows a low peak, as shown in Figure 5D, and because the peak shape is gentle, it may result in an incorrect value that differs from the melting temperature of the target gene in the solution. Furthermore, if the decrease in fluorescence intensity is large, the fluorescence intensity due to the amplification of the target gene may become lower than the detectable intensity and become undetectable.

[0036] (2) DNA detection method In another embodiment, the present invention is: A step of amplifying the nucleic acid to be tested in the presence of a primer pair including a forward primer and a reverse primer and a probe, A step of measuring the binding of nucleic acids amplified by the forward primer and the reverse primer to the probe. The present invention provides a method for detecting target nucleic acids, including Either or both of the forward primer or the reverse primer includes a mutation in its sequence to introduce a mutation into the amplified nucleic acid such that, at the temperature at which the probe is bound to the amplified nucleic acid, the proportion of single-stranded bases in the probe-binding region of the amplified nucleic acid is increased.

[0037] In this specification, detection of a target nucleic acid means determining the presence or absence of the nucleic acid to be detected, measuring the concentration or relative abundance of the nucleic acid to be detected, identifying the nucleic acid to be detected, determining its genotype, and so on.

[0038] Either or both of the forward primer and / or reverse primer are the primers described in the preceding section.

[0039] The probe contains a fluorescent dye, or contains both a fluorescent dye and a quenching dye. If the probe contains a fluorescent dye, this dye is used to measure the binding of the amplified nucleic acid to the probe. In one embodiment, the 3' and 5' terminal sequences of the probe have complementary sequences, and if the probe does not bind to the amplified nucleic acid, the 3' and 5' terminal sequences of the probe bind to form a stem structure. If the probe does not contain a fluorescent dye, a DNA intercalator can be used, for example.

[0040] The amplification step can be carried out by any amplification reaction known in the art. Preferably, the amplification step is carried out by an asymmetric nucleic acid amplification reaction. In this case, the amplification reaction is carried out by increasing the amount of either the forward primer or the reverse primer (for example, a primer containing a mutation). In a preferred embodiment, the amplification reaction is carried out by adding more reverse primers containing mutations than forward primers. For asymmetric nucleic acid amplification reactions, see, for example, Anal. Chem., 92, pp11705-11713, 2020 (Non-Patent Literature 2).

[0041] In one embodiment, the binding of amplified nucleic acid to a probe is measured with respect to temperature changes. Since the binding changes with the melting temperature, the melting temperature of the double hemisphere of the amplified nucleic acid and probe can be calculated from the change in binding with respect to this temperature change.

[0042] In one embodiment, the binding of amplified nucleic acid to a probe is measured for each temperature change cycle, and the target nucleic acid can be detected from the relationship between the number of cycles and the change in binding.

[0043] By using multiple types of primers containing the aforementioned mutations, multiple target nucleic acids can be detected. In this case, the binding of the amplified nucleic acid to the probe is measured for each temperature change cycle, and the ratio of the initial concentrations of multiple target nucleic acids in the test nucleic acid can be calculated from the relationship between the number of cycles and the change in binding.

[0044] In one embodiment, the binding of the amplified nucleic acid to the probe is measured for each temperature change cycle, and the relationship between the number of cycles and the change in binding can be compared with the change in binding of a sample containing a known concentration of the target nucleic acid to calculate the initial concentration of the target nucleic acid in the tested nucleic acid whose concentration is unknown.

[0045] The DNA detection method disclosed herein includes the steps of: performing an asymmetric nucleic acid amplification reaction on a solution containing DNA to be detected using a primer that introduces a mutation so that, at the temperature at which the fluorescent probe is bound, the proportion of double-stranded structures in the fluorescent probe-binding region of the DNA to be detected increases the proportion of single-stranded structures; a fluorescent probe that binds to the DNA to be detected; a primer that pairs with the mutation-introducing primer to amplify the fluorescent probe-binding region of the DNA to be detected; and a DNA polymerase that amplifies the DNA to be detected; measuring the fluorescence intensity in response to a change in temperature of the DNA solution; and calculating the melting temperature of the DNA double helix from the change in fluorescence intensity in response to the change in temperature of the DNA solution. The DNA detection method will be described in detail below with reference to the schematic diagram in Figure 6.

[0046] First, a mutated primer is designed to reduce the proportion of double-stranded structures and increase the proportion of single-stranded structures in the amplified target DNA's fluorescent probe-binding region (S601). The mutated primer may or may not have a tag sequence. Next, the target DNA is amplified using the mutated primer via an asymmetric nucleic acid amplification reaction (S602). The target DNA amplified by the asymmetric nucleic acid amplification reaction forms a secondary structure in which the proportion of double-stranded structures in the probe-binding region decreases and the proportion of single-stranded structures increases (S603). The change in fluorescence intensity of the solution of the target DNA amplified by the asymmetric nucleic acid amplification reaction is measured with respect to temperature changes to create a melting curve, and the melting temperature is calculated from the peak temperature of the differential curve of the melting curve (S604). Finally, the type of target DNA contained in the solution is determined from the fluorescence color and melting temperature of the solution of the target DNA amplified by the asymmetric nucleic acid amplification reaction (S605).

[0047] If there are multiple types of DNA to be detected, multiple primers and probes can be prepared according to the sequences to be detected, added simultaneously, and an asymmetric nucleic acid amplification reaction can be performed on a DNA solution containing multiple target DNAs. By designing the multiple probes with different melting temperatures relative to each target DNA or by changing the type of fluorescent dye, the types of target DNA contained in the solution can be identified after the asymmetric nucleic acid amplification reaction from the fluorescence color and melting temperature of the solution. For example, this can be illustrated by a case where the target gene includes multiple types of alleles, such as wild-type and mutant alleles, but the multiple types of target genes are not limited to these.

[0048] The process of asymmetric nucleic acid amplification using mutagenesis primers can also be monitored in real time with a fluorescently labeled probe. For example, in an asymmetric nucleic acid amplification reaction, the ratio of the initial concentrations of the target DNA in multiple solutions can be calculated by measuring the fluorescence intensity at each temperature change cycle and examining the relationship between the number of cycles and the change in fluorescence intensity. Furthermore, by preparing a control of the target DNA with a known concentration and measuring it simultaneously, it is possible to calculate the initial concentration of the target DNA in a sample with an unknown concentration by comparing the relationship between the fluorescence intensity measurement at each temperature change cycle and the change in fluorescence intensity with the change in fluorescence intensity of the sample with a known concentration.

[0049] Asymmetric nucleic acid amplification reactions using mutagenesis primers can also be applied to digital PCR. The reaction solution containing the mutagenesis primers can be divided into microcompartments, and after the amplification reaction, melting curve analysis can be performed to detect the target gene contained within the microcompartment (e.g., genotyping).

[0050] For example, if the DNA solution to be tested contains both target gene P and target gene Q, in the microcompartment containing target gene P, a fluorescently labeled probe corresponding to target gene P will hybridize with the PCR-amplified DNA and emit fluorescence. By analyzing the resulting fluorescence, the melting temperature corresponding to the fluorescently labeled probe of target gene P can be calculated. Similarly, in the microcompartment containing target gene Q, a fluorescently labeled probe corresponding to target gene Q will hybridize with the PCR-amplified DNA and emit fluorescence. By analyzing the resulting fluorescence, the melting temperature corresponding to the fluorescently labeled probe of target gene Q can be calculated. In this way, the presence or absence of target gene P and target gene Q can be determined based on the fluorescence intensity, the type of fluorescence (e.g., color), and the melting temperature.

[0051] Since the melting temperature of DNA is not affected by the reaction efficiency of PCR or the in-plane measurement variability during fluorescence measurement, using the melting temperature of DNA allows for highly accurate identification of the type of DNA (e.g., genotype) within a microcompartment. For example, by arranging the sequences of fluorescently labeled probes so that each fluorescently labeled probe has a different melting temperature (Tm) for the target gene, and then measuring the change in fluorescence intensity with temperature changes for the DNA within the microcompartment, performing melting curve analysis, and comparing the melting temperatures, it becomes possible to detect DNA within the microcompartment (e.g., genotype identification).

[0052] When detecting multiple target genes simultaneously, they can be distinguished based on a reference melting temperature and a measured melting temperature, but it is preferable to distinguish them based on a reference melting temperature range. For example, if the measured melting temperature for a certain well is within a predetermined range that includes the reference melting temperature recorded in the database for a certain target gene (for example, within ±1°C of the reference melting temperature), it is determined that the target gene is present in that well. Using such a reference melting temperature range allows for more accurate determination, taking into account the acceptable range appropriately.

[0053] Alternatively, the ratio or difference of fluorescence intensities at different temperatures may be used as information on fluorescence intensity. For example, fluorescence intensity can be standardized by using the ratio or difference between the fluorescence intensity at a temperature lower than the reference melting temperature and the fluorescence intensity at a temperature higher than the reference melting temperature. For example, if this ratio or difference for a well falls within a predetermined range, that well is determined to be positive; otherwise, that well is determined to be negative.

[0054] For example, by subtracting the fluorescence intensity at 85°C from the fluorescence intensity at 50°C, the effect of the fluorescence of the fluorescently labeled probe itself, i.e., the background effect, can be eliminated.

[0055] The method for determining the fluorescence intensity range and the reference melting temperature range can be arbitrarily selected. For example, pilot experiments may be conducted in advance, and the operator may statistically determine the values ​​based on the results, or the DNA detection system may automatically determine them. Alternatively, the fluorescence intensity threshold and the predetermined range of the reference melting temperature may be determined statistically using the measurement data from each well in the cartridge each time a digital PCR measurement is performed.

[0056] The data used to statistically identify DNA within a well may include any or all of the following items, or other items. - Fluorescence intensity at temperatures lower than the reference melting temperature - Fluorescence intensity at temperatures higher than the reference melting temperature - The ratio of fluorescence intensity at temperatures lower than the reference melting temperature to fluorescence intensity at temperatures higher than the reference melting temperature. - The difference between the fluorescence intensity at temperatures lower than the reference melting temperature and the fluorescence intensity at temperatures higher than the reference melting temperature. - Feature quantities representing the reference melting temperature - Feature quantities that represent the shape of the melting curve

[0057] By altering the secondary structure of the amplified nucleic acid in the manner described above, and thereby increasing the proportion of single-stranded bases in the probe-binding region, the amount of binding to the probe-binding region in the amplified nucleic acid increases, thus improving measurement sensitivity and accuracy, such as the accuracy of genotype discrimination.

[0058] (3) DNA detection kit The method of the present invention described above can be carried out more easily and simply by using a kit that includes at least a primer for introducing mutations. That is, in one embodiment, the present invention provides a kit for detecting a target nucleic acid, which kit includes A primer pair including a forward primer and a reverse primer, A probe that binds to nucleic acids amplified using forward primers and reverse primers. Includes, Either or both of the forward primer or the reverse primer includes a mutation in its sequence to introduce a mutation into the nucleic acid to be amplified such that, at the temperature at which the probe is bound to the nucleic acid to be amplified, the proportion of single-stranded bases in the probe-binding region of the nucleic acid to be amplified is increased.

[0059] The forward primer and reverse primer are as described in the preceding paragraph. In one embodiment, the concentrations of the forward primer and the reverse primer in the kit of the present invention are different, with the concentration of one of them (preferably the primer for introducing mutations) being higher.

[0060] In one embodiment, the kit of the present invention may include multiple primer pairs, each containing a forward primer and a reverse primer, for multiple target nucleic acids. In another embodiment, the kit of the present invention may include multiple probes, each containing multiple target nucleic acids.

[0061] The kit according to the present invention may further include other components necessary for carrying out the amplification reaction, such as DNA polymerase and substrates. It may also include instructions describing procedures and protocols for detecting target nucleic acids.

[0062] In one embodiment, the DNA detection kit disclosed herein comprises: a first primer that introduces a mutation of a first concentration such that, at the temperature at which the fluorescent probe is bound, the proportion of double-stranded structures in the fluorescent probe-binding region of the DNA to be detected increases the proportion of single-stranded structures; a fluorescent probe that binds to the DNA to be detected; a second primer of a different concentration than the first, paired with the first primer to amplify the fluorescent probe-binding region of the DNA to be detected; and a DNA polymerase for amplifying the DNA to be detected.

[0063] The first primer that introduces the mutation and its corresponding second primer have different concentrations to perform an asymmetric nucleic acid amplification reaction, and either the first or second primer that introduces the mutation may have a higher concentration. Furthermore, the first primer that introduces the mutation may contain not only the mutation but also a tag sequence.

[0064] If there are multiple DNAs to be detected, multiple primers and probes can be prepared according to the sequences to be detected, added simultaneously, and an asymmetric nucleic acid amplification reaction can be performed on the DNA solution containing multiple DNAs. By designing the multiple probes with different melting temperatures relative to each DNA or by using different types of fluorescent dyes, the types of DNA present in the solution can be identified after the asymmetric nucleic acid amplification reaction based on the fluorescence color and melting temperature of the solution.

[0065] (4) Primer design method The present invention also provides a method for designing primers for detecting a target nucleic acid based on the amplification of a test nucleic acid and the measurement of the binding of the amplified product to a probe. Such a method includes designing a primer pair for amplifying a test nucleic acid, determining the percentage of single-stranded bases in the probe-binding region of the nucleic acid amplified by the designed primer pair, and introducing mutations into one or both primers of the designed primer pair such that the percentage of single-stranded bases is at least 50%. Such a method may further include determining the percentage of single-stranded bases in the probe-binding region of the nucleic acid amplified by the primer pair containing the mutated primer, and confirming whether the percentage of single-stranded bases is at least 50%. [Examples]

[0066] [Example 1] This embodiment demonstrates an example of performing an asymmetric nucleic acid amplification reaction using a mutation-introducing primer.

[0067] First, we will explain the results of melting curve analysis performed on an asymmetric nucleic acid amplification reaction without using mutation primers. Genomic DNA of the G13D mutant of the KRAS gene (final concentration 133 molecules / μL) was prepared, and the forward primer (final concentration 0.25 μM), reverse primer (final concentration 2.0 μM), fluorescently labeled probe corresponding to the wild type (final concentration 0.5 μM), fluorescently labeled probe corresponding to the G13D mutant (final concentration 0.5 μM), and 1x master mix (containing DNA polymerase and dNTPs) necessary for PCR were added to prepare the PCR reaction mixture. At this time, the concentrations of the primer pairs were added asymmetrically so that the complementary DNA strand of the fluorescently labeled probe would be over-amplified. The sequences of the primers and probes are as follows. Note that all fluorescently labeled probes have complementary sequences near both ends, and they are designed to form a double helix within the molecule. Furthermore, the fluorescently labeled probe for the wild type has HEX as a fluorescent dye bound to its 5' end and BHQ-1 as a quencher bound to its 3' end, while the fluorescently labeled probe for the mutant type has FAM as a fluorescent dye bound to its 5' end and BHQ-1 as a quencher bound to its 3' end.

[0068] Forward primer: 5'-GTCACATTTTCATTATTTTTATTATAAGG-3' (SEQ ID NO: 1) Reverse primer: 5'-GTATCGTCAAGGCACTCTTGCC-3' (SEQ ID NO: 2) Fluorescently labeled probe for wild-type: 5'-TTGGAGCTGGTGGCGT-3' (SEQ ID NO: 3) Fluorescently labeled probe for variants: 5'-CTGGTGACGTAGGCA-3' (SEQ ID NO: 4)

[0069] Figure 7A shows the positional relationship between the secondary structure 701 (SEQ ID NO: 5) of the amplified G13D mutant single-stranded DNA and the binding region 702 of the fluorescently labeled probe corresponding to the wild type. Figure 7B shows the positional relationship between the secondary structure 701 (SEQ ID NO: 5) of the amplified G13D mutant single-stranded DNA and the binding region 703 of the fluorescently labeled probe corresponding to the mutant. In Figure 7A, the binding region 702 of the fluorescently labeled probe shows a single-strand formation rate of 69%, while in Figure 7B, the binding region 703 of the fluorescently labeled probe shows a single-strand formation rate of 47%.

[0070] Figure 7C shows the differential curve of the melting curve obtained after melting curve analysis following an asymmetric nucleic acid amplification reaction. Compared to peak 704 of the differential melting curve of the amplified G13D mutant DNA 701 and the fluorescently labeled probe 702 corresponding to the wild type, peak 705 of the differential melting curve of the amplified G13D mutant DNA 701 and the fluorescently labeled probe 703 corresponding to the mutant was smaller. From this result, it was found that when the single-strand formation rate of the binding region of the fluorescently labeled probe is low, the amount of binding between the amplified DNA and the fluorescently labeled probe decreases. In such cases, since the mutant may only be present in small amounts, the fluorescence intensity decreases further, making detection difficult.

[0071] Next, Figures 8A and 8B show the results of asymmetric nucleic acid amplification reactions using mutation-introducing primers. Figure 8A illustrates the positional relationship between the secondary structure 801 (SEQ ID NO: 5) of the amplified G13D mutant single-stranded DNA and the binding region 802 of the fluorescently labeled probe corresponding to the mutant, with the position 803 where the mutation is planned to be introduced by the primer, when the mutation-introducing primer shown in Figure 7A was not used. Figure 8B shows the position 805 where the mutation was introduced by the primer, when an asymmetric nucleic acid amplification reaction was performed using a mutation-introducing primer that changes the base sequence CC at position 803 where the mutation is planned to be introduced by the primer to AA, with the positional relationship between the secondary structure 804 (SEQ ID NO: 6) of the amplified G13D mutant single-stranded DNA and the binding region 802 of the fluorescently labeled probe corresponding to the mutant, with the position 805 where the mutation was introduced by the primer. In Figure 8B, the proportion of single-strand formation at the binding region 802 of the fluorescently labeled probe corresponding to the mutant increases, and the amount of fluorescently labeled probe bound increases.

[0072] Thus, by performing an asymmetric nucleic acid amplification reaction using mutation-introducing primers, the secondary structure of the target gene to be amplified is altered, increasing the proportion of single-strand formation. This increases the amount of fluorescently labeled probes that can be bound, thereby improving measurement sensitivity and accuracy.

[0073] [Example 2] This example demonstrates an asymmetric nucleic acid amplification reaction using tagged mutation primers.

[0074] Figures 9A and 9B show the results of asymmetric nucleic acid amplification reactions using tagged mutagenesis primers. Figure 9A illustrates the positional relationship between the secondary structure 901 (SEQ ID NO: 5) of the amplified G13D mutant single-stranded DNA and the binding region 902 of the fluorescently labeled probe corresponding to the mutant, with the position 903 where the mutation is planned to be introduced by the primer, when the mutagenesis primer shown in Figure 7A was not used. Figure 9B shows the position 905 where the mutation was introduced by the primer and the position 906 of the tag added by the primer, when an asymmetric nucleic acid amplification reaction was performed using a designed mutagenesis primer that changed the base sequence CC at position 903 where the mutation is planned to be introduced by the primer to AA and added a tag sequence to the 3' end, with the positional relationship between the secondary structure 904 (SEQ ID NO: 7) of the amplified G13D mutant single-stranded DNA and the binding region 902 of the fluorescently labeled probe corresponding to the mutant, with the position 905 where the mutation was introduced by the primer and the position 906 of the tag added by the primer. In Figure 9B, the proportion of single-strand formation at the binding region 902 of the fluorescently labeled probe corresponding to the mutant increases, and the amount of fluorescently labeled probe bound increases. Furthermore, as shown in Figures 9A and 9B, the secondary structure of single-stranded DNA can change significantly before and after the introduction of mutations.

[0075] Thus, by performing asymmetric nucleic acid amplification reactions using tagged mutagenesis primers, the secondary structure of the target gene to be amplified is altered, increasing the proportion of single-strand formation. This increases the amount of fluorescently labeled probes that can be bound, thereby improving measurement sensitivity and accuracy. [Explanation of Symbols]

[0076] 101…DNA 102...Fluorescently labeled probe 103...Fluorescent dye 104... Quencher 201…Forward Primer 202…Reverse Primer 203...Fluorescently labeled probe 204... DNA to be detected 205…Molecules in which complementary strands of the DNA being detected form a double-stranded structure. 206…Molecules in which a fluorescently labeled probe is bound to a single-strand DNA molecule of the target of detection. 207... One strand of the DNA being detected 301...Forward Primer 302... Mutation-introducing reverse primer 303...Fluorescently labeled probe 304... DNA to be detected 305...Secondary structure of single-stranded target DNA amplified with mutation-free reverse primers. 306... Site where mutation is planned 307...Secondary structure of single-stranded target DNA amplified with mutation-introducing reverse primers 308... Introduced mutation 309... Mutation 401…Forward Primer 402...Tagged Mutagenesis Reverse Primer 403...Fluorescently labeled probe 404... DNA to be detected 405…Tag Array 406...Secondary structure of single-stranded target DNA amplified with mutation-free reverse primers. 407... Site where mutation is planned 408...Secondary structure of single-stranded target DNA amplified with tagged mutation-introducing reverse primers. 409... Introduced mutation 410... Added tag array 411... Mutation 501... Melting temperature 701...Secondary structure of single-stranded target DNA amplified with mutation-free reverse primers. 702...Binding region of fluorescently labeled probe corresponding to wild type 703...Binding region of fluorescently labeled probe corresponding to the variant. 704…Peaks of amplified G13D mutant DNA and fluorescently labeled probes corresponding to the wild type. 705... Peaks of amplified G13D mutant DNA and fluorescently labeled probes corresponding to the mutant. 801...Secondary structure of single-stranded DNA of the G13D mutant amplified without the use of mutation primers 802...Binding region of fluorescently labeled probe 803...Location where mutation is planned to be introduced 804...Secondary structure of single-stranded DNA of the G13D mutant amplified using mutation-introducing primers 805... Mutation site 901...Secondary structure of single-stranded DNA of the G13D mutant amplified without the use of mutation primers 902...Binding region of fluorescently labeled probe 903...Location where mutation is planned to be introduced 904...Secondary structure of single-stranded DNA of the G13D mutant amplified using mutation-introducing primers 905... Mutation site 906... Tag array [Sequence Listing Free Text]

[0077] Sequence IDs 1-7: DNA (artificial sequences, synthetic polynucleotides)

Claims

1. A step of amplifying a region of a test nucleic acid containing a target nucleic acid in the presence of a primer pair including a forward primer and a reverse primer and a probe, A step of measuring the binding of nucleic acids amplified by the forward primer and the reverse primer to the probe. A method for detecting target nucleic acids, including The forward primer and the reverse primer are for specifically binding to and amplifying the target nucleic acid. Either or both of the forward primer or the reverse primer has a mutation in its sequence that introduces a mutation into the region of the amplified nucleic acid containing the target nucleic acid such that, at the temperature at which the probe is bound to the amplified nucleic acid, the proportion of single-stranded bases in the probe-binding region of the amplified nucleic acid. The method wherein the mutation is introduced into the region of the amplified nucleic acid that includes the target nucleic acid by the amplification step.

2. The method according to claim 1, wherein the reverse primer contains the mutation.

3. The method according to claim 1, wherein the probe contains a fluorescent dye or contains a fluorescent dye and a quenching dye, and the binding of the amplified nucleic acid to the probe is measured using the fluorescent dye.

4. The method according to claim 1, wherein the 3' terminal sequence and the 5' terminal sequence of the probe have complementary sequences.

5. The method according to claim 1, wherein the amplification step is carried out by an asymmetric nucleic acid amplification reaction.

6. The method according to claim 1, wherein either or both of the forward primer or the reverse primer further include a tag sequence, and the amplified nucleic acid includes the tag sequence.

7. The method according to claim 1, wherein the proportion of single-stranded bases in the probe-binding region of the amplified nucleic acid is 50% or more.

8. The method according to claim 1, wherein the binding of the amplified nucleic acid to the probe is measured with respect to a change in temperature, and the melting temperature of the double hemisphere of the amplified nucleic acid and the probe is calculated from the change in the binding with respect to the change in temperature.

9. The method according to claim 1, wherein the binding of the amplified nucleic acid to the probe is measured for each temperature change cycle, and the target nucleic acid is detected from the relationship between the number of cycles and the change in the binding.

10. The method according to claim 1, comprising detecting multiple target nucleic acids using multiple types of primers containing the aforementioned mutation.

11. The method according to claim 10, comprising measuring the binding of the amplified nucleic acid to the probe for each temperature change cycle, and calculating the ratio of the initial concentrations of the plurality of target nucleic acids in the test nucleic acid from the relationship between the number of cycles and the change in binding.

12. The method according to claim 1, further comprising measuring the binding of the amplified nucleic acid to the probe for each temperature change cycle, comparing the relationship between the number of cycles and the change in binding with the change in binding of a sample containing a known concentration of target nucleic acid, and calculating the initial concentration of the target nucleic acid in the test nucleic acid whose concentration is unknown.

13. The method according to claim 1, wherein the detection of the target nucleic acid includes determining the genotype of the target nucleic acid.

14. A kit for carrying out the method of claim 1, comprising at least a primer for introducing a mutation into a region of a test nucleic acid containing a target nucleic acid and amplifying it, wherein the primer is for specifically binding to and amplifying the target nucleic acid, and the primer has a mutation in its sequence for introducing a mutation into a region of the test nucleic acid containing the target nucleic acid such that, at the temperature at which the nucleic acid amplified from the test nucleic acid binds to the probe, the proportion of single-stranded bases in the probe binding region of the nucleic acid amplified from the test nucleic acid is increased.

15. The kit according to claim 14, wherein the primer is a reverse primer.

16. The kit according to claim 14, wherein the primer further comprises a tag sequence.

17. A kit for detecting target nucleic acids, A primer pair comprising a forward primer and a reverse primer for amplifying the region containing the target nucleic acid, A probe that binds to nucleic acids amplified using forward primers and reverse primers. Includes, The kit wherein either or both of the forward primers or reverse primers include in their sequences mutations for introducing mutations into a region of the amplified nucleic acid containing the target nucleic acid such that, at the temperature at which the probe is bound to the nucleic acid, the proportion of single-stranded bases in the probe-binding region of the amplified nucleic acid is increased.

18. The kit according to claim 17, wherein either or both of the forward primer or the reverse primer further include a tag sequence.

19. The kit according to claim 17, wherein the concentration of the forward primer and the concentration of the reverse primer are different.

20. The kit according to claim 17, wherein the probe comprises a fluorescent dye, or comprises a fluorescent dye and a quenching dye.

21. The kit according to claim 17, wherein the 3' terminal sequence and the 5' terminal sequence of the probe have complementary sequences.

22. The kit according to claim 17, wherein the proportion of single-stranded bases in the probe-binding region of the amplified nucleic acid is 50% or more.

23. For multiple target nucleic acids, each includes multiple types of primer pairs, each containing the forward primer and the reverse primer, and / or The kit according to claim 17, wherein each of the target nucleic acids comprises multiple types of probes.

24. The kit according to claim 17, further comprising DNA polymerase.

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