DNA detection method and DNA detection kit

Abstract

To provide a DNA detection method and a kit capable of accurately determining an object gene at a melting temperature, with fluorescent dye color, and with fluorescence intensity, in a digital PCR in which melting curve analyses are combined.SOLUTION: A DNA detection method comprises the steps of: preparing a reaction mixture by mixing a fluorescence marker probe to an object gene, a primer for amplifying an area including the object area, a biological sample to be inspected, enzyme, and an additive for inhibiting secondary structure formation of a DNA; dividing the reaction mixture into a plurality of minute zones, and performing an amplification reaction in each of the minute zones; measuring the fluorescence intensity of the plurality of minute zones while changing the temperature to measure coupling between the DNA amplified in the amplification reaction and the probe; performing a melting curve analysis in each of the minute zones on the basis of the measurement result to calculate a melting temperature; and determining the presence / absence and / or the kind of the object gene on the basis of the color of the fluorescence, the fluorescence intensity, and the melting temperature, for each of the minute zones.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a DNA detection method and a DNA detection kit for analyzing genes, and more specifically to a DNA detection method and a DNA detection kit for detecting genes using a fluorescently labeled probe. [Background technology]

[0002] Genetic testing includes methods that use polymerase chain reaction (PCR), real-time PCR, digital PCR, etc. Real-time PCR and digital PCR, which allow for more accurate quantification than PCR, detect DNA using intercalators or fluorescently labeled probes. Two types of DNA detection methods that use fluorescently labeled probes are commonly used: hydrolysis probes and molecular beacons. Unlike hydrolysis probes, which use the nuclease activity of DNA polymerase to break down the probe and release a fluorescent dye from the probe, molecular beacons form stem loops in the free state and bind to the target DNA at the loop. These have the characteristic of not being broken down during PCR, and can be used to determine whether amplification has occurred based on fluorescence intensity after PCR, as well as to perform melting curve analysis.

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

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

[0005] An example of a detection method for digital PCR is shown below. First, DNA polymerase, primers, and fluorescently labeled probes required for PCR are added to a limiting diluted sample to prepare a PCR reaction solution. The PCR reaction solution is divided into microcompartments such as wells or droplets. At this time, each compartment is designed to either contain one molecule of the target gene or not. Next, the target gene in the microcompartment is amplified by PCR. After PCR, the fluorescence intensity of each microcompartment is measured, and the target gene can be quantified by counting the number of microcompartments with fluorescence intensity exceeding a threshold value.

[0006] When an asymmetric nucleic acid amplification reaction using a molecular beacon is applied, after PCR, a melting curve analysis of the target gene amplified in the microcompartment and the molecular beacon is performed, and different melting temperatures (Tm) are observed for each genotype of the target gene, making it possible to distinguish them. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US2019 / 0352699A1 (Patent Publication No. 2018-108063) [Non-patent literature]

[0008] [Non-Patent Document 1] BMC Microbiol.,13:295,2013 [Non-Patent Document 2] Anal.Chem.,92:11705-11713,2020 Summary of the Invention [Problem to be solved by the invention]

[0009] However, the present inventors were the first to recognize that the magnitude of the variance in melting temperature in the measurement data (distribution of melting temperatures measured when the target gene is present or absent) differs depending on the type of target gene and the sequence of the molecular beacon (Example 1). When multiple types of target genes are distinguished by their melting temperatures using molecular beacons modified with the same fluorescent dye and with different sequences, if the variance in melting temperatures is large, the distributions of melting temperatures of the different target genes will overlap. As a result, the measured melting temperature may be erroneously determined to be that of the other target gene, resulting in a false positive.

[0010] Therefore, an object of the present invention is to provide a DNA detection method and a DNA detection kit that can reduce the degree of variation in the melting temperatures of target genes to be distinguished based on differences in melting temperatures, and can accurately determine and quantify the genes. [Means for solving the problem]

[0011] The present inventors compared the melting curves of genes with large and small variations in melting temperature, and found that the melting curve of the gene with small variations in melting temperature has a large change in fluorescence intensity with temperature change, and therefore the peak of the differential curve of the melting curve is large. In addition, when the sequence of a combination of a gene with small variations in melting temperature and a molecular beacon is examined, it was revealed that the proportion of the molecular beacon binding region of one strand of the DNA that was over-amplified by the asymmetric nucleic acid amplification reaction forming a double-stranded structure is small. Therefore, it was found that when an additive that inhibits the formation of secondary structures in nucleic acids such as DNA is added to the reaction solution, the reaction solution is divided into small compartments, and then PCR and melting curve analysis are performed, the change in fluorescence intensity with temperature change in the melting curve is large, and the variation in melting temperature is reduced, leading to the completion of the present invention.

[0012] The present invention, in one aspect, comprises: preparing a reaction solution by mixing a fluorescently labeled probe for a gene of interest, a primer for amplifying a region including the gene of interest, a biological sample to be tested, an enzyme, and an additive that inhibits the formation of secondary structures in DNA; Dividing the reaction solution into a plurality of microcompartments and carrying out an amplification reaction in each of the plurality of microcompartments; measuring the binding between the DNA amplified in the amplification reaction and the probe by measuring the fluorescence intensity of the plurality of micro-compartments while changing the temperature; performing a melting curve analysis for each of the microcompartments based on the results of the measurement, and calculating a melting temperature; determining the presence and / or type of the target gene for each of the microcompartments based on the color, intensity and melting temperature of the fluorescence; The present invention provides a method for detecting DNA comprising the steps of:

[0013] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: A primer pair including a forward primer and a reverse primer for amplifying a region including a gene of interest; a fluorescently labeled probe for the gene of interest that binds to DNA amplified using the forward and reverse primers; Additives that inhibit the formation of secondary structures in DNA A DNA detection kit comprising: Effect of the Invention

[0014] The present invention provides a DNA detection method and a DNA detection kit that can detect or quantify target genes, particularly multiple types of target genes, with high accuracy and high sensitivity based on the difference in melting temperature obtained by melting curve analysis. Therefore, the present invention is useful in fields such as basic research, testing, and drug discovery that involve gene detection and genotyping. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram showing an example of a method for measuring the melting temperature of DNA using a fluorescently labeled probe. [Diagram 2]FIG. 1 shows examples of results obtained by a method for determining and counting the presence or absence of multiple genes in each microcompartment based on the melting temperature measured using a fluorescently labeled probe in digital PCR (dPCR). [Diagram 3] FIG. 1 shows examples of melting curves obtained by measuring genes with large variations in melting temperatures (A and B) and genes with small variations in melting temperatures (C to F). [Figure 4] 1 is a flow chart showing one embodiment of a DNA detection method in which an additive that inhibits the formation of secondary structures in DNA is added to a reaction solution and digital PCR combined with melting curve analysis is performed to quantify the copy number of a target gene. [Diagram 5] FIG. 1 shows the measurement results in Example 1 when digital PCR was performed without adding an additive that inhibits the formation of secondary structures in DNA, followed by melting curve analysis. [Figure 6A] FIG. 1 shows the predicted secondary structure and probe binding region of the KRAS gene amplicon with small melting temperature variation. [Figure 6B] FIG. 1 shows the predicted secondary structure and probe binding region of the hTERT gene amplicon with a large melting temperature variation. [Figure 6C] FIG. 1 shows the predicted secondary structures and probe binding regions of GNAS gene amplicons with large melting temperature variations. [Figure 7A-B] FIG. 13 shows the measurement results in Example 2, in which betaine (trimethylglycine) was added as an additive that inhibits the formation of secondary structures in DNA in real-time PCR, and melting curve analysis was performed. [Figure 7C-D] FIG. 13 shows the measurement results in Example 2, in which DMSO was added as an additive that inhibits the formation of secondary structures in DNA in real-time PCR, and melting curve analysis was performed. [Figure 7E-F] FIG. 13 shows the measurement results in Example 2, in which formamide was added as an additive that inhibits the formation of secondary structures in DNA in real-time PCR, and melting curve analysis was performed. [Figure 8]FIG. 13 shows the measurement results in Example 3, in which an additive that inhibits the formation of secondary structures in DNA was added in digital PCR and melting curve analysis of the hTERT gene was performed. [Figure 9] FIG. 13 shows the measurement results in Example 3, when an additive that inhibits the formation of secondary DNA structures was added in digital PCR and melting curve analysis of the GNAS gene was performed. [Figure 10] 1 shows the measurement results of the relationship between trimethylglycine concentration and the variation in melting temperature when trimethylglycine, a type of betaine, was added to perform melting curve analysis in digital PCR in Example 3. A: hTERT gene, B: GNAS gene. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The objectives, features, advantages, and ideas related thereto of the present invention will be apparent to those skilled in the art from the description of this specification. From the description of this specification, those skilled in the art can easily reproduce the present invention. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustrative or explanatory purposes, and do not limit the present invention thereto. It is apparent to those skilled in the art that various modifications and alterations can be made based on the description of this specification without departing from the intent and scope of the present invention disclosed in this specification.

[0017] (1) DNA detection method In one aspect, the present invention provides a method for DNA detection, the method comprising: preparing a reaction solution by mixing a fluorescently labeled probe for a gene of interest, a primer for amplifying a region including the gene of interest, a biological sample to be tested, an enzyme, and an additive that inhibits the formation of secondary structures in DNA; Dividing the reaction solution into a plurality of microcompartments and carrying out an amplification reaction in each of the plurality of microcompartments; measuring the binding between the DNA amplified in the amplification reaction and the probe by measuring the fluorescence intensity of the plurality of micro-compartments while changing the temperature; performing a melting curve analysis for each of the microcompartments based on the results of the measurement, and calculating a melting temperature; determining the presence and / or type of the target gene for each of the microcompartments based on the color, intensity and melting temperature of the fluorescence; Includes.

[0018] In the present invention, the test biological sample is not particularly limited as long as it is a biological sample containing DNA that contains or may contain the target gene to be detected, and may be, for example, a body fluid sample such as blood or urine, tissue, or cultured cells, or may be DNA (genomic DNA, circulating DNA, etc.) extracted and purified from such samples, or may be DNA (cDNA, etc.) derived from mRNA extracted and purified from a sample. Alternatively, synthesized DNA may be used as the test biological sample for the design of suitable probes and primers. When the test biological sample is a body fluid sample, tissue, cultured cells, etc., it is preferable to pre-treat the DNA, etc., to make it available for the amplification reaction performed by the method of the present invention. Methods for preparing DNA from such samples are known in the art, and kits for easily purifying DNA and kits for extracting mRNA and synthesizing cDNA are also commercially available.

[0019] In the present invention, the target gene may be any specific DNA site or region to be detected, and includes, for example, a gene, a mutation in a gene, a regulatory sequence of a gene, a mutation in a regulatory sequence of a gene, etc. The target gene may be one type, or may include multiple target genes.

[0020] In the method of the present invention, a reaction solution is prepared by mixing a fluorescently labeled probe for a target gene, a primer for amplifying a region containing the target gene, a test biological sample, an enzyme, and an additive that inhibits the formation of secondary structures in DNA.

[0021] The probe is designed so that at least a part of the probe has a sequence specific to the base sequence of the target gene, that is, a sequence complementary to the base sequence of the gene. When multiple target genes are to be detected, a probe is required for each of the multiple target genes, so that the number of probes corresponding to the type of target gene is prepared. When a gene mutation is to be detected, a probe having a sequence complementary to the wild-type sequence is prepared, and the target gene mutation can be detected including a mismatched base, in which case a lower melting temperature is observed than that of a completely complementary wild-type sequence due to the mismatched base. Probe design methods are well known in the art, and probes usable in the present invention are designed to have a length and base composition (melting temperature) that allow specific binding (hybridization). For example, as a length that functions as a probe, the base length of the sequence portion specific to the target gene is preferably 10 bases or more, more preferably 15 to 50 bases, and even more preferably 15 to 30 bases, for example, about 20 bases. In addition, when designing the probe, it is preferable to confirm the GC content and melting temperature (Tm) of the probe. To confirm Tm, known probe design software can be used.

[0022] The probe is fluorescently labeled, specifically, contains a fluorescent dye, or contains a fluorescent dye and a quencher dye. When the probe contains a fluorescent dye, the fluorescence intensity of the fluorescent dye is used to measure the binding between the probe and the DNA amplified in the amplification reaction described below. Fluorescent dyes and combinations of fluorescent dyes and quencher dyes are well known in the art, and a person skilled in the art can select an appropriate one in consideration of the amplification reaction and melting curve analysis. In one embodiment, the 3'-end sequence and the 5'-end sequence of the probe have a complementary sequence portion or an interacting structure, and the fluorescent dye of the probe is quenched in a free state (when not bound to the amplified DNA). In another embodiment, the 3'-end sequence and the 5'-end sequence of the probe have a complementary sequence portion, and the 3'-end sequence and the 5'-end sequence of the probe are bound to form a stem-loop structure. In other words, the probe is preferably a molecular beacon.

[0023] In one embodiment, when the probe comprises multiple types of probes, they may be labeled with the same fluorescent dye.

[0024] The designed probes can be chemically synthesized by known oligonucleotide synthesis techniques, but are usually synthesized using a commercially available chemical synthesis apparatus.

[0025] A primer for amplifying a region containing a target gene is designed so that the above-mentioned probe can bind to a part of the region amplified using the primer. Such primer design methods are also well known in the art, and primers usable in the present invention are designed to satisfy conditions for specific annealing, for example, to have a length and base composition (melting temperature) that allow specific annealing. For example, the length that functions as a primer is preferably 10 bases or more, more preferably 15 to 50 bases, and even more preferably 15 to 30 bases, for example about 20 bases. In addition, when designing, it is preferable to confirm the GC content and melting temperature (Tm) of the primer. To confirm Tm, known primer design software can be used. The designed primer can be chemically synthesized by known oligonucleotide synthesis methods, but is usually synthesized using a commercially available chemical synthesis device.

[0026] The enzyme is a polymerase capable of carrying out an amplification reaction, i.e., an extension reaction using DNA as a template, and any known DNA polymerase can be used, preferably a DNA polymerase used in an amplification reaction involving temperature change cycles.

[0027] The additive that inhibits the formation of secondary structures of DNA is not particularly limited as long as it has the inhibitory effect. Examples of additives that inhibit the formation of secondary structures of DNA include betaine (e.g., trimethylglycine, carnitine, proline betaine, 2-(tripentylammonio)acetic acid, etc.), dimethyl sulfoxide (DMSO), formamide, 7-deaza dGTP (7-deaza-2'-deoxyguanosine), and nonionic surfactants (e.g., Triton X-100, Tween 20, or Nonidet P-40 (NP-40)). Although the mechanism of inhibiting the formation of secondary structures of DNA differs depending on the type of additive, any additive with any mechanism can be used. For example, the zwitterion betaine inhibits DNA secondary structure formation by stabilizing both AT and GC base pairs equally, DMSO and formamide inhibit DNA secondary structure formation by reducing hydrogen bonds between the two DNA strands, and 7-deaza dGTP inhibits DNA secondary structure formation by reducing the stability of double-stranded DNA (Karunanathie et al., Biochimie 197:130-143, 2022; A&T Corporation Internet website: https: / / www.aandt.co.jp / jpn / medical / tree / vol_13_2 / ).

[0028] When preparing a reaction solution by mixing a fluorescently labeled probe for a target gene, a primer for amplifying a region containing the target gene, a biological sample to be tested, an enzyme, and an additive that inhibits the formation of a secondary structure of DNA, the amount of each component to be mixed is appropriately set for the amplification reaction described below. In one embodiment, the amount of either the forward primer or the reverse primer added is increased to perform the amplification reaction (asymmetric nucleic acid amplification reaction). For asymmetric nucleic acid amplification reaction, please refer to, for example, the description in Anal.Chem., 92, 11705-11713, 2020 (Non-Patent Document 2).

[0029] The reaction solution is then divided into a plurality of microcompartments, and an amplification reaction is carried out in each of the plurality of microcompartments. Amplification reactions in microcompartments are known in the art, and are called, for example, digital PCR (dPCR) (Patent Document 1, Non-Patent Document 1). Wells, droplets, etc. are known as microcompartments, and each of the microcompartments is manipulated so that it either contains one molecule of the target gene or does not contain one molecule of the target gene.

[0030] The amplification reaction can be carried out by any amplification reaction known in the art. In one embodiment, the amplification reaction is an amplification reaction carried out with cycles of temperature change, preferably a polymerase chain reaction (PCR). Preferably, the amplification reaction is carried out by an asymmetric nucleic acid amplification reaction.

[0031] Next, the binding between the DNA amplified in the amplification reaction and the probe is measured. Specifically, the binding between the DNA obtained in the amplification reaction and the probe is measured by measuring the fluorescence intensity of a plurality of micro-compartments while changing the temperature. In one embodiment, this step includes measuring the fluorescence intensity while changing the temperature at a sampling interval of less than 1°C, for example, at intervals of 0.01°C to 0.99°C, preferably at intervals of 0.3°C to 0.5°C.

[0032] Then, based on the measurement results, a melting curve analysis is performed for each microcompartment to calculate the melting temperature. Since the binding changes depending on the melting temperature, the melting temperature of the double strand of the amplified DNA and the probe can be calculated from the change in binding (i.e., the change in fluorescence intensity) that accompanies the temperature change of the solution. This operation is called melting curve analysis. Alternatively, the binding between the amplified DNA and the probe can be measured for each cycle of temperature change of the solution, and the target gene can be determined from the relationship between the number of cycles and the change in binding.

[0033] By carrying out the amplification reaction and melting curve analysis as described above, the presence or absence and / or type of the target gene is determined for each microcompartment from the color of the fluorescence (probe, i.e., type of target gene), fluorescence intensity (binding), and melting temperature. In one embodiment, when the target gene includes multiple target genes, melting curve analysis is carried out for each of the multiple target genes to determine the presence or absence and / or type of each target gene. Furthermore, it is also possible to quantify the copy number of the target gene or the abundance ratio relative to the wild type for DNA in the test biological sample. In one embodiment, the method further includes a step of counting the number of target genes by combining the results of the above-mentioned determination of the multiple microcompartments.

[0034] The DNA detection method of the present invention will be specifically described with reference to the schematic diagrams of FIGS.

[0035] Fig. 1 is a schematic diagram showing an example of a method for measuring the melting temperature of DNA using a fluorescently labeled probe. Examples of the fluorescently labeled probe include a molecular beacon and a Pleiades probe, but Fig. 1 will be explained here using a molecular beacon as an example.

[0036] The fluorescently labeled probe 102, which is a molecular beacon, is configured as an oligonucleotide and has a sequence complementary to the sequence (amplified DNA 101) between the primer pair used in the amplification reaction to amplify the target gene. The molecular beacon also has complementary sequence portions at both ends, with a fluorescent dye 103 at one end and a quenching dye (quencher) 104 at the other end. In the initial state (free state) of the amplification reaction, the fluorescently labeled probe (molecular beacon) 102 exists alone and free, as shown in B of FIG. 1. At that time, the fluorescently labeled probe (molecular beacon) 102 forms a stem loop at the complementary sequence portion, and the fluorescent dye 103 and the quencher 104 are close to each other, so no fluorescence is emitted. In the first denaturation step, when the sample solution is heated, it takes on a highly flexible structure as shown in C of FIG. 1, but the fluorescent dye and the quenching dye are not always separated, so the fluorescence remains quenched. In the annealing step, when the temperature is lowered to about room temperature, the loop portion of the fluorescently labeled probe (molecular beacon) 102 anneals to the amplified DNA 101 in the sample solution as shown in A of FIG. 1. As a result, the fluorescent dye 103 and the quencher 104 are always separated, and the fluorescently labeled probe 102 emits strong fluorescence. In the next extension step, the fluorescently labeled probe (molecular beacon) 102 is released, and the state becomes again as shown in B of FIG. 1, and the fluorescence is quenched. In the next denaturation step, the state becomes again as shown in C of FIG. 1, and the fluorescence remains quenched. In the amplification reaction, this step is repeated, so the fluorescence intensity may be measured at some stage during heating or cooling. The same method can be used to measure the fluorescence intensity after the amplification reaction is completed. After the amplification reaction is completed, the fluorescence intensity may be measured by heating or cooling just to measure the fluorescence intensity. A melting curve is created by plotting the change in fluorescence intensity with the temperature change at this time, and the melting temperature can be calculated from the melting curve.

[0037] In the fluorescently labeled probe (molecular beacon) 102 used here, the combination of the fluorescent dye 103 and the quencher 104 is not particularly limited as long as it is a combination that is generally used in real-time PCR. For example, examples of the fluorescent dye 103 include FAM, VIC, ROX, Cy3, Cy5, etc., and examples of the quencher 104 include TAMRA, BHQ1, BHQ2, BHQ3, etc. All of them are commonly used and are commercially available.

[0038] When two types of genes with different sequences are to be analyzed, the sequences of the fluorescently labeled probes (molecular beacons) 102 are prepared that specifically bind to each of the target genes, and by binding different fluorescent dyes, it is possible to distinguish and detect the two types of target genes in one reaction system. Alternatively, by designing the sequences of the molecular beacons so that the melting temperatures of the two types of molecular beacons are different, it is possible to distinguish and detect the two types of target genes in one reaction system even if the same fluorescent dye is bound.

[0039] Figure 2 shows an example of the results obtained by a method of determining and counting the presence or absence of multiple genes in each micro-compartment based on the melting temperature measured using a fluorescently labeled probe in digital PCR (dPCR). As shown in A of Figure 1, when the variation in melting temperature is large, the melting temperature distributions of two types of genes, i.e., melting temperature distribution 201 of the gene with a high melting temperature and melting temperature distribution 202 of the gene with a low melting temperature, are close to each other with a wide area, and the micro-compartment 203 containing the gene with a high melting temperature is erroneously observed within the threshold of the melting temperature distribution 202 of the gene with a low melting temperature, and is counted as a different gene. On the other hand, when the variation in melting temperature is small as shown in B of Figure 1, even if the average value of the melting temperature distributions of the two types of genes is the same as A of Figure 1, the melting temperature distributions 201 and 202 are far apart, and the probability of misjudging the type of gene is reduced.

[0040] FIG. 3 is a diagram showing an example of melting curves of microcompartments containing genes with large or small variations in melting temperature. FIG. 3A shows an example of melting curves of genes with small variations in melting temperature, and FIG. 3C and D show examples of melting curves of genes with large variations in melting temperature. As shown in FIG. 3A, the melting curve 302 of the microcompartment (positive well) containing the target gene shows high fluorescence intensity at low temperatures because the fluorescently labeled probe binds to the corresponding DNA, and the fluorescence intensity decreases as the temperature increases because the fluorescently labeled probe dissociates from the corresponding DNA. The melting curve 303 of the microcompartment (negative well) not containing the target gene does not contain DNA to which the fluorescently labeled probe binds, so there is no change in fluorescence intensity with temperature change. When the differential curve is calculated from FIG. 3A, a high peak is shown at 60° C. as shown in FIG. 3B, and the temperature of the peak is the melting temperature (Tm) 301. On the other hand, an example of the melting curve when a gene with large variations in melting temperature is measured is shown in FIG. 3C, and the differential curve is shown in FIG. 3D. As shown in FIG. 3C, in the melting curve 302 of the microcompartment containing the target gene, the fluorescently labeled probe binds to the corresponding DNA at low temperatures, but the amount of binding is small, and the fluorescence intensity is low compared to FIG. 3A. The reaction occurring at this time is that one strand of the DNA excessively amplified by the asymmetric nucleic acid amplification reaction forms a secondary structure, making it difficult for the fluorescently labeled probe to bind, and the amount of binding of the fluorescently labeled probe decreases. Therefore, as shown in FIG. 3D, the differential curve has a low peak height and is broad (a peak that spreads horizontally and has a low height). Another example of a melting curve when a gene with a large variation in melting temperature is measured is shown in FIG. 3E, and its differential curve is shown in FIG. 3F. As shown in FIG. 3E, in the melting curve 302 of the microcompartment containing the target gene, the fluorescently labeled probe binds to the corresponding DNA at low temperatures and shows high fluorescence intensity, but even if the fluorescently labeled probe dissociates from the corresponding DNA with increasing temperature, the decrease in fluorescence intensity is small, and the change in fluorescence intensity with temperature change is small compared to FIG. 3A. The reaction that occurs at this time is that after the fluorescently labeled probe dissociates from the corresponding DNA, the fluorescently labeled probe forms a secondary structure different from the stem-loop, and the fluorescent dye and quenching dye do not form a structure in close proximity to each other, so the fluorescent dye is not quenched and emits fluorescence.Therefore, the differential curve has a low and broad peak as shown in Figure 3F. Although the melting curve shapes of Figure 3C and E are different, they share the common feature that the change in fluorescence intensity with temperature change is small, and as a result, the differential curves of the melting curves both have broad peaks as shown in Figure 3D and F. A differential curve with a broad peak can easily cause large variations in melting temperature 301 due to measurement errors.

[0041] According to the present invention, when an additive that inhibits the formation of secondary structures in DNA is added to the reaction solution, the melting curve changes from the shapes of C and E in Fig. 3 to the shape of A in Fig. 3. In addition, the differential curve of the melting curve, which had a broad peak, changes to a sharp peak as shown in B in Fig. 3. As a result, the variation in melting temperature 301 due to measurement errors is reduced.

[0042] FIG. 4 is a flow chart showing an embodiment of a DNA detection method in which an additive that inhibits the formation of secondary structures in DNA is added to a reaction solution, digital PCR combined with melting curve analysis is performed, and the copy number of a target gene is quantified. First, a probe corresponding to each of a plurality of target genes is designed (S401). The probe, biological sample, enzyme, primer, and additive that inhibits the formation of secondary structures in DNA are mixed, divided into wells, and PCR is performed (S402). A melting curve between the probe corresponding to each target gene and the amplified product (amplified DNA) is obtained by melting curve analysis (S403). For each well, the presence or absence of a target gene is determined from the color of fluorescence, the fluorescence intensity, and the melting temperature (S404). Finally, the wells containing each target gene and the wells not containing both target genes are counted, and the initial concentration of each is calculated by Poisson correction (S405).

[0043] When there are multiple target genes to be detected, multiple primers or multiple probes may be prepared according to the target genes, and amplification reactions (e.g., asymmetric nucleic acid amplification reactions) may be performed on the test biological sample that contains or may contain the DNA of the multiple target genes at the same time. By designing the multiple probes to have different melting temperatures with the DNA of each target gene or different types of fluorescent dyes, the types of target genes contained in the solution can be identified from the fluorescent color and melting temperature of the solution after the amplification reaction.

[0044] In the DNA detection method of the present invention, when detecting a target gene by a combination of PCR and melting curve analysis, by performing PCR and melting curve analysis in the presence of an additive that inhibits the formation of secondary structures in DNA, or by performing melting curve analysis in the presence of an additive that inhibits the formation of secondary structures in DNA, the variation in melting temperature is reduced and the error bars are also reduced, making it possible to detect or quantitate a target gene, particularly multiple target genes, with greater accuracy and sensitivity.

[0045] (2) DNA detection kit The above-mentioned method of the present invention can be carried out more easily and conveniently by using a kit containing at least an additive that inhibits the formation of secondary structures in DNA.

[0046] That is, in one aspect, the present invention provides a DNA detection kit, the kit comprising: A primer pair including a forward primer and a reverse primer for amplifying a region including a gene of interest; a fluorescently labeled probe for the gene of interest that binds to DNA amplified using the forward and reverse primers; Additives that inhibit the formation of secondary structures in DNA Includes.

[0047] The fluorescently labeled probe is as described in the previous section, and has a different fluorescent dye or melting temperature for each target gene. In one embodiment, the probe contains a fluorescent dye and a quenching dye, and the 3'-end sequence and the 5'-end sequence of the probe have complementary sequence portions or interacting structures, and the fluorescent dye of the probe is quenched in a free state.

[0048] The forward primer and reverse primer are as described in the previous section. In one embodiment, the concentration of the forward primer and the concentration of the reverse primer contained in the kit of the present invention are different, and one of the primers is higher (due to asymmetric nucleic acid amplification reaction). It is preferable that the concentration of the primer that synthesizes the complementary strand of the fluorescently labeled probe is higher.

[0049] The additive that inhibits the formation of secondary structure of DNA is as described in the previous section. In one embodiment, the additive that inhibits the formation of secondary structure of DNA comprises at least one selected from the group consisting of betaine (e.g., trimethylglycine), dimethyl sulfoxide (DMSO), formamide, 7-deaza dGTP, and non-ionic surfactant.

[0050] The kit according to the present invention may further include a DNA polymerase. The DNA polymerase is also as described in the previous section, and any DNA polymerase can be used.

[0051] The kit of the present invention may further include other components necessary for carrying out the amplification reaction, such as substrates, etc. Also, it may include instructions describing the procedure and protocol for determining the target gene.

[0052] In one embodiment, the DNA detection kit of the present invention can be used to carry out the above-mentioned DNA detection method of the present invention. Alternatively, the DNA detection kit of the present invention can be used to detect a target gene by a combination of PCR and melting curve analysis, and an additive that inhibits the formation of secondary structures of DNA may be added when performing PCR and melting curve analysis, or an additive that inhibits the formation of secondary structures of DNA may be added when performing melting curve analysis. The DNA detection kit of the present invention is useful for determining the presence or absence of a target gene (especially multiple target genes) and for determining the positional relationship (cis type or trans type) of the alleles of the target genes.

[0053] Furthermore, the DNA detection kit of the present invention can be used to evaluate traits (eg, susceptibility to a disease or disorder, drug responsiveness, etc.) associated with a target gene (particularly a gene mutation). EXAMPLES

[0054] [Example 1] In this example, a description will be given of the measurement results when digital PCR combined with melting curve analysis was performed without the addition of an additive that inhibits the formation of secondary structures in DNA.

[0055] Wild-type genomic DNA (final concentration 133 molecules / μL, Horizon's genomic DNA standard (KRAS Wild Type Reference Standard, Catalog ID: HD710)) was prepared, and forward primers (final concentration 0.25 μM) corresponding to each target gene required for PCR, reverse primers (final concentration 2.0 μM) corresponding to the target gene, fluorescently labeled probes (final concentration 0.5 μM) corresponding to the target gene, and 1x master mix (containing DNA polymerase and dNTPs) were added to prepare a PCR reaction solution. At this time, the primer pairs were added at asymmetric concentrations so that the complementary DNA strand of the fluorescently labeled probe was over-amplified (the concentration of the reverse primer was set high). The sequences of the primers and probes are as follows:

[0056] Forward primer corresponding to the KRAS gene: 5'-GTCACATTTTCATTATTTTTATTATAAGG-3' (SEQ ID NO: 1) Reverse primer corresponding to the KRAS gene: 5'-GTCAAGGCACTCTTGCCTAC-3' (SEQ ID NO: 2) Fluorescently labeled probe for the KRAS gene: 5'-CTCGATGTGAGTTTCTGC-3' (SEQ ID NO: 3) Forward primer corresponding to the hTERT gene: 5'-GGGTCCTCGCCTGTGTACAG-3' (SEQ ID NO: 4) Reverse primer corresponding to the hTERT gene: 5'-CCTGGGAGCTCTGGGAATTT-3' (SEQ ID NO: 5) Fluorescently labeled probe corresponding to the hTERT gene: 5'-CACACCTTTGGTCACTC-3' (SEQ ID NO: 6) Forward primer corresponding to the GNAS gene: 5'-GCTTTGGTGAGATCCATTGAC-3' (SEQ ID NO: 7) Reverse primer corresponding to the GNAS gene: 5'-TCCACCTGGAACTTGGTCTC-3' (SEQ ID NO: 8) Fluorescently labeled probe corresponding to the GNAS gene: 5'-TTCGCTGCCGTGTCC-3' (SEQ ID NO: 9)

[0057] All fluorescently labeled probes used EasyBeacon (PentaBase), which has hydrophobic special bases HyNA(TM) near both ends, and are designed to form hydrophobic bonds within the free fluorescently labeled probes. In addition, all fluorescently labeled probes have HEX as a fluorescent dye bound to the 5' end and BHQ-1 as a quencher bound to the 3' end.

[0058] After that, 15μL of PCR reaction solution was divided into 20,000 wells, and DNA was amplified by PCR. The PCR reaction was performed at 96℃ for 10 minutes, followed by 59 cycles of (60℃, 2 minutes → 98℃, 30 seconds), and finally at 60℃ for 2 minutes. After the reaction, the chip with the wells was heated from 45℃ to 80℃ on the temperature control stage, while fluorescent images of the chip were obtained at 0.35℃ intervals using a digital PCR device compatible with melting curve analysis, and the changes in fluorescent intensity of each well were observed from the obtained fluorescent images, and the melting curve was measured and analyzed.

[0059] Figure 5 shows the results of measuring the melting curves and melting temperature distributions of each gene when no additives that inhibit the formation of secondary structures in DNA were added. Figures 5A and 5B show the melting curves and melting temperature distributions of the KRAS gene, Figures 5C and 5D show the melting curves and melting temperature distributions of the hTERT gene, and Figures 5E and 5F show the melting curves and melting temperature distributions of the GNAS gene. Figure 5A shows that the melting curve of the KRAS gene shows a large change in fluorescence intensity with temperature change, and as shown in Figure 5B, the variation in melting temperature is small, with the variation being less than 0.33°C. On the other hand, Figure 5C shows that the melting curve of the hTERT gene shows a small change in fluorescence intensity with temperature change, and as shown in Figure 5D, the variation in melting temperature is large, with the variation being 0.90°C. Furthermore, in Fig. 5E, the melting curve of the GNAS gene shows a large difference in fluorescence intensity between the positive well containing the target gene and the negative well not containing the target gene at low temperatures, but the decrease in fluorescence intensity in the positive well with increasing temperature is small, and the fluorescence intensity remains high even at high temperatures. As a result, the change in fluorescence intensity with temperature change is small, and the variation in melting temperature is large, as shown in Fig. 5F, with a variation of 0.97°C.

[0060] The sequences of the amplicons and fluorescently labeled probes of the KRAS gene, which has a small variation in melting temperature, and the hTERT and GNAS genes, which have a large variation in melting temperature, are examined. Figures 6A, 6B, and 6C show the sequences of the amplicons (SEQ ID NOs: 10 to 12, respectively) corresponding to the KRAS gene, the hTERT gene, and the GNAS gene, respectively, along with the predicted secondary structure and the probe binding region 601. As shown in Figures 6A to 6C, the amplicons may form double-stranded structures in themselves. In Figures 6A to 6C, the region 601 to which the fluorescently labeled probe binds is shown in a circle. As shown in Figure 6A, it was found that the KRAS gene, which has a small variation in melting temperature, has a small proportion of the fluorescently labeled probe binding region of one strand of the over-amplified DNA formed into a double-stranded structure by the asymmetric nucleic acid amplification reaction. On the other hand, when looking at the sequences of the amplicons and fluorescently labeled probes of the hTERT and GNAS genes, which have a large variation in melting temperature, as shown in Figures 6B and 6C, the fluorescently labeled probe binding region of one strand of the over-amplified DNA was often at a high rate of forming a double-stranded structure due to the asymmetric nucleic acid amplification reaction. Furthermore, it was found that the fluorescence intensity at low temperatures decreased when the fluorescently labeled probe binding region of the KRAS gene was redesigned so that the rate of forming a double-stranded structure of one strand of the over-amplified DNA was changed from a region with a low rate to a region with a high rate (PCT / JP2022 / 024591).

[0061] These results suggest that the formation of secondary DNA structures may be a factor in increasing the variability in melting temperatures.

[0062] [Example 2] From the results of Example 1, it was predicted that the variation in melting temperature would be reduced by inhibiting the formation of secondary DNA structures. Therefore, in this Example, an example is shown in which the variation in melting temperature is reduced and the target gene is measured with high accuracy by performing real-time PCR in combination with melting curve analysis after adding an additive that inhibits the formation of secondary DNA structures.

[0063] As in Example 1, wild-type genomic DNA (final concentration 133 molecules / μL) was prepared, and a forward primer (final concentration 0.25 μM) corresponding to the hTERT gene, a reverse primer (final concentration 2.0 μM) corresponding to the hTERT gene, a fluorescently labeled probe (final concentration 0.5 μM) corresponding to the hTERT gene, and a 1x master mix (containing DNA polymerase and dNTPs) were added, which are necessary for PCR. Trimethylglycine, a type of betaine, DMSO, or formamide was added to this PCR reaction solution at different concentrations as additives. At this time, the primer pair was added at asymmetric concentrations so that the complementary DNA strand of the fluorescently labeled probe was excessively amplified (the concentration of the reverse primer added was set high). The primers and probes were the same as those used in Example 1.

[0064] The PCR reaction was carried out at 95°C for 20 seconds, followed by 60 cycles of (95°C, 1 second → 60°C, 20 seconds). After the reaction, the change in fluorescence intensity was observed using a real-time PCR device while heating from 50°C to 95°C, and the melting curve was measured and analyzed.

[0065] FIG. 7A shows the melting curves when betaine (trimethylglycine) was added at concentrations ranging from 0M to 1M, and FIG. 7B shows the differential curve of the melting curve. FIG. 7C shows the melting curves when DMSO was added at concentrations ranging from 0% to 4%, and FIG. 7D shows the differential curve of the melting curve. FIG. 7E shows the melting curves when formamide was added at concentrations ranging from 0% to 4%, and FIG. 7F shows the differential curve of the melting curve. In FIG. 7A to 7F, PC represents the melting curve of a well containing a target gene and its differential curve, and NC represents the melting curve of a well not containing a target gene and its differential curve. The numbers in parentheses after PC and NC represent the concentrations of the additives.

[0066] Regardless of which additive was used, the shape of the melting curve changed in the presence of the additive, and the higher the additive concentration, the higher the fluorescence intensity at low temperatures (Figures 7A-7F). As a result, when looking at the differential curve, the higher the additive concentration, the higher the peak value of the differential curve. Among the three types of additives, the addition of trimethylglycine caused the largest change in the shape of the melting curve and its differential curve.

[0067] [Example 3] In this example, trimethylglycine, a type of betaine, was added to the PCR reaction solution, and digital PCR combined with melting curve analysis was performed to evaluate the variation in melting temperature.

[0068] As in Example 1, wild-type genomic DNA (final concentration 133 molecules / μL) was prepared, and a forward primer (final concentration 0.25 μM) corresponding to the target gene required for PCR, a reverse primer (final concentration 2.0 μM) corresponding to the target gene, a fluorescently labeled probe (final concentration 0.5 μM) corresponding to the target gene, 0.5 M trimethylglycine, and 1x master mix (containing DNA polymerase and dNTPs) were added to prepare a PCR reaction solution. At this time, the primer pair was added at an asymmetric concentration so that the complementary DNA strand of the fluorescently labeled probe was excessively amplified (the concentration of the reverse primer added was set high). The primers and probes were the same as those used in Example 1.

[0069] After that, 15μL of PCR reaction solution was divided into 20,000 wells, and DNA was amplified by PCR. The PCR reaction was performed at 96℃ for 10 minutes, followed by 59 cycles of (60℃, 2 minutes → 98℃, 30 seconds), and finally at 60℃ for 2 minutes. After the reaction, the chip with the wells was heated from 45℃ to 80℃ on the temperature control stage, while fluorescent images of the chip were obtained at 0.35℃ intervals using a digital PCR device compatible with melting curve analysis, and the changes in fluorescent intensity of each well were observed from the obtained fluorescent images, and the melting curve was measured and analyzed.

[0070] Figure 8 shows the results of performing digital PCR combined with melting curve analysis in the presence or absence of trimethylglycine, and measuring the melting curve and melting temperature variation of the hTERT gene. Figure 9 shows the results of measuring the melting curve and melting temperature variation of the GNAS gene in a similar manner. In Figures 8 and 9, A shows the melting curve in the absence of trimethylglycine, B shows the melting curve in the presence of trimethylglycine, C shows the distribution of melting temperatures in the absence of trimethylglycine, and D shows the distribution of melting temperatures in the presence of trimethylglycine. It can be seen that the variation in melting temperature is reduced in both the hTERT gene and the GNAS gene.

[0071] Figures 10A and 10B show graphs summarizing the variation in the trimethylglycine concentration and the melting temperature for the hTERT gene and the GNAS gene, respectively. For both the hTERT gene and the GNAS gene, the higher the trimethylglycine concentration, the smaller the distribution of the melting temperature. By adding 0.5 M trimethylglycine, the distribution of the melting temperature of the hTERT gene decreased from 0.90°C to 0.48°C (47% decrease) (Figure 10A), and the distribution of the Tm value of the GNAS gene decreased from 0.97°C to 0.72°C (26% decrease) (Figure 10B). Furthermore, the variation between measurements when multiple measurements were performed is shown as error bars in Figures 10A and 10B. The higher the trimethylglycine concentration, the smaller the error bars on the graph, suggesting that the addition of betaines such as trimethylglycine reduces the inter-measurement difference in the distribution of the melting temperature, and increases the reproducibility of the measurement results.

[0072] In this way, by performing digital PCR in combination with melting curve analysis using an additive that inhibits the formation of secondary DNA structures, the variability in melting temperature is reduced, enabling the target gene to be accurately identified based on the melting temperature, the color of the fluorescent dye, and the fluorescence intensity. [Explanation of symbols]

[0073] 101...DNA 102...Fluorescently labeled probe 103...Fluorescent dye 104…Quencher 201...Melting temperature distribution of genes with high melting temperatures 202...Melting temperature distribution of genes with low melting temperatures 203...Microcompartment containing genes with high melting temperature 301...Melting temperature 302…Melt curve of a microcompartment containing a gene of interest 303…Melt curve of a microcompartment that does not contain the gene of interest 601... Fluorescently labeled probe binding region [Sequence List Free Text]

[0074] SEQ ID NOs: 1 to 12: DNA (synthetic constructs), synthetic polynucleotides

Claims

1. preparing a reaction solution by mixing a fluorescently labeled probe for a gene of interest, a primer for amplifying a region including the gene of interest, a biological sample to be tested, an enzyme, and an additive that inhibits the formation of secondary structures in DNA; Dividing the reaction solution into a plurality of microcompartments and carrying out an amplification reaction in each of the plurality of microcompartments; measuring the binding between the DNA amplified in the amplification reaction and the probe by measuring the fluorescence intensity of the plurality of micro-compartments while changing the temperature; performing a melting curve analysis for each of the microcompartments based on the results of the measurement, and calculating a melting temperature; determining the presence or absence and / or type of the target gene for each of the microcompartments based on the color, intensity and melting temperature of the fluorescence; A DNA detection method comprising:

2. the probe comprises a fluorescent dye and a quencher dye; The method of claim 1 , wherein the binding of the amplified DNA to the probe is measured using the fluorescence intensity of the fluorescent dye.

3. The method according to claim 1, wherein the 3'-terminal sequence and the 5'-terminal sequence of the probe have complementary sequence portions or an interacting structure, and the fluorescent dye of the probe is quenched in a free state.

4. The method according to claim 1 , further comprising a step of combining the determination results of the plurality of micro-compartments by the determination and counting the number of the target genes.

5. 2. The method of claim 1, wherein measuring the fluorescence intensity while varying the temperature comprises measuring the fluorescence intensity while varying the temperature at sampling intervals of less than 1°C.

6. the gene of interest comprises a plurality of genes of interest; The method according to claim 1 , wherein a melting curve analysis is performed for each of a plurality of target genes to determine the presence or absence and / or type of each target gene.

7. The method according to claim 1, wherein the additive that inhibits the formation of a secondary structure of DNA comprises at least one selected from the group consisting of betaine, dimethyl sulfoxide (DMSO), formamide, 7-deaza dGTP, and a nonionic surfactant.

8. The method of claim 1 , wherein the step of performing an amplification reaction is performed by an asymmetric nucleic acid amplification reaction.

9. The method of claim 1 , wherein the amplification reaction is carried out with cycles of temperature change.

10. The method of claim 1 , wherein when the probes comprise multiple types of probes, the probes are labeled with the same fluorescent dye.

11. A primer pair including a forward primer and a reverse primer for amplifying a region including a gene of interest; a fluorescently labeled probe for the gene of interest that binds to DNA amplified using the forward and reverse primers; An additive that inhibits the formation of secondary structures in DNA; A DNA detection kit comprising:

12. The kit of claim 11 further comprising a DNA polymerase.

13. The kit according to claim 11, wherein the additive that inhibits secondary structure formation of DNA comprises at least one selected from the group consisting of betaine, dimethyl sulfoxide (DMSO), formamide, 7-deaza dGTP, and a nonionic surfactant.

14. A kit according to claim 11 for carrying out the method according to claim 1.

15. the probe comprises a fluorescent dye and a quencher dye; The kit according to claim 11, wherein the 3'-terminal sequence and the 5'-terminal sequence of the probe have complementary sequence portions or an interacting structure, and the fluorescent dye of the probe is quenched in a free state.

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