Nucleic acid amplification method

Abstract

To provide a nucleic acid amplification method that can easily amplify nucleic acids.SOLUTION: Provided is a method for amplifying nucleic acid, comprising mixing the following components (A) to (F): component (A): single-stranded DNA; component (B): a primer; component (C): a base-specific nicking enzyme; component (D): a base material consisting of dATP, dTTP, dCTP, and dGTP; component (E): a base material containing an atypical base recognized by the base-specific nicking enzyme; and component (F): a DNA polymerase.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for amplifying nucleic acids. [Background technology]

[0002] BACKGROUND ART Conventionally, methods have been proposed for amplifying nucleic acid samples in order to analyze the details of the samples in academic research, clinical testing, and the like.

[0003] Among these, PCR (Polymerase Chain Reaction) is a widely known nucleic acid amplification method.

[0004] However, the PCR method involves a thermal denaturation step to convert double-stranded DNA into single-stranded DNA, an annealing step to hybridize a primer to a target sequence in the single-stranded DNA, and an extension step to extend the double-stranded DNA starting from the primer sequence, and each step requires a different reaction temperature.

[0005] In order to carry out such a reaction based on the PCR method, special equipment capable of accurately controlling the reaction temperature is required, and this, combined with the complexity of the primer design described above, tends to make the PCR method expensive and complicated.

[0006] In recent years, the so-called "isothermal nucleic acid amplification method" has been proposed, which does not require as strict temperature control as the PCR method. Although the isothermal nucleic acid amplification method does not require the expensive equipment that the PCR method requires, there is still a strong demand for a simpler method.

[0007] Isothermal nucleic acid amplification methods such as LAMP, RCA, RPA, and SDA have been developed, and various improvements have been implemented in these methods to achieve efficient amplification.

[0008] For example, a method has been reported in which two types of primers are added to the four types of primers in the LAMP method, and a method has been developed in which amplification is promoted by inserting a recognition sequence for sequence-specific nickase into the amplified nucleic acid in the RCA method.

[0009] However, these methods are only effective when the target nucleic acid sequence is known. Furthermore, they require the procurement of target sequence-specific primers and sequence-specific enzymes. Therefore, a method for efficiently promoting nucleic acid amplification that is independent of the target nucleic acid sequence is desired. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Molecular and Cellular Probes vol.16,2002, 223-229 [Non-patent document 2] Analyst vol.140, 2015, 74-78 Summary of the Invention [Problem to be solved by the invention]

[0011] In view of the above circumstances, an object of the present invention is to provide a simple method for amplifying nucleic acids. [Means for solving the problem]

[0012] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that nucleic acid amplification can be easily achieved by utilizing a nicking enzyme that recognizes specific bases and forms nicks. Based on this finding, the present inventors have conducted further research and have completed the present invention.

[0013] That is, the present invention provides the following nucleic acid amplification method. Section 1. A method for amplifying nucleic acid, comprising the step of mixing the following components (A) to (F): Component (A): Single-stranded DNA Component (B): Primer Component (C): Base-specific nicking enzyme Component (D): base material consisting of dATP, dTTP, dCTP, and dGTP Component (E): a base material containing an atypical base recognized by the base-specific nicking enzyme Component (F): DNA polymerase Section 2. Item 2. The nucleic acid amplification method according to Item 1, wherein the component (C) is an enzyme that recognizes and cleaves uracil and / or hypoxanthine in a nucleic acid sequence. Section 3. Item 2. The nucleic acid amplification method according to Item 1, wherein the component (F) is a strand-displacing DNA polymerase. Section 4. A nucleic acid amplification kit for carrying out the nucleic acid amplification method according to any one of Items 1 to 3. Section 5. Item 5. The nucleic acid amplification kit according to Item 4, comprising the components (C) and (E). [Effects of the Invention]

[0014] According to the nucleic acid amplification method of the present invention described above, nucleic acids can be amplified easily. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is an explanatory diagram of the nucleic acid amplification method of the present invention. [Figure 2] A / Structure of single-stranded DNA (M13mp18). B / Results of nucleic acid amplification confirmation test by electrophoresis. C / Results of nucleic acid amplification monitoring test. D / Results of nucleic acid amplification confirmation test by fluorescence intensity measurement. E / Results of restriction enzyme treatment test of nucleic acid amplification products. [Figure 3] A / Results of nucleic acid amplification confirmation test by electrophoresis. B / Results of nucleic acid amplification monitoring test. C / Results of nucleic acid amplification confirmation test by fluorescence intensity measurement. [Figure 4]A / Structure of a single-stranded 91-mer oligonucleotide. B / Results of a nucleic acid amplification monitoring test. C / Results of a nucleic acid amplification confirmation test using fluorescence intensity measurement. D / Results of a restriction enzyme digestion test of nucleic acid amplification products. DETAILED DESCRIPTION OF THE INVENTION

[0016] In this specification, the term "containing" is a concept that encompasses all of "comprise," "consist essentially of," and "consist only of." Furthermore, in this specification, when a numerical range is expressed as "A to B," it means A or more and B or less.

[0017] (1. Nucleic Acid Amplification Method) The nucleic acid amplification method of the present invention comprises the step of mixing the following components (A) to (F): Component (A): Single-stranded DNA Component (B): Primer Component (C): Base-specific nicking enzyme Component (D): base material consisting of dATP, dTTP, dCTP, and dGTP Component (E): a base material containing an atypical base recognized by the base-specific nicking enzyme Component (F): DNA polymerase

[0018] Component (A) is, for example, a nucleic acid to be amplified. Component (A) is single-stranded DNA and can be obtained by widely employing known methods. For example, it may be obtained by heat denaturing double-stranded DNA to be amplified, or by synthesis. The single-stranded DNA may be linear single-stranded DNA or circular single-stranded DNA, and is not particularly limited.

[0019] The concentration of component (A) added to the entire reaction system obtained by mixing components (A) to (F) is preferably 0.1 fM or more, more preferably 0.5 nM or more, and even more preferably 2 nM or more.

[0020] On the other hand, the concentration of component (A) added to the entire reaction system obtained by mixing components (A) to (F) is preferably 20 nM or less, more preferably 10 nM or less, and even more preferably 7 nM or less.

[0021] Component (B) serves as a starting point for forming double-stranded DNA using the single-stranded DNA as a template. The method for designing the primers is not particularly limited and may be any known method employed in known nucleic acid amplification methods.

[0022] The concentration of component (B) added to the entire reaction system obtained by mixing components (A) to (F) is preferably 1.0 nM or more, more preferably 3.2 nM or more, and even more preferably 80 nM or more.

[0023] Furthermore, the concentration of component (B) added to the entire reaction system obtained by mixing components (A) to (F) is preferably 100 nM or less, more preferably 80 nM or less, and even more preferably 100 μM or less.

[0024] Component (C) is a base-specific nicking enzyme that recognizes and cuts a specific atypical base in one of the single-stranded DNA strands of double-stranded DNA.

[0025] Such base-specific nicking enzymes are not particularly limited as long as they are enzymes that can recognize and attack uracil, hypoxanthine, xanthine, 5-hydroxyuracil, dihydrouracil, 5-hydroxycytosine, etc., and form nicks, and a wide variety of known enzymes can be used.

[0026] More specifically, examples include Endonuclease Q described in Journal of Bacteriology, 2020, Volume 202 (2), Endonuclease IV described in Nature, 2002, Volume 415, Endonuclease V described in Journal of Biological Chemistry, 1994, Volume 269 (23), and Exonuclease III described in Nucleic Acids Research, 2006, Volume 34 (18). Among these, Endonuclease Q (SEQ ID NO: 1) is preferably used because it can more efficiently recognize atypical bases such as uracil and hypoxanthine present on one single-stranded DNA of a double-stranded DNA and form nicks. As such Endonuclease Q, those having 30% or more homology with the sequence shown in SEQ ID NO: 1, more preferably 50% or more homology, even more preferably 70% or more homology, and particularly preferably 90% or more homology can be used.

[0027] The concentration of component (C) added to the entire reaction system obtained by mixing components (A) to (F) is preferably 1 nM or more, more preferably 10 nM or more, even more preferably 20 nM or more, and particularly preferably 40 nM or more.

[0028] Furthermore, the concentration of component (C) added to the entire reaction system obtained by mixing components (A) to (F) is preferably 50 μM or less, more preferably 5 μM or less, even more preferably 100 nM or less, and particularly preferably 70 nM or less.

[0029] Component (D) is a base material consisting of dATP, dTTP, dCTP, and dGTP, which are base materials for nucleic acid bases such as adenine, thymine, cytosine, and guanine, respectively.

[0030] The concentration of component (D) added to the entire reaction system obtained by mixing components (A) to (F) is preferably 0.03 mM or more, more preferably 0.10 mM or more, and even more preferably 0.20 mM or more for each base material.

[0031] Furthermore, the concentration of component (D) added to the entire reaction system obtained by mixing components (A) to (F) is preferably 1 mM or less for each base material, more preferably 0.80 mM or less, and even more preferably 0.40 mM or less.

[0032] Component (E) is a base material containing an atypical base recognized by the base-specific nicking enzyme. In this specification, the atypical base recognized by the base-specific nicking enzyme is defined as a base that is recognized by the base-specific nicking enzyme described below, and is attacked and damaged to form a nick.

[0033] Examples of atypical bases recognized by such base-specific nicking enzymes include, but are not limited to, uracil, hypoxanthine, xanthine, 5-hydroxyuracil, dihydrouracil, and 5-hydroxycytosine, with uracil and hypoxanthine being particularly preferred.

[0034] The base material of the atypical base recognized by the base-specific nicking enzyme is a reagent added to form the atypical base, and examples thereof include dUTP or UTP for uracil, dITP for hypoxanthine, dXTP for xanthine, 5-hydroxyuracil triphosphate for 5-hydroxyuracil, dihydrouracil triphosphate for dihydrouracil, and 5-hydroxycytosine triphosphate for 5-hydroxycytosine.

[0035] The concentration of component (E) added to the entire reaction system obtained by mixing components (A) to (F) is preferably 2 to 10,000, more preferably 3 to 100, even more preferably 5 to 20, and particularly preferably 8 to 12, in terms of the molar ratio of component (D) to component (E) (component (D) / component (E)) relative to the total concentration of component (D).

[0036] Component (F) is a DNA polymerase. There are no particular limitations on the DNA polymerase, as long as it can incorporate a base material of an atypical base to form the atypical base in a DNA base sequence. DNA polymerases that have such properties and also have strand displacement properties (hereinafter also referred to as strand displacement DNA polymerases in this specification) can be preferably used.

[0037] As used herein, a strand-displacing DNA polymerase is defined as a DNA polymerase that, when it encounters another DNA strand bound to a template DNA during DNA extension along the template DNA, peels the other DNA strand off the template DNA and continues the extension reaction. In other words, with a general DNA polymerase that does not have this property, when it encounters another DNA strand during DNA extension along the template DNA, the DNA extension reaction stops at that point.

[0038] The strand-displacing DNA polymerase used in the present invention is not particularly limited and may be any known DNA polymerase having the above-mentioned functions, including φ29 DNA polymerase, Bst DNA polymerase, and Vent (exo-) DNA polymerase.

[0039] The concentration of component (F) added to the entire reaction system obtained by mixing components (A) to (F) is preferably 0.01 U / μL or more, more preferably 0.02 U / μL or more, even more preferably 0.04 U / μL or more, and particularly preferably 0.1 U / μL or more.

[0040] On the other hand, the concentration of component (F) added to the entire reaction system obtained by mixing components (A) to (F) is preferably 50 U / μL or less, more preferably 10 U / μL or less, and even more preferably 5 U / μL or less.

[0041] In addition to the above-mentioned components (A) to (F), water (preferably pure water, more preferably ultrapure water), buffer solution, serum, surfactant, molecular crowding agent, cell extract, protein, or salt may be added or mixed within a range that does not impair the object and effect of the present invention.

[0042] In the nucleic acid amplification method of the present invention, after the above-mentioned various components are mixed, the reaction system is preferably maintained at a predetermined temperature for a predetermined period of time.

[0043] The temperature conditions are preferably determined based on the optimum temperatures at which components (A) to (F), particularly components (C) and (F), exhibit their activity. Specific temperature conditions depend on the type of enzyme, but are preferably 4 to 100°C, more preferably 55 to 75°C, and even more preferably 30 to 37°C.

[0044] The time for maintaining the temperature is not particularly limited and may be determined appropriately depending on the specific types of components used and the extent to which nucleic acid amplification is to be performed, for example, preferably 5 minutes to 24 hours, more preferably 1 hour to 2 hours.

[0045] In the nucleic acid amplification method of the present invention, the nucleic acid amplification reaction is thought to proceed through the reaction shown in Figure 1. The reaction mechanism will be explained below with reference to Figure 1.

[0046] As shown in step 1, a primer is hybridized (annealed) to the single-stranded DNA of the nucleic acid to be amplified.

[0047] Next, as shown in step 2, a nucleic acid extension reaction by DNA polymerase proceeds to form double-stranded DNA.

[0048] In the presence of the base-specific nicking enzyme, the atypical base recognized by the base-specific nicking enzyme is attacked to form a nick (step 3).

[0049] Starting from the nick formed in step 3, a DNA polymerase (preferably a strand-displacing DNA polymerase) peels off one of the two DNA strands, causing a new nucleic acid elongation reaction to proceed (step 4). A new primer anneals to the peeled single-stranded DNA, and the reactions from step 1 onwards are repeated.

[0050] (2. Nucleic Acid Amplification Kit) The present invention includes an invention relating to a nucleic acid amplification kit. The nucleic acid amplification kit of the present invention is a kit for carrying out the nucleic acid amplification method of the present invention described above.

[0051] The kit may, for example, be a kit containing some or all of the enzymes or reagents required for the nucleic acid amplification method described above.

[0052] Specifically, an example of a kit includes some or all of the base materials such as dATP, dTTP, dCTP, and dGTP, a DNA polymerase (preferably, a strand-displacing DNA polymerase), a base-specific nicking enzyme, and atypical bases such as dUTP or dITP.

[0053] Although the embodiments of the present invention have been described above, the present invention is not limited to these examples, and it goes without saying that the present invention can be embodied in various forms without departing from the spirit of the present invention. [Example]

[0054] Hereinafter, the embodiments of the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

[0055] Using the components (A) to (F) in Table 1 below, reaction solutions for nucleic acid amplification were prepared for Example 1 and each of the comparative examples.

[0056] [1. Nucleic acid amplification test of single-stranded DNA (M13mp18) using dUTP] Endonuclease Q wild type (WT) was prepared according to the method described in Nucleic Acids Research 43, 2015, 2853-2863.

[0057] The structure of the single-stranded DNA (M13mp18) of component (A) is shown in Figure 2A. This single-stranded DNA consists of 7,249 bases, and when double-stranded, restriction enzyme sites appear, allowing cleavage with EcoRI and BglII. The arrows in Figure 2A indicate the annealing positions of primers M13-F and M13-R used in rolling circle amplification (hereinafter simply referred to as "RCA"). The primers used are the forward and reverse primers shown in Table 1 below (SEQ ID NOs: 2 and 3, respectively).

[0058] [Table 1]

[0059] (1.1. Nucleic acid amplification confirmation test by electrophoresis) Reaction solutions for nucleic acid amplification of Comparative Examples 1 to 7 and Examples 1 to 4 were prepared based on the compositions of components (A) to (F) shown in Table 2 below.

[0060] [Table 2]

[0061] The "other components" in Table 1 were formulated as shown in Table 1 in all Examples and Comparative Examples (hereinafter, the same applies to all Examples and Comparative Examples). Specifically, component (B) and the following components in Table 1 were mixed to prepare a 2x master mix. 4 μL of component (A) was added to this to make a total of 8 μL of reaction solution. All preparation procedures were carried out on ice. No fluorescent dye was added in the nucleic acid amplification confirmation test. The reaction solutions of each Example and Comparative Example were incubated at 30°C for 2 hours using a T100 thermal cycler (Bio-Rad). The resulting DNA products were electrophoresed on a 1% (w / v) non-denaturing agarose gel and stained with ethidium bromide.

[0062] As shown in FIG. 2B, in Examples 1 to 4, bands due to cleavage products produced by nucleic acid amplification were observed, but in Comparative Examples 1 to 7, no such bands were observed.

[0063] (1.2. Nucleic Acid Amplification Monitoring Test)

[0064] [Table 3]

[0065] The results of the fluorescence intensity obtained in each comparative example and comparative example are shown in Figure 2C. A comparison of the fluorescence intensity at 180 minutes for Comparative Examples 13 to 15 and Example 5 is shown in Figure 2D. In Example 5, the fluorescence intensity increased over time, suggesting that the nucleic acid amplification reaction was promoted. On the other hand, no clear improvement in fluorescence intensity was observed in each comparative example compared to Example 5.

[0066] (1.3. Restriction enzyme digestion test of amplified products) The amplified product was digested with a restriction enzyme specific to the M13mp18 double-stranded DNA sequence. The nucleic acid amplification reaction solution from Example 6, containing dUTP and EndoQ, was incubated at 30°C for 4 hours, after which 10 U each of EcoRI (New England BioLabs) and BglII (New England BioLabs) were added as restriction enzymes, and the mixture was further incubated at 37°C for 2 hours. As a control, a reaction without enzymes was also incubated in the same manner as above. The product was subjected to 1% agarose gel electrophoresis and stained with ethidium bromide.

[0067] As shown in Figure 2E, when the M13mp18 double-stranded DNA-specific restriction enzyme was added to the amplified product, a shorter DNA strand was generated from the product. This suggests that the amplified product contains a sequence derived from the target DNA. In other words, Example 5 is considered to be a product specifically amplified with the target sequence.

[0068] [2. Nucleic acid amplification test of single-stranded DNA (M13mp18) using dITP] The tests described in items 1.1 to 1.2 above were carried out in the same manner as in items 1.1 to 1.2, except that dITP was used instead of dUTP as the atypical base material, incubation was carried out at 30°C in the nucleic acid amplification monitoring test, and incubation was carried out at 30°C for 240 minutes in the nucleic acid amplification confirmation test by fluorescence intensity measurement. The components used to prepare the nucleic acid amplification reaction solution are listed in Table 4 below.

[0069] [Table 4]

[0070] The compositions of the reaction solutions for nucleic acid amplification prepared for the nucleic acid amplification confirmation test and nucleic acid amplification monitoring test by electrophoresis in each comparative example and example are shown in Tables 5 and 6 below, respectively.

[0071] [Table 5]

[0072] [Table 6]

[0073] The test results using dITP as the atypical base material are shown in Figures 3A, 3B, and 3C. The fluorescence intensity of some comparative examples and examples at 200 minutes in the nucleic acid amplification monitoring test is shown in Figure 3D. When the nucleic acid amplification reaction solution of each example was used, results suggesting that nucleic acid amplification was promoted were obtained. On the other hand, when the nucleic acid amplification reaction solution of each comparative example was used, such results were not obtained.

[0074] [3. Examination of the contribution of nicking enzymes to nucleic acid amplification] A nucleic acid amplification monitoring test and a restriction enzyme treatment test of the amplified products were carried out in the same manner as in items 1.2 to 1.3 above, except that a single-stranded 91-mer oligonucleotide (the sequence of the padlock probe described in Analytical Biochemistry, 519, 2017, pp. 15-18) was used as the nucleic acid to be amplified.

[0075] (Preparation of single-stranded 91-mer oligonucleotide) A single-stranded 91-mer oligonucleotide (sequence of the padlock probe described in Analytical Biochemistry, 519, 2017, pp. 15–18) was used. The oligonucleotide had a phosphate group attached to the 5′ end. A 50 μL intramolecular ligation reaction mixture (composition shown in Table 7) was prepared in a tube on ice. The resulting mixture was incubated overnight (16–24 hours) at 16°C. The enzyme was then inactivated by incubation at 65°C for 15 minutes. To remove unreacted single-stranded DNA, 1 μL of 2 U / μL Exonuclease I (New England Biolabs) was added to the 50 μL reaction mixture after ligation, and the mixture was incubated at 37°C for 15 minutes. The enzyme was then inactivated by incubation at 80°C for 15 minutes. Assuming a 100% efficiency of the circularization reaction, a final concentration of 275 nM single-stranded circular 91-mer-PP was obtained.

[0076] [Table 7]

[0077] (Preparation of endonuclease Q mutants without nicking activity) Endonuclease Q wild-type (WT) and E76A mutant were prepared according to the method described in Nucleic Acids Research 43, 2015, pp. 2853-2863. The E76A mutant is a mutant that has no nicking activity, as described in Journal of Bacteriology 202, 2020, e00542-19.

[0078] Based on the above, the components were prepared as shown in Table 8 below.

[0079] [Table 8]

[0080] The same test as in item 1.2 above was carried out using the components prepared above. The comparative examples and examples prepared for the nucleic acid amplification monitoring test are listed in order in Table 9 below. The nucleic acid amplification monitoring test was carried out by incubation at 30°C for 250 minutes, and the nucleic acid amplification confirmation test by fluorescence intensity measurement was carried out by incubation at 30°C for 250 minutes.

[0081] [Table 9]

[0082] (Restriction enzyme digestion test of amplified products) The sequence specificity of the amplified product was examined using a restriction enzyme that specifically acts on the 91mer-PP sequence. Restriction enzyme digestion was performed. The above amplification reaction mixture was incubated at 30°C for 4 hours, after which 10 U of MluI (New England Biolabs) was added and the mixture was further incubated at 37°C for 2 hours. As a control experiment, a reaction without the enzyme was also incubated in the same manner as above. The product was subjected to 1% agarose gel electrophoresis and stained with ethidium bromide.

[0083] The results of the above tests are shown in Figures 4B to 4D. The amplification monitoring results shown in Figure 4B demonstrate that nucleic acid amplification proceeded in the presence of target nucleic acid and polymerase, and DNA amplification over time was observed (Comparative Example 37). In Example 11, in which EndoQ WT and dUTP were added simultaneously, a rapid amplification enhancement was observed starting 50 minutes after incubation. On the other hand, this amplification enhancement was not observed in Comparative Examples 37 to 39, in which EndoQ WT, dUTP, or both were lacking. Furthermore, no amplification enhancement was observed in the reaction using the inactive mutant E76A instead of the wild-type (Comparative Example 40), indicating that this enhancement was due to the nicking activity of EndoQ. In Comparative Examples 31 to 36, which did not contain target nucleic acid or polymerase, no DNA amplification was observed with the addition of EndoQ and dUTP. These results suggest that EndoQ WT and dUTP enhance DNA amplification in a target nucleic acid-dependent manner.

[0084] Figure 4C shows the fluorescence intensity ratio at 70 minutes after the start of incubation. The fluorescence intensity of the reaction without EndoQ and dUTP (Comparative Example 37) is shown as 1. Comparing Comparative Example 37 with the reaction with EndoQ WT and dUTP (Example 11), a 7.8-fold increase in amplification was detected.

[0085] Figure 4D examines the sequence dependency of the amplification products obtained from the nucleic acid amplification reaction solutions of Comparative Example 37 and Example 11. When a restriction enzyme specific to the 91mer-PP sequence was added to the amplification product, a DNA strand thought to be an even shorter 91mer was generated from the product. This indicates that the amplification product contains a sequence derived from the target DNA. In other words, the product of Example 11, like Comparative Example 37, is thought to be a product specifically amplified with the target sequence.

Claims

1. A method for amplifying nucleic acid, comprising the step of mixing the following components (A) to (F): Component (A): Single-stranded DNA Component (B): Primer Component (C): Base-specific nicking enzyme Component (D): base materials consisting of dATP, dTTP, dCTP, and dGTP Component (E): a base material containing an atypical base recognized by the base-specific nicking enzyme Component (F): DNA polymerase

2. 2. The nucleic acid amplification method according to claim 1, wherein the component (C) is an enzyme that recognizes and cleaves uracil and / or hypoxanthine in a nucleic acid sequence.

3. The nucleic acid amplification method according to claim 1 , wherein the component (F) is a strand-displacing DNA polymerase.

4. A nucleic acid amplification kit for carrying out the nucleic acid amplification method according to any one of claims 1 to 3.

5. The nucleic acid amplification kit according to claim 4, comprising the components (C) and (E).

Images