A method for detecting single-base mutation of a gene by using Tth Ago enzyme cleavage and its application

Abstract

The present invention discloses a method for detecting single-base mutation of a gene by using Tth Ago enzyme shearing and its application. The method comprises the following steps: (1) designing 2 gDNAs for identifying mutation ends and 2 gDNAs for identifying non-mutation ends according to the target gene or DNA sequence to be detected, both of which are 16-19 nt; (2) using the designed 4 gDNAs and Tth Ago enzyme to shear the target gene or DNA fragment; (3) designing constant temperature amplification primers for the 45-65bp sequence between the shearing sites of the Tth Ago enzyme at both ends of the target gene or DNA fragment, and performing PCR constant temperature amplification; (4) judging whether there is a single-base mutation in the sample to be tested according to the Ct value of the sample to be tested relative to the wild-type sample during the constant temperature amplification reaction. The method of the present invention has simple steps, simple operation, high specificity and high sensitivity, and can be used for the detection of low-abundance single-base mutations.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene mutation detection, and particularly relates to a method for detecting single-base gene mutations by utilizing Tth Ago enzyme cleavage and its application. Background Art

[0002] Single nucleotide variation (SNV) refers to a change in a single nucleotide at a specific DNA position. It is widely present in the genetic information encoding of organisms. When the frequency of the less common allele is greater than or equal to 1%, it is called a SNP. The study of point mutations helps to deepen our understanding of the genetic characteristics of higher organisms, explain individual phenotypic differences, and reveal the normal functions of genes and proteins, the causes of variation, and the mechanisms by which different individuals respond to environmental changes. It is also of great significance for functional genomics research and the establishment of molecular markers. The detection of rare polymorphic alleles is increasingly important for the early diagnosis and monitoring of various tumors. Therefore, the detection of extremely rare variant alleles in complex mixtures of DNA molecules has attracted increasing attention. The main goal is to solve the problems associated with accurate single nucleotide resolution and simplify multiplex detection technologies, especially for the detection of cancer-related DNA biomarkers in patients. With the development of genome sequencing, the detection and screening of point mutations are becoming the focus of attention, and detection methods are developing rapidly. In addition, when the changes in the physical and chemical properties of the gene caused by the point mutation are relatively weak, the detection of point mutations becomes very difficult, which has prompted people to explore and establish new detection methods from many aspects.

[0003] Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was the first method used to analyze known point mutations. If a point mutation occurs at a restriction enzyme recognition site, resulting in an increase or decrease in the restriction site, the presence of the point mutation can be determined by digesting the DNA fragment specifically amplified by PCR with a specific restriction enzyme and separating the digestion products by agarose gel electrophoresis. This test has high specificity and good reproducibility, but is only suitable for detecting mutations involving specific restriction enzyme recognition sites and polymorphisms with a mutation frequency greater than 1%. Furthermore, it cannot determine the specific base sequence involved in the mutation. In the past few years, new nucleic acid editing methods and PCR protocols have also been explored. However, these methods have been unsatisfactory in terms of detection sensitivity, cost, time, and simplicity. For example, conventional PCR cannot detect single-base mutations. The amplification retardation mutation system (ARMS) method, which includes the mutant base sequence in the primer sequence, can selectively amplify variant sequences, but cannot identify or verify the mutant allele. Other methods, such as blocker displacement amplification and low-temperature co-amplification, require strict target annealing temperatures. Restriction endonuclease-mediated selective polymerase chain reaction analysis allows for simultaneous amplification of mutation signals and suppression of wild-type gene amplification, but is limited by the availability of thermostable restriction enzymes. Digital PCR is currently the most advanced single-base mutation analysis technology, capable of detecting mutations at a rate of 0.01%. While it offers several advantages over traditional methods, the high cost of digital PCR equipment hinders its widespread adoption.

[0004] Endonucleases with sequence-specific cleavage capabilities are powerful tools for identifying nucleic acid targets and designing subsequent assays. In recent years, researchers have identified various endonucleases from archaea and bacteria that can be guided by small nucleic acid fragments to cleave complementary double-stranded DNA. CRISPR-Cas has garnered considerable attention as a genome editing tool, but its application has been significantly limited, in part due to its reliance on a PAM recognition site, which is absent in many reported biological gene sequences. Similar to CRISPR-Cas, Argonaute (Ago) proteins are also endonucleases guided by small nucleic acid fragments. However, unlike Cas nucleases, Ago nucleases do not require a PAM recognition site, making them more versatile. Under appropriate conditions, Ago nucleases cleave target DNA or RNA using a small nucleic acid (gDNA) complementary to the target DNA or RNA as a guide.

[0005] The structure of the Ago enzyme from Thermus thermophiles (Tth) includes six domains: N-terminal, L1, PAZ, L2, MID and PIWI. The PIWI domain contains a catalytic tetrad DEDX (X is D, H or K), which can bind to divalent metal ions involved in shear catalysis. When guided by gDNA, the Tth Ago enzyme can shear the complementary target between the 10th and 11th positions (g10 / g11) of the gDNA. Although Mg 2+ As a catalyst for activating Tth Ago enzyme activity, its catalytic effect on double-strand cleavage is not very ideal.

[0006] The development of the polymerase chain reaction (PCR) has provided a powerful tool for nucleic acid amplification, enabling the exponential amplification of minute nucleic acid targets. However, most PCR-based techniques require electrically powered thermal cycling equipment for repeated heating and cooling, limiting their application outside the laboratory. The emergence of isothermal nucleic acid amplification methods has overcome the limitations of traditional PCR, offering the possibility of performing nucleic acid amplification without the need for thermal cycling equipment. Furthermore, one of the future needs of bioinformatics analysis is the development of simple, sensitive, and reliable isothermal nucleic acid amplification methods for detecting emerging infectious diseases in remote or isolated areas and developing countries. Strand exchange reactions (SEAs) in nucleic acids are a key natural process in living organisms for homologous recombination, DNA replication, and DNA repair. The weak interactions between base pairs in dsDNA allow for transient opening, resulting in intermittent base pair breakage at certain temperatures, leading to the formation of single-strand denatured bubbles in the DNA. This DNA phenomenon allows SEA to replicate without the assistance of recombinases.

[0007] The location of each single-base mutation, the fixed length of the excised nucleic acid fragment to be amplified (45-65 bp), and the requirement for isothermal amplification from the tips of the excised nucleic acid fragments dictate the fixed sequence of primers designed for amplification of the nucleic acid fragments. This results in a high degree of nonspecific amplification. Because SEA requires high primer specificity for amplification, it is necessary to find suitable methods to reduce nonspecific amplification and increase SEA amplification efficiency. Summary of the Invention

[0008] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for detecting single-base mutations in genes using Tth Ago enzyme cleavage.

[0009] Another object of the present invention is to provide an application of the method for detecting single-base mutations in genes by utilizing Tth Ago enzyme cleavage.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A method for detecting single-base mutations in genes using Tth Ago enzyme cleavage comprises the following steps:

[0012] (1) Design of 4 gDNAs

[0013] According to the sequence of the target gene or DNA fragment to be detected, two 16-19 nt gDNAs (guide DNAs) for recognizing the mutant end and two 16-19 nt gDNAs for recognizing the non-mutant end are designed respectively; wherein the first base at the 5' end of the gDNA recognizing the mutant end and the gDNA recognizing the non-mutant end is T, and the base T is modified by a phosphate group; except for the first phosphorylated T base, the remaining sequence of the gDNA recognizing the mutant end is complementary to the mutant gene sequence of the target gene and forms a single base mismatch with the wild-type gene sequence of the target gene; except for the first phosphorylated T base, the remaining sequence of the gDNA recognizing the non-mutant end is complementary to the sequence 45-65 bp upstream or downstream of the gDNA shearing site at the mutant end; the four gDNA sequences are different;

[0014] (2) Tth Ago enzyme cleavage

[0015] The Tth Ago enzyme, the two gDNAs designed in step (1) for recognizing the mutant end, and the two gDNAs for recognizing the non-mutant end are incubated in a buffer system for 15 to 30 minutes respectively. After the incubation is completed, they are mixed together, and then the sample to be tested containing the target gene or DNA fragment is added, and the Tth Ago enzyme cleavage reaction is carried out at 66 to 90° C., while the wild-type gene containing the target gene or DNA fragment is used as a control sample, and the reaction obtains the enzyme cleavage product of the sample to be tested and the enzyme cleavage product of the control sample; wherein the buffer system is Mg-containing 2+ and Mn 2+ A buffer system comprising at least one of:

[0016] (3) Fluorescence quantitative PCR

[0017] ① Design PCR constant temperature amplification primers

[0018] Based on the 45-65 bp DNA fragment sequence between the two end cleavage sites of the target gene or DNA fragment guided by the Tth Ago enzyme on the mutant and non-mutant ends (if there is a single base mutation, the sequence is the 45-65 bp enzyme-cleaved fragment), design a pair of 20-25 nt long PCR isothermal amplification primers (forward primer, reverse primer) that are sufficient to amplify from the top ends of the 45-65 bp DNA fragment;

[0019] ②PCR constant temperature amplification

[0020] The enzyme digestion products of the test sample and the control sample obtained in step (2) are first treated with nuclease exonuclease, and then added to a reaction system containing DNA polymerase, PEG 200 and betaine, respectively. Fluorescence quantitative PCR isothermal amplification is performed using the PCR isothermal amplification primers designed in step ①, and real-time fluorescence curves of the reaction systems during the reaction process are obtained, thereby determining whether there is a single base mutation in the test sample of the target gene;

[0021] (4) Judgment:

[0022] If the real-time fluorescence curve obtained during the reaction of the control sample tends to be stable over time (extended) or its Ct value is lower than the Ct value of the test sample, and the real-time fluorescence curve obtained during the reaction of the test sample shows an exponential amplification process over time (the mutant gene is successfully cleaved and effectively amplified), it indicates that the test sample has a single base mutation; if the real-time fluorescence curve obtained during the reaction of the test sample changes with time and is consistent with that of the control sample, it indicates that the test sample does not have a single base mutation.

[0023] The gDNA that recognizes the mutant end in step (1) is complementary to the mutant target gene or DNA fragment (except that the first base at the 5' end is fixed as T), forms a single base mismatch with the wild-type target gene or DNA fragment, and the mismatch site between the gDNA that recognizes the mutant end and the wild-type target gene or DNA fragment is located at the 9th, 10th, 11th or 15th position of the complementary sequence between the gDNA and the target gene; preferably, the mismatch site between the gDNA that recognizes the mutant end and the wild-type target gene or DNA fragment is located at the 10th position of the complementary sequence between the gDNA and the target gene.

[0024] The sequence of the gDNA for identifying the mutant end described in step (1) is as follows:

[0025] gDNA-1:

[0026] gDNA-2:

[0027] Note: The black bold and underlined characters above are the mismatch sites between gDNA and wild-type double-stranded DNA.

[0028] The sequence of the gDNA for identifying the non-mutated end described in step (1) is as follows:

[0029] gDNA-3: 5'-p TGGCTGGAATCCGAGTTA-3';

[0030] gDNA-4: 5'-pTAATAACTCGGATTCCAG-3'.

[0031] The molar ratio of the four gDNAs in the buffer system described in step (2) is 1:1:1:1 (same concentration), and the molar ratio of the total molar ratio of the four gDNAs to the Tth Ago enzyme is greater than 5:1; preferably 5 to 10:1; more preferably 10:1.

[0032] Mg in the buffer system described in step (2) 2+ The concentration of Mg is 4 to 24 mmol / L; preferably 8 to 24 mmol / L; more preferably Mg 2+ The concentration is 20mmol / L.

[0033] Mn in the buffer system described in step (2) 2+ The concentration is 1 to 6 mmol / L; preferably 4 to 6 mmol / L; more preferably 5 mmol / L.

[0034] The formula of the buffer system described in step (2) is: 2mM Tris-HCl, 1mM KCl, 1mM (NH4)2SO4, 0.01% (v / v) Triton X-100, and a certain concentration of metal ions; wherein the metal ions are 4 to 24mmol / L Mg 2+ and 1-6 mmol / L Mn 2+ At least one of .

[0035] The buffer system in step (2) is preferably formulated as follows: 2 mM Tris-HCl, 1 mM KCl, 1 mM (NH4)2SO4, 0.01% (v / v) Triton X-100, 5 mM Mn 2+ .

[0036] In the reaction system described in step (2), the mutant type of the target gene or DNA fragment can be cleaved at both ends by the Tth Ago enzyme, while the wild type of the target gene or DNA fragment can only be cleaved at one end by the Tth Ago enzyme.

[0037] The temperature of the Tth Ago enzyme cleavage reaction in step (2) is preferably 75°C to 85°C; more preferably 80°C to 85°C; and even more preferably 85°C.

[0038] The exonuclease described in step (3) is an enzyme having the activity of degrading single-stranded DNA, preferably exonuclease I.

[0039] The DNA polymerase described in step (3) is a DNA polymerase with strand displacement activity (ability), and the temperature of the isothermal amplification reaction using it needs to be lower than the Tm value (melting temperature) of the 45-65 bp DNA fragment sheared in step (2). That is, in the present invention, a suitable DNA polymerase is selected according to the Tm value of the 45-65 bp DNA fragment sheared in step (2), and the temperature of the isothermal amplification reaction is the optimal temperature of the DNA polymerase (for each different DNA polymerase, its optimal temperature is known); the DNA polymerase is preferably Bst 2.0 warmstart DNA polymerase, and for Bst 2.0 warmstart DNA polymerase, the temperature range of its isothermal amplification reaction is 37°C to 65°C; preferably 58°C to 65°C; more preferably 61°C.

[0040] The primer sequences used in the isothermal amplification reaction described in step (3) are as follows:

[0041] Primer-1: 5'-CGAGTTATTATTTGATGTGTC-3';

[0042] Primer-2: 5'-GGCCCCTGTCTTGCTGTCATG-3'.

[0043] The amount of PEG 200 added to the reaction system in step (3) is 1 to 4% by volume, preferably 1% by volume.

[0044] The concentration of betaine in the reaction system in step (3) is 0.9 to 1.8 mol / L, preferably 1.8 mol / L.

[0045] The reaction system in step (3) is as follows: 1 μl each of 5 μM PCR constant temperature amplification primers (forward primer, reverse primer); 1 μl of enzyme digestion product (template); 2 μl of 10× bst DNA polymerase buffer; 1.6 μl of 10 mM dNTP; 1 μl of Bst DNA polymerase; 100 mM MgCl2 2+ 1.2 μl; 10× Eva Green (nucleic acid dye for PCR) 1 μl; 5 M betaine 3.6-7.2 μl; polyethylene glycol 200 (PEG 200) 0.2-0.8 μl; H2O to 20 μl.

[0046] The reaction system in step (3) is preferably formulated as follows: 1 μl each of 5 μM PCR constant temperature amplification primers (forward primer, reverse primer); 1 μl of enzyme digestion product (template); 2 μl of 10× bst DNA polymerase buffer; 1.6 μl of 10 mM dNTP; 1 μl of Bst DNA polymerase; 100 mM MgCl2 2+ 1.2 μl; 10× Eva Green (nucleic acid dye for PCR) 1 μl; 5 M betaine 7.2 μl; polyethylene glycol 200 (PEG 200) 0.2 μl; H2O to 20 μl.

[0047] The method for detecting single-base mutations in genes by utilizing Tth Ago enzyme cleavage is used in detecting single-base mutations in genes in vitro.

[0048] The present invention has the following advantages and effects compared to the prior art:

[0049] (1) The present invention provides a method for detecting single-base mutations in genes using Tth Ago enzyme cleavage, the method comprising the following steps: 1) designing a corresponding guide DNA for the mutant gene sequence of the target gene; 2) using Thermus thermophilus argonaute (Tth Ago) enzyme to cleave the mutant gene; 3) designing primers for isothermal amplification of the sequence between the two end cleavage sites on the target gene or DNA fragment guided by the Tth Ago enzyme at the mutant end and the non-mutant end;

[0050] 4) Fluorescence quantitative isothermal amplification; this method is simple in steps, easy to operate, and has high specificity and high sensitivity (the minimum detection limit of a single base mutation is 1×10 -17 M) and other characteristics, and can be used for low-abundance mutation detection.

[0051] (2) The method of the present invention can quickly and accurately detect single-base mutation gene sequences, and has a high degree of selectivity for single-base mutation gene sequences; in addition, the PCR isothermal amplification method changes the previous cumbersome PCR experimental steps, the experimental conditions are relatively simple, and no laboratory electric thermal cycler is required. It is easy to promote to non-professional operation, the detection cost is low, easy to prepare, and has high repeatability. It can be applied to monitoring points and nursing points for real-time detection.

[0052] (3) The Ago enzyme from Thermus thermophiles (Tth) in the present invention has the ability to efficiently cleave nucleic acid fragments. We use this feature to design a method to identify single-base mutation sites in target genes and cleave the mutated target genes.

[0053] (4) The present invention utilizes the property of dsDNA to generate denaturation bubbles at a single temperature, introduces a short DNA primer fragment to invade the denaturation bubble, and then uses the strand displacement activity and polymerase activity of DNA enzyme to achieve extension; this process can be carried out at a single temperature lower than the Tm value of dsDNA, thereby achieving amplification without the need for traditional thermal denaturation process.

[0054] (5) The present invention has found through experiments that adding a certain concentration of Mn 2+ When catalyzing Tth Ago enzyme, the cleavage activity of Tth Ago enzyme is greatly improved, and the optimum temperature of Tth Ago enzyme is relatively low compared to Mg 2+ During catalysis, the temperature will drop by 5℃, ranging from 75℃ to 85℃.

[0055] (6) The present invention found through experiments that the simultaneous addition of betaine and PEG 200 can reduce nonspecific amplification and increase the amplification efficiency of SEA. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Schematic diagram of the principle of detecting single base mutations by the method of the present invention.

[0057] Figure 2 1 is a schematic diagram of the results of detecting single-base mutations using the method of the present invention in Example 1; wherein A is the electrophoresis result (lane 1: experimental group containing Tth Ago enzyme, the target used is the mutant gene; lane 2: control group without Tth Ago enzyme, the target used is the mutant gene; lane 3: experimental group containing Tth Ago enzyme, the target used is the wild-type gene; lane 4: control group without Tth Ago enzyme, the target used is the wild-type gene); B is the real-time fluorescence curve obtained by fluorescence quantitative isothermal amplification of the wild-type gene sequence after cleavage by Tth Ago enzyme using different template concentrations; C is the real-time fluorescence curve obtained by fluorescence quantitative isothermal amplification of the mutant gene sequence after cleavage by Tth Ago enzyme using different template concentrations.

[0058] Figure 3 This is a statistical diagram showing how different mismatch sites between gDNA and the wild-type target gene affect the cleavage efficiency of Tth Ago enzyme in Example 2 (in the figure, ssDNA MT is mutant single-stranded DNA; ssDNA WT is wild-type single-stranded DNA).

[0059] Figure 4 Schematic diagram of the metal ion optimization results for Tth Ago enzyme cleavage in Example 3; wherein A is the electrophoresis results (lanes 1 to 9 are respectively added with H2O (control group) and added with Ca 2+ 、Cu 2+ 、Zn2+ Mg 2+ 、Ba 2+ 、Ni 2+ 、Mn 2+ and Co 2+ Lane N is the control group without Tth Ago enzyme (No Tth Ago), and lane T is the control group containing only the mutant target gene target sequence and no enzyme digestion system); B is the experimental system with different concentrations of Mn 2+ (Lanes 1 to 7 were supplemented with 0, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, and 6 mM Mn 2+ Lane N is the control group without Tth Ago enzyme (No Tth Ago), and lane T is the control group containing only the mutant target gene target sequence and no enzyme digestion system); C is the experimental system with different concentrations of Mg added. 2+ (Lanes 1 to 7 were supplemented with 0, 4mM, 8mM, 12mM, 16mM, 20mM, and 24mM Mg 2+ Lane N is a control group without Tth Ago enzyme (No Tth Ago), and lane T is a control group containing only the mutant target gene target sequence and no enzyme digestion system).

[0060] Figure 5 This is a schematic diagram of the optimization results of the temperature used for Tth Ago enzyme cleavage in Example 4.

[0061] Figure 6 This is a schematic diagram of the optimization results of PEG 200 concentration in SEA isothermal amplification in Example 5.

[0062] Figure 7 This is a schematic diagram of the optimization results of betaine concentration in SEA isothermal amplification in Example 6. DETAILED DESCRIPTION

[0063] The present invention will be described in further detail below in conjunction with the examples, but embodiments of the present invention are not limited thereto. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are generally based on conventional experimental conditions or the experimental conditions recommended by the manufacturer. Unless otherwise stated, the reagents and raw materials used in the present invention can be obtained commercially or by conventional methods.

[0064] The present invention combines the cleavage function of Tth Ago enzyme with PCR isothermal amplification to obtain the method for detecting single-base mutations with high specificity and sensitivity. Figure 1This is a schematic diagram of the principle of detecting single-base mutations in genes using the method described in the present invention. The specific principle is: the gDNA bound to the Tth Ago enzyme involved in this application contains a 5'-terminal phosphate group, and the gDNA (except the first phosphorylated T base) is completely complementary to the mutant gene sequence of the target gene. The complementary fragment is 15 to 18 nt (nt = nucleotide), forming a single-base mismatch with the wild-type gene sequence of the target gene. The mismatch site is located at the 9th, 10th, 11th or 15th position at the 5' end of the complementary fragment formed by the gDNA and the wild-type gene of the target gene; the mutant end gDNA guides the Tth Ago enzyme to bind to the target gene sequence and cut a DNA fragment containing a single-base mutation, while the mutant end corresponding to the wild-type gene sequence of the target gene cannot be effectively cut by the Tth Ago enzyme; 45 to 65 bp upstream or downstream of the site where the Tth Ago enzyme cuts the single-base mutation end, both the wild type and mutant types of the target gene can be cut by the Tth Ago enzyme guided by the non-mutant end gDNA. Ago enzyme cleavage can only cut out a 45-65bp-long nucleic acid segment of the mutant target gene. The nucleic acid segment is then isothermally amplified to obtain a real-time fluorescence curve during the reaction process, thereby determining whether there is a single-base mutation in the target gene in the test sample.

[0065] Specifically, the method for detecting a single base mutation involved in the present invention comprises the following steps:

[0066] (1) Designing gDNA: The gDNA is a single-stranded oligonucleotide sequence with a length of 16 to 19 nt. The gDNA is designed according to the sequence of the target gene or DNA fragment to be detected. The gDNA designed to recognize the mutant end (except the first phosphorylated T base) is complementary to the mutant gene sequence of the target gene and forms a single base mismatch with the wild-type gene sequence of the target gene; the gDNA designed to recognize the non-mutant end (except the first phosphorylated T base) is complementary to the sequence 45-65 bp upstream or downstream of the gDNA cleavage site at the mutant end, thereby achieving the purpose of cleaving both ends of the mutant gene and only cleaving one end of the wild-type gene by distinguishing single base mutations;

[0067] (2) adding a sample of the target gene to a solution containing Tth Ago enzyme and gDNA to form a reaction system, and shearing the sample using Tth Ago enzyme; wherein the gDNA comprises four strands, two of which recognize the mutant end and the other two recognize the non-mutant end; the Tth Ago enzyme shears the complementary target corresponding to the 10th and 11th positions of the gDNA; in the reaction system, the mutant type of the target gene can be sheared by the Tth Ago enzyme into a 45-65 bp long nucleic acid fragment, while the wild type of the target gene cannot;

[0068] (3) Design constant temperature amplification primers for the 45-65 bp long DNA fragment between the two end cleavage sites of the target gene or DNA fragment guided by the Tth Ago enzyme on the mutant end and the non-mutant end. After the cleavage product is treated with nuclease I (Exonuclease I), constant temperature fluorescence quantitative PCR amplification is performed;

[0069] (4) Obtain the real-time fluorescence curve of the reaction system during the reaction process to determine whether there is a single-base mutation in the target gene sample to be tested: if the real-time fluorescence curve of the control sample (wild type) obtained during the reaction process tends to be stable over time (extended) or its Ct value is lower than the Ct value of the test sample, and the real-time fluorescence curve of the test sample obtained during the reaction process has an exponential amplification process over time (the mutant gene is successfully cut and effectively amplified), it indicates that the test sample has a single-base mutation; if the real-time fluorescence curve of the test sample obtained during the reaction process changes with time and is consistent with that of the control sample, it indicates that the test sample does not have a single-base mutation.

[0070] Example 1: Detecting single-base mutations in target genes using the method of the present invention

[0071] (1) The mutant type and wild type of the double-stranded target sequence used in this embodiment are both 99 bp in length. The mutant gene sequence forms a single base mismatch at position 22 relative to the wild type gene sequence. The wild type has a T at position 22, while the mutant type has a G at position 22. The 5' end T base of the four gDNAs used (gDNA-1, gDNA-2, gDNA-3, and gDNA-4) is modified with a phosphate group. The lengths are all 18 nt. gDNA-1 and gDNA-2 form complementarity at the 5' end of the mutant gene sequence target gene. The mismatch site is located at the 10th position at the 5' end of the complementary fragment formed by the gDNA and the wild type gene of the target gene. gDNA-3 and gDNA-4 form complementarity at the 3' end of the target gene. In this way, the Tth Ago enzyme can cut both ends of the mutant sequence of the target gene, but can only cut one end of the wild type sequence. The gDNA used was synthesized at Shanghai Shenggong Biotechnology Co., Ltd., and the double-stranded target sequence used was synthesized at Huzhou Hippo Biotechnology Co., Ltd. The sequence is as follows:

[0072] Wild type-Forward:

[0073]

[0074] Wild Type-Reverse:

[0075]

[0076] Mutation-Forward:

[0077]

[0078] Mutant-Reverse:

[0079]

[0080] Note: The above bold and underlined black fonts are mutation sites.

[0081] Mutation end gDNA sequence:

[0082] gDNA-1:

[0083] gDNA-2:

[0084] Non-mutated end gDNA sequence:

[0085] gDNA-3: 5'-p TGGCTGGAATCCGAGTTA-3';

[0086] gDNA-4: 5'-pTAATAACTCGGATTCCAG-3'.

[0087] Note: The black bold and underlined characters above are the mismatch sites between gDNA and wild-type double-stranded DNA.

[0088] (2) The Tth Ago enzyme used in this example was purchased from NEB, and the 10× Tth Ago Buffer used was prepared by: containing 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% (v / v) Triton X-100, and 50 mM MnCl2.

[0089] (3) adding a wild-type target sequence and a mutant target sequence sample containing the target gene to a solution containing Tth Ago enzyme and gDNA to form a reaction system, and performing shearing using Tth Ago enzyme; wherein,

[0090] The first reaction system is:

[0091] <![CDATA[H2O]]> 25 μl gDNA-3 (5 μM) 2.5 μl 10×Tth Ago Buffer 5μl gDNA-4 (5 μM) 2.5 μl gDNA-1 (5 μM) 2.5 μl Tth Ago enzyme (1 μM) 5μl gDNA-2 (5 μM) 2.5 μl Target sequence (1 μM) 5μl

[0092] The specific steps are:

[0093] ① Incubate equal amounts of Tth Ago enzyme (1.25 μl each) with four gDNAs (2.5 μl each) in 1.25 μl of 10× Tth Ago Buffer for 15–30 minutes. After incubation, mix the two gDNAs. A control without Tth Ago enzyme was used.

[0094] ② After mixing, add the mutant and wild-type target sequences to the system respectively, then add H2O to make a 50μl system and react at 85℃ for 40 minutes;

[0095] ③After the reaction is complete, stain with SYBR Green and then perform electrophoresis using 10% PAGE gel.

[0096] ④ Electrophoresis results are as follows Figure 2 As shown in Figure A, the target sequence used in the experimental system in lanes 1 and 2 is a mutant, and the target sequence used in lanes 3 and 4 is a wild-type. Lanes 1 and 3 represent the experimental group containing Tth Ago enzyme, and lanes 2 and 4 represent the control group without Tth Ago enzyme. Comparing lanes 1 and 3, it can be seen that the mutant gene is cleaved at both ends by the Tth Ago enzyme, producing a 60bp long intermediate fragment (the cleavage product also contains two nucleic acid fragments of 21bp and 18bp in length), while the wild-type gene is cleaved at one end by the Tth Ago enzyme, producing an 81bp cleavage product (the cleavage product also contains an 18bp long nucleic acid fragment).

[0097] (4) The 60 bp fragment generated after target sequence shearing was used as a template for designing isothermal amplification primers. The sheared product was treated with exonuclease I and then subjected to isothermal fluorescence quantitative PCR amplification. The primers used were 21 nt and synthesized at Shanghai Sangon Biotechnology Co., Ltd. The sequences are as follows:

[0098] Primer-1: 5'-CGAGTTATTATTTGATGTGTC-3';

[0099] Primer-2: 5'-GGCCCCTGTCTTGCTGTCATG-3'.

[0100] The Bst 2.0 warmstart DNA polymerase used was purchased from NEB, the 10× Bst DNA polymerase Buffer used was provided by NEB, and the nuclease I and 10× nuclease I Buffer used were purchased from Biolabs.

[0101] The second reaction system is:

[0102] <![CDATA[H2O]]> 2.8 μl Primer-2 (5 μM) 1 μl Primer-1 (5 μM) 1 μl template 1 μl 10×bst DNA polymerase Buffer 2 μl Bst 2.0 warmstart DNA polymerase 1 μl dNTP (10mM) 1.6 μl Betaine (5M) 7.2 μl Polyethylene glycol 200 (PEG 200) 0.2 μl <![CDATA[Mg 2+ (100mM)]]> 1.2 μl 10× Eva Green (nucleic acid dye for PCR) 1 μl

[0103] The specific steps are:

[0104] ① Take 10ul of the first reaction system (template concentration is 100nM), add 2ul of nuclease I, 2ul of 10× nuclease I buffer and 6ul of H2O, place in a PCR instrument and incubate at 37℃ for 30 minutes, then heat inactivate at 80℃ for 10 minutes.

[0105] ② Dilute the system from step ① (template concentration is 50 nM) so that the final concentrations of the template mixed with other reagents in the second reaction system are: 100 fM, 10 fM, 1 fM, 100 aM, and 10 aM (NTC is the abbreviation for No target control, which is a control group without amplified template). Incubate the reaction system at 61°C in a fluorescent quantitative PCR instrument for 120 min.

[0106] ③ Obtain the real-time fluorescence curve of the second reaction system during the reaction process to determine whether there is a single base mutation in the target sequence in the sample to be tested.

[0107] ④Constant temperature quantitative PCR results are as follows Figure 2 As shown, Figure 2 B is the real-time fluorescence curve obtained by quantitative isothermal amplification of the wild-type gene sequence after Tth Ago enzyme cleavage with different template concentrations. It can be seen that the real-time fluorescence curves of the fragments of the wild-type gene sequence with different concentrations after Tth Ago enzyme cleavage tend to be stable over time, indicating that the wild-type gene of the target sequence is not cleaved at both ends, resulting in ineffective amplification; Figure 2 C shows the real-time fluorescence curves obtained by quantitative isothermal amplification of the mutant gene sequence after Tth Ago enzyme cleavage with different template concentrations. It can be seen that the real-time fluorescence curves of the five mutant gene sequences with different concentrations after Tth Ago enzyme cleavage all show an exponential amplification process over time, indicating that the mutant gene of the target sequence is successfully cleaved at both ends and effectively amplified. This method can effectively detect the presence of single-base mutations in samples with a sensitivity of up to 1×10 -17 M.

[0108] Example 2: Investigating the effect of single-base mismatch between gDNA and target gene on Tth Ago enzyme cleavage of wild-type and mutant target genes

[0109] (1) The mutant (ssDNA MT) and wild-type (ssDNA WT) target sequences used in this example are both single-stranded and 99 nt in length. The mutant gene sequence forms a single base mismatch at position 78 relative to the wild-type gene sequence. The wild-type has an A at position 78, while the mutant has a C at position 78. The 17 gDNAs (m1-m17) used have 5'-terminal T bases modified with phosphate groups. The lengths of the 17 gDNAs (m1-m17) are all 18 nt. The mismatch sites with the wild-type correspond to g1-g17, respectively. The gDNAs used were synthesized at Shanghai Sangon Biotechnology Co., Ltd., and the sequences are as follows:

[0110] Wild-type single-stranded DNA sequence:

[0111]

[0112] Mutant single-stranded DNA sequence:

[0113]

[0114] Note: The above bold and underlined black fonts are mutation sites.

[0115] gDNA sequence:

[0116] gDNA m1:

[0117] gDNA m2:

[0118] gDNA m3:

[0119] gDNA m4:

[0120] gDNA m5:

[0121] gDNA m6:

[0122] gDNA m7:

[0123] gDNA m8:

[0124] gDNA m9:

[0125] gDNA m10:

[0126] gDNA m11:

[0127] gDNA m12:

[0128] gDNA m13:

[0129] gDNA m14:

[0130] gDNA m15:

[0131] gDNA m16:

[0132] gDNA m17:

[0133] Note: The black bold and underlined characters above are the mismatch sites between gDNA and wild-type single-stranded DNA.

[0134] (2) The Tth Ago enzyme used in this example was purchased from NEB. The 10× Tth Ago Buffer used was prepared by: containing 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% (v / v) Triton X-100, and 50 mM MnCl2.

[0135] (3) Adding solutions of gDNA (gDNA m1-m17) with different mismatch sites to a solution containing Tth Ago enzyme, and then adding the wild-type single-stranded target sequence and mutant single-stranded target sequence test samples of the target gene to form a reaction system and using Tth Ago enzyme for shearing; wherein,

[0136] The reaction system is:

[0137]

[0138] The specific steps are:

[0139] ① First, incubate 3 μl of Tth Ago enzyme with 6 μl of gDNA from m1 to m17 in 3 μl of 10×Tth Ago buffer for 15 to 30 minutes, for a total of 17 systems. After incubation, divide each system into 2 equal parts;

[0140] ② Add mutant (ssDNA MT) and wild-type (ssDNA WT) single-stranded target sequences to the same two systems, then add H2O to form a 15μl system, and react at 85℃ for 40 minutes;

[0141] ③After the reaction is complete, stain with SYBR Green and then perform electrophoresis using 10% PAGE gel.

[0142] ④ After repeating the results three times, the cleavage percentages of wild-type and mutant templates by Tth Ago enzyme in the gDNA experimental groups with different mismatch sites were counted.

[0143] ⑤Statistical results such as Figure 3 As shown, when the mismatch sites between gDNA and the wild-type target gene were located at m9-m11 and m15 around the Tth Ago enzyme cleavage site (g10 / g11), better shearing discrimination was achieved; among them, the best result was achieved when the mismatch site was at m10.

[0144] Example 3: Adding metal ions to improve the cleavage efficiency of Tth Ago enzyme

[0145] (1) The mutant type target sequence and gDNA sequence used in this example are the same as those in Example 1.

[0146] (2) The Tth Ago enzyme used in this example was ordered from New England Biolabs. The 10× Tth Ago Buffer used was provided by New England Biolabs and contained 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , and 0.1% (v / v) Triton X-100.

[0147] (3) Add the target gene mutant sequence to the solution containing Tth Ago enzyme and gDNA to form a reaction system, and then add different metal divalent cations (Me 2+ ), cleaved using Tth Ago enzyme; wherein,

[0148] The reaction system is:

[0149]

[0150]

[0151] The specific steps are:

[0152] ① First, incubate equal amounts of Tth Ago enzyme (0.375 μl each) with four gDNAs (0.75 μl each) in 0.375 μl of 10× Tth Ago Buffer for 15-30 minutes. After incubation, mix them together and then add the mutant target sequence, for a total of 9 systems;

[0153] ② Add H2O (control group) and different metal ions (Ca 2+ 、Cu 2+ 、Zn 2+ Mg 2+ 、Ba 2+ 、Ni2+ 、Mn 2+ 、Co 2+ ), and then add H2O to form a 15μl system, and react at 85℃ for 40 minutes;

[0154] ③After the reaction is complete, stain with SYBR Green and then perform electrophoresis using 10% PAGE gel.

[0155] ④ Electrophoresis results are as follows Figure 4 As shown, Figure 4 In A, lanes 1-9 represent the addition of H2O (control group) and the addition of Ca alone. 2 + 、Cu 2+ 、Zn 2+ Mg 2+ 、Ba 2+ 、Ni 2+ 、Mn 2+ and Co 2+ The lane N is the control group without Tth Ago enzyme (No Tth Ago), and the lane T is the control group containing only the mutant target gene target sequence and no enzyme cleavage system. It can be clearly seen from the results that metal divalent cations must be added to enable Tth Ago enzyme to have cleavage activity, and it was found that only Mg 2+ and Mn 2+ Only then can the Tth Ago enzyme have cleavage activity. Figure 4 B is the experimental system with different concentrations of Mn added 2+ Lanes 1-7 were added with 0, 1mM, 2mM, 3mM, 4mM, 5mM, and 6mM Mn, respectively. 2+ Lane N is a control group without Tth Ago enzyme (No Tth Ago), and lane T is a control group containing only the mutant target gene target sequence and no enzyme digestion system; Figure 4 C represents the experimental system with different concentrations of Mg added. 2+ Lanes 1-7 were supplemented with 0, 4mM, 8mM, 12mM, 16mM, 20mM, and 24mM Mg, respectively. 2+ Lane N is the control group without Tth Ago enzyme (No Tth Ago), and lane T is the control group containing only the mutant target gene sequence and no enzyme digestion system. It can be observed that Mn 2+ The effect on Tth Ago enzyme cleavage activity is much better than Mg 2+ , and when 5mM Mn was added 2+ , the activity of TthAgo enzyme cleavage reaches the best.

[0156] Example 4: Changing the experimental temperature to improve the cleavage efficiency of Tth Ago enzyme

[0157] (1) The mutant type target sequence and gDNA sequence used in this example are the same as those in Example 1.

[0158] (2) The Tth Ago enzyme used in this example was purchased from NEB. The 10× Tth Ago Buffer used was prepared by: containing 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 50 mM MnCl2, and 0.1% (v / v) Triton X-100.

[0159] (3) adding a sample of the target gene mutant sequence to a solution containing Tth Ago enzyme and gDNA to form a reaction system, changing the temperature used in the experiment, and using Tth Ago enzyme for shearing; wherein,

[0160] Reaction system:

[0161]

[0162]

[0163] The specific steps are:

[0164] ① First, incubate equal amounts of Tth Ago enzyme (0.375 μl each) with four gDNAs (0.75 μl each) in 0.375 μl of 10× Tth Ago Buffer for 15-30 minutes. After incubation, mix them together.

[0165] ② After mixing, add the mutant target sequence and H2O to the system to form a 15 μl system. A total of 6 identical systems were then reacted at different temperatures (66°C, 71°C, 75°C, 80°C, 85°C, and 90°C) for 40 minutes.

[0166] ③After the reaction is complete, stain with SYBR Green and then perform electrophoresis using 10% PAGE gel.

[0167] ④ Electrophoresis results are as follows Figure 5 As shown: Figure 5 In the figure, the temperatures used in lanes 1-6 are 66°C, 71°C, 75°C, 80°C, 85°C and 90°C, respectively. Lane N is a control group without Tth Ago enzyme (No Tth Ago), and lane T is a control group containing only the mutant target gene target sequence and no enzyme cleavage system. It can be clearly seen from the results that when the experimental temperature is controlled at 80°C~85°C, the experimental effect is better; when the experimental temperature is controlled at 85°C, the experimental effect reaches the best.

[0168] Example 5: Adding PEG 200 to improve the efficiency of SEA isothermal amplification

[0169] (1) The Primer (Primer-1, Primer-2) sequences used in this example are the same as those in Example 1; the template is the double-stranded target gene mutant sequence in Example 1, which was optimized by mismatch site optimization (m10) in Example 2, and metal ion catalysis optimization (5 mM Mn in Example 3). 2+ ) and the final product after shearing at optimized shearing temperature (85° C.) in Example 4, the specific sequence is as follows:

[0170] Template sequence (60 bp):

[0171] Mutant splicing product-Forward:

[0172] 5'-GGCCCCTGTCTTGCTGTCATGAAATCAGCAAGAGAGGATGACACATCAAATAATAACTCG-3';

[0173] Mutant splicing product-Reverse:

[0174] 5'-CGAGTTATTATTTGATGTGTCATCCTCTCTTGCTGATTTCATGACAGCAAGACAGGGGCC-3';

[0175] (2) The Bst 2.0 warmstart DNA polymerase used in this example was purchased from New England Biolabs, the 10× bst DNA polymerase buffer used was purchased from New England Biolabs, and the PEG 200 used was purchased from Sangon Biotech Co., Ltd. and was of analytical grade.

[0176] Reaction system:

[0177] <![CDATA[H2O]]> 2.2 μl 10×bst DNA polymerase Buffer 2 μl dNTP (10mM) 1.6 μl Betaine (5M) 7.2 μl <![CDATA[Mg 2+ (100mM)]]> 1.2 μl Primer-1 (5 μM) 1 μl Primer-2 (5 μM) 1 μl Template (200aM) 1 μl Bst 2.0 warmstart DNA polymerase 1 μl 10×Eva Green 1 μl

[0178] After adding the above reagents, mix them thoroughly (take 5 times the amount of each reagent and then divide it into 5 equal parts), the total volume is 19.2μl. Then add PEG 200 according to the proportions in the table below:

[0179]

[0180] Specific experimental steps:

[0181] ① After adding the above reagents, transfer them to an eight-tube strip and place them in a real-time fluorescence PCR instrument at a constant temperature of 61°C for 125 minutes.

[0182] ②Observe its fluorescence quantitative curve and analyze the results.

[0183] ③The results of constant temperature amplification are Figure 6 As shown (NTC is the abbreviation of No Target control, which is a control group without amplification template): The results show that adding a certain concentration of PEG 200 in the amplification experiment can increase the efficiency of amplification, among which when 1% PEG 200 is added, the experimental effect is best.

[0184] Example 6: Adding betaine to improve the efficiency of SEA isothermal amplification

[0185] (1) The Primer (Primer-1, Primer-2) sequences used in this example are the same as those in Example 1; the template is the double-stranded target gene mutant sequence in Example 1, which was optimized by mismatch site optimization (m10) in Example 2, and metal ion catalysis optimization (5 mM Mn in Example 3). 2+ ) and the final product after shearing with optimized shearing temperature (85°C) in Example 4, the sequence is the same as that in Example 5.

[0186] (2) The Bst 2.0 warmstart DNA polymerase used in this example was purchased from New England Biolabs. The 10× Bst DNA polymerase buffer used was also purchased from New England Biolabs. The PEG 200 used was purchased from Sangon Biotech Co., Ltd. and was of analytical grade.

[0187] Reaction system:

[0188] <![CDATA[H2O]]> 1 μl 10×bst DNA polymerase Buffer 2 μl dNTP (10mM) 1.6 μl PEG 200 0.2 μl <![CDATA[Mg 2+ (100mM)]]> 1.2 μl Primer-1 (5 μM) 1 μl Primer-2 (5 μM) 1 μl Template (200aM) 1 μl Bst 2.0 warmstart DNA polymerase 1 μl 10×Eva Green 1 μl

[0189] After adding the above reagents, mix them evenly (take 5 times the amount of each reagent and then divide it into 5 equal parts), the system is 11μl; then add betaine to each of the 5 systems according to the proportions in the following table:

[0190] Experimental group 1 2 3 4 5 Betaine final concentration 0.45 M 0.9M 1.35M 1.8M 2.25M Betaine (5M) 1.8 μl 3.6 μl 5.4 μl 7.2 μl 9 μl <![CDATA[H2O]]> 7.2 μl 5.4 μl 3.6 μl 1.8 μl 0μl

[0191] Specific experimental steps:

[0192] ① After adding the above reagents, transfer them to an eight-tube strip and place them in a real-time fluorescence PCR instrument at a constant temperature of 61°C for 125 minutes.

[0193] ②Observe its fluorescence quantitative curve and analyze the results.

[0194] ③ Constant temperature quantitative results are obtained by Figure 7As shown (NTC is the abbreviation of No Target control, which is a control group without amplification template): It can be seen from the figure that betaine is an essential reagent for this amplification experiment. It is not good to add too much or too little. When the final concentration of the added betaine is 0.9-1.8M, the experimental effect is better; when the added betaine working concentration is 1.8M, the experimental effect is best.

[0195] It should be noted that the implementation cases described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the present specification and claims, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same function. Sequence Listing <110> South China Normal University <120> A method for detecting single-base mutations in genes using Tth Ago enzyme cleavage and its application <160> 28 <170> SIPOSequenceListing 1.0 <210> 1 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Wild type-Forward <400> 1 ttgttttgaa actcagtatg ctgcccctgt cttgctgtca tgaaatcagc aagagaggat 60 gacacatcaa ataataactc ggattccagc ccacattgg 99 <210> 2 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Wild Type-Reverse <400> 2 ccaatgtggg ctggaatccg agttatattatt tgatgtgtca tcctctcttg ctgatttcat 60 gacagcaaga caggggcagc atactgagtt tcaaaacaa 99 <210> 3 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Mutation-Forward <400> 3 ttgttttgaa actcagtatg cggcccctgt cttgctgtca tgaaatcagc aagagaggat 60 gacacatcaa ataataactc ggattccagc ccacattgg 99 <210> 4 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Mutant-Reverse <400> 4 ccaatgtggg ctggaatccg agttatattatt tgatgtgtca tcctctcttg ctgatttcat 60 gacagcaaga caggggccgc atactgagtt tcaaaacaa 99 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA-1 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 5 tgacaggggc cgcatact 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA-2 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 6 ttcagtatgc ggcccctg 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA-3 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 7 tggctggaat ccgagtta 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA-4 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 8 taataactcg gattccag 18 <210> 9 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primer-1 <400> 9 cgagttatta tttgatgtgt c 21 <210> 10 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primer-2 <400> 10 ggcccctgtc ttgctgtcat g 21 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m1 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 11 tggcccctgt cttgctgt 18 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m2 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 12 tcggcccctg tcttgctg 18 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m3 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 13 tgcggcccct gtcttgct 18 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m4 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 14 ttgcggcccc tgtcttgc 18 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m5 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 15 tatgcggccc ctgtcttg 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m6 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 16 ttatgcggcc cctgtctt 18 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m7 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 17 tgtatgcggc ccctgtct 18 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m8 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 18 tagtatgcgg cccctgtc 18 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m9 <220> <222> (1) <223> Base T is modified with a phosphate group <400> 19 tcagtatgcg gcccctgt 18 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA sequences <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 20 tctcagtatg cggcccct 18 <210> twenty one <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m12 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> twenty one tactcagtat gcggcccc 18 <210> twenty two <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m13 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> twenty two taactcagta tgcggccc 18 <210> twenty three <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m14 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> twenty three taaactcagt atgcggcc 18 <210> twenty four <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m15 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> twenty four tgaaactcag tatgcggc 18 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m16 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 25 ttgaaactca gtatgcgg 18 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gDNA m17 <220> <222> (1)..(1) <223> Base T is modified with a phosphate group <400> 26 tttgaaactc agtatgcg 18 <210> 27 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Mutant splicing product-Forward <400> 27 ggcccctgtc ttgctgtcat gaaatcagca agagaggatg acacatcaaa taataactcg 60 <210> 28 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Mutant splicing product-Reverse <400> 28 cgagttatta tttgatgtgt catcctctct tgctgatttc atgacagcaa gacaggggcc 60

Claims

1. A method for detecting single-base mutations in genes using Tth Ago enzyme cleavage for non-disease diagnosis purposes, It is characterized in that The steps include: (1) Design of 4 gDNAs According to the sequence of the target gene or DNA fragment, two 16-19 nt gDNAs were designed, one for recognizing the mutant end and the other for recognizing the non-mutant end, the first base of which was T at the 5' end and modified by a phosphate group; among them, except for the first base, the remaining sequence of the gDNA recognizing the mutant end was complementary to the mutant gene sequence of the target gene, and the mismatch site with the wild-type target gene or DNA fragment was located at the 9th, 10th, 11th or 15th position of the complementary sequence; except for the first base, the remaining sequence of the gDNA recognizing the non-mutant end was complementary to the sequence 45-65 bp upstream or downstream of the gDNA shearing site at the mutant end; the sequences of the four gDNAs were different; (2) Tth Ago enzyme cleavage Tth Ago enzyme, gDNA for recognizing the mutant end and gDNA for recognizing the non-mutant end in step (1) were respectively placed in a solution containing 1-6 mmol / LMn 2+ The mixture is incubated in a buffer system for 15 to 30 minutes, and then mixed and added to the sample to be tested containing the target gene or DNA fragment, and a shearing reaction is performed at 75°C to 85°C. At the same time, a wild-type gene containing the target gene or DNA fragment is used as a control sample to obtain an enzyme cleavage product; (3) Fluorescence quantitative PCR ① Design a pair of PCR isothermal amplification primers with a length of 20 to 25 nt and that meet the requirements of amplification from the top of both ends of the DNA fragment according to the 45 to 65 bp DNA fragment sequence between the two end cleavage sites of the target gene or DNA fragment guided by the Tth Ago enzyme on the mutant end and the non-mutant end; ② The enzyme cleavage products in step (2) are first treated with nuclease exonuclease, and then added to a reaction system containing a DNA polymerase with strand displacement activity, 1-4% by volume PEG 200 and 0.9-1.8 mol / L betaine, and amplified using the PCR isothermal amplification primers in step ① to obtain real-time fluorescence curves to determine whether there is a single base mutation in the sample to be tested; (4) Judgment If the real-time fluorescence curve of the control sample tends to be stable over time or its Ct value is lower than the Ct value of the sample to be tested, and the real-time fluorescence curve of the sample to be tested has an exponential amplification process as time goes on, it indicates that the sample to be tested has a single base mutation; if the real-time fluorescence curve of the sample to be tested changes with time and is consistent with that of the control sample, it indicates that the sample to be tested does not have a single base mutation.

2. The method according to claim 1, Features: The mismatch site between the gDNA identifying the mutant end and the wild-type target gene or DNA fragment described in step (1) is located at the 10th position of the complementary sequence between the gDNA and the target gene.

3. The method according to claim 1, Features: The molar ratio of the four gDNAs in the buffer system described in step (2) is 1:1:1:1, and the molar ratio of the total molar ratio of the four gDNAs to the TthAgo enzyme is 5 to 10:

1.

4. The method according to claim 1, Features: The exonuclease in step (3) is exonuclease I.

5. The method according to claim 1, Features: The Mn in the buffer system described in step (2) 2+ The concentration is 4-6 mmol / L; The temperature of the Tth Ago enzyme cleavage reaction in step (2) is 80°C to 85°C; The amount of PEG 200 added to the reaction system described in step (3) is 1% by volume; The concentration of betaine in the reaction system described in step (3) is 1.8 mol / L.

6. The method according to claim 5, Features: The Mn in the buffer system described in step (2) 2+ The concentration is 5 mmol / L; The temperature of the Tth Ago enzyme cleavage reaction in step (2) is 85°C.

7. The method according to claim 1, Features: The formula of the buffer system described in step (2) is: 2 mM Tris-HCl, 1 mM KCl, 1 mM (NH 4 ) 2 SO 4 , 0.01% (v / v) Triton X-100, 1~6 mmol / L Mn 2+ ; The formula of the reaction system described in step (3) is as follows: 1 μl of each forward and reverse primer in 5 μM PCR isothermal amplification primer; 1 μl of enzyme digestion product; 2 μl of 10×bst DNA polymerase buffer; 1.6 μl of 10 mM dNTP; 1 μl of Bst DNA polymerase; 100 mM Mg 2+ 1.2 μl; 10× Eva Green 1 μl; 5 M betaine 3.6~7.2 μl; polyethylene glycol 2000.2~0.8 μl; H 2 0 to make up to 20 μl.

8. The method according to claim 7, Features: The formula of the buffer system described in step (2) is: 2 mM Tris-HCl, 1 mM KCl, 1 mM (NH 4 ) 2 SO 4 , 0.01% (v / v) Triton X-100, 5 mM Mn 2+ ; The formula of the reaction system described in step (3) is as follows: 1 μl of each forward and reverse primer in 5 μM PCR isothermal amplification primer; 1 μl of enzyme digestion product; 2 μl of 10×bst DNA polymerase buffer; 1.6 μl of 10 mM dNTP; 1 μl of Bst DNA polymerase; 100 mM Mg 2+ 1.2 μl; 10× Eva Green 1 μl; 5 M betaine 7.2 μl; polyethylene glycol 200 0.2 μl; H 2 Make up to 20 μl with 0.1% HO.

9. Use of the method for detecting single-base mutation of a gene by utilizing Tth Ago enzyme cleavage as claimed in any one of claims 1 to 8 in detecting single-base mutation of a gene for purposes other than disease diagnosis.

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