CN114921529A

CN114921529A - Method for detecting single base mutation of gene by utilizing Tth Ago enzyme shearing and application thereof

Info

Publication number: CN114921529A
Application number: CN202210527142.8A
Authority: CN
Inventors: 常好才; 朱梦媛; 欧阳雯雯
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2022-05-16
Filing date: 2022-05-16
Publication date: 2022-08-19

Abstract

The invention discloses a method for detecting single base mutation of a gene by utilizing Tth Ago enzyme shearing and application thereof. The method comprises the following steps: (1) designing 2 gDNAs for identifying a mutation end and 2 gDNAs for identifying a non-mutation end according to a target gene or DNA sequence to be detected, wherein the gDNAs are 16-19 nt; (2) shearing a target gene or a DNA fragment by using 4 pieces of designed gDNA and Tth Ago enzyme; (3) designing a primer for constant temperature amplification aiming at a 45-65bp sequence between the Tth Ago enzyme two-end shearing sites on a target gene or a DNA fragment, and carrying out PCR constant temperature amplification; (4) and judging whether the single base mutation exists in the sample to be detected according to the Ct value of the sample to be detected relative to the wild sample in the constant-temperature amplification reaction process. The method has the advantages of simple steps, simple operation, high specificity and high sensitivity, and can be used for detecting low-abundance single base mutation.

Description

Method for detecting single base mutation of gene by using Tth Ago enzyme shearing and application thereof

Technical Field

The invention belongs to the technical field of gene mutation detection, and particularly relates to a method for detecting single base mutation of a gene by utilizing Tth Ago enzyme shearing and application thereof.

Background

A single nucleotide variation is a nucleotide change occurring at a specific position in DNA, and is widely present in the coding of genetic information of an organism, and is called a single nucleotide polymorphism when the frequency of a less frequently occurring allele is 1% or more. The research on point mutation is helpful for deeply knowing the genetic characteristics of higher organisms, explaining the phenotypic difference of individuals, revealing the normal functions of genes and proteins, the cause of variation and the response mechanism of different individuals to environmental change, and has important significance for functional genomics research and establishment of molecular markers. The detection of rare polymorphic alleles is increasingly important for the early diagnosis and monitoring of a wide variety of tumors, and therefore the detection of extremely rare variant alleles in complex mixtures of deoxyribonucleic acid molecules has attracted increasing attention, mainly with the aim of solving the problems associated with accurate single nucleotide resolution and simplifying multiplex detection techniques, in particular for the detection of cancer-associated deoxyribonucleic acid biomarkers in patients. With the development of genome sequencing work, the detection and screening of point mutation become the focus of attention of people, and the detection method is rapidly developed; in addition, when the change of the physicochemical properties of genes caused by point mutation is weak, the detection of point mutation becomes very difficult, which has prompted many studies and the establishment of new detection methods.

Restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) was the earliest method used for analyzing known point mutations. If the point mutation happens to occur on the recognition site of a certain restriction enzyme, so that the restriction enzyme cutting site is increased or disappeared, the DNA which is specifically amplified by utilizing PCR is digested by a certain restriction enzyme, and the restriction enzyme cutting product is separated by agarose gel electrophoresis, so that whether the point mutation exists can be determined. The detection has higher specificity and good repeatability, but is only suitable for mutation detection involving specific restriction enzyme recognition sites and polymorphism detection with mutation frequency of more than 1 percent; in addition, this method cannot determine which base is mutated. Over the past few years, new nucleic acid editing methods and polymerase chain reaction procedures have also been explored. However, these methods are unsatisfactory in terms of detection sensitivity, cost, time and simplicity. For example, conventional PCR, cannot detect single base mutations. The mutant amplification retardation system (ARMS) approach allows the inclusion of mutant base sequences in the primer sequence, allowing selective amplification of variant sequences, but not the determination or validation of mutant alleles. Other methods, such as blocker displacement amplification and low temperature co-amplification, require stringent requirements on the annealing temperature of the target. Restriction enzyme mediated selective polymerase chain reaction analysis allows for simultaneous amplification of mutant signals and suppression of wild type gene amplification, but is limited by the variety of thermostable restriction enzymes. The digital PCR method is the most advanced single base mutation analysis technology at present, can realize the detection when the single base mutation rate is 0.01 percent, has various advantages compared with the traditional method, and has the defect that the high cost of digital PCR equipment hinders the wide application of the method.

Endonucleases with sequence-specific cleavage capabilities are powerful tools for the recognition of nucleic acid targets and subsequent design of detection. In recent years, researchers have identified various endonucleases from archaea and bacteria that can be guided by small fragments of nucleic acids to cleave complementary double strands, where CRISPR-Cas has attracted considerable attention as a genome editing tool, but its application has been greatly limited because one of the reasons is its dependence on PAM recognition sites, which are not present in many of the reported biological gene sequences. Similar to CRISPR-Cas, the argonaute (ago) protein is also a small fragment nucleic acid-guided endonuclease. Unlike Cas nucleases, however, Ago nucleases do not require the presence of a recognition site for the PAM sequence and are therefore more versatile. Under appropriate conditions, cleavage of the target DNA or RNA by Ago nuclease is achieved, guided by a small fragment of nucleic acid (gDNA) complementary to the target DNA or RNA.

The structure of Ago enzyme from thermophilic bacteria (thermophiles, Tth) comprises six domains of N-terminal, L1, PAZ, L2, MID and PIWI, with catalytic tetrad DEDX (X is D, H or K) in the PIWI domain, capable of binding divalent metal ions involved in the cleavage catalysis, and when primed with gDNA, Tth Ago enzyme is capable of cleaving complementary targets between positions 10 and 11 (g10/g11) of gDNA. Although Mg is present ²⁺ The double-strand cleavage effect is not ideal as a catalyst for activating the activity of Tth Ago enzyme.

The development of the Polymerase Chain Reaction (PCR) provides a powerful tool for nucleic acid amplification, and minute amounts of nucleic acid targets can be amplified by PCR. However, PCR-based techniques mostly require electrically powered thermocycling equipment to repeatedly heat and cool, which limits their application in an environment outside the laboratory. The advent of isothermal nucleic acid amplification methods overcomes the limitations of traditional PCR and offers the potential for nucleic acid amplification without the need for thermocycling equipment. In addition, one of the future demands for bioinformatic analysis is the development of simple, sensitive and reliable isothermal nucleic acid amplification methods for the detection of new infectious diseases in remote or remote areas and developing countries. Strand exchange reaction (SEA) of nucleic acids is a key natural process for genetic homologous recombination, DNA replication and DNA repair in vivo. The interaction forces between each base pair of dsDNA are weak, allowing instantaneous opening, which can cause intermittent fragmentation of the base pairs at a certain temperature, resulting in single-stranded denatured bubbles of DNA. Due to this phenomenon of DNA, SEA can replicate DNA without the aid of a gene recombinase.

Aiming at the determination of the position of each single base mutation, the fixation (45-65 bp) of the length of the sheared nucleic acid fragment to be amplified and the requirement of isothermal amplification from the top ends of two ends of the sheared nucleic acid fragment determine the immobilization of a primer design sequence when the nucleic acid fragment is amplified, so that high non-specific amplification can be generated. Also, because of the high specificity of primer amplification required by SEA, it is necessary to find a suitable method to reduce non-specific amplification and increase the efficiency of SEA amplification.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a method for detecting single base mutation of a gene by utilizing Tth Ago enzyme shearing.

Another purpose of the invention is to provide an application of the method for detecting single base mutation of the gene by utilizing Tth Ago enzymatic cleavage.

The purpose of the invention is realized by the following technical scheme:

a method for detecting single base mutation of a gene by using Tth Ago enzymatic cleavage comprises the following steps:

(1) design 4 gDNAs

Respectively designing 2 gDNAs (guide DNAs) for identifying a mutation end with 16-19 nt and 2 gDNAs for identifying a non-mutation end with 16-19 nt according to a sequence of a target gene or DNA fragment to be detected; wherein, the first base of the 5' end of the gDNA for identifying the mutant end and the gDNA for identifying the non-mutant end is T, and the base T is modified by a phosphate group; the gDNA for identifying the mutant end is complementary with the mutant gene sequence of the target gene except the first phosphorylated T base, and forms single base mismatch with the wild type gene sequence of the target gene; the gDNA for identifying the non-mutation end is complementary with the sequence at the upstream or downstream 45-65bp of the gDNA shearing site of the mutation end except the first phosphorylated T basic group; the 4 gDNA sequences are different;

(2) tth Ago enzymatic cleavage

Respectively incubating the Tth Ago enzyme, the 2 gDNAs for identifying the mutant ends and the 2 gDNAs for identifying the non-mutant ends, which are designed in the step (1), in a buffer system for 15-30 minutes, mixing the two after incubation is finished, adding a sample to be tested containing a target gene or a DNA fragment, carrying out a Tth Ago enzyme shearing reaction at the temperature of 66-90 ℃, and reacting to obtain an enzyme digestion product of the sample to be tested and an enzyme digestion product of the control sample by taking a wild type gene containing the target gene or the DNA fragment as the control sample; wherein, the buffer system contains Mg ²⁺ And Mn ²⁺ A buffer system of at least one of (a);

(3) fluorescent quantitative PCR

Designing PCR constant temperature amplification primer

Designing a pair of PCR constant temperature amplification primers (forward primers and reverse primers) which have the length of 20-25 nt and can be amplified from the top ends of two ends of a 45-65bp DNA fragment according to a 45-65bp DNA fragment sequence (if single base mutation exists, the sequence is a cut enzyme digestion fragment with the length of 45-65 bp) of Tth Ago enzyme guided by a mutation end and a non-mutation end gDNA (deoxyribonucleic acid) between two end shearing sites on a target gene or a DNA fragment;

② PCR constant temperature amplification

Performing exonuclease treatment on the enzyme digestion product of the sample to be detected and the enzyme digestion product of the reference sample obtained in the step (2), then respectively adding the enzyme digestion products into a reaction system containing DNA polymerase, PEG 200 and betaine, performing fluorescent quantitative PCR constant temperature amplification by using the PCR constant temperature amplification primer designed in the step (I), respectively obtaining a real-time fluorescent curve of the reaction system in the reaction process, and judging whether the sample to be detected of the target gene has single base mutation or not according to the real-time fluorescent curve;

(4) and (3) judging:

if the real-time fluorescence curve obtained by the control sample in the reaction process always tends to be stable along with the change (extension) of time or the Ct value of the real-time fluorescence curve is lower than the Ct value of the sample to be detected, and the real-time fluorescence curve obtained by the sample to be detected in the reaction process has an exponential amplification process along with the extension of time (the mutant gene is successfully sheared and effectively amplified), the single base mutation of the sample to be detected is indicated; if the real-time fluorescence curve obtained by the sample to be detected in the reaction process changes with time and is consistent with that of the control sample, the fact that the sample to be detected does not have single base mutation is shown.

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

The sequence of gDNA recognizing the mutant ends described in step (1) is as follows:

gDNA-1：

gDNA-2：

note: the black font is bold and underlined is the mismatch site of gDNA and wild-type double-stranded DNA.

The sequence of gDNA recognizing the non-mutated end described in step (1) is as follows:

gDNA-3：5'-p TGGCTGGAATCCGAGTTA-3'；

gDNA-4：5'-p TAATAACTCGGATTCCAG-3'。

the molar ratio of 4 pieces of gDNA in the buffer system in the step (2) is 1:1:1:1 (the concentration is the same), and the molar ratio of the total mole of the 4 pieces of gDNA to the mole of Tth Ago enzyme is more than 5: 1; preferably 5-10: 1; more preferably 10: 1.

Mg in the buffer system in the step (2) ²⁺ The concentration of (A) is 4-24 mmol/L; preferably 8-24 mmol/L; more preferably Mg ²⁺ The concentration of (3) is 20 mmol/L.

Mn in the buffer system described in step (2) ²⁺ The concentration of (a) is 1-6 mmol/L; preferably 4-6 mmol/L; more preferably 5 mmol/L.

The formula of the buffer system in the step (2) is as follows: 2mM Tris-HCl, 1mM KCl, 1mM (NH) ₄ ) ₂ SO ₄ 0.01% (v/v) Triton X-100, a certain concentration of metal ions; wherein the metal ion is 4-24 mmol/L Mg ²⁺ And 1 to 6mmol/L of Mn ²⁺ At least one of (1).

The formula of the buffer system in the step (2) is preferably as follows: 2mM Tris-HCl, 1mM KCl, 1mM (NH) ₄ ) ₂ SO ₄ ，0.01％(v/v)Triton X-100，5mM Mn ²⁺ 。

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

The temperature of the Tth Ago enzyme shearing reaction in the step (2) is preferably 75-85 ℃; further preferably 80 to 85 ℃; more preferably 85 deg.c.

The exonuclease in the step (3) is an enzyme with the activity of degrading single-stranded DNA, and preferably exonuclease I (exoclease I).

The DNA polymerase in the step (3) is a DNA polymerase with strand displacement activity (capability), the temperature for performing isothermal amplification reaction by using the DNA polymerase needs to be lower than the Tm value (melting temperature) of the 45-65bp DNA fragment cut in the step (2), namely, in the invention, a proper DNA polymerase is selected according to the Tm value of the 45-65bp DNA fragment cut in the step (2), and the temperature for isothermal amplification reaction is the optimal temperature of the DNA polymerase (the optimal temperature is known for each different DNA polymerase); the DNA polymerase is preferably Bst 2.0warmstart DNA polymerase, and the temperature range of isothermal amplification reaction of the Bst 2.0warmstart DNA polymerase is 37-65 ℃; preferably 58 to 65 ℃; more preferably 61 ℃.

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

Primer-1：5’-CGAGTTATTATTTGATGTGTC-3’；

Primer-2：5’-GGCCCCTGTCTTGCTGTCATG-3’。

the addition amount of PEG 200 in the reaction system in the step (3) is 1-4% by volume; preferably 1% by volume.

The concentration of betaine in the reaction system in the step (3) is 0.9-1.8 mol/L; preferably 1.8 mol/L.

The formula of the reaction system in the step (3) is as follows: 1 mul of 5 mul PCR constant temperature amplification primers (forward primer and reverse primer) respectively; 1 mul of enzyme digestion product (template); 10 × bst DNA polymerase Buffer 2 μ l; 1.6. mu.l of 10mM dNTP; bst DNA polymerase 1 u l; 100mM Mg ²⁺ 1.2 μ l; 10 × Eva Green (nucleic acid dye for PCR) 1 μ l; 3.6-7.2 mul of 5M betaine; 0.2-0.8 mul of polyethylene glycol 200(PEG 200); h ₂ Make up to 20. mu.l of O.

The formula of the reaction system described in the step (3) is preferably: mu.l of each 5 mu.M PCR constant-temperature amplification primer (forward primer and reverse primer); 1 mul of enzyme digestion product (template); 10 XBst DNA polymerase Buffer 2 μ l; 1.6. mu.l of 10mM dNTP; bst DNA polymerase 1 u l; 100mM Mg ²⁺ 1.2 μ l; 10 × Eva Green (nucleic acid dye for PCR) 1 μ l; 7.2. mu.l of 5M betaine; polyethylene glycol 200(PEG 200) 0.2. mu.l; h ₂ O make up to 20. mu.l.

The method for detecting the single base mutation of the gene by utilizing the Tth Ago enzyme shearing is applied to the in-vitro detection of the single base mutation of the gene.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention provides a method for detecting single base mutation of a gene by utilizing Tth Ago enzyme shearing, which comprises the following steps: 1) designing corresponding guide DNA aiming at the mutant gene sequence of the target gene; 2) shearing the mutant gene by using Thermus thermophilus argonaute (Tth Ago) enzyme; 3) designing primers for isothermal amplification according to sequences of Tth Ago enzymes guided by gDNA at a mutation end and a non-mutation end between two end shearing sites on a target gene or a DNA fragment;

4) fluorescence quantitative isothermal amplification; the method has simple steps, simple operation, high specificity and high sensitivity (the minimum detection limit of single base mutation is 1 × 10) ^-17 M), and the like, and can be used for low-abundance mutation detection.

(2) The method can quickly and accurately detect the single base mutant gene sequence and has high selectivity on the single base mutant gene sequence; in addition, the PCR isothermal amplification method changes the previous complicated PCR experimental steps, has simpler experimental conditions, does not need to use an electric thermal cycler in a laboratory, is easy to popularize to non-professional personnel for operation, has low detection cost, easy preparation and high repeatability, and can be applied to real-time detection of monitoring points and nursing points.

(3) The Ago enzyme from thermophilic bacteria (Tth) has the capability of efficiently cutting nucleic acid fragments, and the characteristic design method is utilized to identify single base mutation sites of target genes and cut the mutated target genes.

(4) In the invention, the property that dsDNA generates denatured bubbles at a single temperature is utilized, a short-fragment DNA primer is introduced to invade the denatured bubbles, and then the extension is obtained by virtue of the strand displacement activity and the polymerase activity of DNase; this process can be performed at a single temperature below the Tm of the dsDNA, thus allowing amplification without the need for conventional thermal denaturation processes.

(5) Experiments show that Mn with a certain concentration is added ²⁺ Catalyzing Tth Ago enzymeWhen the temperature is higher than that of Mg, the cleavage activity of the Tth Ago enzyme is greatly improved, and the optimum temperature of the Tth Ago enzyme is higher than that of Mg ²⁺ The temperature can be reduced by 5 ℃ during catalysis, and the range is 75-85 ℃.

(6) Experiments show that the non-specific amplification can be reduced and the amplification efficiency of SEA can be improved by simultaneously adding betaine and PEG 200.

Drawings

FIG. 1 is a schematic diagram of the method of the present invention for detecting single base mutations.

FIG. 2 is a graph showing the results of detecting single base mutations in example 1 using the method of the present invention; wherein A is the result of electrophoresis (lane 1: an experimental group containing Tth Ago, the target used is a mutant gene, lane 2: a control group not containing Tth Ago, the target used is a mutant gene, lane 3: an experimental group containing Tth Ago, the target used is a wild-type gene, lane 4: a control group not containing Tth Ago, the target used is a wild-type gene); b is a real-time fluorescence curve obtained by carrying out fluorescence quantitative isothermal amplification on templates with different concentrations of wild type gene sequences after being cut by Tth Ago enzyme; c is a real-time fluorescence curve obtained by carrying out fluorescence quantitative isothermal amplification on templates with different concentrations after the mutant gene sequences are sheared by Tth Ago enzyme.

FIG. 3 is a statistical chart showing the effect of different mismatch sites between gDNA and a Wild-type target gene on the cleavage efficiency of the Tth Ago enzyme in example 2 (in the figure, ssDNA MT is a mutant (Mutation type) single-stranded DNA; ssDNA WT is a Wild type (Wild type) single-stranded DNA).

FIG. 4 is a graph showing the results of optimizing metal ions for cleavage by the Tth Ago enzyme in example 3; wherein A is the result of electrophoresis (lanes 1-9 are sequentially added with H) ₂ O (control group) and Ca alone ²⁺ 、Cu ²⁺ 、Zn ²⁺ 、Mg ²⁺ 、Ba ²⁺ 、Ni ²⁺ 、Mn ²⁺ And Co ²⁺ A control group (No Tth Ago) containing No Tth Ago, and a control group (Lane T) containing only the target sequence of the mutant target gene and No enzyme cleavage system); b is Mn with different concentrations added in an experimental system ²⁺ (lanes 1 to 7, each containing 0, 1mM, 2mM, 3mM, 4mM, 5mM, and 6mM of Mn ²⁺ Lane N is a control group (No Tth Ago) containing No Tth Ago enzyme, and lane T is a control group containing only the target sequence of the mutant target gene and containing No enzyme digestion system; c is Mg with different concentrations added into an experimental system ²⁺ (lanes 1 to 7, each containing 0, 4mM, 8mM, 12mM, 16mM, 20mM, and 24mM of Mg) ²⁺ Lane N is a control group containing No Tth Ago enzyme (No Tth Ago), and lane T is a control group containing only the target sequence of the mutant target gene and containing No enzyme cleavage system).

FIG. 5 is a graph showing the optimization results of the temperature used for the cleavage by the Tth Ago enzyme in example 4.

FIG. 6 is a graph showing the optimized results regarding the concentration of PEG 200 in the isothermal amplification of SEA in example 5.

FIG. 7 is a graph showing the results of the optimization with respect to betaine concentration in the isothermal amplification of SEA in example 6.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise indicated, reagents and starting materials for use in the invention may be commercially or conventionally prepared.

The method for detecting single base mutation with high specificity and sensitivity is obtained by combining the shearing function of the Tth Ago enzyme and PCR (polymerase chain reaction) isothermal amplification. FIG. 1 is a schematic diagram of the method of the present invention for detecting single base mutation of a gene. The specific principle is as follows: the gDNA binding to the Tth Ago enzyme according to the present application contains a 5 '-terminal phosphate group, the gDNA (excluding the first phosphorylated T base) is completely complementary to a mutant gene sequence of a target gene, the complementary fragment is 15 to 18nt (nt-nucleotide), a single-base mismatch is formed with a wild-type gene sequence of the target gene, and the mismatch site is located at the 9 th, 10 th, 11 th or 15 th position of the 5' -terminal in 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 be combined with a target gene sequence and cuts a DNA fragment containing single base mutation, and the mutant end corresponding to the wild type gene sequence of the target gene cannot be effectively cut by the Tth Ago enzyme; at the upstream or downstream 45-65bp of a single base mutation end site cut by the Tth Ago enzyme, the wild type and the mutant type of the target gene can be cut by the Tth Ago enzyme guided by non-mutant end gDNA, only the mutant type target gene can be cut into a nucleic acid with the length of 45-65bp, and the nucleic acid of the fragment is amplified at the constant temperature to obtain a real-time fluorescence curve in the reaction process so as to judge whether the target gene has the single base mutation in a sample to be detected.

Specifically, the invention relates to a method for detecting single base mutation, which comprises the following steps:

(1) design of gDNA: the gDNA is a single-stranded oligonucleotide sequence, the length of the gDNA is 16-19 nt, the gDNA is designed according to the sequence of a target gene or a DNA fragment to be detected, the gDNA (except for the first phosphorylated T base) at a designed identification mutation end is complementary with a mutant gene sequence of the target gene, and forms single base mismatch with a wild-type gene sequence of the target gene; the gDNA (except the first phosphorylated T basic group) of the non-mutation end is designed to be complementary with the sequence at the upstream or downstream 45-65bp of the gDNA shearing site of the mutation end, so that the aim of only shearing one end of the wild-type gene by shearing two ends of the mutant gene is achieved by distinguishing single basic group mutation;

(2) adding a sample to be detected of a target gene into a solution containing the Tth Ago enzyme and the gDNA to form a reaction system, and shearing by using the Tth Ago enzyme; wherein the gDNA has 4 pieces, 2 pieces are gDNA for identifying a mutation end, and the other 2 pieces are gDNA for identifying a non-mutation end; the Tth Ago enzyme cleaves a complementary target corresponding between positions 10 and 11 of gDNA; in a reaction system, a mutant type of a target gene can be cut off by Tth Ago enzyme to form a nucleic acid fragment with the length of 45-65bp, but a wild type of the target gene cannot;

(3) designing a constant temperature amplification primer aiming at a DNA fragment of 45-65bp length between Tth Ago enzyme guided by gDNA at a mutation end and a non-mutation end on a target gene or a DNA fragment, and performing fluorescent quantitative PCR constant temperature amplification after a cleavage product is treated by exonuclease I (Exonaclease I);

(4) acquiring a real-time fluorescence curve of a reaction system in a reaction process, and judging whether a single base mutation exists in a sample to be detected of a target gene: if the real-time fluorescence curve obtained by the control sample (wild type) in the reaction process always tends to be stable along with the change (extension) of time or the Ct value of the real-time fluorescence curve is lower than the Ct value of the sample to be detected, and the real-time fluorescence curve obtained by the sample to be detected in the reaction process has an exponential amplification process along with the extension of time (the mutant gene is successfully sheared and effectively amplified), the fact that the sample to be detected has single base mutation is indicated; if the real-time fluorescence curve obtained by the sample to be detected in the reaction process changes with time and is consistent with that of the control sample, the fact that the sample to be detected does not have single base mutation is indicated.

Example 1: detection of Single base mutations in target genes Using the methods of the invention

(1) The length of the mutant type (Mutation type) and the Wild type (Wild type) of the double-stranded target sequence used in this example were both 99bp, the mutant type gene sequence formed a single base mismatch at position 22 relative to the Wild type gene sequence, the Wild type gene sequence was T at position 22, and the 22 nd position of the mutant type is G, the 5 'end T basic group of 4 gDNAs (gDNA-1, gDNA-2, gDNA-3 and gDNA-4) is modified by phosphate group, the length is 18nt, the gDNA-1 and gDNA-2 form complementation at the 5' end of the target gene of the mutant type gene sequence, the mismatch site is located at the 10 th position of the 5 'end in the complementary segment formed by gDNA and wild type gene of the target gene, the gDNA-3 and gDNA-4 form complementation at the 3' end of the target gene, thus, the Tth Ago enzyme can cleave both ends of the mutant sequence of the target gene, but can cleave only one end of the wild-type sequence. The gDNA used was synthesized by Shanghai Biotechnology Inc., and the double-stranded target sequence used was synthesized by Huzhou river horse Biotechnology, Inc., with the following sequence:

wild type-Forward:

wild type-Reverse:

mutant-Forward:

mutant-Reverse:

note: the black font is bold and underlined is the mutation site.

Mutant terminal gDNA sequence:

gDNA-1：

gDNA-2：

non-mutated terminal gDNA sequence:

gDNA-3：5'-p TGGCTGGAATCCGAGTTA-3'；

gDNA-4：5'-p TAATAACTCGGATTCCAG-3'。

note: the black font bold underlines the mismatch sites between gDNA and wild-type double-stranded DNA.

(2) The Tth Ago enzyme used in this example was purchased from NEB, and 10 × Tth Ago Buffer used was prepared by: containing 20mM Tris-HCl, 10mM KCl, 10mM (NH) ₄ ) ₂ SO ₄ ，0.1％(v/v)Triton X-100，50mM MnCl ₂ 。

(3) Adding a wild type target sequence containing a target gene and a mutant type target sequence to-be-detected sample into a solution containing the Tth Ago enzyme and the gDNA to form a reaction system, and shearing by using the Tth Ago enzyme; wherein the content of the first and second substances,

the first reaction system is:

H ₂ O	25μl	gDNA-3(5μM)	2.5μl
				10×Tth Ago Buffer	5μl	gDNA-4(5μM)	2.5μl
gDNA-1(5μM)	2.5μl	tth Ago enzyme (1. mu.M)	5μl
				gDNA-2(5μM)	2.5μl	Target sequence (1. mu.M)	5μl

The method comprises the following specific steps:

firstly, respectively incubating equal amounts of Tth Ago enzyme (1.25 mu l of each) and 4 pieces of gDNA (2.5 mu l of each) in 1.25 mu l of 10 XTth Ago Buffer for 15-30 minutes, and mixing the two together after incubation is finished; no Tth Ago enzyme is added as a control;

② after mixing, adding mutant and wild target sequence into the system respectively, then using H ₂ O is supplemented into a 50 mu l system and is reacted at a constant temperature of 85 DEG CThe time is 40 minutes;

③ after the reaction, SYBR Green staining is carried out, and then 10% PAGE gel is used for electrophoresis.

The electrophoresis result is shown in fig. 2A, in which the target sequences used in the experimental systems of

lanes

1 and 2 are mutant types, the target sequences used in

lanes

3 and 4 are wild types,

lanes

1 and 3 are experimental groups containing Tth Ago enzyme, and

lanes

2 and 4 are control groups not containing Tth Ago enzyme. As can be seen from comparison of

lanes

1 and 3, the mutant type gene was cleaved at both ends by the Tth Ago enzyme to give a cleavage product of an intermediate fragment 60bp long (the cleavage product also has two nucleic acid fragments 21bp and 18bp long), and the wild type gene was cleaved at one end by the Tth Ago enzyme to give a cleavage product of 81bp long (the cleavage product also has a nucleic acid fragment 18bp long).

(4) Using a 60bp long fragment generated after the target sequence is cut as a template, designing a constant temperature amplification primer for the fragment, and carrying out fluorescent quantitative PCR constant temperature amplification after the cut product is treated by exonuclease I (Exonuclease I); the primer used is 21nt, synthesized by Shanghai Biotechnology company, and has the following sequence:

Primer-1：5’-CGAGTTATTATTTGATGTGTC-3’；

Primer-2：5’-GGCCCCTGTCTTGCTGTCATG-3’。

bst 2.0warmstart DNA polymerase used was from NEB, 10 XBst DNA polymerase Buffer was from NEB, and exonuclease I and 10 exonuclease I Buffer were both from Poolebo.

The second reaction system is:

H ₂ O	2.8μl	Primer-2(5μM)	1μl
				Primer-1(5μM)	1μl	form panel	1μl
10 XBst DNA polymerase Buffer	2μl	Bst 2.0warmstart DNA polymerase	1μl
				dNTP(10mM)	1.6μl	Betaine (5M)	7.2μl
Polyethylene glycol 200(PEG 200)	0.2μl	Mg ²⁺ (100mM)	1.2μl
				10 × Eva Green (nucleic acid dye for PCR)	1μl

The method comprises the following specific steps:

taking 10ul of the first reaction system (the concentration of the template is 100nM), adding 2ul of exonuclease I, 2ul of 10 Xexonuclease I Buffer and 6ul of H ₂ O, placing the mixture in a PCR instrument to incubate for 30 minutes at a constant temperature of 37 ℃, and then thermally inactivating the mixture for 10 minutes at 80 ℃.

And secondly, diluting the system (the template concentration is 50nM) in the step I, so that the final concentrations of the template and other reagents mixed in the second reaction system are respectively as follows: 100fM, 10fM, 1fM, 100aM and 10aM (NTC is an abbreviation for No target control, and is a control group without an amplification template), and the reaction system was reacted in a fluorescent quantitative PCR instrument at an isothermal temperature of 61 ℃ for 120 minutes.

And thirdly, acquiring a real-time fluorescence curve of the second reaction system in the reaction process, and judging whether the target sequence has single base mutation in the sample to be detected.

The result of the isothermal quantitative PCR is shown in FIG. 2, FIG. 2B is a real-time fluorescence curve obtained by performing fluorescent quantitative isothermal amplification on templates with different concentrations of a wild-type gene sequence subjected to Tth Ago enzyme shearing, and it can be seen that the real-time fluorescence curves of 5 wild-type gene sequences with different concentrations subjected to Tth Ago enzyme shearing in the graph always tend to be stable along with the extension of time, which indicates that the wild-type gene of a target sequence is not sheared at two ends to cause ineffective amplification; fig. 2C is a real-time fluorescence curve obtained by performing fluorescent quantitative isothermal amplification on templates with different concentrations of the mutant gene sequence after being cut by the Tth Ago enzyme, and it can be seen that the real-time fluorescence curves of 5 mutant gene sequences with different concentrations after being cut by the Tth Ago enzyme in the graph have an exponential amplification process along with the time extension, which indicates that the mutant gene of the target sequence is successfully subjected to both-end cutting and effective amplification. The method can effectively detect whether single base mutation exists in the sample, and the sensitivity can reach 1 multiplied by 10 ^-17 M。

Example 2: the influence of the single base mismatching position of the gDNA and the target gene on the wild type and the mutant type of the Tth Ago enzyme-cut target gene is researched

(1) In this example, the target sequence of both mutant (ssDNA MT) and wild type (ssDNA WT) were single-stranded and had a length of 99nt, the mutant gene sequence formed a single-base mismatch at position 78 with respect to the wild type gene sequence, the wild type gene sequence was A at position 78, the mutant gene sequence was C at position 78, the 5' -end T base of 17 gDNAs (m1-m17) used was modified with a phosphate group and had a length of 18nt, and the mismatch sites formed with the wild type were g1-g17, respectively, and the gDNAs used were synthesized by Shanghai Biotech having the following sequences:

wild-type single-stranded DNA sequence:

mutant single-stranded DNA sequence:

note: the black font is bold and underlined to indicate the mutation site.

The gDNA sequence:

gDNA m1：

gDNA m2：

gDNA m3：

gDNA m4：

gDNA m5：

gDNA m6：

gDNA m7：

gDNA m8：

gDNA m9：

gDNA m10：

gDNA m11：

gDNA m12：

gDNA m13：

gDNA m14：

gDNA m15：

gDNA m16：

gDNA m17：

note: the black font is bold and underlined is the mismatch site of gDNA and wild-type single-stranded DNA.

(3) Adding gDNA (gDNA m1-m17) solutions with different mismatch sites into a solution containing the Tth Ago enzyme respectively, then adding a wild single-stranded target sequence of a target gene and a mutant single-stranded target sequence to-be-detected sample, and forming a reaction system to cut by using the Tth Ago enzyme; wherein the content of the first and second substances,

the reaction system is as follows:

the method comprises the following specific steps:

firstly, respectively incubating 3 mu l of Tth Ago enzyme and 6 mu l m1-m17 gDNA in 3 mu l of 10 XTth Ago buffer for 15-30 minutes, wherein 17 parts of the system are formed, and each part of the system is averagely divided into 2 parts after incubation is completed;

② adding mutant (ssDNA MT) and wild type (ssDNA WT) single-stranded target sequence into the same two systems respectively, and adding H respectively ₂ Forming a 15-microliter system by O, and reacting for 40 minutes at the constant temperature of 85 ℃;

Fourthly, after repeating the results for three times, the shearing percentage of the Tth Ago enzyme to the wild type template and the mutant type template in the gDNA experiment group with different mismatching sites is counted.

The statistical result is shown in figure 3, when the mismatch site of gDNA and the wild target gene is positioned at m9-m11 and m15 around the Tth Ago enzyme cleavage site (g10/g11), better shearing discrimination is generated; among these, the mismatch site at m10 gave the best results.

Example 3: increasing the shear efficiency of Tth Ago enzymes by adding metal ions

(1) The mutant (Mutation type) target sequence and gDNA sequence used in this example were the same as those in example 1.

(2) The Tth Ago enzyme used in this example was ordered from NEB, and 10 XTth Ago Buffer was supplied from NEB, containing 20mM Tris-HCl, 10mM KCl, 10mM (NH) ₄ ) ₂ SO ₄ ，0.1％(v/v)Triton X-100。

(3) Adding a sample to be detected of a target gene mutant type target sequence into a solution containing Tth Ago enzyme and gDNA to form a reaction system, and adding different metal divalent cations (Me) with the same concentration ²⁺ ) Cutting by using Tth Ago enzyme; wherein the content of the first and second substances,

the reaction system is as follows:

the method comprises the following specific steps:

firstly, respectively incubating equal amounts of Tth Ago enzyme (0.375 mu l each) and 4 gDNAs (0.75 mu l each) in 0.375 mu l of 10 XTth Ago Buffer for 15-30 minutes, mixing the incubated products together after incubation is finished, and then adding a mutant type target sequence into the mixed product to obtain 9 parts of system;

② adding H into 9 parts of system respectively ₂ O (control group) and different metal ions (Ca) ²⁺ 、Cu ²⁺ 、Zn ²⁺ 、Mg ²⁺ 、Ba ²⁺ 、Ni ²⁺ 、Mn ²⁺ 、Co ²⁺ ) Then supplementing H ₂ O respectively forms a system of 15 mul and reacts for 40 minutes at the constant temperature of 85 ℃;

③ after the reaction, staining with SYBR Green, and then performing electrophoresis with 10% PAGE gel.

The electrophoresis result is shown in FIG. 4, in FIG. 4A, lanes 1-9 are sequentially added with H ₂ O (control group) and Ca alone ² ⁺ 、Cu ²⁺ 、Zn ²⁺ 、Mg ²⁺ 、Ba ²⁺ 、Ni ²⁺ 、Mn ²⁺ And Co ²⁺ In the group, the control group (No Tth Ago) containing No Tth Ago was shown in the lane N, and the control group containing only the target sequence of the mutant target gene and No enzyme cleavage system was shown in the lane T, and it is obvious from the results that the Tth Ago had to be cleaved by adding a metal divalent cation, and only Mg was found ²⁺ And Mn ²⁺ The Tth Ago enzyme can be made active for cleavage. FIG. 4B shows the experimental system with different concentrations of Mn added ²⁺ Lanes 1 to 7, to which Mn was added at 0mM, 1mM, 2mM, 3mM, 4mM, 5mM, and 6mM, respectively ²⁺ Lane N is a control group (No Tth Ago) containing No Tth Ago enzyme, and lane T is a control group containing only the target sequence of the mutant target gene and containing No enzyme digestion system; FIG. 4C shows the experimental system with Mg added at different concentrations ²⁺ Lanes 1-7 with 0, 4mM, 8mM, 12mM, 16mM, 20mM, and 24mM Mg, respectively ²⁺ In the case of the control group containing No Tth Ago (No Tth Ago), in the case of lane N, and the control group containing only the target sequence of the mutant target gene and containing No enzyme digestion system, Mn was observed ²⁺ The effect on the shearing activity of the Tth Ago enzyme is far better than that of Mg ²⁺ And when 5mM Mn is added ²⁺ The activity of Tth Ago enzymatic cleavage was optimized.

Example 4: the temperature used in the experiment was varied to increase the shearing efficiency of the Tth Ago enzyme

(1) The mutant (Mutation type) target sequence and gDNA sequence used in this example were the same as in example 1.

(2) The Tth Ago enzyme used in this example was ordered from NEB and the 10 × Tth Ago Buffer used was prepared by: containing 20mM Tris-HCl, 10mM KCl, 10mM (NH) ₄ ) ₂ SO ₄ ，50mM MnCl ₂ ，0.1％(v/v)Triton X-100。

(3) Adding a sample to be detected of a target gene mutant type target sequence into a solution containing the Tth Ago enzyme and the gDNA to form a reaction system, changing the temperature used in the experiment, and shearing by using the Tth Ago enzyme; wherein, the first and the second end of the pipe are connected with each other,

reaction system:

the method comprises the following specific steps:

firstly, respectively incubating equivalent Tth Ago enzyme (0.375 mu l each) and 4 gDNAs (0.75 mu l each) in 0.375 mu l of 10 XTthago Buffer for 15-30 minutes, and mixing the two together after incubation is finished;

② adding mutant target sequence and H into the system after mixing ₂ O forms a system of 15 mu l, and 6 parts of the same system are reacted for 40 minutes at constant temperature at different temperatures (66 ℃, 71 ℃, 75 ℃, 80 ℃, 85 ℃ and 90 ℃);

FIG. 5 shows the results of electrophoresis: in fig. 5, the temperatures used in lanes 1 to 6 are 66 ℃, 71 ℃, 75 ℃, 80 ℃, 85 ℃ and 90 ℃, respectively, lane N is a control group (No Tth Ago) containing No Tth Ago enzyme, and lane T is a control group containing only the target sequence of the mutant target gene and containing No enzyme digestion system, and it is obvious from the results that the experimental effect is better when the experimental temperature is controlled to 80 ℃ to 85 ℃; when the experiment temperature is controlled at 85 ℃, the experiment effect is optimal.

Example 5: addition of PEG 200 to improve SEA isothermal amplification efficiency

(1) The sequences of primers (Primer-1, Primer-2) used in this example were the same as those used in example 1; the template was the double-stranded target gene mutant sequence of example 1 optimized by mismatch site of example 2 (m10), metal ion catalysis of example 3 (5mM Mn) ²⁺ ) And example 4 final product post-sheared with optimized shearing temperature (85 ℃), specific sequences are given below:

template sequence (60 bp):

mutant cleavage product-Forward:

5’-GGCCCCTGTCTTGCTGTCATGAAATCAGCAAGAGAGGATGACACATCAAATAATAACTCG-3’；

mutant cleavage product-Reverse:

5’-CGAGTTATTATTTGATGTGTCATCCTCTCTTGCTGATTTCATGACAGCAAGACAGGGGCC-3’；

(2) bst 2.0warmstart DNA polymerase used in this example was purchased from NEB, 10 XBstDNA polymerase Buffer was purchased from NEB, and PEG 200 was purchased from Biotechnology engineering, Inc. and was analyzed.

Reaction system:

H ₂ O	2.2μl	10 XBst DNA polymerase Buffer	2μl
				dNTP(10mM)	1.6μl	Betaine (5M)	7.2μl
Mg ²⁺ (100mM)	1.2μl	Primer-1(5μM)	1μl
				Primer-2(5μM)	1μl	Stencil (200aM)	1μl
Bst 2.0warmstart DNA polymerase	1μl	10×Eva Green	1μl

The above reagents were added and mixed (5 times the amount of each reagent was measured and then divided into 5 parts on average) to give a system of 19.2. mu.l. Then PEG 200 was added in the proportions shown in the following table:

the specific experimental steps are as follows:

adding the reagents, transferring the reagents into an eight-connected tube, and placing the eight-connected tube into a real-time fluorescent PCR instrument to react at a constant temperature of 61 ℃ for 125 minutes.

And observing the fluorescence quantitative curve and analyzing the result.

Results of isothermal amplification are shown in FIG. 6 (NTC is an abbreviation for No Target control, and is a control group without amplification template): from the results, it can be seen that the efficiency of amplification can be increased by adding a certain concentration of PEG 200 in the amplification experiment, wherein the experiment effect is the best when 1% of PEG 200 is added.

Example 6: increasing SEA isothermal amplification efficiency by adding betaine

(1) The sequences of primers (Primer-1, Primer-2) used in this example were the same as those used in example 1; the template was the double-stranded target gene mutant sequence of example 1 optimized by mismatch site of example 2 (m10) and metal ion catalysis of example 3 (5mM Mn) ²⁺ ) Example 4 final product of shear temperature optimization (85 ℃), sequence the same as example 5.

(2) Bst 2.0warmstart DNA polymerase used in this example was purchased from NEB, 10 XBst DNA polymerase Buffer was purchased from NEB, and PEG 200 was purchased from Biotechnology, Inc. and was analytically pure.

Reaction system:

H ₂ O	1μl	10 XBst DNA polymerase Buffer	2μl
				dNTP(10mM)	1.6μl	PEG	200	0.2μl
Mg ²⁺ (100mM)	1.2μl	Primer-1(5μM)	1μl
				Primer-2(5μM)	1μl	Stencil (200aM)	1μl
Bst 2.0warmstart DNA polymerase	1μl	10×Eva Green	1μl

Adding the reagents, and uniformly mixing (measuring each reagent by 5 times, and then averagely dividing into 5 parts) to obtain a system of 11 mu l; then, 5 parts of system are added with betaine according to the proportion in the following table:

experimental group	1	2	3	4	5
						Final concentration of betaine	0.45 M	0.9M	1.35M	1.8M	2.25M
Betaine (5M)	1.8μl	3.6μl	5.4μl	7.2μl	9μl
						H ₂ O	7.2μl	5.4μl	3.6μl	1.8μl	0μl

The specific experimental steps are as follows:

firstly, after the reagents are added, the mixture is transferred into an eight-connected tube and put into a real-time fluorescent PCR instrument to react at the constant temperature of 61 ℃ for 125 minutes.

And observing the fluorescence quantitative curve and analyzing the result.

The results of the constant temperature quantification are shown in FIG. 7 (NTC is an abbreviation for No Target control, and is a control group without an amplification template): according to the figure, betaine is an essential reagent for the amplification experiment, the addition amount is too high or too low, and the experiment effect is good when the final concentration of the added betaine is 0.9-1.8M; the experimental effect was best when the working concentration of betaine added was 1.8M.

It should be noted that the above-mentioned embodiments are only used for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the description and claims of the invention, and modifications can be made without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Sequence listing

<110> university of south China

<120> method for detecting single base mutation of gene by using Tth Ago enzyme shearing and application thereof

<160> 28

<170> SIPOSequenceListing 1.0

<210> 1

<211> 99

<212> DNA

<213> Artificial Sequence (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 (Artificial Sequence)

<220>

<223> wild type-Reverse

<400> 2

ccaatgtggg ctggaatccg agttattatt tgatgtgtca tcctctcttg ctgatttcat 60

gacagcaaga caggggcagc atactgagtt tcaaaacaa 99

<210> 3

<211> 99

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mutant-Forward

<400> 3

ttgttttgaa actcagtatg cggcccctgt cttgctgtca tgaaatcagc aagagaggat 60

gacacatcaa ataataactc ggattccagc ccacattgg 99

<210> 4

<211> 99

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mutant-Reverse

<400> 4

ccaatgtggg ctggaatccg agttattatt tgatgtgtca tcctctcttg ctgatttcat 60

gacagcaaga caggggccgc atactgagtt tcaaaacaa 99

<210> 5

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA-1

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 5

tgacaggggc cgcatact 18

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA-2

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 6

ttcagtatgc ggcccctg 18

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA-3

<220>

<222> (1)..(1)

<223> group T is modified with a phosphate group

<400> 7

tggctggaat ccgagtta 18

<210> 8

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA-4

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 8

taataactcg gattccag 18

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer-1

<400> 9

cgagttatta tttgatgtgt c 21

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer-2

<400> 10

ggcccctgtc ttgctgtcat g 21

<210> 11

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m1

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 11

tggcccctgt cttgctgt 18

<210> 12

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m2

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 12

tcggcccctg tcttgctg 18

<210> 13

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m3

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 13

tgcggcccct gtcttgct 18

<210> 14

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m4

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 14

ttgcggcccc tgtcttgc 18

<210> 15

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m5

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 15

tatgcggccc ctgtcttg 18

<210> 16

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m6

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 16

ttatgcggcc cctgtctt 18

<210> 17

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m7

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 17

tgtatgcggc ccctgtct 18

<210> 18

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m8

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 18

tagtatgcgg cccctgtc 18

<210> 19

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m9

<220>

<222> (1)

<223> modification of base T with phosphate group

<400> 19

tcagtatgcg gcccctgt 18

<210> 20

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA sequence

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 20

tctcagtatg cggcccct 18

<210> 21

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m12

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 21

tactcagtat gcggcccc 18

<210> 22

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m13

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 22

taactcagta tgcggccc 18

<210> 23

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m14

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 23

taaactcagt atgcggcc 18

<210> 24

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m15

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 24

tgaaactcag tatgcggc 18

<210> 25

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m16

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 25

ttgaaactca gtatgcgg 18

<210> 26

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> gDNA m17

<220>

<222> (1)..(1)

<223> modification of base T with phosphate group

<400> 26

tttgaaactc agtatgcg 18

<210> 27

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mutant cleavage product-Forward

<400> 27

ggcccctgtc ttgctgtcat gaaatcagca agagaggatg acacatcaaa taataactcg 60

<210> 28

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mutant cleavage product-Reverse

<400> 28

cgagttatta tttgatgtgt catcctctct tgctgatttc atgacagcaa gacaggggcc 60

Claims

1. A method for detecting single base mutation of a gene by utilizing Tth Ago enzyme shearing is characterized by comprising the following steps:

(1) design 4 gDNAs

Respectively designing 2 gDNAs (deoxyribonucleic acids) for identifying a mutation end with 16-19 nt and 2 gDNAs for identifying a non-mutation end with 16-19 nt according to a sequence of a target gene or DNA fragment to be detected; wherein, the first base of the 5' end of the gDNA for identifying the mutant end and the gDNA for identifying the non-mutant end is T, and the base T is modified by a phosphate group; the gDNA for identifying the mutant end is complementary with the mutant gene sequence of the target gene except the first phosphorylated T base, and forms single base mismatch with the wild type gene sequence of the target gene; the gDNA for identifying the non-mutation end is complementary with the sequence at the upstream or downstream 45-65bp of the gDNA shearing site of the mutation end except the first phosphorylated T basic group; the 4 gDNA sequences are different;

(2) tth Ago enzymatic cleavage

Respectively incubating the Tth Ago enzyme, the 2 gDNAs for identifying the mutant ends and the 2 gDNAs for identifying the non-mutant ends, which are designed in the step (1), in a buffer system for 15-30 minutes, mixing the two after incubation is finished, adding a sample to be tested containing a target gene or a DNA fragment, carrying out a Tth Ago enzyme shearing reaction at the temperature of 66-90 ℃, and reacting to obtain an enzyme digestion product of the sample to be tested and an enzyme digestion product of the control sample by taking a wild type gene containing the target gene or the DNA fragment as the control sample; wherein the buffer system contains Mg ²⁺ And Mn ²⁺ A buffer system of at least one of (a);

(3) fluorescent quantitative PCR

Firstly, designing PCR constant temperature amplification primer

Designing a pair of PCR constant-temperature amplification primers which have the length of 20-25 nt and meet the requirement of starting amplification from the top ends of two ends of a 45-65bp DNA fragment according to a 45-65bp DNA fragment sequence between two end shearing sites of a target gene or a DNA fragment of Tth Ago enzyme guided by gDNA at a mutation end and a non-mutation end;

② PCR constant temperature amplification

Treating the enzyme digestion product of the sample to be detected obtained in the step (2) and the enzyme digestion product of the control sample by exonuclease, then respectively adding the treated enzyme digestion products into a reaction system containing DNA polymerase, PEG 200 and betaine, performing fluorescent quantitative PCR constant temperature amplification by using the PCR constant temperature amplification primer designed in the step (i), respectively obtaining a real-time fluorescent curve of the reaction system in the reaction process, and judging whether the sample to be detected of the target gene has single base mutation or not according to the real-time fluorescent curve;

(4) and (3) judging:

if the real-time fluorescence curve obtained by the control sample in the reaction process always tends to be stable along with the change of time or the Ct value of the real-time fluorescence curve is lower than the Ct value of the sample to be detected, and the real-time fluorescence curve obtained by the sample to be detected in the reaction process has an exponential amplification process along with the extension of time, the single base mutation of the sample to be detected is indicated; if the real-time fluorescence curve obtained by the sample to be detected in the reaction process changes with time and is consistent with that of the control sample, the fact that the sample to be detected does not have single base mutation is shown.

2. The method for detecting a single base mutation in a gene using Tth Ago enzymatic cleavage according to claim 1, wherein:

the mismatch site of the gDNA for identifying the mutant end in the step (1) and the wild-type target gene or the DNA fragment is located at the 9 th, 10 th, 11 th or 15 th position of the complementary sequence of the gDNA and the target gene.

3. The method for detecting a single base mutation in a gene using Tth Ago enzymatic cleavage according to claim 2, wherein:

the mismatch site of gDNA of the mutation end and the wild-type target gene or DNA fragment is identified in the step (1) and is positioned at the 10 th position of the complementary sequence of the gDNA and the target gene.

4. The method for detecting a single base mutation in a gene using Tth Ago enzymatic cleavage according to claim 1, wherein:

the molar ratio of 4 pieces of gDNA in the buffer system in the step (2) is 1:1:1:1, and the molar ratio of the total mole of the 4 pieces of gDNA to the Tth Ago enzyme is 5-10: 1;

and (3) the DNA polymerase in the step (3) is DNA polymerase with strand displacement activity, and the temperature for carrying out isothermal amplification reaction by using the DNA polymerase is lower than the Tm value of the DNA fragment of 45-65bp sheared in the step (2).

5. The method for detecting a single base mutation in a gene using Tth Ago enzymatic cleavage according to claim 1, wherein:

mg in the buffer system in the step (2) ²⁺ The concentration of (A) is 4-24 mmol/L;

mn in the buffer system described in step (2) ²⁺ The concentration of (A) is 1-6 mmol/L;

the temperature of the Tth Ago enzyme shearing reaction in the step (2) is 75-85 ℃;

the exonuclease in the step (3) is exonuclease I;

the addition amount of PEG 200 in the reaction system in the step (3) is 1-4% by volume;

the concentration of the betaine in the reaction system in the step (3) is 0.9-1.8 mol/L.

6. The method for detecting a single base mutation in a gene using Tth Ago enzymatic cleavage according to claim 5, wherein:

mg in the buffer system in the step (2) ²⁺ The concentration of (A) is 8-24 mmol/L;

mn in the buffer system described in step (2) ²⁺ The concentration of (A) is 4-6 mmol/L;

the temperature of the Tth Ago enzyme shearing reaction in the step (2) is 80-85 ℃;

the addition amount of PEG 200 in the reaction system in the step (3) is 1 percent by volume;

the concentration of betaine in the reaction system in the step (3) is 1.8 mol/L.

7. The method for detecting a single base mutation in a gene by using Tth Ago enzymatic cleavage according to claim 6, wherein:

mg in the buffer system in the step (2) ²⁺ The concentration of (A) is 20 mmol/L;

mn in the buffer system described in step (2) ²⁺ The concentration of (A) is 5 mmol/L;

the temperature of the Tth Ago enzymatic cleavage reaction described in the step (2) was 85 ℃.

8. The method for detecting a single base mutation in a gene using Tth Ago enzymatic cleavage according to claim 1, wherein:

the formula of the buffer system in the step (2) is as follows: 2mM Tris-HCl, 1mM KCl, 1mM (NH) ₄ ) ₂ SO ₄ 0.01% (v/v) Triton X-100, a certain concentration of metal ions; wherein the metal ion is 4-24 mmol/L Mg ²⁺ And 1 to 6mmol/L of Mn ²⁺ At least one of;

the formula of the reaction system in the step (3) is as follows: 1 mul of each of forward and reverse primers in the 5 mul PCR constant temperature amplification primer; 1 mul of enzyme digestion product; 10 × bst DNA polymerase Buffer 2 μ l; 1.6. mu.l of 10mM dNTP; bst DNA polymerase 1 u l; 100mM Mg ²⁺ 1.2 μ l; 10 × Eva Green 1 μ l; 3.6-7.2 mul of 5M betaine; 2000.2-0.8 mu l of polyethylene glycol; h ₂ Make up to 20. mu.l of O.

9. The method for detecting a single base mutation in a gene using Tth Ago enzymatic cleavage according to claim 8, wherein:

the formula of the buffer system in the step (2) is as follows: 2mM Tris-HCl, 1mM KCl, 1mM (NH) ₄ ) ₂ SO ₄ ，0.01％(v/v)Triton X-100，5mM Mn ²⁺ ；

The formula of the reaction system in the step (3) is as follows: 1 mul of each of forward and reverse primers in the 5 mul PCR constant temperature amplification primer; 1 mul of enzyme digestion product; 10 XBst DNA polymerase Buffer 2 μ l; 1.6. mu.l of 10mM dNTP; bst DNA polymerase 1 u l; 100mM Mg ²⁺ 1.2 μ l; 10 × Eva Green 1 μ l; 7.2. mu.l of 5M betaine; polyethylene glycol 2000.2. mu.l; h ₂ O supplementThe volume was 20. mu.l.

10. The use of the method for detecting single base mutation of a gene by Tth Ago enzymatic cleavage as set forth in any one of claims 1 to 9 for detecting single base mutation of a gene in vitro.