Gene mutation detection method based on flap endonuclease 1 assisted ligase chain reaction

Abstract

The invention discloses a gene mutation detection method based on a flap endonuclease 1 assisted ligase chain reaction. The method is characterized in that the mutant DNA is efficiently amplified by using a flap endonuclease 1-assisted ligase chain reaction, and an amplification product is analyzed by an electrochemical end-point quantitative detection method or a real-time fluorescent quantitative method, so that the ultra-sensitive quantitative detection of the low-abundance mutant DNA is realized. According to the flap endonuclease 1 (FEN1)-assisted ligase chain reaction (LCR) provided by the invention, by virtue of the identification capability of the FEN1 on a rocking base pair, the problem of false positive easily occurring when the traditional LCR is used for detecting'conversion mutation 'is effectively solved, and the flap endonuclease 1 (FEN1)-assisted ligase chain reaction (LCR) has higher universality on gene mutation detection. In addition, according to the method, phosphorylation nicking is generated only through the enzyme digestion effect of FEN1 when the target exists, so that phosphorylation modification does not need to be performed on the probe in advance, the problem of non-specific amplification is effectively avoided, and the detection sensitivity is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of gene mutation detection, and in particular to a method based on flap endonuclease 1-assisted ligase chain reaction, by which mutant DNA is selectively amplified and combined with an electrochemical detection method or a real-time fluorescence detection method to achieve rapid and low-cost detection of ultra-low abundance gene mutations. Background Art

[0002] Single nucleotide polymorphism (SNP), also known as point mutation, is a valuable genetic marker for the diagnosis and prognosis of many diseases, and is also one of the important indicators for predicting drug resistance and drug efficacy. [1-2] Because the difference of a single nucleotide has a minimal effect on the stability of the DNA double helix and there is a large amount of interference from wild-type DNA in clinical samples, the detection of point mutations is more challenging than the detection of structural variations (such as fusion genes).

[0003] At present, the detection method of gene mutation used in clinical practice is mainly based on PCR amplification technology. [3-4] and sequencing technology [5-6] However, these methods are cumbersome to operate, the detection equipment is expensive, and professional technicians are required. At the same time, the detection performance of low-abundance point mutations is poor. As the role of gene mutations in personalized medicine becomes increasingly prominent, it is increasingly important to develop low-cost, rapid and simple, high-sensitivity point mutation analysis technologies that can be used as routine clinical tests. Ligase chain reaction (LCR) is a new DNA in vitro exponential amplification technology that emerged after PCR. Its amplification efficiency is comparable to that of PCR, and its sensitivity to point mutations is better than that of PCR. [7] , which can achieve quantitative detection of extremely low-abundance point mutations. When LCR is used for mutation detection, the upstream and downstream probes that are complementary to the target nucleic acid must first be designed. The downstream probe RP is completely complementary to the wild-type or mutant template, while the 3' end of the upstream probe FP is complementary to the point mutation site of the template and has a single base mismatch with the wild-type template. The sensitivity of DNA ligase to base mismatches is used to distinguish point mutations. T4 DNA ligase [8] and ampligase [9]These are the two most commonly used DNA ligases in analytical science. However, since T4 DNA ligase is not heat-resistant and can ligate blunt-ended DNA, it cannot be used in LCR. Amplidase is heat-resistant and very sensitive to single-base mismatches at the DNA nicking site, which has significant advantages in LCR applications. However, when LCR is used to detect "conversion mutations" (G>A), the 3' end of the upstream probe will form a GT wobble base pair with the wild-type template. Numerous crystal structure analysis studies have shown that [10-11] The stability of the wobble base pair GT is comparable to that of AT, resulting in a stronger tolerance of ampligase to GT mismatches.

[12] , so false positive results are prone to occur, which limits the versatility of LCR for mutation detection. In addition, since ampligase catalyzes the formation of phosphodiester bonds between the 5' phosphate and 3' hydroxyl groups at the DNA nicking site, the LCR probe must be 5' phosphorylated during synthesis, which not only increases the cost of probe modification, but also because ampligase has a weak "non-template-dependent" connection effect (i.e., the connection between the 5' phosphate and 3' hydroxyl groups between free probes), resulting in a certain degree of non-specific amplification in traditional LCR (i.e., the source of background signals), limiting its detection sensitivity. Therefore, there is an urgent need to effectively solve the problem of non-specific amplification of LCR and improve its versatility for gene mutation detection.

[0004] Thermostable flap endonuclease 1 (FEN1) is a structure-specific nuclease required for DNA replication and repair. It recognizes specific structures and is not dependent on sequences. It can catalyze the removal of the flanking sequence (flap) in the forked double-stranded DNA substrate to generate free flap sequences and DNA cuts with 5' phosphate ends. DNA ligase can recognize and connect the DNA cuts to produce complete double-stranded DNA.

[13] . Based on this feature, the present invention discloses a nucleic acid amplification method based on flap endonuclease 1 (FEN1) assisted ligase chain reaction (LCR). On the one hand, this method can effectively solve the false positive problem of detecting "conversion mutations" and has higher versatility for gene mutation detection; on the other hand, this method only produces phosphorylation cleavage through the enzymatic cleavage effect of FEN1 when the target is present, so there is no need to pre-phosphorylate the probe, thereby effectively avoiding the non-specific amplification problem of the above-mentioned traditional LCR, significantly inhibiting the generation of background signals, and thus improving the detection sensitivity. Finally, ultra-sensitive quantitative detection of low-abundance mutant DNA can be achieved through electrochemical endpoint quantitative detection or real-time fluorescence quantitative detection.

[0005] References

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[0024]

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[0025]

[13] Tsutakawa SE,Thompson MJ,Arvai AS,et al.Phosphate steering by Flap Endonuclease 1

[0026] promotes 5'-flap specificity and incision to prevent genome instability[J].Nature Communications,2017,8:15855. Summary of the Invention

[0027] 1. The purpose of the present invention is to provide a gene mutation detection method for non-disease diagnosis and treatment purposes. By introducing the design of FEN1 into the traditional LCR, the non-specific amplification problem of LCR is effectively solved and the versatility of mutation detection is improved.

[0028] 2. To achieve the purpose of the invention, the gene mutation detection method of the present invention has the following operating steps:

[0029] 1) determining a target DNA sequence and a mutation site, wherein the target DNA sequence includes a mutant DNA sequence and a wild-type DNA sequence; the mutation site is a site where a base difference between the mutant DNA sequence and the wild-type DNA sequence is located; designing a flap endonuclease 1-assisted ligase chain reaction for detecting the mutation site, wherein the ligase chain reaction has four probes, including an upstream probe FP, a downstream probe RP, an upstream complementary probe cFP and a downstream complementary probe cRP; the upstream probe FP is completely complementary to the target DNA sequence, and has a melting temperature Tm The value is controlled at 55°C to 65°C, the position of the first base at the 3' end of the upstream probe FP is designed to be on the mutation site; the downstream probe RP sequence comprises a hybridization sequence at the 3' end and a flanking sequence greater than or equal to 1 nt at the 5' end, the hybridization sequence is completely complementary to the target DNA sequence and the melting temperature Tm value of the hybridization sequence is controlled at 55°C to 65°C, and after hybridization with the target DNA sequence, the downstream probe RP can form a three-base overlap with the first base at the 3' end of the upstream probe FP at the mutation site, and the three-base overlap structure is ATT, TAA, CGG or GCC;

[0030] 2) preparing an amplification reaction system: mixing the four probes described in step 1), the flap endonuclease 1, ampligase DNA ligase and 1×ampligase buffer with the target DNA sequence to obtain an amplification reaction system;

[0031] 3) subjecting the amplification reaction system obtained in step 2) to temperature denaturation and thermal cycling, wherein the flap-shaped nuclease 1 recognizes the three-base overlapping structure, cuts and generates free flanking sequences and double-stranded DNA containing a 5' phosphate group at the nicking site, and the ampligase DNA ligase catalyzes the formation of a phosphodiester bond between the 5' phosphate group and the 3' hydroxyl group at the nicking site, thereby forming a large number of complete double-stranded DNA amplification products;

[0032] 4) Detecting the double-stranded DNA amplification product described in step 3).

[0033] The target DNA sequence described in the above step 1) is genomic DNA or cDNA. When the present method is used to analyze genomic DNA, a kit is required to extract and purify the genomic DNA in the genomic sample; when the present method is used to analyze cDNA, a kit is required to extract and purify the RNA in the RNA sample, and then reverse transcribe the RNA to obtain cDNA.

[0034] In the above step 1), preferably, the 5' end of the upstream probe FP is modified with biotin, and the 3' end of RP is modified with 6-carboxyfluorescein FAM.

[0035] In the above step 1), preferably, the 5' end of the upstream probe FP is modified with biotin, and the 3' end of RP is modified with 6-carboxyfluorescein FAM. The double-stranded DNA amplification product in step 4) is detected by electrochemical endpoint quantification method.

[0036] In the above step 4), preferably, the double-stranded DNA amplification product is detected by electrochemical endpoint quantification, and the steps of the electrochemical endpoint quantification method are: 1) using streptavidin-labeled magnetic beads as the fixed carrier of the double-stranded DNA amplification product; 2) using the immune reaction between FAM and anti-FAM antibody labeled horseradish peroxidase (anti-FAM-HRP) to obtain magnetic beads loaded with horseradish peroxidase (HRP); 3) adsorbing the magnetic beads loaded with HRP in step 2) on the surface of the glassy carbon electrode through the action of a magnetic field; 4) using HRP to catalyze H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB), and the generated oxidized TMB+ obtains electrons on the surface of the glassy carbon electrode to generate a reduction current, and the detection of the target DNA sequence is achieved by collecting the current signal. Preferably, step 4) uses the Ampere IT curve method to collect the current signal.

[0037] In the above step 1), preferably, the 3'-end flanking sequence of the downstream probe RP is modified with a fluorescent group and the 5'-end hybridization sequence is modified with a quenching group, and the fluorescent group and quenching group combination is selected from: FAM-BHQ1, TAMRA-BHQ2, VIC-MGB, CY5-Dabcyl, Texas Red-QSY7, Alexa Fluor 488-BHQ1, Quasar670-ZEN, HEX-NFQ or JOE-BHQ3.

[0038] In the above step 4), preferably, the double-stranded DNA amplification product is detected by real-time fluorescence quantitative method. Preferably, the real-time fluorescence quantitative method is detected by real-time fluorescence quantitative PCR instrument.

[0039] When the electrochemical endpoint quantitative detection method is used for detection, preferably, the thermal cycle parameters in the above step 2) are set as follows: 30 temperature cycles after pre-denaturation at 94°C for 5 minutes, wherein the temperature cycles are 60°C for 2 minutes and 94°C for 1 minute.

[0040] The specific operation of the electrochemical endpoint quantitative detection method is as follows: 1) using the specific affinity between streptavidin and biotin, the double-stranded DNA connection product in step 2) is efficiently captured using streptavidin-modified magnetic beads (MBs) to obtain FAM-dsDNA-MBs; the FAM-dsDNA-MBs in step 3) are reacted with anti-FAM antibody labeled-horse radish peroxidase (6-Carboxyfluorescein antibody labeled-horse radish peroxidase, anti-FAM-HRP) for incubation, and HRP is fixed on the surface of magnetic beads by antigen-antibody immune reaction to obtain HRP-FAM-dsDNA-MBs; the HRP-FAM-dsDNA-MBs in step 4) are adsorbed on the surface of glassy carbon electrode by magnetic field, and 3,3',5,5'-tetramethylbenzidine (TMB) is oxidized by hydrogen peroxide (H2O2) catalyzed by HRP, and the generated oxidized TMB+ obtains electrons at the electrode interface to generate reduction current, and the current signal is collected by the ampere IT curve method to realize the quantitative analysis of mutant DNA.

[0041] The real-time fluorescence quantitative method uses a real-time fluorescence quantitative PCR instrument for detection. When the real-time fluorescence quantitative method is used for detection, preferably, the temperature cycle parameters described in the above step 2) are 94°C pre-denaturation for 5 minutes; 94°C denaturation for 1 minute, 60°C enzyme digestion / ligation / fluorescence collection for 2 minutes, and 35 cycles.

[0042] The specific operation of the real-time fluorescence quantitative detection method is to mix the sample to be tested with the four reaction probes in step 1), add ampligase and FEN1, put it into the real-time fluorescence quantitative PCR instrument for thermal cycle amplification and fluorescence monitoring, and obtain a real-time fluorescence curve by setting the thermal cycle parameters and monitoring the fluorescence signal at the end of each amplification cycle. Preferably, data analysis is performed by cycle threshold (Ct) value to achieve quantitative detection of mutant DNA.

[0043] Compared with the traditional LCR, the FEN1-assisted LCR proposed in the present invention has obvious advantages, which are specifically described as follows:

[0044] When traditional LCR detects "conversion mutation" types (G>A), the 3' end of the upstream probe and the wild-type template will form a GT wobble base pair. Since the stability of the wobble base pair GT is equivalent to that of AT, the tolerance of ampligase to GT mismatch is high, and false positive results are prone to occur, limiting the versatility of LCR for mutation detection; the 3' and 5' ends of conventionally synthesized probes are both hydroxyl groups, and the substrates for ampligase-catalyzed ligation are the 5' phosphate and 3' hydroxyl groups at the DNA nicking site. Therefore, the LCR probe must be additionally 5' phosphorylated, which not only increases the cost, but also easily generates non-specific amplification background signals (weak non-template-dependent ligation effect of ampligase), resulting in low sensitivity of LCR; when real-time fluorescence quantification is used to monitor the amplification process of traditional LCR in real time, the temperature of fluorescence collection must be optimized, and the fluorescence background is easily unstable due to incomplete unwinding of the unreacted probe (short double-stranded DNA). The FEN1-assisted LCR disclosed in the present invention can effectively solve the shortcomings of the above-mentioned traditional LCR: this method can effectively solve the false positive problem of detecting "conversion mutations" by relying on the recognition ability of FEN1 for G:T wobble base pairs, thereby improving the versatility of gene mutation detection; this method only produces phosphorylation cleavage through the enzymatic cleavage effect of FEN1 when the target is present, so there is no need to pre-phosphorylate the probe, thereby effectively avoiding the non-specific amplification problem of the above-mentioned traditional LCR, significantly inhibiting the generation of background signals, and thereby improving the detection sensitivity; by modifying the fluorescent group and the quenching group on the probe, combined with the cutting effect of FEN1 during the amplification process, the fluorescent signal can be collected after each temperature cycle, without the need to optimize the signal collection temperature, and the background is lower. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the highly specific amplification of mutant target strands by flap endonuclease 1-assisted ligase chain reaction.

[0046] Figure 2 The time-current curves (A) and the linear relationship between the current signal and the logarithm of the target concentration (B) of FEN1-assisted LCR combined with electrochemical endpoint detection for analyzing mutant DNA at different concentrations. -17 M~1×10 -14 In the range of M, the corresponding current signal values ​​are respectively related to the logarithm of mutant DNA concentration lgC target(M) There is a good linear relationship.

[0047] Figure 3The current signal values ​​of mutant DNA (a), wild-type DNA (b), single-base mismatch DNA 1 (c) and single-base mismatch DNA 2 (d) of the same concentration were analyzed by FEN1-assisted LCR combined with electrochemical endpoint detection. As can be seen from the figure, the gene mutation detection method proposed in the present invention can effectively distinguish conversion mutations from other transversion mutations. The mutant DNA has a G>A conversion mutation with the wild-type DNA, a C>A transversion mutation with the single-base mismatch DNA 1, and a T>A transversion mutation with the single-base mismatch DNA 2.

[0048] Figure 4 The real-time fluorescence curves of FEN1-assisted LCR combined with real-time fluorescence quantitative analysis of mutant DNA (a), wild-type DNA (b), single-base mismatch DNA 1 (c) and single-base mismatch DNA 2 (d) of the same concentration are shown. As can be seen from the figure, the FEN1-assisted LCR proposed in the present invention can use real-time fluorescence quantitative method as a signal readout, and can effectively distinguish conversion mutations from other transversion mutations. The mutant DNA has a G>A conversion mutation with the wild-type DNA, a C>A transversion mutation with the single-base mismatch DNA 1, and a T>A transversion mutation with the single-base mismatch DNA 2.

[0049] Figure 5 This is a denaturing polyacrylamide gel electrophoresis diagram of the amplification products of traditional LCR and FEN1-assisted LCR in Example 3 under different ampligase enzyme amounts. DETAILED DESCRIPTION

[0050] The present invention will be further described below by specific embodiments in conjunction with the accompanying drawings. Those skilled in the art should understand that these embodiments are only used to illustrate the present invention and do not limit the scope of the present invention.

[0051] Example 1: Quantitative analysis of mutant DNA using flap endonuclease 1-assisted ligase chain reaction combined with electrochemical endpoint detection

[0052] In this example, a conversion mutation was selected as the detection object, and the conversion mutation was the sequence where the point mutation (T315I) at codon 315 of exon 6 of the ABL1 proto-oncogene was located.

[0053] Table 1. Sequences of nucleic acid chains used in this example

[0054]

[0055] The sequences of ABL1 T315I wild-type DNA and ABL1 T315I mutant DNA are shown in Table 1. To verify the universality of this method for other mutation types, two single-base mismatch DNAs were also designed, including single-base mismatch DNA (C>A) and single-base mismatch DNA (T>A). The T at the 11th and 25th positions in the downstream probe (RP) sequence in Table 1 are mutation sites; the A at the 26th position in the mutant DNA sequence is the mutation site; the G at the 26th position in the wild-type DNA sequence is the mutation site; the C at the 26th position in the single-base mismatch DNA1 (C>A) sequence is the mutation site; and the T at the 26th position in the single-base mismatch DNA2 (T>A) sequence is the mutation site.

[0056] Four probes for FEN1-assisted LCR were designed for the target DNA sequence, including the upstream probe FP, the downstream probe RP, the upstream complementary probe cFP, and the downstream complementary probe cRP. After hybridization with the mutant DNA sequence, FP and RP can form a three-base overlapping structure of A:T:T at the mutation site, and after hybridization with the wild-type DNA sequence, a mismatch structure of G:T:T is formed at the mutation site. In order to achieve exponential amplification, the complementary sequence cFP of the upstream probe FP (i.e., the upstream complementary probe cFP) and the complementary sequence cRP of the downstream probe RP (i.e., the downstream complementary probe cRP) were designed. The 5' end of FP was modified with biotin, and the 3' end of RP was modified with a FAM fluorescent group.

[0057] The specific implementation steps are as follows:

[0058] (1) The instruments and equipment used in the present invention are as follows:

[0059] Table 2. Instruments and equipment used in the present invention

[0060]

[0061]

[0062] Table 3. Reagents used in the present invention

[0063]

[0064] (2) FEN1-assisted LCR for specific amplification of mutant DNA: 5 μL FP (0.1 μM), 5 μL RP (0.1 μM), 5 μL cFP (0.1 μM), 5 μL cRP (0.1 μM), 0.4 μL ampligase (5 U / μL), 2.5 μL FEN1 (1.28 U / μL), 5 μL ampligase buffer (10×), 5 μL target gene (mutant DNA or wild-type DNA in Table 1) solution and 17.1 μL enzyme-free water were fully mixed and placed in a gene amplification instrument for amplification. The reaction conditions were: 61°C, 2 min-94°C 1 min, 30 cycles. After the reaction was completed, the amplified product was obtained and stored at 4°C for later use.

[0065] (3) Electrode pretreatment: A magnetic glassy carbon electrode (MGCE, 3 mm diameter) was polished on suede with a suspension of 0.05 μm Al2O3 powder for 3 min, and then ultrasonicated in anhydrous ethanol and ultrapure water for 1 min each to remove residual Al2O3 powder and other impurities.

[0066] (4) Gently shake the streptavidin-labeled magnetic beads (10 mg / ml) to disperse them evenly, and then take 0.4 μL into a 0.2 mL centrifuge tube.

[0067] (5) Add 100 μL of magnetic bead washing buffer (10 mM PB + 0.5 mM EDTA + 1 M NaCl + 0.01% Tween-20) to the centrifuge tube in step (4), vortex and mix well, then place on a small magnetic rack. After the magnetic beads aggregate on one side of the centrifuge tube, discard the magnetic bead washing buffer.

[0068] (6) After repeating step (5) twice, add 50 μL of the amplified product obtained in step (2) to resuspend the magnetic beads and vortex to mix, rotate and assemble at room temperature for 30 minutes, place the centrifuge tube on a small magnetic rack, and discard the reaction solution after the magnetic beads aggregate on one side of the centrifuge tube. Repeat step (5) three times to wash the magnetic beads to obtain the FAM-dsDNA-MBs complex, and store at 4°C for later use.

[0069] (7) Add 100 μL of 0.5 U / mL anti-6-carboxyfluorescein antibody-labeled horseradish peroxidase (anti-FAM-HRP) to resuspend the FAM-dsDNA-MBs complex obtained in step (6), and incubate with rotation at room temperature for 15 min. After magnetic separation, discard the reaction solution, repeat step (5) three times to wash the magnetic beads, and add 10 μL of PBS to resuspend to obtain HRP-FAM-dsDNA-MBs complex.

[0070] (8) Electrochemical detection: The HRP-FAM-dsDNA-MBs complex obtained in step (7) was dropped onto the surface of the magnetic glassy carbon electrode obtained in step (3), and the HRP-FAM-dsDNA-MBs complex was fixed on the electrode surface under the action of a magnetic field to obtain an MBs modified electrode. The measurement was performed using an electrochemical workstation with a three-electrode system, with the MBs modified electrode as the working electrode, Ag / AgCl as the reference electrode, and a platinum wire electrode as the auxiliary electrode. The measurement was performed using a time-current curve in a TMB (containing H2O2) solution, with a potential of 0.1 V and a scanning time of 100 s. The working curve was plotted based on the relationship between the stable current value obtained at the 100th s and the concentration of the target gene (mutant DNA or wild-type DNA in Table 1) standard solution.

[0071] Example 2: Quantitative analysis of mutant DNA using flap endonuclease 1-assisted ligase chain reaction combined with real-time fluorescence quantitative detection

[0072] When real-time fluorescence quantitative method is used to monitor the amplification process of traditional LCR in real time, the current main strategy is to use the difference in melting temperature (Tm) between the amplified product (long DNA double-strand) and the unreacted probe (short DNA double-strand) to collect the fluorescence signal generated by the double-stranded DNA-specific nucleic acid dye (such as SYBR Green I) combined with the amplified product (long double-stranded DNA) at a specific temperature. The disadvantage of this strategy is that the temperature of fluorescence collection must be optimized, and the fluorescence background is easily unstable due to incomplete melting of the unreacted probe (short double-stranded DNA). In this method, by modifying the fluorescent group and the quenching group on the probe, combined with the cleavage of FEN1 during the amplification process, the fluorescence signal can be collected after each temperature cycle, without the need to optimize the signal collection temperature, and the background is lower.

[0073] The wild-type DNA, mutant DNA, single-base mismatch DNA (C>A), single-base mismatch DNA (T>A) of ABL1 T315I used in this example are listed in Table 1. The fluorescent group FAM and the quenching group BHQ1 were modified on the 11th and 25th T bases in the RP sequence, respectively. The instruments and equipment used in this example are listed in Table 2, and the reagents used are listed in Table 3.

[0074] The specific implementation steps are as follows:

[0075] (1) Preparation of real-time fluorescence FEN1-assisted LCR reaction system: 2 μL FP (4 μM), 2 μL RP (4 μM), 2 μL cFP (4 μM), 2 μL cRP (4 μM), 0.4 μL ampligase (5 U / μL), 5 μL FEN1 (1.28 U / μL), 2 μL ampligase buffer (10×), 0.1 μL ROX reference dye, 2 μL test gene solution and 2.5 μL enzyme-free water were thoroughly mixed to obtain a reaction mixture.

[0076] (2) The reaction mixture in step (1) was placed in a Q5 fluorescence quantitative PCR instrument for PCR amplification and fluorescence signal collection. The program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, enzyme digestion / ligation / fluorescence collection at 60°C for 2 min, and 35 cycles.

[0077] (3) Performing data analysis on the real-time fluorescence curve obtained in step (2), including but not limited to performing data analysis by cycle threshold (Ct) value, to achieve quantitative detection of mutant DNA. The specific detection results are shown in Figure 4 , Figure 4 The real-time fluorescence curves of FEN1-assisted LCR combined with real-time fluorescence quantitative analysis of mutant DNA (a), wild-type DNA (b), single-base mismatch DNA 1 (c) and single-base mismatch DNA 2 (d) of the same concentration are shown. As can be seen from the figure, the FEN1-assisted LCR proposed in the present invention can use real-time fluorescence quantitative method as a signal readout, and can effectively distinguish conversion mutations from other transversion mutations. The mutant DNA has a G>A conversion mutation with the wild-type DNA, a C>A transversion mutation with the single-base mismatch DNA 1, and a T>A transversion mutation with the single-base mismatch DNA 2.

[0078] Example 3: Comparison of non-specific amplification between conventional LCR and FEN1-assisted LCR by denaturing polyacrylamide gel electrophoresis

[0079] In order to prove that the introduction of FEN1 design in LCR can effectively eliminate nonspecific amplification, we used traditional LCR and FEN1-assisted LCR to amplify mutant DNA, respectively. The mutant DNA sequence is shown in Table 1, and the amplified products were analyzed and compared by denaturing polyacrylamide gel electrophoresis.

[0080] The nucleic acid chain sequence used by FEN1-assisted LCR is shown in Table 1, and the nucleic acid chain sequence used by traditional LCR is shown in Table 4. Among them, T-RP and T-cFP must be 5' phosphorylated, and the 3' end of T-RP is modified with a FAM group.

[0081] Table 4. Nucleic acid chain sequences used in traditional LCR

[0082]

[0083] The specific steps are as follows:

[0084] (1) Preparation of FEN1-assisted LCR reaction products: 2 μL FP (1 μM), 2 μL RP (1 μM), 2 μL cFP (1 μM), 2 μL cRP (1 μM), 1 μL FEN1 (3.2 U / μL), 2 μL ampligase buffer (10×), 2 μL mutant DNA (1 nM) or enzyme-free water, and different volumes of ampligase (0.5 U / μL), add enzyme-free water to the total volume to 20 μL, mix well, and place in a gene thermal cycler for amplification. The reaction conditions are: 61°C, 2 min-94°C 1 min, 30 cycles. After the reaction is completed, store at 4°C for future use.

[0085] (2) Preparation of traditional LCR reaction products: 2 μL T-FP (1 μM), 2 μL T-RP (1 μM), 2 μL T-cFP (1 μM), 2 μL T-cRP (1 μM), 2 μL ampligase buffer (10×), 2 μL mutant DNA (1 nM) or enzyme-free water and different volumes of ampligase (0.5 U / μL), add enzyme-free water to the total volume to 20 μL, mix well and place in a gene amplification instrument for amplification, reaction conditions: 61°C, 2 min-94°C 1 min, 30 cycles. After the reaction is completed, store at 4°C for future use.

[0086] (3) Determine the DNA band distribution of the product solution in step (1) and step (2) using 15 wt% denaturing polyacrylamide gel electrophoresis.

[0087] The electrophoresis results are as follows Figure 5 As shown, lane 9 is RP or T-RP labeled with FAM as a band position control. Figure 5 B in the figure shows the product bands of traditional LCR under different ampligase dosages. The top band is the amplified product band. By comparing lanes 2, 4, 6, and 8, it can be seen that in the presence of mutant DNA, as the amount of ampligase enzyme gradually increases, the amount of amplified product also gradually increases. However, when no mutant DNA is added, gradually increasing amplified product bands are also observed in lanes 1, 3, 5, and 7, indicating that there is obvious nonspecific amplification in traditional LCR. Figure 5A in the figure shows the product bands of FEN1-assisted LCR under different ampligase dosages. By comparing lanes 2, 4, 6, and 8, it can be seen that as the amount of ampligase enzyme gradually increases, the amount of amplified product also gradually increases, and the amplification efficiency is comparable to that of traditional LCR. However, no nonspecific amplification bands were observed in lanes 1, 3, 5, and 7, indicating that the introduction of FEN1 design in LCR can effectively eliminate nonspecific amplification.

Claims

1. A method for detecting gene mutations for non-disease diagnosis and treatment purposes, characterized in that: The following steps are involved: 1) determining a target DNA sequence and a mutation site, wherein the target DNA sequence includes a mutant DNA sequence and a wild-type DNA sequence; the mutation site is a site where a different base between the mutant DNA sequence and the wild-type DNA sequence is located; A flap endonuclease 1-assisted ligase chain reaction is designed for detecting the mutation site, wherein the ligase chain reaction has four probes, including an upstream probe FP, a downstream probe RP, an upstream complementary probe cFP and a downstream complementary probe cRP; the upstream probe FP is completely complementary to the target DNA sequence, and the melting temperature Tm value is controlled at 55°C to 65°C, and the position of the first base at the 3' end of the upstream probe FP is designed to be on the mutation site; the downstream probe RP sequence comprises a hybridization sequence at the 3' end and a flanking sequence greater than or equal to 1 nt at the 5' end, the hybridization sequence is completely complementary to the target DNA sequence, and the melting temperature Tm value of the hybridization sequence is controlled at 55°C to 65°C, and after hybridization with the target DNA sequence, the downstream probe RP can form a three-base overlapping structure with the first base at the 3' end of the upstream probe FP at the mutation site, and the three-base overlapping structure is ATT, TAA, CGG or GCC; 2) preparing an amplification reaction system: mixing the four probes described in step 1), the flap endonuclease 1, ampligase DNA ligase and 1×ampligase buffer with the target DNA sequence to obtain an amplification reaction system; 3) subjecting the amplification reaction system obtained in step 2) to temperature denaturation and thermal cycling, wherein the flap-shaped nuclease 1 recognizes the three-base overlapping structure, cuts and generates free flanking sequences and double-stranded DNA containing a 5' phosphate group at the nicking site, and the ampligase DNA ligase catalyzes the formation of a phosphodiester bond between the 5' phosphate group and the 3' hydroxyl group at the nicking site, thereby forming a large number of complete double-stranded DNA amplification products; 4) Detecting the double-stranded DNA amplification product described in step 3).

2. The gene mutation detection method according to claim 1, characterized in that: The target DNA sequence described in step 1) is genomic DNA or cDNA.

3. The gene mutation detection method according to claim 1, characterized in that: The 5' end of the upstream probe FP in step 1) is modified with biotin, and the 3' end of RP is modified with 6-carboxyfluorescein FAM. The double-stranded DNA amplification product in step 4) is detected by electrochemical endpoint quantitative method.

4. The gene mutation detection method according to claim 3, characterized in that: The steps of the electrochemical endpoint quantitative method are: 1) using streptavidin-labeled magnetic beads as the fixed carrier of the double-stranded DNA amplification product; 2) using the immune reaction between FAM and anti-FAM-HRP to obtain magnetic beads loaded with HRP; 3) adsorbing the magnetic beads loaded with HRP in step 2) on the surface of the glassy carbon electrode through the action of a magnetic field; 4) HRP is used to catalyze H2O2 to oxidize TMB, and the generated oxidized TMB+ obtains electrons on the surface of the glassy carbon electrode to generate a reduction current. The target DNA sequence is detected by collecting the current signal.

5. The method for detecting gene mutation according to claim 4, characterized in that: Step 4) Use the Ampere IT curve method to collect current signals.

6. The gene mutation detection method according to claim 1, characterized in that: Step 1) The 3'-end flanking sequence of the downstream probe RP is modified with a fluorescent group and the 5'-end hybridization sequence is modified with a quenching group, and the fluorescent group and quenching group combination is selected from: FAM-BHQ1, TAMRA-BHQ2, VIC-MGB, CY5-Dabcyl, Texas Red-QSY7, Alexa Fluor488-BHQ1, Quasar 670-ZEN, HEX-NFQ, JOE-BHQ3; Step 4) The double-stranded DNA amplification product is detected by real-time fluorescence quantitative method.

7. The method for detecting gene mutation according to claim 6, characterized in that: The real-time fluorescence quantitative method uses a real-time fluorescence quantitative PCR instrument for detection.

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