A SNP detection method and product based on a nanopore constriction region

Abstract

The application provides a SNP detection method and product based on a nanopore constriction area, and belongs to the technical field of gene detection. The application constructs a nanomembrane clamp system, combines the unique structure of a glass nanopore, guides a double-stranded nucleic acid molecule containing a SNP site to a constriction area, uses the hydrogen bond or electrostatic interaction between a mismatched base and a lysine residue in the nanopore to cause characteristic ion current change, and thus realizes high-sensitivity detection with single-base resolution. The hydrogen bond or electrostatic interaction between a mismatched base and a positively charged lysine residue in the nanopore constriction area is used, when a nucleic acid molecule containing a SNP site passes through the nanopore, the mismatched base will destroy the stability of the DNA double helix structure, cause the conformation of the nucleic acid molecule to change in the constriction area, and then affect the path and efficiency of ions passing through the nanopore, cause characteristic current fluctuation, and realize single-base level resolution.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a method and product for detecting SNPs based on the contraction zone of nanopores. Background Technology

[0002] Single nucleotide polymorphisms (SNPs) are variations in a single nucleotide in the genome, and they hold core value in disease association studies, personalized medicine, and forensic individual identification. Current SNP mutation detection technologies (such as PCR-RFLP, gene chips, and sequencing) suffer from technical bottlenecks including operational complexity, high cost, low sensitivity, and insufficient specificity. While traditional sequencing technology is considered the "gold standard" for SNP detection, its expensive equipment, complex data processing, and poor sensitivity when detecting low-frequency mutations make it difficult to meet the needs of rapid clinical diagnosis and large-scale screening. These problems severely restrict the widespread application and in-depth development of SNP mutation detection.

[0003] Nanopore sensing technology is a single-molecule detection technique based on nanoscale pores (typically 1-100 nanometers in diameter). Its core principle is to identify and analyze target molecules by utilizing the changes in electrical, mechanical, or optical signals caused by molecules passing through the pores. In nanopatch-clamp devices, the nanopores serve as the sole channel between the two chambers of an electrolytic cell; when a molecule passes through the pore, it causes a momentary interruption of the ionic current. The amplitude and duration of the current change can reflect the size, conformation, or interactions of the molecule. In 2018, Cherie S. Tan's team first used γ-hemolysin nanopores (HlgC-HlgB) for double-stranded DNA structure analysis, successfully distinguishing the G→I base change. However, there is currently no existing technology to apply it to the detection of single nucleotide polymorphisms.

[0004] The use of nanopore sensing technology to detect SNP sites presents several challenges: (1) Due to the small amplitude of current changes caused by SNPs, the sensitivity of nanopore technology is far lower than that of conventional SNP detection methods such as digital PCR; (2) The DNA strand passes through the pore quickly and only briefly lingers in the contraction zone, making signal resolution difficult; (3) The specific interaction mechanism between mismatched bases and the contraction zone of the nanopore is not clearly understood, making it impossible to achieve high-precision SNP detection. Therefore, although nanopore technology has the potential for single-molecule detection, achieving high-precision single-base differentiation remains a technical bottleneck. Thus, providing a simple, low-cost, efficient, and accurate SNP detection method is crucial. Summary of the Invention

[0005] The purpose of this invention is to provide a SNP detection method and SNP detection product based on the nanopore contraction region, which can be used for SNP site detection. It has high detection efficiency, high sensitivity and good specificity. A single experiment can detect hundreds of thousands to millions of SNP sites. It is suitable for genome-wide association studies (GWAS) or population genetic analysis, and can also be used for SNP site detection for specific diseases (such as cancer, cardiovascular disease) or populations.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for SNP detection based on the contraction region of nanopores, comprising the following steps:

[0008] (1) Constructing a nanoporous patch clamp system: Solid glass nanopores are set between the cis-side electrolytic cell and the trans-side electrolytic cell of the nanoporous patch clamp device, and a phospholipid layer is attached to the glass nanopores to construct nanopores.

[0009] (2) SNP site detection: A first type of DNA with a poly(dA)30 tail and an extended sequence was designed for the target SNP site, and a second type of RNA with a complementary sequence to the first type of DNA was designed. The first type of DNA and the second type of RNA were added to the cis-side electrolytic cell, and a voltage was applied to detect the blocking current generated by the mismatched base pairs in the contraction region of the nanopore. The mismatched base pairs were identified based on the measured blocking current, so as to achieve qualitative and quantitative identification of SNP sites.

[0010] Preferably, in step (1), the nanopores are nanopores based on γ-hemolysin.

[0011] Preferably, in step (2), the design method for the first DNA of the target sequence includes:

[0012] A poly(dA)30 tail was designed at the 5' end of the ssDNA to guide the pore. The SNP site was placed at the base position immediately after the poly(dA)30 tail. Then, an extension sequence was designed to obtain the first type of DNA with the target sequence.

[0013] Preferably, in step (2), the conditions for designing the extended sequence are: the ratio of cytosine and guanine is controlled below 40% to prevent excessive ratios from leading to the formation of G-quadruplexes or i-motif structures, and to avoid situations where they can form complementary sequences or hairpin structures.

[0014] This invention also provides an SNP detection product prepared based on a SNP detection method for the contraction region of nanopores.

[0015] The beneficial effects of this invention compared to the prior art are as follows:

[0016] (1) The present invention provides a method for SNP detection based on the contraction region of nanopores, including: construction of nanopatch clamp device, formation of γ-hemolysin nanopores, nucleic acid sample hybridization, voltage application and current analysis.

[0017] (2) Based on the known SNP sites in the DNA single strand to be detected, this invention designs an RNA chain that is complementary to the sequence near the site. The RNA chain controls the position of the mismatched base pairs in the nanoprotein channel, dissolves the nucleic acid chain in water and forms a double-stranded molecule containing the complementary sequence. Combined with the unique structure of the nanopore (the diameter of the contraction region is about 2.6 nm), the double-stranded nucleic acid molecule containing the SNP site is guided to the contraction region. The hydrogen bond or electrostatic interaction between the mismatched bases and the lysine residues in the channel prolongs the residence time of the mismatched double strand in the contraction region and induces a characteristic ion current change, thereby achieving high-sensitivity detection with single-base resolution. By utilizing the hydrogen bond or electrostatic interaction between mismatched bases and positively charged lysine residues in the contraction region of nanopores, when nucleic acid molecules containing SNP sites pass through nanopores, the mismatched bases disrupt the stability of the DNA double helix structure, causing conformational changes in the contraction region of the nucleic acid molecule. This, in turn, affects the path and efficiency of ions passing through the nanopores, triggering characteristic current fluctuations, thereby enabling the precise identification of SNP site mutations. This directly solves the technical bottleneck of traditional technologies (such as gene chips) being unable to distinguish differences in SNP sites.

[0018] (3) The SNP detection method of this invention can directly identify the presence and type of SNP point mutations in nucleic acid molecules by measuring the blocking current characteristics passing through the nanopores, without the need for complex signal processing procedures, thus significantly shortening the detection time. Furthermore, the simple assembly method of the solid pores and phospholipid layer (such as the pneumatic impaction method) further reduces equipment costs. It avoids the use of expensive labeling reagents (such as fluorescent probes) and high-throughput sequencing equipment; the core cost is concentrated on γ-hemolysin protein and the electrochemical detection device, significantly reducing detection expenses. It has advantages such as label-free operation, high sensitivity, high accuracy, simple operation, and low cost, and can be widely used in fields such as genetic disease diagnosis and drug development, meeting the needs of rapid, precise single-molecule detection in genetic disease diagnosis and personalized medicine.

[0019] (4) The DNA-RNA hybridization of this invention forms an A-type double helix (approximately 2.3 nm in diameter) that is highly matched with the size of the contraction region (2.6 nm), ensuring that the mismatch site is precisely aligned with the detection area of ​​the contraction region and avoiding signal drift. It can also cover multiple SNP types (such as GG and CC mismatches) and distinguish different mutation types through current signal spectrum.

[0020] (5) This invention directly detects characteristic current changes at SNP sites through nanopores, eliminating the need for labeling steps such as fluorescent probes. Combined with automated signal analysis (such as QuB and Originlab software), SNP mutation analysis can be completed within hours, significantly shortening the detection cycle. Furthermore, a single probe can identify multiple SNP mutation points and can also perform quantitative identification (quantitative: the frequency of blocking current events is recorded using QUB software to generate a current peak distribution histogram. Peak diagrams are plotted using software such as Originlab, and the frequency of each SNP mutation is determined by combining the peak diagrams). Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a graph showing the detection results of GG mismatch sites (SNP sites) in the DNA-RNA hybridization strand in Example 1 of the present invention;

[0023] Figure 2 This is a diagram showing the detection results of CG pairing sites (normal base complementary pairing) in Example 1 of the present invention. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Glossary of technical terms related to this invention

[0030] 1. γ-hemolysin

[0031] Definition: An octamer β-barrel protein structure formed by the assembly of F-class polypeptides (such as HlgB) and S-class polypeptides (such as HlgC) secreted by Staphylococcus aureus, containing a vestibular region and a contractile region, with the inner diameter of the contractile region being approximately 2.6 nm.

[0032] Function: As a single-molecule detection channel, it interacts with nucleic acid molecules through its contraction region (narrow channel) to detect current changes induced by single nucleotide polymorphisms (SNPs).

[0033] 2. Constriction zone of nanopores

[0034] Definition: The connection region between the vestibular and β-barrel structures in the γ-hemolysin nanopore. The contraction region of the γ-hemolysin nanopore is any combination of amino acid sequences 104 to 112 and 141 to 152 of the F-type polypeptide shown in SEQ ID NO.1 and amino acid sequences 100 to 109 and 132 to 141 of the S-type polypeptide shown in SEQ ID NO.2.

[0035] Function: By forming hydrogen bonds or electrostatic interactions between positively charged lysine residues and mismatched bases (such as unpaired cytosine or adenine), DNA retention time is prolonged and characteristic current signals are generated.

[0036] 3. SNP (Single Nucleotide Polymorphism)

[0037] Definition: Variations in a single nucleotide (A, T, C, G) in the genome that result in differences in the DNA sequence.

[0038] Significance: SNP mutations are closely related to genetic diseases and drug responses, and detecting their sites is crucial for precision medicine.

[0039] 4. Mismatch base

[0040] Definition: Mismatched base pairs (such as GG, CC) in a double-stranded nucleic acid molecule, caused by SNP mutations.

[0041] Function: It disrupts the stability of the DNA double helix structure, triggers local conformational changes (such as "bulges"), and interacts with the contraction region of the nanopore to generate characteristic current signals.

[0042] 5. Nanopatch-clamp device

[0043] Definition: An electrochemical device for single-molecule detection, comprising a cis / trans detection cell, a solid pore (such as a glass nanopore), a phospholipid layer, and nanopore channels.

[0044] Function: By applying voltage to drive nucleic acid molecules through nanopores, changes in ion current are monitored in real time to analyze molecular characteristics.

[0045] 6. Glass nanopores

[0046] Definition: Nanoscale pores (e.g., 2 μm in diameter) made of glass, serving as the sole channel for electrolyte flow.

[0047] Function: To support the construction of phospholipid layers and nanopores, ensuring the stability of ion current detection.

[0048] 7. Phospholipid bilayer

[0049] Definition: A bilayer membrane structure formed by the self-assembly of lipid molecules such as diaphytylphosphatidylcholine (DPhPC) attached to the surface of a solid glass nanopore.

[0050] Function: It simulates the biomembrane environment, provides embedding sites for γ-hemolysin nanopores, isolates the direct connection between the two detection cells, and becomes the core component of detection.

[0051] 8. A-form duplex

[0052] Definition: A right-handed helical structure formed by the hybridization of RNA or PNA with DNA, which is shorter and larger in diameter (approximately 2.3 nm) than B-type DNA.

[0053] Function: Matches the size of the γ-hemolysin contraction zone to ensure that mismatched sites are accurately located in the detection area.

[0054] 9. Blockade current

[0055] Definition: When nucleic acid molecules pass through nanopores, they partially or completely block ion channels, resulting in a decrease in current amplitude (current blocking).

[0056] Significance: By analyzing the amplitude, duration, and other characteristics of the current signal, we can determine the molecular type and whether there are any mutations.

[0057] 10. Salt solution (e.g., 3M KCl buffer)

[0058] Definition: High-concentration salt solutions (2-4M KCl / NaCl) provide an ionicly conductive environment, and pH 5.0 HOAc / KOAc buffers optimize γ-hemolysin stability.

[0059] Function: Salt concentration and pH value affect the migration rate of nucleic acid molecules and the signal-to-noise ratio of current signals.

[0060] 11. Complementary sequences

[0061] Definition: A sequence that forms a double helix with a target nucleic acid (DNA or RNA) through base pairing, such as DNA-RNA, DNA-PNA, or RNA-DNA combinations.

[0062] Function: It converts SNP sites into mismatched bases, forms an A-type double helix structure through hybridization, and guides the mismatched sites into the nanopore shrinkage region.

[0063] 12. PNA (Peptide Nucleic Acid)

[0064] Definition: A DNA analogue that replaces the sugar-phosphate backbone with a neutral peptide backbone, and whose bases are complementary to DNA.

[0065] Advantages: It has a higher affinity for DNA when hybridizing and is not degraded by nucleases, making it suitable as a complementary sequence.

[0066] 13. Time Domain and Frequency Domain Characteristics

[0067] Software such as QuB, Matlab, and Originlab are used to analyze the amplitude, duration (time domain), and frequency characteristics (frequency domain) of the blocking current, distinguish different SNP types (such as GG mismatch and CC mismatch), and achieve high-precision determination of mutation type.

[0068] Related sequences of this invention:

[0069] HlgB-SEQ ID NO.1:

[0070] MNMNKLVKSSVATSMALLLLSNTANAEGKITPVSVKKVDDKVTLYKTTATADSDKFKISQILTFNFIKDKSYDKDTLVLKAAGNINSGYERPNPKDYDFSKIYWGAKYNVSISSQSNDSVNVVDYAPKNQNEEFQVQNTLGYTFGGDISISNGLSGGLNGNTA FSETINYKQESYRTTLSRNTNYKNVGWGVEAHKIMNNGWGPYGRDSFHPTYGNELFLAGRQSSAYAGQNFIAQHQMPLLSRSNFNPEFLSVLSHRQDGAKKSKITVTYQREMDLYQIRWNGFYWAGANYKNFKTRTFKSTYEIDWENHKVKLLDTKENENNK;

[0071] HlgC-SEQ ID NO.2:

[0072] MLKNKILATTLSVSLLAPLANPLLENAKAANDTEDIGKGNDVEIIKRTEDKTSNKWGVTQNIQFDFVKDKKYNKDALILKMQGFISSRTTYYNYKNTNHIKSMRWPFQYNIGLKTNDKYVSLINYLPKNKNKIESTNVSQTLGYNIGGNFQSAPSLGGNG SFNYSKSISYTQQNYVSEVEQQNSKSVLWGVKANSFATESGQKSAFDSDLFVGYKPHSKDPRDYFVPDSELPPLVQSGFNPSFIATVSHEKGSSDTSEFEITYGRNMDVTHAIKRSTHYGNSYLDGHRVHNAFKNRNYTVKYEVNWKTHEIKVKGQN.

[0073] This invention provides a method for SNP detection based on the contraction region of nanopores, comprising the following steps:

[0074] (1) Constructing a nanoporous patch clamp system: Solid glass nanopores are set between the cis-side electrolytic cell and the trans-side electrolytic cell of the nanoporous patch clamp device, and a phospholipid layer is attached to the glass nanopores to construct nanopores.

[0075] (2) SNP site detection: A first type of DNA with a poly(dA)30 tail and an extended sequence was designed for the target SNP site, and a second type of RNA with a complementary sequence to the first type of DNA was designed. The first type of DNA and the second type of RNA were added to the cis-side electrolytic cell, and a voltage was applied to detect the blocking current generated by the mismatched base pairs in the contraction region of the nanopore. The mismatched base pairs were identified based on the measured blocking current, so as to achieve qualitative and quantitative identification of SNP sites.

[0076] The steps for constructing nanopores on glass nanopores coated with a phospholipid layer are as follows:

[0077] 5 μg of γ-hemolysin protein was added to the cis-reaction cell, and a gas pressure of 20-40 Pa (80 mm H2O) and a voltage of 100-350 mV (trans vs cis) were applied to assist the γ-hemolysin protein in embedding into the phospholipid layer. The device was then scanned with positive and negative voltages. Under the influence of the applied gas pressure and voltage, γ-hemolysin nanopores were constructed on the phospholipid layer, allowing the salt solution between the cis-reaction cell and the trans-reaction cell to communicate. When the pressure increased to the point where an opening current appeared, and the signal disappeared after the gas pressure returned to zero, it proved that the γ-hemolysin nanopores had been formed on the phospholipid layer.

[0078] Example 1

[0079] Example 1 of this invention provides a highly sensitive and accurate SNP detection method based on the contraction region of γ-hemolysin nanopores. The specific steps are as follows:

[0080] (1) Construction of nanoporous patch-clamp system

[0081] Preparation of glass nanopores: The initial voltage was set to 30 mV (trans vs cis), the temperature was controlled at 20.0 ± 0.5 °C, and the electrical signal was 0 pA. A solid glass nanopore with a diameter of 2 μm was placed between the cis-side and trans-side electrolytic cells of the nanopatch clamp apparatus, connecting the two reaction cells. 3M potassium chloride solution (the solvent of the potassium chloride solution was 20 mM HOAc / KOAc buffer solution at pH 5.0, or any concentration of potassium chloride solution or sodium chloride solution between 2M and 4M) was injected into the cis-side and trans-side electrolytic cells respectively using a syringe until the liquid level covered the glass nanopore. The electrical signal disappeared. The liquid level was then aspirated below the height of the glass nanopore using a syringe, and the electrical signal reappeared. Repeating this process multiple times showed the same phenomenon, indicating that the glass nanopore was clean and unobstructed, and subsequent experiments could proceed.

[0082] Attaching a phospholipid layer to glass nanopores: Diphytylphosphatidylcholine (DPhPC) powder was dissolved in decane at a concentration of 10 mg / mL to form Solution 1. Then, Solution 1 was added dropwise to the salt solution in the cis-reaction cell to bring the final concentration of DPhPC to 0.0025 mg / mL. The liquid level was adjusted by repeated aspiration using a syringe to promote the passage and attachment of DPhPC through the glass nanopores. Regardless of whether the salt solution level covered the glass nanopores, a current signal of 0 pA was observed under the applied initial voltage, indicating that the phospholipid layer was successfully attached to the glass nanopores. Then apply a pressure of 100 Pa (or any value between 100-130 Pa). If the electrical signal suddenly disappears after applying a pressure within a certain range, it indicates that the phospholipid layer has been broken and is of appropriate thickness. If it is not broken, it is too thick, and the page position needs to be adjusted by suction, the phospholipid concentration needs to be adjusted, and the layers need to be reattached to block the glass nanopores between the cis and trans reaction cells. When the sampling rate is 10 kHz, the noise is less than 2 pA, and subsequent experiments can continue.

[0083] Preparation of γ-hemolysin protein: F-type polypeptide HlgB (SEQ ID NO.1) and S-type polypeptide HlgC (SEQ ID NO.2) were mixed with 100 mM deoxycholate (DOC) solution (HlgB / HlgC / DOC mass ratio 1:1:10) in 100 mM Tris-HCl buffer (pH 8.3) with gradual stirring until a final concentration of 6.25 mM DOC and 3.5 μM (a mixture of S-type polypeptide HlgC and F-type polypeptide HlgB) was obtained. The mixture was incubated at room temperature for two hours. Monomers were removed using a 100 kDa cutoff centrifuge filter (Amicon Ultra 100K device), and the purified γ-hemolysin protein octamer was obtained. The contraction region of the γ-hemolysin nanopore is formed by any combination of amino acid sequences 104 to 112 and 141 to 152 of the F-type polypeptide shown in SEQ ID NO.1 and amino acid sequences 100 to 109 and 132 to 141 of the S-type polypeptide shown in SEQ ID NO.2.

[0084] Constructing γ-hemolysin nanopores on a phospholipid layer: 5 μg of γ-hemolysin protein was added to the cis-reaction cell, and a pressure of 30 Pa and a voltage of 200 mV (trans vs cis) were applied to assist the γ-hemolysin protein in embedding into the phospholipid layer. The device was then scanned with positive and negative voltages. Under the influence of the applied pressure and voltage, γ-hemolysin nanopores were constructed on the phospholipid layer, allowing the salt solution between the cis-reaction cell and the trans-reaction cell to connect. When the pressure increased to the point where an opening current appeared, and the signal disappeared after the pressure returned to zero, it proved that the γ-hemolysin nanopores had formed on the phospholipid layer. In a 3M potassium chloride solution, an opening current of 750 ± 20 pA was obtained by applying a voltage of 120 mV (trans vs cis). Detection of the opening current of the γ-hemolysin nanopores showed that the γ-hemolysin nanopores had good stability, maintained an open state, and could support experiments for several hours.

[0085] (2) SNP site detection: A solid-phase synthesis method was used to target the SNP site. A poly(dA)30 tail was designed at the 5' end of the ssDNA to guide the flow through the well. The SNP site was placed at the base position immediately following the poly(dA)30 tail. An additional length (e.g., 23 bp) extension sequence was then designed (SEQ ID NO.3: 5'-(A)30-GCATCAGTAGAACTCAGAAACTC-3') to obtain the first DNA of the target sequence. During the design of the first DNA, it is important to control the ratio of cytosine and guanine below 40% to prevent the formation of G-quadruplexes or i-motif structures due to excessive ratios, and to avoid situations where they can form complementary sequences or hairpin structures. Next, using an RNA sequence of equal length (SEQ ID NO.4: 5'-GAGUUUCUGAGUUCUACUGAUGG, where the GG pairing at the end of the DNA-RNA complementary strand is a mismatch site different from base pairing, i.e., the SNP site to be detected) as the complementary nucleic acid strand to the first DNA sequence, the synthesized nucleic acid was purified using anion exchange HPLC for 30 minutes per sample, while monitoring the UV absorbance at 260 nm. The samples were dialyzed in ddH2O at 4°C for 36 hours to remove purification salts, and then lyophilized. The concentration of the first DNA or second RNA molecule resuspended in ddH2O was obtained by measuring the absorbance at 260 nm and estimating the extinction coefficient using primary sequence. The two can hybridize to form a type A DNA-RNA double helix structure, yielding an RNA strand including a poly(dA)30 tail, an extended sequence, and a complementary sequence.

[0086] A first type of DNA and a second type of RNA (concentration ratio 3:4) are added to a cis-side electrolytic cell to form complementary sequences in the solution, forming an A-type double helix structure. A voltage of 120mV (trans vs cis) is applied, and the blocking current generated by mismatched base pairs in the contraction region of the nanopores is detected. The mismatched base pairs are identified based on the measured blocking current, thus achieving qualitative and quantitative identification of SNP sites.

[0087] Signal Acquisition and Processing: The acquired blocking current signal was processed using QUB software to obtain time-domain and frequency-domain signal characteristics. MATLAB software was then used to further statistically analyze signal frequency, peak value, and other information. OriginLab software was used for data analysis and plotting. Based on the characteristic signal shape, degree of obstruction, and duration of the measured blocking current, different SNP sites can be distinguished. Peak plots based on the frequency of characteristic current occurrences can be used to count the occurrence frequency of SNP sites, ultimately achieving accurate qualitative and quantitative identification. The results are as follows: Figure 1 , 2 As shown.

[0088] Figure 1 , 2 The test results graph shows that the two have different degrees of current signal blocking (I). block There are obvious differences that can be distinguished by the naked eye, which can be used to determine the existence of a mismatch. Further statistical analysis can be performed on information such as the current value and duration of the current signal to confirm that the current signal is a mismatch signal, thus achieving accurate identification of the mismatch site.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A non-diagnostic SNP detection method based on the contraction region of nanopores, characterized in that, The detection method includes the following steps: (1) Constructing a nanoporous patch-clamp system: Solid glass nanopores are set between the cis-side electrolytic cell and the trans-side electrolytic cell of the nanoporous patch-clamp device, and a phospholipid layer is attached to the glass nanopores. Nanopores are constructed on the glass nanopores with the attached phospholipid layer. (2) SNP site detection: A first type of DNA with a poly(dA)30 tail and an extended sequence is designed for the target SNP site, and a second type of RNA with a complementary sequence corresponding to the extended sequence of the first type of DNA is designed. The first type of DNA and the second type of RNA are added to the cis-side electrolytic cell, a voltage is applied, and the blocking current generated by the mismatch base pairs in the contraction region of the nanopore is detected. The mismatch base pairs are identified based on the measured blocking current, so as to achieve qualitative and quantitative identification of SNP sites. In step (1), the nanopores are nanopores based on γ-hemolysin; In step (2), the design method for the first DNA of the target sequence includes: A poly(dA)30 tail was designed at the 5' end of the ssDNA to guide the pore. The SNP site was placed at the base position immediately after the poly(dA)30 tail. Then, an extension sequence was designed to obtain the first type of DNA with the target sequence. In step (2), the conditions for designing the extended sequence are: the ratio of cytosine and guanine is controlled below 40% to prevent excessive ratios from leading to G-quadruplexes or i-motif structures, and to avoid situations where they can form complementary sequences or hairpin structures. In step (2), the first type of DNA and the second type of RNA form a DNA-RNA complementary strand. The end of the DNA-RNA complementary strand is a mismatch site that is different from the base complementary pairing, i.e., the SNP site to be detected.

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