A nucleic acid detection method based on ternary DNA encoding library

Abstract

A method for nucleic acid detection based on a ternary DNA coding library belongs to the technical field of in vitro nucleic acid detection. The method aims to quickly determine whether a specific gene fragment is present in a solution through the coding library. By setting a certain number of nucleic acid structures in the coding region on the carrier, the size and number of nucleic acid structures in different locations determine the intensity of the nanopore electrical signal, thereby forming a multi-level electrical signal, and then creating a multi-binary coding library. Probes and marker nucleic acid structures are set in the sensing area of ​​the carrier to detect specific gene fragments. This method is based on DNA data coding structure for library construction and is an emerging rapid detection method with the characteristics of high sensitivity and high throughput.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro nucleic acid detection, and in particular relates to a nucleic acid detection method based on a ternary DNA encoding library. Background Art

[0002] Nucleic acid testing is crucial for clinical diagnosis and genotyping. Its high sensitivity and specificity enable accurate virus identification in the early stages of infection, providing opportunities for early intervention. Furthermore, it provides crucial data for epidemiological research, helping to understand infection rates and transmission pathways. It also plays a key role in monitoring viral mutations, providing a scientific basis for public health policymaking.

[0003] Early nucleic acid testing widely used techniques such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and DNA microarray testing. PCR is a molecular biology technique used to amplify specific DNA fragments. It is based on the principle of semiconservative DNA replication. It exponentially increases the number of target DNA fragments through a three-step cycle: high-temperature denaturation (unwinding double-stranded DNA into single strands), low-temperature annealing (primers bind to the single-stranded DNA template), and thermophilic extension (synthesis of new DNA strands using the primers as a starting point under the action of DNA polymerase). The procedure is relatively complex, requiring specialized equipment (such as a PCR machine) and technicians. Testing is lengthy, typically taking several hours. Contamination is also a risk, and false-positive results can occur if exogenous nucleic acids contaminate the sample or reaction system. DNA microarray testing is technically demanding, with complex chip fabrication and testing processes. Costs are high, including both chip production and testing equipment costs. Data interpretation is also complex, requiring specialized bioinformatics expertise. While existing methods offer high specificity and sensitivity, they are time-consuming, expensive, require additional modifications, and involve cumbersome sample processing, resulting in limited immediacy. Furthermore, these early methods have limited applicability, typically only detecting specific pathogens and lacking the ability to detect multiple targets simultaneously. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a nucleic acid detection method based on a ternary DNA encoding library; the DNA encoding library prepared by the present invention makes full use of the characteristics of DNA molecules and encodes the corresponding DNA vectors according to the detection requirements of different pathogens. By analyzing the characteristic current peak signals generated by each DNA vector in the DNA encoding library in the nanopore, the type of pathogen detected can be effectively distinguished. This method not only provides a high-density information storage method, but also supports high-throughput detection, significantly improving the efficiency and accuracy of pathogen identification. Due to its unique encoding mechanism, the DNA encoding library can process a large number of samples at the same time, meeting the needs of large-scale screening.

[0005] The first object of the present invention is to provide a rapid detection method based on a ternary DNA encoding library.

[0006] The second purpose of the present invention is to propose a ternary coding structure, which increases the data density compared with the classic binary coding and can encode the mark 3 3 =27 different DNA vectors.

[0007] The third object of the present invention is to propose a detection method using a DNA encoding library, which has the characteristics of high throughput and high specificity.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0009] A ternary-based DNA coding structure includes a coding region and a sensor on a DNA carrier. The coding region includes two markers and three coding positions, and the three coding positions are located between the two markers.

[0010] A group of DNA dumbbell sequences from SEQ ID NO: 1 to SEQ ID NO: 11 are used to construct the first marker in the coding region of a DNA vector. The nucleic acid sequences are shown in Table 1:

[0011] Table 1

[0012]

[0013] A group of DNA dumbbell sequences from SEQ ID NO: 12 to SEQ ID NO: 22 are used to construct a second marker in the coding region of a DNA vector. The nucleic acid sequences are shown in Table 2:

[0014] Table 2

[0015]

[0016] A set of DNA dumbbell sequences from SEQ ID NO: 23 to SEQ ID NO: 29 are used to form the first coding position "1" in the ternary encoding at the coding position of the DNA vector. The sequences are shown in Table 3:

[0017] Table 3

[0018]

[0019] A set of DNA dumbbell sequences from SEQ ID NO:30 to SEQ ID NO:36 are used to form the second coding position "1" in the ternary encoding at the coding position of the DNA vector. The sequences are shown in Table 4:

[0020] Table 4

[0021]

[0022] A set of DNA dumbbell sequences from SEQ ID NO:37 to SEQ ID NO:43 are used to form the third coding position "1" in the ternary encoding at the coding position of the DNA vector. The sequences are shown in Table 5:

[0023] Table 5

[0024]

[0025] A group of DNA sequences from SEQ ID NO: 44 to SEQ ID NO: 53 were combined with the WI(0) sequence of SEQ ID NO: 54 to form 10 DNA three-way structure sequences, which were used to form the first coding position "2" in the ternary encoding at the coding position of the DNA vector. The nucleic acid sequences are shown in Table 6:

[0026] Table 6

[0027]

[0028] A group of DNA sequences from SEQ ID NO: 55 to SEQ ID NO: 64 were combined with the WII(0) sequence of SEQ ID NO: 65 to form 10 DNA three-way structure sequences, which were used to form the second coding position "2" in the ternary code at the coding position of the DNA vector. The nucleic acid sequences are shown in Table 7:

[0029] Table 7

[0030]

[0031] A group of DNA sequences from SEQ ID NO: 66 to SEQ ID NO: 75 were respectively combined with the WIII(0) sequence of SEQ ID NO: 76 to form 10 DNA three-way structure sequences, which are used to form the third coding position "2" in the ternary encoding at the coding position of the DNA vector. The nucleic acid sequences are shown in Table 8:

[0032] Table 8

[0033]

[0034] If the coding position of the DNA vector does not form a sequence of "1" in the ternary code and "2" in the ternary code, then the coding position is "0" in the ternary code.

[0035] The sensing area includes at least one sensing unit, and the sensing unit includes corresponding probe sites and label sites; the probe is used to capture target DNA;

[0036] The nucleotide sequences of SEQ ID NO:77 and SEQ ID NO:78 are used to form probe sites on the DNA structure. In SEQ ID NO:78, "TTCCAGTGTG GCTGGTCATC" is used to capture the target DNA, and "GTAGCACCAT TACCATTAGC" is a portion of SEQ ID NO:195, complementary to the DNA template strand. SEQ ID NO:77 is the remaining portion of SEQ ID NO:195, used to fill the position. The corresponding marker sites are formed by SEQ ID NO:83-SEQ ID NO:88, forming a sensing unit.

[0037] The nucleotide sequences of SEQ ID NO:79 and SEQ ID NO:80 form a probe site on the DNA structure, wherein "GTATTCCTCC AGATCTCTAC" in SEQ ID NO:80 is used to capture the target DNA, and "ACCGATATAT TCGGTCGCTG" is a portion of SEQ ID NO:225, which is complementary to the DNA template strand; SEQ ID NO:79 is the remaining portion of SEQ ID NO:225 and is used to fill the position. The corresponding label site is formed by SEQ ID NO:89-SEQ ID NO:93, forming a sensing unit;

[0038] The nucleotide sequences of SEQ ID NO:81 and SEQ ID NO:82 form a probe site on the DNA structure, where "GCTCTGACGA ACGCTACAGG" in SEQ ID NO:82 is used to capture the target DNA, and "GGTCAGGATT AGAGAGTACC" is a portion of SEQ ID NO:255, which is complementary to the DNA template chain; SEQ ID NO:81 is the remaining portion of SEQ ID NO:255 and is used to fill the position. The corresponding label site is formed by SEQ ID NO:94-SEQ ID NO:98, forming a sensing unit.

[0039] The DNA sequences of SEQ ID NO: 77 to SEQ ID NO: 82 are used to form probes at the three sensing units of the DNA carrier sensing region, respectively. The nucleic acid sequences are shown in Table 9:

[0040] Table 9

[0041]

[0042] A set of DNA dumbbell sequences of SEQ ID NO:83 to SEQ ID NO:88 is used to form a marker at the first sensing unit of the DNA carrier sensing region. The nucleic acid sequences are shown in Table 10:

[0043] Table 10

[0044]

[0045] A set of DNA dumbbell sequences of SEQ ID NO:89 to SEQ ID NO:93 is used to form a marker site at the second sensing unit of the DNA carrier sensing region. The nucleic acid sequences are shown in Table 11:

[0046] Table 11

[0047]

[0048] A set of DNA dumbbell sequences of SEQ ID NO: 94 to SEQ ID NO: 98 is used to form a marker site at the third sensing unit of the DNA carrier sensing region. The nucleic acid sequences are shown in Table 12:

[0049] Table 12

[0050]

[0051] A ternary DNA coding library has three options: "0", "1" and "2" at each coding position in the coding structure. The coding library has 27 codes: 000, 001, 002, 010, 011, 012, 020, 021, 022, 100, 101, 102, 110, 111, 112, 120, 121, 122, 200, 201, 202, 210, 211, 212, 220, 221, 222.

[0052] The present invention provides a DNA vector scheme as follows:

[0053] The coding area uses ternary coding to represent 3 3 = 27 different detection sequences. The DNA vector is divided into a coding region and a sensor region:

[0054] The two ends of the coding region are composed of 11 DNA dumbbell structures. These special DNA structures can generate current peaks when passing through the nanopore, thereby determining the location of the coding region.

[0055] The coding region consists of two markers and three coding sites. Six DNA dumbbell structures attached to a coding site represent a "1" in the ternary code and generate a current peak when passing through the nanopore. Ten DNA tee structures, on the other hand, represent a "2" in the ternary code and generate a larger current peak when passing through the nanopore. Any portion of the coding site without the aforementioned structures that form "1" and "2" codes is considered a "0" in the ternary code.

[0056] The sensing region on the carrier consists of a probe site and a labeling site. The probe site is connected to a probe sequence that specifically recognizes and captures the target nucleic acid, enabling it to bind to the carrier and be retained. The labeling site, adjacent to the probe sequence, also contains a DNA dumbbell structure that serves as a marker. This DNA dumbbell structure can be used to mark the specific location of the target nucleic acid on the carrier, providing a basis for subsequent detection and identification.

[0057] A DNA vector comprising the coding region and sensing region according to claim 1, wherein the sensing region comprises a probe site and a labeling site; the probe is used to capture target DNA, and the label is used to mark different detection sites.

[0058] A method for preparing a DNA vector comprises the following steps:

[0059] (1) Take the M13mp18 circular DNA solution, add buffer and a single-stranded DNA solution that is partially complementary to it; raise the temperature to 65°C and then cool it to room temperature at a rate of 1-3°C / min;

[0060] (2) Use 1 μL BamHI-HF enzyme and 1 μL EcoRI-HF enzyme to digest the mixed solution;

[0061] (3) Purify the DNA using the DNA CleanUp Kit to obtain the cleaved M13mp18 single-stranded DNA;

[0062] (4) Using linear M13mp18 single-stranded DNA as the basic template, there are a total of 190 complete complementary short chains (Nicholas A. W. Bell and Ulrich F. Keyser; NATURE NANOTECHNOLOGY, VOL 11, JULY 2016; nucleotide sequence table in the supporting information of Digitally encoded DNA nanostructures for multiplexed, single-molecule proteins ensing with nanopores), the complementary short chains remaining after the functional site template (i.e., the complementary short chains remaining after removing the short chains at the functional site position from the 190 short chains) are mixed with the short chains of the functional site, and 500 μL of 100 mM Tris buffer and 25 μL of 50 mM MgCl2 solution are added to prepare a mixed solution; the temperature is raised to 70°C, and then cooled to room temperature at a rate of 1-3°C / min to obtain a linear double-stranded DNA vector containing the coding region and the sensing region;

[0063] The short chain of functional bits is a short chain of flag bits, probe bits, marker bits, and coding bits.

[0064] An application of a DNA vector, wherein the DNA vector is applied to the detection of target nucleic acid.

[0065] A DNA vector application and detection method comprises the following steps:

[0066] (1) The target nucleic acid to be detected is mixed with a linear double-stranded DNA vector containing a coding region and a sensing region; the target nucleic acid will be recognized and captured by the corresponding complementary probe on the vector;

[0067] (2) Inject LiCl solution into the nanopore chip, extract the LiCl solution and inject it into the DNA carrier after capturing the target nucleic acid and ensure that there are no bubbles in the chip;

[0068] (3) The pore-crossing event is collected by a patch clamp device. By applying voltage to the chip, the DNA molecule passes through the nanopore, causing a current change. The detection of the target nucleic acid is confirmed by identifying the current peak.

[0069] The present invention provides a method for establishing a code library for detection based on ternary coding, the steps of which are as follows:

[0070] (1) Perform nucleic acid pretreatment on the sample to be tested;

[0071] (2) Select relevant probes for the gene to be detected; one end of the probe sequence can be synthesized into the probe position, and the other end can be paired with the gene to be detected.

[0072] (3) Determine a set of codes according to the coding rules of the coding library;

[0073] (4) Synthesize DNA vector according to the coding scheme;

[0074] (5) Loading the sample and hybridizing the DNA carrier with the sample to be tested;

[0075] (6) Fabrication of nanopore chips;

[0076] (7) By applying voltage to the chip, the DNA molecules pass through the nanopore, causing a change in the current peak. The detection sample code is confirmed by identifying the current peak.

[0077] In the above steps, by applying voltage to the chip, the DNA molecules pass through the nanopore, causing the current to change. The peak height and integrated area of ​​the current peak with a coding value of 2 are significantly higher than the peak height and integrated area of ​​the current peak with a coding value of 1, and can be accurately distinguished by the set threshold.

[0078] Furthermore, by applying voltage on the chip, the DNA molecule passes through the nanopore, causing a change in current. When the coding value is 0 and the DNA molecule passes through the nanopore, no change in current is generated.

[0079] The present invention also provides an application of the above sequence group or the method of establishing a coding library for detection: using ternary coding to prepare an identification probe library of the gene to be detected and detecting the target nucleic acid with high throughput and high specificity.

[0080] The beneficial effects of the present invention are:

[0081] This encoding library increases data density and can encode tags 3 3 = 27 different DNA vectors. A multi-level encoding architecture and sensing structure are constructed into a DNA encoding library. DNA nanopore chips are used to read the DNA encoding and then use the encoding to distinguish the DNA vectors to be detected. Based on technical research, this invention is committed to developing a high-throughput, high-discrimination, sensitive, low-cost, and rapid nucleic acid detection method.

[0082] By placing a certain number of nucleic acid structures within the coding region of the vector, the size and number of nucleic acid structures in different locations determine the strength of the nanopore electrical signal, thereby forming a multi-level electrical signal and creating a multi-binary encoding library. Probes and marker nucleic acid structures are placed within the sensing region of the vector to detect specific gene fragments. This method, based on DNA data encoding structures for library construction, is an emerging rapid detection method with high sensitivity and high throughput. Multi-binary DNA encoding is a technology that uses the properties of DNA molecules to encode information into DNA nanostructures of different sizes at different locations. Multi-binary DNA encoding has a high information density and can store more data within the same DNA length. This makes multi-binary DNA encoding have the potential for widespread application in fields such as data storage and biosensor detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Schematic diagram of different DNA structures in DNA vectors.

[0084] Figure 2 It is a schematic diagram of the self-assembly of a single-stranded DNA template, complementary short-stranded DNA and its functional DNA structure to form a DNA vector.

[0085] Figure 3 This is a schematic diagram of the coding region of a DNA vector.

[0086] Figure 4 Schematic diagram of the sensing region of the DNA vector.

[0087] Figure 5 This is a photo of the nanopore chip before sample addition.

[0088] Figure 6 The graph shows the characteristic current peaks generated by 27 encoding carriers passing through the nanopore chip.

[0089] Figure 7 The graph shows the characteristic current peaks generated by the DNA carrier C unit coded as 012 before and after capturing the target DNA through the nanopore chip. DETAILED DESCRIPTION

[0090] A ternary-based DNA coding structure includes a coding region and a sensing region. The coding region includes two flag bits and three coding bits, with the three coding bits located between the two flag bits. The three coding bits are the first coding bit, the second coding bit, and the third coding bit. Each coding bit can encode the values ​​0, 1, or 2, allowing for flexible combinations.

[0091] The nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 11 are used to construct the first marker site in the coding region of the DNA vector;

[0092] A group of DNA dumbbell sequences from SEQ ID NO: 12 to SEQ ID NO: 22 are used to construct a second marker site in the coding region of a DNA vector;

[0093] A set of DNA dumbbell sequences of SEQ ID NO: 23 to SEQ ID NO: 29 is used to form the first coding position "1" in the ternary encoding at the coding position of the DNA vector;

[0094] A set of DNA dumbbell sequences of SEQ ID NO:30 to SEQ ID NO:36 is used to form a "1" of the second coding position in the ternary code at the coding position of the DNA vector;

[0095] A set of DNA dumbbell sequences of SEQ ID NO:37 to SEQ ID NO:43 is used to form a "1" of the third coding position in a ternary code at the coding position of a DNA vector;

[0096] A group of DNA sequences from SEQ ID NO:44 to SEQ ID NO:53 are respectively combined with the WI(0) sequence of SEQ ID NO:54 to form 10 DNA three-way structure sequences, which are used to form the first coding position "2" in the ternary code at the coding position of the DNA vector;

[0097] A group of DNA sequences from SEQ ID NO:55 to SEQ ID NO:64 are respectively combined with the WII(0) sequence of SEQ ID NO:65 to form 10 DNA three-way structure sequences, which are used to form the second coding position "2" in the ternary code at the coding position of the DNA vector;

[0098] A group of DNA sequences from SEQ ID NO:66 to SEQ ID NO:75 are respectively combined with the WIII(0) sequence of SEQ ID NO:76 to form 10 DNA three-way structure sequences, which are used to form the third coding position "2" in the ternary code at the coding position of the DNA vector;

[0099] If the coding position of the DNA vector is in a simple double-stranded state and does not form a sequence of "1" in the ternary code or "2" in the ternary code, then the coding position is "0" in the ternary code.

[0100] In a specific implementation, each coding bit has three options: "0", "1" and "2", so the coding structure contains 27 codes: 000, 001, 002, 010, 011, 012, 020, 021, 022, 100, 101, 102, 110, 111, 112, 120, 121, 122, 200, 201, 202, 210, 211, 212, 220, 221, 222.

[0101] The sensing area includes at least one sensing unit, and the sensing unit includes corresponding probe positions and label positions; the probe is used to capture target DNA.

[0102] In the first sensing unit, the nucleotide sequences of SEQ ID NO: 77 and SEQ ID NO: 78 are used to form a probe site on the DNA structure, and the nucleotide sequences of SEQ ID NO: 83 to SEQ ID NO: 88 form a label site on the DNA structure;

[0103] In the second sensing unit, the nucleotide sequences of SEQ ID NO:79 and SEQ ID NO:80 are used to form a probe site on the DNA structure, and the nucleotide sequences of SEQ ID NO:89 to SEQ ID NO:93 form a label site on the DNA structure;

[0104] In the third sensing unit, the nucleotide sequences of SEQ ID NO:81 and SEQ ID NO:82 are used to form a probe site on the DNA structure, and the nucleotide sequences of SEQ ID NO:94 to SEQ ID NO:98 form a label site on the DNA structure;

[0105] In a specific embodiment, three target nucleic acids can be detected on each encoded DNA vector, so 81 types of DNA to be detected can be detected simultaneously; the DNA code is read by a DNA nanopore chip, and the DNA vectors to be detected are distinguished by the coding and sensing units.

[0106] The present invention provides a detection method based on ternary DNA coding, an example of which is as follows:

[0107] Example 1

[0108] Synthesize a DNA vector with the coding region coded as "012" and the sensing region detecting genes "A", "B", and "C":

[0109] (1) Take 44 μL of M13mp18 circular DNA solution, add 8 μL of 10x shearing buffer and 2 μL of oligo solution, and then add appropriate amount of deionized water to make 80 μL of solution. Then, quickly raise the temperature to 65°C and slowly cool it down to 25°C at a rate of 1°C / min.

[0110] (2) Use 1 μL BamHI-HF enzyme and 1 μL EcoRI-HF enzyme to digest the mixed solution at 37°C for 1 hour. Purify the DNA using the DNA CleanUp Kit to obtain the cut M13mp18 single-stranded DNA.

[0111] (3) Using the linear M13mp18 single-stranded DNA as the basic template, the complementary short chains remaining after the functional site template is reserved (i.e., the complementary short chains remaining after removing the short chains at the functional site position from the 190 short chains) are mixed with the short chains at the functional site mentioned above. (Schematic diagram as shown) Figure 1 To the mixture (shown), 2.9 μL of 10 mM Tris-HCl buffer and 1 μL of 10 mM MgCl₂ solution were added to prepare a 40 μL mixture with a final vector concentration of 0.25 nM. The mixture was rapidly heated to 75°C and then slowly cooled to 25°C at a rate of 1°C / minute. This yielded a double-stranded DNA vector coded "012" with sensing regions for detecting three different genes: "A," "B," and "C."

[0112] Using this method, a DNA vector with the coding region coded as "012" and the sensing region detecting "A, B, C" genes was synthesized; the schematic diagram of the synthesis process is shown in Figure 2 shown. Figure 3 The ternary coding region of this example is presented. This coding region uses six DNA dumbbell structures and 10 DNA tee structures to represent the codes "1" and "2," respectively. The 11 DNA dumbbell structures, represented by two groups of gray blocks, serve as reference points to mark the coding region. Figure 4 The sensor area of ​​this embodiment is shown. The sensor area consists of three sensing units A, B, and C. Each unit consists of a probe and five DNA dumbbell structures. The five DNA dumbbell structures are used to mark different detection sites, while the probes are used to capture target DNA.

[0113] The above preparation method can be used to prepare other vectors with different codes or different probes. Each coding vector needs to be independently assembled due to the different short chain mixture combinations.

[0114] Example 2

[0115] Results collection for 27 coding vectors

[0116] A mixture of 27 encoding vectors was diluted in 4 M LiCl buffer to a final concentration of 0.25 nM and introduced into the tip of a glass nanopore. Electrodes were placed on opposite sides of the nanopore, and a 600 mV potential was applied to promote the transfer of negatively charged vector molecules through the nanopore, generating a characteristic current peak after the vectors passed through the pore. Current signals were recorded using an Axopatch 200B patch clamp amplifier (Axon Instruments) at a sampling rate of 1 MHz and filtered using an external 50 kHz low-pass Bessel filter (Model 900CT, frequency converter). Signals were digitized using a 16-bit resolution data acquisition card (PCIe-6251 or PCIe-6351, National Instruments). Current analysis was performed using custom LabVIEW software and Python programs. Figure 6 The characteristic current peak results of 27 types of coding carriers after passing through the hole are shown as a standard chart for subsequent detection comparison. Figure 5 This is a physical picture of the nanopore chip before sample addition.

[0117] Example 3

[0118] In the following experiments, the encoding DNA vector prepared in Example 2 was used, and the generated encoding signal was detected.

[0119] (1) First, order artificially synthesized dry powder of gene fragments that simulate real pathogens.

[0120] (2) Dissolve the simulated target sequence dry powder in nuclease-free water to prepare a 100 μM storage solution and store it at -20°C.

[0121] (3) The vector assembled in Example 1 was centrifuged and washed using a 100 kDa ultrafiltration membrane to remove excess unhybridized short chains, and the purified product was quantified and frozen for later use.

[0122] (4) The target nucleic acid to be detected and the DNA vector were incubated in TM buffer (10 mM Tris-HCl, 10 mM MgCl2, pH 8.0) at room temperature for 10 minutes, and the target nucleic acid was recognized and captured by the corresponding complementary probe on the vector.

[0123] (5) The mixture was diluted into 4 M LiCl buffer to a final carrier concentration of 0.25 nM and introduced into the tip of the glass nanopore. Electrodes were placed on both sides of the nanopore and a 600 mV voltage was applied to promote the negatively charged carrier molecules to pass through the nanopore, generating a characteristic electrical signal.

[0124] (6) Using a patch clamp amplifier to collect the electrical signal of the through-hole, and obtain the characteristic electrical signal of the carrier after the target nucleic acid is captured and passed through the hole; Figure 7 As shown, compared with the standard graph coded as "012" obtained in Example 3, the marker position C changes from a single peak to a double peak after capturing the target nucleic acid, proving that the DNA vector detects the target nucleic acid C.

Claims

1. A DNA coding structure based on ternary system, characterized by: The coding structure includes a coding region and a sensing region. The coding region includes two flag bits and three coding bits, and the three coding bits are located between the two flag bits. The three coding bits are respectively a first coding bit, a second coding bit, and a third coding bit. The value of each coding bit is selected from 0, 1, and 2. The nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 11 are used to construct the first marker site in the coding region of the DNA vector; A group of DNA dumbbell sequences from SEQ ID NO: 12 to SEQ ID NO: 22 are used to construct a second marker site in the coding region of a DNA vector; A set of DNA dumbbell sequences of SEQ ID NO:23 to SEQ ID NO:29 is used to form the first coding position "1" in the ternary encoding at the coding position of the DNA vector; A set of DNA dumbbell sequences of SEQ ID NO:30 to SEQ ID NO:36 is used to form the second coding position "1" in the ternary encoding at the coding position of the DNA vector; A set of DNA dumbbell sequences of SEQ ID NO:37 to SEQ ID NO:43 is used to form the third coding position "1" in the ternary encoding at the coding position of the DNA vector; A group of DNA sequences from SEQ ID NO:44 to SEQ ID NO:53 are respectively combined with the WI(0) sequence of SEQ ID NO:54 to form 10 DNA three-way structure sequences, which are used to form the first coding position "2" in the ternary code at the coding position of the DNA vector; A group of DNA sequences from SEQ ID NO:55 to SEQ ID NO:64 are respectively combined with the WII(0) sequence of SEQ ID NO:65 to form 10 DNA three-way structure sequences, which are used to form the second coding position "2" in the ternary code at the coding position of the DNA vector; A group of DNA sequences from SEQ ID NO:66 to SEQ ID NO:75 are respectively combined with the WIII(0) sequence of SEQ ID NO:76 to form 10 DNA three-way structure sequences, which are used to form the third coding position "2" in the ternary code at the coding position of the DNA vector; If the coding position of the DNA vector is in a simple double-stranded state and does not form a sequence of "1" in the ternary code or "2" in the ternary code, then the coding position is "0" in the ternary code.

2. The ternary-based DNA encoding structure according to claim 1, characterized in that: The sensing area includes at least one sensing unit, and the sensing unit includes corresponding probe sites and label sites; the probe is used to capture target DNA; The nucleotide sequences of SEQ ID NO:77-SEQ ID NO:78 are used to form a probe site on the DNA structure, and the corresponding label site is formed by SEQ ID NO:83-SEQ ID NO:88, constituting a sensing unit; The nucleotide sequences of SEQ ID NO:79-SEQ ID NO:80 are used to form a probe site on the DNA structure, and the corresponding label sites are formed by SEQ ID NO:89-SEQ ID NO:93, constituting a sensing unit; The nucleotide sequence of SEQ ID NO:81-SEQ ID NO:82 is used to form a probe site on the DNA structure, and the corresponding label site is formed by SEQ ID NO:94-SEQ ID NO:98, forming a sensing unit.

3. A ternary-based DNA encoding library, characterized by: The coding library contains the DNA coding structure according to claim 1 or 2, and the coding library contains 27 codes: 000, 001, 002, 010, 011, 012, 020, 021, 022, 100, 101, 102, 110, 111, 112, 120, 121, 122, 200, 201, 202, 210, 211, 212, 220, 221, and 222.

4. A DNA vector, characterized in that: The DNA vector is a DNA vector comprising the coding structure according to claim 1 or 2.

5. The method for preparing a DNA vector according to claim 4, characterized in that: The following steps are involved: (1) Take the M13mp18 circular DNA solution, add buffer and oligo solution; raise the temperature to 50-70°C, and then reduce it to room temperature at a rate of 1-3°C / min; (2) Using BamHI-HF enzyme and EcoRI-HF enzyme, the mixed solution was enzymatically digested; (3) Purify the DNA using the DNA CleanUp Kit to obtain the cleaved M13mp18 single-stranded DNA; (4) Using the linear M13mp18 single-stranded DNA as the basic template, the template complementary short chain with the functional site reserved and the short chain of the functional site are mixed, and a buffer solution and MgCl2 solution are added to prepare a mixed solution; the temperature is raised to 60-80°C, and then cooled to room temperature at a rate of 1-3°C / min to obtain a linear double-stranded DNA vector containing the coding region and the sensing region; The short chain of functional bits is a short chain of flag bits, probe bits, marker bits, and coding bits.

6. The use of a DNA vector for non-disease diagnosis and treatment purposes according to claim 4, characterized in that: The DNA vector is used for detecting target nucleic acid.

7. The use of a DNA vector for non-disease diagnosis and treatment purposes according to claim 6, characterized in that: The detection method includes the following steps: (1) The target nucleic acid to be detected is mixed with a linear double-stranded DNA vector containing a coding region and a sensing region; the target nucleic acid is recognized and captured by the corresponding complementary probe on the vector; (2) Inject 4M LiCl solution into the nanopore chip, and inject the DNA carrier diluted with 4M LiCl solution after capturing the target nucleic acid into the middle sample pool, and ensure that there are no bubbles in the chip; (3) The patch clamp device is used to collect the pore-passing event, and the characteristic electrical signal of the carrier after the target nucleic acid is captured and passed through the pore is obtained. The detection of the target nucleic acid is confirmed by identifying the electrical signal.

8. The use of a DNA vector for non-disease diagnosis and treatment purposes according to claim 7, characterized in that: In step (3), by applying voltage to the chip, the DNA molecule passes through the nanopore, causing the current to change, and the peak height and integrated area of ​​the current peak with a coding value of 2 are higher than the peak height and integrated area of ​​the current peak with a coding value of 1.

Images