Nucleic acid sequence separation equipment based on capillary electrophoresis, separation method and application

Abstract

The invention relates to nucleic acid sequence separation equipment based on capillary electrophoresis, a separation method and application, the equipment comprises a sample container and one or more sorting containers, after nucleic acid is extracted, PCR amplification is performed, a specific nucleic acid sequence is labeled with a fluorescent probe, and the fluorescent probe serves as a sample to be added into the sample container; the switching circuit is respectively connected with a cathode electrode in the sample container and an anode electrode in the sorting container, a capillary group is arranged between the sample container and the sorting container, a detection unit is arranged on the capillary group in front of the sorting container, and the switching circuit works to drive a nucleic acid sequence to migrate to a positive electrode; gating the corresponding sorting container to enable the target nucleic acid sequence to enter, closing the corresponding switching circuit after sorting is completed, and repeating until sorting is completed; the method is applied to purification of one or more nucleic acid sequences. The purification of a specific nucleic acid sequence is not influenced by the abundance of a target nucleic acid sequence in a sample, and the separation purity is high; the equipment is integrally simple in structure and convenient to operate.

Description

Technical Field

[0001] This invention relates to the technical field of testing or analyzing materials by measuring their chemical or physical properties, and particularly to a nucleic acid sequence separation device, separation method and application based on capillary electrophoresis. Background Technology

[0002] Nanopore sequencing is a cutting-edge third-generation single-molecule sequencing technology. Due to its ease of sequencing and instrument operation, it has been widely used in scientific research and medical testing. It determines the sequence of DNA molecules by detecting changes in electrical current induced by the molecules within a nanopore, making it a highly efficient sequencing technology. The accuracy and efficiency of nanopore sequencing analysis depend on the purity of the target nucleic acid sequence in the sample. Higher abundance of the target nucleic acid sequence in the sample increases the probability of sequencing within the nanopore. Furthermore, repeating multiple sequencing runs of the same sequence allows for software correction and the acquisition of optimal, accurate sequencing results, enabling rapid sequencing and improved efficiency.

[0003] In fact, the abundance of the target nucleic acid sequence in the sample is uncertain, and there are no publicly available techniques and methods for purifying specific target nucleic acid sequences.

[0004] For example, Chinese patent CN101665785A discloses a method for extracting and purifying nucleic acids from samples using magnetic beads, including (1) magnetic bead treatment; (2) cell lysis; (3) nucleic acid adsorption; (4) impurity removal; and (5) nucleic acid recovery to obtain extracted and purified nucleic acids. However, the nucleic acid purification described here is more for microorganisms such as bacteria and viruses, including but not limited to cell membrane lysis, using magnetic beads to adsorb nucleic acids and then washing away impurities such as proteins to obtain the full-sequence nucleic acid of the microorganisms, rather than for nucleic acid molecules of a specific sequence.

[0005] Given that the identification of most pathogens or diseases currently requires identifying their subtypes or discovering unknown mutation points, meaning that the nucleic acid sequence of the pathogen or tissue cell is mostly known and only the mutation of its unknown part needs to be measured, existing technologies cannot purify specific target nucleic acid sequences and cannot meet the current development needs. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a nucleic acid sequence separation device, separation method, and application based on capillary electrophoresis.

[0007] The technical solution adopted in this invention is a nucleic acid sequence separation device based on capillary electrophoresis. The device includes a sample container for holding pre-treated samples, one or more sorting containers for holding separated nucleic acid sequences, an anode electrode in each sorting container, a cathode electrode in each sample container, and a switching circuit between the cathode electrode and the anode electrode. A capillary assembly is provided between the sample container and the sorting container, and a detection unit is provided on the capillary assembly in front of the sorting container.

[0008] Preferably, the switching circuit includes a high-voltage generator, the negative terminal of which is connected to the cathode electrode, and the positive terminal of which is connected to the anode electrode through a corresponding selector switch.

[0009] Preferably, the cathode electrode is submerged in the cathode buffer in the sample container, and each anode electrode is submerged in the anode buffer in the corresponding sorting container.

[0010] Preferably, the capillary assembly includes an inlet capillary with one end disposed in the cathode buffer of the sample container, and the other end of the inlet capillary is configured with an output capillary corresponding to each sorting container via one or more tee fittings, with the outlet of each output capillary disposed in the anodic buffer of the sorting container.

[0011] Preferably, the detection unit includes a fluorescence detection window disposed on the sample injection capillary, and a fluorescence probe is disposed on the sample in conjunction with the fluorescence detection window; the fluorescence detection window is connected to a control terminal.

[0012] Preferably, the control terminal is configured in conjunction with a switching circuit.

[0013] Preferably, any two tee fittings are connected in series via an output capillary tube.

[0014] Preferably, both the inlet capillary and the outlet capillary are provided with gel layers of the same or different thicknesses.

[0015] A method for nucleic acid sequence separation using a capillary electrophoresis-based nucleic acid sequence separation device, the method comprising the following steps: S1 performs PCR amplification on the extracted full-sequence nucleic acid, and labels the specific nucleic acid sequence that matches the template with a fluorescent probe, adding it to the sample container as a pretreated sample; S2 is equipped with a switching circuit, which is connected to the cathode electrode in the sample container and the anode electrode in each sorting container respectively; When the S3 switch circuit is activated, it drives the negatively charged nucleic acid sequence to migrate toward the positive electrode. When the target fluorescent group passes through the detection unit, it selects the corresponding sorting container, allowing the target nucleic acid sequence to enter this sorting container. S4 The current target nucleic acid sequence sorting is completed. Turn off the switch circuit corresponding to the current sorting container. If there are still nucleic acid sequences to be sorted, repeat S3; otherwise, proceed to the next step. S5 completes sorting; waste liquid is cleaned up.

[0016] An application of a nucleic acid sequence separation device based on capillary electrophoresis, used for the purification of one or more nucleic acid sequences.

[0017] This invention relates to a nucleic acid sequence separation device, method, and application based on capillary electrophoresis. The device includes a sample container and one or more sorting containers. After extraction, the whole-sequence nucleic acid is amplified by PCR. A fluorescent probe labeled with a specific nucleic acid sequence matching the template is added to the sample container as a pretreated sample. A switching circuit is set up to connect to the cathode electrode in the sample container and the anode electrode in each sorting container. A capillary assembly is provided between the sample container and the sorting containers. A detection unit is provided on the capillary assembly before the sorting containers. When the switching circuit is activated, it drives the negatively charged nucleic acid sequence to migrate towards the positive electrode. When the target fluorescent group passes through the detection unit, the corresponding sorting container is selected, allowing the target nucleic acid sequence to enter. Once the current target nucleic acid sequence is sorted, the switching circuit corresponding to the current sorting container is turned off. This process is repeated until the sorting is complete. The method is applied to the purification of one or more nucleic acid sequences.

[0018] The beneficial effects of this invention are as follows: (1) The purification of specific nucleic acid sequences is not affected by the abundance of the target nucleic acid sequence in the sample. The electric field applied by the high voltage module makes it easier to control the nucleic acid sequences labeled with different fluorescence to enter the corresponding specific container, complete the separation and use it for subsequent processing, and the separation purity is high. (2) The equipment has a simple overall structure and is easy to operate. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the device structure of the present invention; Figure 2 This is a schematic diagram of the capillary assembly in this invention; Figure 3 This is a flowchart of the method of the present invention. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0021] This invention relates to a nucleic acid sequence separation device based on capillary electrophoresis. The device includes a sample container 1 for holding a pre-treated sample, one or more sorting containers 2 for holding the separated nucleic acid sequences, an anode electrode 3 is provided in each of the sorting containers 2, a cathode electrode 4 is provided in the sample container 1, and a switching circuit 5 is arranged between the cathode electrode 4 and the anode electrode 3. A capillary assembly is provided between the sample container 1 and the sorting container 2, and a detection unit 7 is provided on the capillary assembly in front of the sorting container 2.

[0022] In this invention, the device is generally equipped with a sample container 1, from which the nucleic acid sequence is electrophoresed to prevent inaccurate results caused by mixing multiple sample containers 1. The sorting container 2 corresponding to a sample container 1 can be one or more, but in practical applications, at least two should be set up to ensure that in addition to holding at least one nucleic acid sequence that needs to be sorted out, it also holds the waste liquid that may be exported during the process. Considering the application logic of nanopore sequencing, an anode electrode 3 and a cathode electrode 4 are respectively set in the sorting container 2 and the sample container 1, and an anode buffer 8 and a cathode buffer 9 are respectively configured. The on / off state between the cathode electrode 4 and the anode electrode 3 is controlled by the switching circuit 5. In the specific implementation process, both the anode electrode 3 and the cathode electrode 4 are submerged in the anode buffer 8 and the cathode buffer 9 of the sorting container 2 and the sample container 1. Generally, the bottom ends of the two are located at the bottom of the corresponding sorting container 2 or sample container 1 to ensure the conduction of the circuit.

[0023] In this invention, a capillary array is set between the sample container 1 and the sorting container 2. Different circuits of the switching circuit 5 are connected through the capillary array, which then cooperates with the detection unit 7 to complete the sorting of one or more specific nucleic acid sequences.

[0024] In the specific implementation process, the switching circuit 5 includes a high voltage generator 51, the negative terminal of the high voltage generator 51 is connected to the cathode electrode 4, and the electrode of the high voltage generator 51 is connected to the anode electrode 3 through a corresponding selector switch 10.

[0025] The capillary assembly includes an inlet capillary 61 with one end located in the cathode buffer 9 of the sample container 1, and the other end of the inlet capillary 61 is configured with an output capillary 63 corresponding to each sorting container 2 via one or more three-way connectors 62. The outlet of each output capillary 63 is located in the anode buffer 8 of the sorting container 2.

[0026] The detection unit 7 includes a fluorescence detection window disposed on the sample injection capillary 61, and a fluorescence probe is disposed on the sample in conjunction with the fluorescence detection window; the fluorescence detection window is connected to the control terminal 11.

[0027] The control terminal 11 is configured in conjunction with the switching circuit 5.

[0028] Any two T-joints 62 are connected in series through the output capillary tube 63.

[0029] In this invention, nucleic acid sequences begin to enter the capillary group sequentially from the inlet of the sample injection capillary 61. When the required nucleic acid sequence is detected by the fluorescent probe at the fluorescence detection window, it feeds back a corresponding signal to the control terminal 11 (controller). The control terminal 11 controls the different loops of the conduction switch circuit 5, thereby the specified nucleic acid sequence is output from the outlet of the corresponding loop's output capillary 63 to the corresponding sorting container 2.

[0030] In this invention, in order to facilitate the application of capillary groups in different sorting scenarios, different schemes for setting up output capillary tubes of multiple sorting channels are realized by connecting three-way connectors 62 in series from the end of the sample inlet capillary 61. The basic principle is the same. Taking two three-way connectors 62 as an example, the sample inlet capillary 61 is connected to the inlet of the first three-way connector 62, and the two outlets of the first three-way connector 62 correspond to the sorting container 2 and the capillary connected to the second three-barrel connector 62, respectively. At this time, the capillary will serve as the "sample inlet capillary" of the second three-barrel connector 62, realizing the "series connection" of the first three-way connector 62 and the second three-barrel connector 62, and so on.

[0031] In this invention, considering that different nucleic acid sequences move at different speeds within the capillary group when the three-way connector 62 is connected in series, in order to avoid sorting failure caused by different movement speeds, gel layers of the same or different thicknesses are distributed in the sample inlet capillary 61 and the output capillary 63. The different resistance forces caused by the different thicknesses of the gel layers enable speed control of the output of different nucleic acid sequences.

[0032] This invention also relates to a nucleic acid sequence separation method using a nucleic acid sequence separation device based on capillary electrophoresis, the method comprising the following steps: S1 performs PCR amplification on the whole sequence of nucleic acid after extraction, and adds the fluorescent probe labeled with a specific nucleic acid sequence that matches the template as a pretreated sample into sample container 1. S2 sets up a switch circuit 5, which is connected to the cathode electrode 4 in the sample container 1 and the anode electrode 3 in each sorting container 2 respectively; When the S3 switch circuit 5 is activated, it drives the negatively charged nucleic acid sequence to migrate toward the positive electrode. When the target fluorescent group passes through the detection unit 7, it selects the corresponding sorting container 2, allowing the target nucleic acid sequence to enter this sorting container 2. S4 The current target nucleic acid sequence sorting is completed. Turn off the switch circuit 5 corresponding to the current sorting container 2. If there are still nucleic acid sequences to be sorted, repeat S3; otherwise, proceed to the next step. S5 completes sorting; waste liquid is cleaned up.

[0033] The present invention also relates to the application of a nucleic acid sequence separation device based on capillary electrophoresis, which is used for the purification of one or more nucleic acid sequences.

[0034] Those skilled in the art will understand that embodiments of the present invention can be provided as methods, systems, or computer program products. Therefore, the present invention can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, the present invention can take the form of a computer program product embodied on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0035] This invention is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the invention. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, generate instructions for implementing the flowchart illustrations and / or block diagrams. Figure 1 One or more processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0036] These computer program instructions may also be stored in a computer-readable storage medium that can direct a computer or other programmable data processing device to function in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of manufacture including instruction means, which are implemented in a process Figure 1 One or more processes and / or boxes Figure 1 The function specified in one or more boxes.

[0037] These computer program instructions may also be loaded onto a computer or other programmable data processing equipment to cause a series of operational steps to be performed on the computer or other programmable equipment to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable equipment for implementing the process. Figure 1 One or more processes and / or boxes Figure 1 The steps of the function specified in one or more boxes.

[0038] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0039] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A nucleic acid sequence separation device based on capillary electrophoresis, characterized by: The device comprises a sample container for containing pre-processed sample, one or more sorting containers for containing separated nucleic acid sequences, an anode electrode provided in any of the sorting containers, a cathode electrode provided in the sample container, and a switching circuit configured between the cathode electrode and the anode electrode; A capillary group is provided between the sample container and the sorting containers, and a detection unit is provided on the capillary group in front of the sorting containers.

2. A nucleic acid sequence separation device based on capillary electrophoresis according to claim 1, characterized in that: The switching circuit comprises a high-voltage generator, a negative electrode of the high-voltage generator is connected to the cathode electrode, and a positive electrode of the high-voltage generator is connected to the anode electrode through a corresponding gating switch.

3. The nucleic acid sequence separation apparatus based on capillary electrophoresis according to claim 1, characterized in that: The cathode electrode is immersed in a cathode buffer in the sample container, and any anode electrode is immersed in an anode buffer in the corresponding sorting container.

4. The nucleic acid sequence separation apparatus based on capillary electrophoresis according to claim 1, characterized in that: The capillary group comprises a sample inlet capillary provided in the cathode buffer in the sample container, and the other end of the sample inlet capillary is configured with an output capillary corresponding to each sorting container through one or more three-way joints, and the outlet of each output capillary is provided in the anode buffer in the sorting container.

5. A nucleic acid sequence separation device based on capillary electrophoresis according to claim 4, characterized in that: The detection unit comprises a fluorescence detection window provided on the sample inlet capillary, and a fluorescent probe is provided on the sample corresponding to the fluorescence detection window; and the fluorescence detection window is connected to a control end.

6. A nucleic acid sequence separation device based on capillary electrophoresis according to claim 5, characterized in that: The control end is provided in cooperation with the switching circuit.

7. A nucleic acid sequence separation device based on capillary electrophoresis according to claim 4, characterized in that: Any two three-way joints are connected in series through the output capillary.

8. A nucleic acid sequence separation device based on capillary electrophoresis according to claim 4, characterized in that: Gel layers with the same thickness or different thicknesses are distributed in the sample inlet capillary and the output capillary.

9. A method for separating a nucleic acid sequence by using the nucleic acid sequence separating apparatus based on capillary electrophoresis according to any one of claims 1 to 8, characterized by: The method comprises the following steps: S1: performing PCR amplification on the whole sequence nucleic acid after extracting the nucleic acid, labeling a specific nucleic acid sequence matched with the template with a fluorescent probe, and adding the pre-processed sample into the sample container; S2: setting the switching circuit to be connected to the cathode electrode in the sample container and the anode electrode in each sorting container, respectively; S3: the switching circuit is operated to drive the negatively charged nucleic acid sequence to migrate to the positive electrode, when the target fluorescent group passes through the detection unit, the corresponding sorting container is gated, and the target nucleic acid sequence enters the sorting container; S4: after the sorting of the current target nucleic acid sequence is completed, the switching circuit corresponding to the current sorting container is closed, if there is still nucleic acid sequence to be sorted, S3 is repeated, otherwise, the next step is performed; S5: completing the sorting; and cleaning the waste liquid.

10. Use of a nucleic acid sequence separation device based on capillary electrophoresis according to one of claims 1 to 8, characterized in that: The device is applied to the purification of one or more nucleic acid sequences.

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