A method for fabricating a gene chip by using a gene chip to transfer and produce a printing plate and then mass-producing gene chips

Abstract

The present invention belongs to the field of biochips, and in particular relates to a method for converting a gene chip into a printing plate and then mass-producing gene chips. Specifically, taking the finished initial gene chip as the master plate, using DNA polymerase to convert the probe sequences on the master plate into template sequences at corresponding positions on the printing plate according to the principle of base complementary pairing. The probe sequences on the master plate correspond one by one to the template sequences on the printing plate. Then, the template sequences on the printing plate are converted into probe sequences on the new gene chip by using DNA polymerase. The probe sequences on the new gene chip correspond one by one to the template sequences on the printing plate. Through two high-fidelity DNA replication processes catalyzed by DNA polymerase, the probe sequences on the initial gene chip are converted into probe sequences on the new gene chip. The present invention can, like plate-making printing, use the initial gene chip to manufacture a printing plate, and then use the printing plate to mass-produce new gene chips quickly and at low cost.

Description

Technical Field

[0001] The present invention relates to the field of biochips, and particularly to a method for converting a gene chip into a printing plate and then mass-producing gene chips. Background Art

[0002] Biochip technology has been widely applied in many fields such as clinical disease diagnosis, health management, drug research and development, animal and plant quarantine, food detection, environmental monitoring, scientific research, forensic detection, etc. It has broad application prospects and a very large market demand.

[0003] A gene chip is a type of biochip, which is prepared by immobilizing a series of probes with known sequences on the substrate of the chip. It can be used for the hybridization detection of specific labeled nucleic acids, and can report the nucleic acid information in the detection object through recognition detection and information processing, effectively solving the "bottleneck" problem in the gene chip industry. The key technology in gene chip manufacturing includes how to effectively immobilize the probes at specific positions on the gene chip. Currently, the methods for fabricating probes on gene chips are mainly divided into two categories: one is the in-situ chemical synthesis method, in which nucleotides are connected one by one at specific positions on the substrate according to the designed procedure by chemical synthesis to form the designed probes; the other is the post-synthesis cross-linking method, in which the probes are first synthesized and then immobilized at specific positions on the substrate.

[0004] The in-situ chemical synthesis method directly synthesizes probes on the substrate. After activating specific positions on the substrate, nucleotides are added one by one to prepare. It can manufacture high-density gene chips, and the probes are covalently cross-linked on the substrate very stably, with significant advantages. Its disadvantages are that the cost is expensive, protecting groups need to be removed at specific positions in each step, and for each additional nucleotide in the probe length, the equipment needs to perform four rounds of operations, and each round completes the site-specific synthesis of one of the four nucleotides, occupying a long machine working time. At the same time, the nucleic acid chemical synthesis method has no correction mechanism, and the synthesis process cannot guarantee 100% accuracy like the biological system. In the chemical synthesis method, if the yield of each step is calculated at 95%, the yield of two-step synthesis is 90.3%, and the yield of three-step synthesis is only 85.7%. Manufacturing high-density gene chips by the in-situ synthesis method results in expensive high-density gene chips due to low synthesis yield, many synthesis steps, high cost of raw materials (nucleotides with protecting groups), and low efficiency of removing protecting genes, making it impossible to be applied on a large scale.

[0005] The post-synthesis cross-linking method first completes the preparation of the probes and then prints them on the substrate, greatly reducing the chip manufacturing cost, but the probe density of the chip is limited. Summary of the Invention

[0006] To solve the deficiencies in the prior art, the present invention utilizes the enzymatic synthesis technology for nucleic acid synthesis in biological systems. Using DNA polymerase, the probe sequence of the gene chip is converted into the template sequence at the tip of the microneedles on the printing plate. The proofreading activity of DNA polymerase is utilized to ensure the fidelity of sequence conversion, and the processive synthesis ability of DNA polymerase is used to complete the synthesis of the template sequence in one go, thereby obtaining a printing plate for mass-producing the gene chip with the existing gene chip manufacturing method. With this technology, as long as one printing plate or one gene chip is completed, the mutual replication between the gene chip and the printing plate can be achieved, enabling the mass production of printing plates to initiate the large-scale production and manufacturing of gene chips.

[0007] The inventive concept of the present invention is: Utilizing the high-fidelity activity and the characteristics of automatic continuous synthesis of DNA polymerase, guiding the synthesis of the template sequence on the printing plate according to the base complementary pairing principle with the probe sequence of the initial gene chip, and then guiding the synthesis of the probe on the new gene chip according to the base complementary pairing principle with the template sequence on the printing plate.

[0008] To solve the above problems, the technical solution of a method for converting a gene chip into a printing plate and then mass-printing gene chips is as follows:

[0009] S1 Obtain the initial gene chip product and related information, including: the probe sequence and its related arrangement order, and the dot density of the initial gene chip.

[0010] S2 Prepare a microneedle cluster on the printing plate material by etching technology. The microneedle cluster matches the dot density of the initial gene chip to ensure that each microneedle position corresponds to a probe sequence.

[0011] S3 Activate the surface of the microneedle tip, chemically modify and protect the surface activation group, and then passivate the surface of the printing plate material to eliminate the influence of the printing plate material on the subsequent synthesis of the template sequence;

[0012] S4 Remove the chemical modification and protection groups on the microneedles of the four printing plate materials respectively, and fix a nucleotide on the microneedles by covalent connection. The nucleotide is connected together through the template linker and the covalent connection point of the printing plate; and only one type of nucleotide is connected to each printing plate material, and only one nucleotide is connected to the activation group at the tip of each microneedle.

[0013] S5 According to the probe sequence and its related arrangement order on the initial gene chip product, draw the complementary base distribution map of the 3'-terminal base of the probe sequence based on the complementary base of the 3'-terminal base of the probe sequence.

[0014] S6 According to the complementary base distribution map of the 3'-terminal base of the probe sequence, corresponding blocks are intercepted from four plate materials covalently linked with nucleotides respectively and pasted onto the plate fixing carrier, and finally a microneedle cluster corresponding to the probe on the initial gene chip product is obtained, and the base at the tip of each microneedle is complementary to the 3'-terminal base of the probe sequence at the corresponding position.

[0015] S7 Cover the initial gene chip product with DNA polymerase and its corresponding reaction system (meeting the relevant elements for probe synthesis), and use precise positioning technology to accurately place the plate fixing carrier on the gene chip product and ensure that the nucleotides covalently linked on the tip of the microneedle can be complementary paired with the 3'-terminal base of the probe sequence on the initial gene chip product.

[0016] S8 DNA polymerase assembles a DNA replication initiation complex with the assistance of relevant proteins. The DNA replication initiation complex includes: the DNA polymerase of bacteriophage Φ29, the terminal protein of bacteriophage Φ29, the p6 single-stranded DNA binding protein of bacteriophage Φ29, four DNA synthesis substrates dNTP, and the corresponding components required for the DNA polymerase to synthesize DNA. The DNA replication initiation complex can automatically and faithfully synthesize a template sequence according to the base complementary pairing principle with the nucleotide at the tip of the microneedle as the starting point under the guidance of the probe sequence on the initial gene chip product, and this template sequence is completely complementary to the probe sequence.

[0017] S9 Use the method of heating and denaturation to separate the newly synthesized template sequence from the probe sequence on the initial gene chip product, and separate the initial gene chip from the plate fixing carrier by hydraulic pressure. The initial gene chip can be used for the next round of plate preparation, while the plate fixing carrier together with the microneedle cluster fixed on it and the newly synthesized template cluster at the tip of the microneedle will be transferred to subsequent processing procedures such as cleaning, and finally packaged and stored for corresponding quality and performance detection. The qualified product (plate) is used for gene chip production.

[0018] S10 According to a method for providing a substrate for in-situ synthesis of gene chip probes by a combination method, use a standard-mode initial substrate or a customized-mode initial substrate to manufacture a substrate for in-situ synthesis of gene chip probes by a combination method; make a gene chip according to a method for in-situ synthesis of gene chip probes using DNA polymerase; the specific operation process is as follows:

[0019] (1) Transfer the substrate with the first nucleotide at the 5'-end of all gene chip probes into a gene chip synthesis instrument, and add a DNA polymerase reaction system on the substrate (including: the DNA polymerase of bacteriophage Φ29, the terminal protein of bacteriophage Φ29, the p6 single-stranded DNA binding protein of bacteriophage Φ29, four DNA synthesis substrates dNTP, and the corresponding components required for the DNA polymerase to synthesize DNA).

[0020] (2) Immerse the printing plate into the DNA polymerase reaction buffer of bacteriophage Φ29 (containing the p6 single-stranded DNA binding protein of bacteriophage Φ29). The p6 single-stranded DNA binding protein of bacteriophage Φ29 binds to the template sequence structure at the tip of the microneedles on the printing plate, preventing the formation of secondary structures of the template sequence and promoting the assembly of the template to form a DNA replication initiation complex.

[0021] (3) Use the printing plate fixing carrier to accurately position and assemble the printing plate and the substrate together, ensuring that the first base at the 3' end of the template sequence bound to each microneedle tip pairs with the starting base at the corresponding position on the substrate, and effectively assembling the DNA replication initiation complex. Use the DNA polymerase of bacteriophage Φ29 to automatically and faithfully synthesize the probe sequence on the substrate according to the base complementary pairing principle under the guidance of the template sequence. The newly synthesized probe sequence is completely complementary to the template sequence on the printing plate.

[0022] (4) Use the method of heating and denaturation to unpair the newly synthesized probe sequence and the template sequence on the gene chip, and separate the newly synthesized gene chip from the printing plate (together with the printing plate fixing carrier) by the method of hydraulic pressurization. The printing plate can be used continuously for the next round of gene chip preparation, while the substrate that has completed probe synthesis will be transferred to subsequent processing procedures such as cleaning, and finally encapsulated and stored for corresponding quality and performance detection. Qualified gene chips are put on the market.

[0023] Preferably, use DNA polymerase to complete the conversion of the probe sequence on the initial gene chip to the template sequence on the microneedle cluster of the printing plate, and then use DNA polymerase again to convert the template sequence on the microneedle cluster of the printing plate to the probe sequence on the newly synthesized gene chip, and achieve the accurate transfer of the probe sequence on the initial gene chip to the probe sequence on the newly synthesized gene chip through the characteristic that DNA polymerase can faithfully replicate DNA sequences.

[0024] Preferably, use etching technology to prepare a microneedle cluster on the printing plate that matches the probe distribution on the initial gene chip, which can ensure that each microneedle corresponds to a specific probe.

[0025] Preferably, divide the microneedles into the microneedle tip and the microneedle assembly part, which not only ensures the matching of the microneedle tip and the probe, but also ensures that the microneedles can be effectively assembled to form a printing plate.

[0026] Preferably, use the method of the complementary base distribution map of the 3' terminal base of the probe sequence to intercept the corresponding blocks from four printing plate materials that have been covalently linked with nucleotides and paste them on the printing plate fixing carrier, and finally obtain a microneedle cluster corresponding to the probes on the finished product of the initial gene chip, and ensure that the base at each microneedle tip is complementary to the 3' terminal base of the probe sequence at the corresponding position.

[0027] Preferably, the gene chip and the printing plate can be replicated with each other. Whether there is initially a gene chip or a printing plate, they can be replicated with each other, so as to be able to manufacture a large number of printing plates to initiate the large-scale production and manufacturing of gene chips.

[0028] Preferably, the present invention can cooperate with an invention of a method for providing a substrate for in-situ synthesizing gene chip probes by a combination method and an invention of a method for fabricating a gene chip by using a method for in-situ synthesizing gene chip probes with DNA polymerase to form a gene chip industrial chain similar to the photocopier mode. The printing plate is equivalent to the selenium drum of the photocopier (carrying the template), DNA polymerase and its reaction system are like ink, and the substrate with different starting bases fixed thereon is like paper. Each rotation of the selenium drum of the photocopier can produce a gene chip. The direct consumptions in the production are mainly four kinds of bases (dNTP) in addition to electric energy.

[0029] Preferably, the linker arm is preferably in the structure of mimicking peptide nucleic acid.

[0030] Preferably, a pattern-guided operation mode is used in both the substrate combination and the microneedle combination, which is automatically completed by the device.

[0031] Compared with the prior art, the present invention mass-produces printing plates from gene chips in a manner similar to photocopying, and then uses the printing plates to mass-produce gene chips in a manner similar to photocopying, completing the closed-loop of the gene chip manufacturing chain. Cooperating with the other two technologies (an invention of a method for providing a substrate for in-situ synthesizing gene chip probes by a combination method and an invention of a method for fabricating a gene chip by using a method for in-situ synthesizing gene chip probes with DNA polymerase), a gene chip industrial chain similar to the photocopier mode is formed, and gene chips can be mass-produced quickly like printing, greatly reducing the manufacturing cost of gene chips. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of using DNA polymerase to complete the conversion between probe sequence and template sequence

[0033] In the figure: substrate 1, covalent cross-linking site 2 on the substrate, starting base linker arm 3, starting base 4, probe middle base 5, 3'-end base 6 of the probe, first 5'-end base 7 of the template, template linker arm 8, covalent cross-linking site 9 between the template and the microneedle, microneedle tip 10, assembly part 11 of the microneedle, printing plate fixing carrier 12, middle base 13 of the template, 3'-end base 14 of the template, DNA polymerase 15. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention will be further described in detail below in conjunction with the accompanying drawings and through specific embodiments. The following embodiments are only descriptive and not restrictive, and the protection scope of the present invention cannot be limited thereby. The chemical reagents and instruments used in the present invention can be purchased from commercial channels without special instructions.

[0035] Example 1 (Existing gene chip)

[0036] S1 Obtain the initial gene chip product and related information, including: probe sequences and their related arrangement orders, and the dot density of the initial gene chip.

[0037] S2 Prepare a micro-needle cluster on the printing plate material by etching technology according to the dot density of the initial gene chip. The micro-needle cluster matches the dot density of the initial gene chip to ensure that each micro-needle tip corresponds to a probe sequence (including: 4 starting bases, 5 middle bases of the probe, and 6 3'-terminal bases of the probe).

[0038] S3 Activate the surface of the micro-needle tip, chemically modify and protect the surface activation group, and then passivate the surface of the printing plate material to eliminate the influence of the printing plate material on the subsequent template sequence synthesis.

[0039] S4 Remove the chemical modification protection groups on the micro-needles of the four printing plate materials respectively, and fix a nucleotide (carrying one of the bases A, T, C, G) on the micro-needle tip by covalent connection. The nucleotide is connected to the covalent connection point 9 of the printing plate tip through the template linker 8 (similar to the structure of peptide nucleic acid); and only one type of nucleotide is connected to each printing plate material, and only one nucleotide is connected to the activation group of each micro-needle tip.

[0040] S5 According to the probe sequences and their related arrangement orders on the initial gene chip product, draw a complementary base distribution map of the 3'-terminal base of the probe sequence based on the complementary base of the 3'-terminal base of the probe sequence.

[0041] S6 According to the complementary base distribution map of the 3'-terminal base of the probe sequence, intercept the corresponding blocks from the four printing plate materials that have been covalently connected with nucleotides and paste them on the printing plate fixing carrier, and finally obtain a micro-needle cluster corresponding to the probes on the initial gene chip product, and the base of each micro-needle tip is complementary to the 3'-terminal base of the probe sequence at the corresponding position (for example: the 3'-terminal base 6 of the probe can be complementary paired with the first base 7 at the 5'-end of the template).

[0042] On the initial finished gene chip, spread DNA polymerase 15 and its corresponding reaction system (meeting the relevant elements for probe synthesis). Using precise positioning technology, accurately place the plate-fixing carrier on the finished gene chip and ensure that the nucleotides covalently linked to the tip of the microneedle can be complementary base-paired with the 3'-terminal base of the probe sequence on the initial finished gene chip (for example: the 3'-terminal base 6 of the probe is complementary base-paired with the first base 7 at the 5'-end of the template).

[0043] With the assistance of related proteins, DNA polymerase assembles a DNA replication initiation complex. The DNA replication initiation complex includes: the DNA polymerase 12 of bacteriophage Φ29, the terminal protein of bacteriophage Φ29, the p6 single-stranded DNA binding protein of bacteriophage Φ29, four DNA synthesis substrates dNTP, and the corresponding components required for the DNA polymerase to synthesize DNA. The DNA replication initiation complex can automatically and faithfully synthesize the template sequence according to the base complementary pairing principle, guided by the probe sequence on the initial finished gene chip and starting from the nucleotides at the tip of the microneedle. This template sequence (including: the first base 7 at the 5'-end of the template, the middle base 13 of the template, the 3'-terminal base 14 of the template) is completely complementary to the probe sequence (including: the starting base 4, the middle base 5 of the probe, the 3'-terminal base 6 of the probe).

[0044] Use the method of heating denaturation to separate the newly synthesized template sequence (including: the first base 7 at the 5'-end of the template, the middle base 13 of the template, the 3'-terminal base 14 of the template) from the probe sequence (including: the starting base 4, the middle base 5 of the probe, the 3'-terminal base 6 of the probe) on the initial finished gene chip. Separate the initial gene chip from the plate-fixing carrier 12 by the method of hydraulic pressurization. The initial gene chip can be continued to be used for the next round of plate preparation, while the plate-fixing carrier 12 together with the microneedle cluster fixed on it and the newly synthesized template cluster at the tip of the microneedle will be transferred to subsequent processing procedures such as cleaning, and finally encapsulated and stored for corresponding quality and performance detection. The qualified product (plate) is for gene chip production.

[0045] According to a method for providing a substrate for in-situ synthesizing gene chip probes by a combination method, use a customized mode initial substrate to manufacture a substrate for in-situ synthesizing gene chip probes by a combination method; make a gene chip according to a method for in-situ synthesizing gene chip probes using DNA polymerase. The specific operation process is as follows.

[0046] (1) Transfer the substrate with the first nucleotide (starting base 4) at the 5' end of all gene chip probes into the gene chip synthesis instrument, and set up a DNA polymerase reaction system on the substrate (including: DNA polymerase of bacteriophage Φ29 15, terminal protein of bacteriophage Φ29, p6 single-stranded DNA binding protein of bacteriophage Φ29, four DNA synthesis substrates dNTP, and corresponding components required for DNA polymerase to synthesize DNA).

[0047] (2) Immerse the printing plate into the DNA polymerase reaction buffer of bacteriophage Φ29 (containing the p6 single-stranded DNA binding protein of bacteriophage Φ29). The p6 single-stranded DNA binding protein of bacteriophage Φ29 binds to the template sequence at the tip 10 of the microneedles on the printing plate (including: the first base 7 at the 5' end of the template, the middle base 13 of the template, and the base 14 at the 3' end of the template), preventing the template sequence from forming secondary structures and promoting the assembly of the template to form a DNA replication initiation complex.

[0048] (3) Use the printing plate fixing carrier 12 to accurately position and assemble the printing plate and the substrate together, ensuring that the first base 14 at the 3' end of the template sequence (including: the first base 7 at the 5' end of the template, the middle base 13 of the template, and the base 14 at the 3' end of the template) bound to each microneedle tip 10 pairs with the starting base 4 at the corresponding position on the substrate, and effectively assemble the DNA replication initiation complex. Use the DNA polymerase 15 of bacteriophage Φ29 to automatically and faithfully synthesize the probe sequence on the substrate (including: starting base 4, probe middle base 5, and base 6 at the 3' end of the probe) according to the base complementary pairing principle under the guidance of the template sequence. The newly synthesized probe sequence (including: starting base 4, probe middle base 5, and base 6 at the 3' end of the probe) is completely complementary to the template sequence on the printing plate (including: the first base 7 at the 5' end of the template, the middle base 13 of the template, and the base 14 at the 3' end of the template).

[0049] (4) Use the method of heating and denaturation to dissociate the newly synthesized probe sequence on the gene chip from the template sequence, and separate the newly synthesized gene chip from the printing plate (together with the printing plate fixing carrier 12) by hydraulic pressure. The printing plate can be used for the next round of gene chip preparation, while the substrate that has completed probe synthesis will be transferred to subsequent processing procedures such as cleaning, and finally packaged and stored for corresponding quality and performance testing. Qualified gene chips are put on the market.

[0050] Example 2 (Existing printing plate, gene chip cloned from the printing plate)

[0051] S1 Prepare a microneedle cluster on the printing plate material by etching technology according to the relevant information of the printing plate.

[0052] S2 performs surface activation on the micro-needle tip 10, chemically modifies and protects the surface activation groups, and then performs surface passivation treatment on the printing plate material to eliminate the influence of the printing plate material on the subsequent template sequence synthesis.

[0053] S3 Removes the chemically modified protection groups on the micro-needle tips 10 of the four printing plate materials respectively, and fixes a nucleotide (carrying one of the bases A, T, C, G) on the micro-needle tips 10 through a covalent connection method. The nucleotide is connected to the covalent connection point 9 of the printing plate tip 10 through the template linker 8 (similar to the structure of peptide nucleic acid); and only one type of nucleotide is connected to each printing plate material, and only one nucleotide is connected to the activation group of each micro-needle tip.

[0054] S4 Forms a template 5'-end base distribution map according to the printing plate information, intercepts the corresponding blocks from the four printing plate materials that have been covalently connected with nucleotides respectively and pastes them on the printing plate fixing carrier 12, and finally obtains a micro-needle cluster consistent with the printing plate, and each micro-needle tip 10 carries the 5'-end base 7 of the template sequence consistent with the printing plate information.

[0055] S5 Covers the initial gene chip product with DNA polymerase 15 and its corresponding reaction system (meeting the relevant elements for probe synthesis), and uses precise positioning technology to accurately place the printing plate fixing carrier 12 on the initial gene chip product and ensure that the nucleotide covalently connected on the micro-needle tip can be complementary paired with the 3'-end base of the probe sequence on the initial gene chip product (for example: the 3'-end base 6 of the probe is complementary paired with the first base 7 at the 5'-end of the template).

[0056] S6 DNA polymerase assembles a DNA replication initiation complex with the assistance of related proteins. The DNA replication initiation complex includes: the DNA polymerase 12 of bacteriophage Φ29, the terminal protein of bacteriophage Φ29, the p6 single-stranded DNA binding protein of bacteriophage Φ29, four DNA synthesis substrates dNTP, and the corresponding components required for DNA polymerase to synthesize DNA. The DNA replication initiation complex can automatically and faithfully synthesize the template sequence according to the base complementary pairing principle with the nucleotide at the micro-needle tip as the starting point under the guidance of the probe sequence on the initial gene chip product. This template sequence (including: the first base 7 at the 5'-end of the template, the middle base 13 of the template, and the 3'-end base 14 of the template) is completely complementary to the probe sequence (including: the starting base 4, the middle base 5 of the probe, and the 3'-end base 6 of the probe).

[0057] S7 separates the newly synthesized template sequences (including the first base 7 at the 5ʹ end of the template, the middle base 13 of the template, and the base 14 at the 3ʹ end of the template) from the probe sequences (including the starting base 4, the middle base 5 of the probe, and the base 6 at the 3ʹ end of the probe) on the initial gene chip finished product by means of heating denaturation, and separates the initial gene chip from the printing plate fixing carrier 12 by means of hydraulic pressing. The initial gene chip can continue to be used for the next round of printing plate preparation, while the printing plate fixing carrier 12 together with the micro-needle clusters fixed thereon and the newly synthesized template clusters at the micro-needle tips will be transferred to subsequent processing steps such as cleaning, and finally encapsulated and stored for corresponding quality and performance testing. The qualified product (printing plate) is used for gene chip production.

[0058] S8 provides a method for manufacturing a substrate for in-situ synthesized gene chip probes by a combination method, and manufactures a substrate for in-situ synthesized gene chip probes by a combination method using a standard-mode initial substrate; manufactures a gene chip by a method for in-situ synthesizing gene chip probes using DNA polymerase; the specific operation process is as follows:

[0059] (1) Transfer the substrate with the first nucleotide (starting base 4) at the 5ʹ end of all gene chip probes into a gene chip synthesizer, and set up a DNA polymerase reaction system on the substrate (including: DNA polymerase 15 of bacteriophage Φ29, terminal protein of bacteriophage Φ29, p6 single-stranded DNA binding protein of bacteriophage Φ29, four DNA synthesis substrates dNTP, and corresponding components required for DNA polymerase to synthesize DNA).

[0060] (2) Immerse the printing plate into the DNA polymerase reaction buffer of bacteriophage Φ29 (containing the p6 single-stranded DNA binding protein of bacteriophage Φ29). The p6 single-stranded DNA binding protein of bacteriophage Φ29 binds to the template sequences (including the first base 7 at the 5ʹ end of the template, the middle base 13 of the template, and the base 14 at the 3ʹ end of the template) at the micro-needle tips 10 on the printing plate, preventing the template sequences from forming secondary structures and promoting the assembly of templates to form a DNA replication initiation complex.

[0061] (3) Use the printing plate fixing carrier 12 to accurately position and assemble the printing plate and the substrate together, ensuring that the first base 14 at the 3' end of the template sequence (including: the first base 7 at the 5' end of the template, the middle base 13 of the template, and the base 14 at the 3' end of the template) bound to each micro-needle tip 10 pairs with the starting base 4 at the corresponding position on the substrate, and effectively assemble the DNA replication initiation complex. Use the DNA polymerase 15 of bacteriophage Φ29 to automatically and faithfully synthesize the probe sequence (including: the starting base 4, the middle base 5 of the probe, and the base 6 at the 3' end of the probe) on the substrate according to the base complementary pairing principle under the guidance of the template sequence. The newly synthesized probe sequence (including: the starting base 4, the middle base 5 of the probe, and the base 6 at the 3' end of the probe) is completely complementary to the template sequence on the printing plate (including: the first base 7 at the 5' end of the template, the middle base 13 of the template, and the base 14 at the 3' end of the template).

[0062] (4) Use the method of heating denaturation to dissociate the newly synthesized probe sequence and the template sequence on the gene chip, and separate the newly synthesized gene chip from the printing plate (together with the printing plate fixing carrier 12) by means of hydraulic pressure. The printing plate can be continuously used for the next round of gene chip preparation, while the substrate that has completed probe synthesis will be transferred to subsequent processing steps such as cleaning, and finally encapsulated and stored for corresponding quality and performance detection. Qualified gene chips will be put on the market.

[0063] As described above, it is only the preferred specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a gene chip, characterized in that, Transfer the initial gene chip to a printing plate and then print the gene chip. The method includes the following steps: S1 Obtain the initial gene chip product and related information, where the information at least includes: the probe sequences of the initial gene chip and their related arrangement order, and the dot density of the initial gene chip; S2 Prepare micro-needle clusters on four printing plate materials by etching technology, such that the micro-needle clusters match the dot density of the initial gene chip, and each micro-needle position corresponds to a probe sequence; S3 Respectively perform surface activation on the tips of the micro-needles of the four printing plate materials, chemically modify and protect the surface activation groups, and perform surface passivation treatment on the printing plate materials to eliminate the influence of the printing plate materials on the subsequent template sequence synthesis; S4 Remove the chemically modified protecting groups on the micro-needles of the four printing plate materials respectively, and fix a nucleotide on the micro-needles by covalent connection, such that the nucleotide is covalently connected to the covalent connection point of the printing plate through the linker on the template, where each printing plate material is only connected with one nucleotide, and only one nucleotide is connected to the activation group at the tip of each micro-needle; S5 According to the probe sequences of the initial gene chip product and their related arrangement order, taking the complementary base of the 3'-terminal base of the probe sequence of the initial gene chip as a reference, draw the complementary base distribution map of the 3'-terminal base of the probe sequence; S6 According to the complementary base distribution map, respectively intercept the corresponding blocks from the four printing plate materials covalently connected with nucleotides obtained in S4, and paste them on the printing plate fixing carrier to obtain micro-needle clusters, where the micro-needle clusters correspond to the probes on the initial gene chip, and the base at the tip of each micro-needle is complementary to the 3'-terminal base of the probe sequence at the corresponding position; S7 Lay the DNA polymerase and the reaction system of the DNA polymerase on the initial gene chip product, and accurately place the printing plate fixing carrier carrying the micro-needle clusters on the gene chip product, such that the nucleotide covalently connected to the tip of the micro-needle is complementary paired with the 3'-terminal base of the probe sequence on the initial gene chip product; S8 Make the DNA polymerase assemble into a DNA replication initiation complex with the assistance of related proteins. The DNA replication initiation complex includes: the DNA polymerase of bacteriophage Φ29, the terminal protein of bacteriophage Φ29, the p6 single-stranded DNA binding protein of bacteriophage Φ29, four DNA synthesis substrates dNTP, and other components required for the DNA polymerase to synthesize DNA, The DNA replication initiation complex uses the probe sequence of the initial gene chip product as a guide, starts with the nucleotide at the tip of the micro-needle, and synthesizes the template sequence according to the base complementary pairing principle. The newly synthesized template sequence is completely complementary to the probe sequence; S9 Heat up to denature, separate the newly synthesized template sequence from the probe sequence on the initial gene chip product, separate the initial gene chip product from the printing plate fixing carrier by hydraulic pressure, and optionally use the initial gene chip product for the next round of printing plate preparation, Transfer the printing plate fixing carrier together with the micro-needle clusters fixed on the printing plate fixing carrier and the newly synthesized template clusters at the tips of the micro-needles, and perform post-treatment processes including a cleaning process to obtain a printing plate.

2. The method according to claim 1, the method comprising step S10, S10 manufactures a substrate for in-situ synthesizing gene chip probes in a combined manner using a standard-mode initial substrate or a customized-mode initial substrate, and obtains an initial substrate with the first nucleotide at the 5'-end of all gene chip probes; Uses DNA polymerase to in-situ synthesize gene chip probes and manufactures a gene chip. The specific operation process includes: (1) Transfer the initial substrate with the first nucleotide at the 5'-end of all gene chip probes into a gene chip synthesizer, Lay a DNA polymerase reaction system on the substrate. The DNA polymerase reaction system includes: DNA polymerase of bacteriophage Φ29, terminal protein of bacteriophage Φ29, p6 single-stranded DNA binding protein of bacteriophage Φ29, four DNA synthesis substrates dNTP, and other components required for the DNA polymerase to synthesize DNA; (2) Immerse the printing plate obtained in S9 into the DNA polymerase reaction buffer of bacteriophage Φ29. The reaction buffer contains the p6 single-stranded DNA binding protein of bacteriophage Φ29, The p6 single-stranded DNA binding protein of bacteriophage Φ29 binds to the template sequence structure at the tip of the microneedles on the printing plate to prevent the template sequence from forming a secondary structure; (3) Use a printing plate fixing carrier to accurately position and assemble the printing plate and the substrate together, so that the first base at the 3'-end of the template sequence bound to each microneedle tip pairs with the starting base at the corresponding position on the substrate, and is effectively assembled into a DNA replication initiation complex, The assembly includes that the DNA polymerase of bacteriophage Φ29 automatically synthesizes the probe sequence on the substrate under the guidance of the template sequence according to the base complementary pairing principle. Among them, the newly synthesized probe sequence is completely complementary to the template sequence on the printing plate; (4) Use heating denaturation to unpair the newly synthesized probe sequence on the gene chip from the template sequence, and separate the newly synthesized gene chip from the printing plate together with the printing plate fixing carrier by hydraulic pressurization to obtain a gene chip, Optionally, use the printing plate for the next round of gene chip preparation, Transfer the substrate that has completed probe synthesis to a post-treatment process including cleaning, Obtain a gene chip that has completed probe synthesis.

3. The method according to claim 1, wherein The method includes: using DNA polymerase to transcribe the probe sequence on the initial gene chip into the template sequence on the microneedle cluster of the printing plate, and then using DNA polymerase again to convert the template sequence on the microneedle cluster of the printing plate into the probe sequence on the newly synthesized gene chip, The method faithfully replicates the DNA sequence through DNA polymerase.

4. The method according to claim 1 or 2, characterized in that, Use an etching technique to prepare a microneedle cluster of the printing plate that matches the probe distribution on the initial gene chip, so that each microneedle corresponds to a specific probe.

5. The method according to claim 1 or 2, characterized in that, The microneedles are divided into microneedle tips and microneedle assembly parts.

6. The method according to claim 1 or 2, characterized in that, The gene chip and the printing plate can replicate each other. The method includes starting from the gene chip or the printing plate, and manufacturing a large number of printing plates through the mutual replication between the gene chip and the printing plate and between the printing plate and the gene chip to carry out the production of gene chips.

7. The method according to claim 1, characterized in that, The linking arm is a structure simulating peptide nucleic acid.

8. A printing plate obtained by the method according to any one of claims 1-7.

Images