A substrate for in-situ synthesized gene chip probes manufactured by a combination method

Abstract

The present invention belongs to the field of biochips, and in particular relates to a substrate for in-situ synthesized gene chip probes manufactured by a combination method. Specifically, a single starting base is densely planted on the substrate and surface passivation is completed. Then, according to the actual needs of the starting bases for future in-situ synthesized gene chip probes, a combination is intercepted from the substrate with a single starting base densely planted, and a substrate for in-situ synthesized gene chip probes is made on a support carrier by a combination method. This invention has more advantages when used in conjunction with the technology of in-situ synthesizing gene chip probes using DNA polymerase. This invention can avoid the difficulty in site selection during substrate activation, perform the same treatment on all activation sites, and reduce the manufacturing cost of the substrate for gene chip probe synthesis.

Description

Technical Field

[0001] The present invention relates to the field of biochips, and particularly to a substrate for in-situ synthesized gene chip probes manufactured by a combination method. 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., with broad application prospects and a very large market demand.

[0003] A gene chip is a type of biochip, which is prepared by colonizing a series of probes with known sequences on the substrate of the chip. It can be used for hybridization detection of specific labeled nucleic acids and can report nucleic acid information in the detection object through recognition detection and information processing.

[0004] The key technology in gene chip manufacturing includes how to effectively colonize 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 in a specific position on the substrate according to a designed program 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 colonized at specific positions on the substrate.

[0005] 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 for preparation. It can manufacture high-density gene chips, and the probes are covalently cross-linked to 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, with each round completing 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%. When manufacturing high-density gene chips by the in-situ synthesis method, due to the low synthesis yield, many synthesis steps, high cost of raw materials (nucleotides with protecting groups), and low efficiency of removing protecting genes, the cost of high-density gene chips is expensive and they cannot be applied on a large scale.

[0006] The post-synthesis cross-linking method first completes the preparation of probes and then prints them onto the substrate, greatly reducing the chip manufacturing cost, but the probe density of the chip is limited. Summary of the Invention

[0007] In order to solve the deficiencies existing in the prior art and address the high substrate cost caused by the activation of selective sites on the substrate during the production of gene chips, the present invention proposes a design of separately activating and combinatorially using the substrate, which can effectively reduce the process complexity of manufacturing the substrate for gene chip production and effectively reduce the manufacturing cost of gene chips.

[0008] The inventive concept of the present invention is a substrate for in-situ synthesizing gene chip probes manufactured by a combinatorial method, which is characterized by including the following steps:

[0009] S1 Activate the surface groups of the initial substrate for gene chip production according to the standard density, and protect the activated surface groups of the initial substrate with a protecting group.

[0010] S2 Perform surface passivation treatment on the initial substrate to eliminate background interference.

[0011] S3 Remove the protecting groups on the activated surface groups of the initial substrate, and connect the nucleotide with a linker to the activated surface groups of the initial substrate by covalent cross-linking; only one type of nucleotide is connected to each initial substrate, and all the nucleotides connected to the activated surface groups are the same; moreover, there is only one nucleotide (specifically called the starting base, which is the first nucleotide for synthesizing the probe) on each activated surface group of the initial substrate; four groups of initial substrates with different starting bases need to be produced and completed for selection during gene chip manufacturing.

[0012] S4 According to the types and distribution characteristics of the starting bases of the probes on the gene chip designed in advance, draw a combined diagram of the substrate for manufacturing the gene chip, and set the probes with the same first nucleotide at the 5' end in the same area in advance, which can greatly reduce the combination difficulty of the subsequent substrate for manufacturing the gene chip.

[0013] S5 According to the combined diagram of the substrate for manufacturing the gene chip and the distribution diagram of the first nucleotide and the starting base at the 5' end of the planned synthesized probe, intercept the corresponding small pieces of the substrate from the corresponding initial substrates that have completed the cross-linking of the starting bases, and assemble and manufacture the substrate for in-situ synthesizing gene chip probes on the support slide according to the combined diagram of the substrate for manufacturing the gene chip; in mass production, the specific work of intercepting the corresponding small pieces of the substrate according to the combined diagram of the substrate for manufacturing the gene chip can be entrusted to the company that manufactures the initial substrates with the corresponding starting base cross-linking, and only need to be combined onto the support carrier for gene chip manufacturing during gene chip production.

[0014] S6 Cooperate with the technology of in-situ synthesizing gene chip probes using DNA polymerase to synthesize the corresponding gene chip probes with the starting bases of the combined substrates on the support carrier, and complete the manufacturing of the gene chip.

[0015] Preferably, the substrate for in-situ synthesized gene chip probes is made by combining small substrates with different single starting bases.

[0016] Preferably, the feature that the initial substrate only connects the same starting base is utilized to avoid the manufacturing difficulties caused by selective activation. All the starting bases with protecting groups are synchronously planted using linkers and the protecting groups are removed synchronously, greatly simplifying the substrate processing procedure and effectively reducing the cost.

[0017] Preferably, each starting base is covalently cross-linked to the initial substrate through a linker.

[0018] Preferably, the initial substrate adopts a modular design and is composed of a series of strip-shaped modules. When combining to make the substrate for in-situ synthesized gene chip probes, only automatic combination according to the number of modules is required, and no later cutting process is needed, which is beneficial to realizing the automation of industrial production.

[0019] Preferably, four types of initial substrates that only connect the same starting base and are composed of a series of modules are first made according to the four starting bases; corresponding numbers of modules can be taken from the corresponding initial substrates that have completed the cross-linking of the starting bases according to the combination diagram of the substrate for making the gene chip, and combined on the support slide to make the substrate for in-situ synthesized gene chip probes, decoupling the substrate production from the gene chip production and realizing production organization through the blockchain mode.

[0020] Preferably, the substrate for in-situ synthesized gene chip probes made by the combination method can cooperate with the technology of in-situ synthesizing gene chip probes using DNA polymerase to complete the printing manufacture of gene chips, greatly reducing the manufacturing cost of gene chips.

[0021] Compared with the prior art, the present invention can avoid the difficulties in site selection during substrate activation, perform the same treatment on all activation sites, reduce the manufacturing cost of the substrate for gene chip probe synthesis; and the present invention can cooperate with the technology of in-situ synthesizing gene chip probes using DNA polymerase to realize the large-scale and rapid preparation of gene chips like printing, greatly reducing the manufacturing cost of gene chips. Description of the Drawings

[0022] Figure 1 Combination diagram of the substrate for making gene chips on the support slide (standard mode)

[0023] Figure 2 Combination diagram of the substrate for making gene chips on the support slide (customized mode)

[0024] Figure 3 Covalent cross-linking of the starting base on the initial substrate (showing the linker)

[0025] Figure 4Schematic diagram of automatic continuous synthesis of probes using DNA polymerase

[0026] In the figure: support slide 1, initial substrate 2 in standard mode (connected with a starting base, such as A), initial substrate 3 in standard mode (connected with a starting base, such as T), initial substrate 4 in standard mode (connected with a starting base, such as C), initial substrate 5 in standard mode (connected with a starting base, such as G), initial substrate 6 in customized mode (connected with a starting base, such as G), initial substrate 7 in customized mode (connected with a starting base, such as C), initial substrate 8 in customized mode (connected with a starting base, such as T), initial substrate 9 in customized mode (connected with a starting base, such as A), covalent cross-linking site 10 on the initial substrate, starting base linker 11, starting base 12, first base at the 3′ end of the template 13, middle base of the template 14, base at the 5′ end of the template 15, linker of the template 16, covalent cross-linking site 17 between the template and the microneedle, tip of the microneedle 18, assembly part of the microneedle 19, DNA polymerase 20, probe 21, last base of the probe 22. Specific implementation mode

[0027] The present invention will be further described in detail below with reference to 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. Chemical reagents and instruments used in the present invention can be purchased from commercial channels without special instructions.

[0028] Example 1

[0029] A substrate for in-situ synthesized gene chip probes manufactured by a combination method includes the following steps:

[0030] S1 Activate the surface groups of the initial substrate 2 for gene chip fabrication at a standard density, and protect the activated surface groups of the initial substrate with a protecting group;

[0031] S2 Perform surface passivation treatment on the initial substrate 2 to eliminate background interference;

[0032] S3 Remove the protecting groups on the activated surface groups of the initial substrate 2, and connect the nucleotide with a linker to the activated surface groups of the initial substrate by covalent cross-linking method (see Figure 3, the covalent cross-linking sites 10 on the initial substrate, the starting base linker arm 11, and the starting base 12); only one kind of nucleotide (such as A) is linked to the initial substrate 2, and all the nucleotides linked to the activated surface groups are the same; moreover, there is only one nucleotide (specifically called the starting base, which is the first nucleotide for synthesizing the probe, such as A) on each activated surface group of the initial substrate; four groups of initial substrates need to be fabricated, namely initial substrate 2, initial substrate 3, initial substrate 4, and initial substrate 5, each with a different starting base for selection during the manufacture of the gene chip;

[0033] S4 According to the types and distribution characteristics of the starting bases of the probes on the pre-designed gene chip, draw a combined diagram of the substrates for fabricating the gene chip, and pre-set the probes with the same first nucleotide at the 5' end in the same area (see the first base 13 at the 3' end of the template), which can greatly reduce the combination difficulty of fabricating the subsequent substrates for the gene chip;

[0034] S5 Based on the combined diagram of the substrates for fabricating the gene chip, according to the first nucleotide (i.e., the starting base) at the 5' end of the planned synthesized probe and the starting base distribution map, intercept the corresponding small substrate pieces from the corresponding initial substrate 4 that has completed the cross-linking of the starting base 12, and assemble and manufacture the substrate for in-situ synthesizing gene chip probes on the support slide 1 according to the combined diagram of the substrates for fabricating the gene chip; Preferred solution: In mass production, produce the four initial substrates according to the production method of the initial substrates in the standard mode. Each initial substrate is composed of a certain number of long strip modules. Take out the corresponding long strip modules according to the combined diagram of the substrates for fabricating the gene chip and assemble them on the support slide to form the substrate for in-situ synthesizing gene chip probes.

[0035] S6 Cooperate with the technology of in-situ synthesizing gene chip probes using DNA polymerase, and synthesize the corresponding gene chip probes with the starting bases of the combined substrates on the support carrier, and perform performance detection on the substrate for in-situ synthesizing gene chip probes fabricated by the combination method. The corresponding detection process is as follows:

[0036] On the substrate for in-situ synthesizing gene chip probes fabricated by the combination method (see Figure 1)The surface is covered with DNA polymerase and its corresponding reaction system (meeting the relevant elements for probe synthesis). Using precise positioning technology, the printing plate is accurately placed on the substrate for gene chip production. The tip 18 of the microneedle carries the first base 13 at the 3' end of the template and completes complementary pairing with the starting base 12 on the substrate 4. And a DNA replication initiation complex is assembled at the position of the starting base 12. The DNA replication initiation complex can automatically complete the synthesis of the probe 21 according to the principle of base complementary pairing, starting from the starting base 12, in the 5' to 3' direction, automatically and faithfully; the DNA polymerase 20 is the DNA polymerase of phage Φ29, and the corresponding enzyme reaction system includes: the terminal protein of phage Φ29, the p6 single-stranded DNA binding protein of phage Φ29, four DNA synthesis substrates dNTP, and the corresponding components and conditions required for the DNA polymerase to synthesize DNA.

[0037] The newly synthesized probe 21 is separated from the template carried by the tip 18 of the printing plate microneedle by using the method of heating and denaturation. The gene chip is separated from the printing plate by applying pressure. The gene chip with probe synthesis completed is removed from the system, washed and then transported to the subsequent processing procedure. After relevant inspections, it is encapsulated and stored for detecting corresponding standard samples to verify the performance of the substrate.

[0038] A large number of gene chips are produced by copying in a similar way to detect the application performance of the substrate for in-situ synthesized gene chip probes manufactured by a combination method in the connection production.

[0039] As mentioned above, it is only the preferred specific implementation manner 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 within the protection scope of the present invention.

Claims

1. A method for manufacturing a substrate for in-situ synthesized gene chip probes by a combination method, the method comprising the following steps: S1 Activate the surface groups of the initial substrate according to a standard density, and protect the surface groups on the activated initial substrate with a protecting group; S2 Perform surface passivation treatment on the initial substrate to eliminate background interference; S3 Remove the protecting group on the activated surface group of the initial substrate, and connect the nucleotide with a linker arm to the activated surface group of the initial substrate by covalent cross-linking; Wherein, the nucleotides connected to all the activated surface groups on each initial substrate are of the same kind; and, only one nucleotide is connected to each activated surface group of the initial substrate, and the nucleotide is the starting base, and the starting base is the first nucleotide for synthesizing the probe; Fabricate four groups of initial substrates with different starting bases; S4 According to the type and distribution characteristics of the starting bases of the probes on the gene chip designed in advance, draw a combination diagram of the substrate for fabricating the gene chip, wherein the probes with the same first nucleotide at the 5' end are arranged in the same area; S5 According to the combination diagram of the substrate for fabricating the gene chip, cut corresponding small pieces of the substrate from the corresponding initial substrates that have completed the cross-linking of the starting bases, and assemble the small pieces of the substrate on the support slide according to the combination diagram of the substrate for fabricating the gene chip to obtain the substrate for in-situ synthesized gene chip probes.

2. A method for manufacturing a gene chip, the method comprising steps S1-S5 in claim 1, and further comprising: S6 Starting from the starting base on the substrate for in-situ synthesized gene chip probes obtained in S5, use DNA polymerase to synthesize gene chip probes on the substrate according to complementary base pairing to obtain a gene chip with the probes.

3. The method according to claim 1 or 2, characterized in that, In S3, the planting of all the nucleotides with linker arms on one initial substrate is carried out synchronously.

4. The method according to claim 1 or 2, characterized in that The initial substrate comprises a series of long strip modules.

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