DNA Synthesis Methods

Abstract

A DNA synthesis chip and a DNA synthesis method, wherein the DNA synthesis chip is a thin film transistor semiconductor array, the thin film transistor semiconductor array comprises: a plurality of reaction sites and a plurality of reaction site control units composed of thin film transistors, and each reaction site control unit comprises at least one pixel circuit, the pixel circuit is composed of the thin film transistors for pixel addressing and control, and each reaction site is configured to be able to synthesize in parallel a DNA chain that is consistent with or different from other reaction sites. The DNA synthesis chip of the present application is designed based on thin film transistors, so that the DNA synthesis reaction can be carried out in a specific area (pixel) as needed, and is a truly programmable, high-throughput DNA synthesis chip.

Description

Technical Field

[0001] This article relates to, but is not limited to, the field of synthetic genomics, and in particular to a method for synthesizing DNA. Background Art

[0002] Since synthetic biology was proposed as a new branch of biological science at the beginning of this century, it has shown more and more extensive application potential in the fields of biomedicine, new energy, new materials, etc. As one of the most important and basic research directions of synthetic biology, synthetic genomics focuses on the de novo design and synthesis of the basic components of life, with core technologies such as deoxyribonucleic acid (DNA) synthesis, shearing, assembly and transfer as technical means. On the one hand, these effective DNA synthesis and editing methods can be used as research methods to explore the role of genes in disease treatment, drug production and other fields, help overcome diseases and serve mankind; secondly, DNA can naturally be used as a 4-base storage medium (A / T / C / G), with many advantages such as high information density, long storage time, mild environment during storage without excessive energy consumption, etc. In the face of the current situation of information explosion and the demand for massive information storage, DNA storage is also a better way to supplement silicon-based solid-state information storage.

[0003] At present, the replication of natural DNA sequences in vivo and in vitro is usually achieved through polymerase chain reaction (PCR) technology, which can quickly and high-throughput obtain DNA chains. However, this DNA synthesis method relies on template chains and can only obtain templates from existing biological genomes to synthesize complementary DNA chains, but cannot synthesize new DNA chains from scratch without transcription templates. Therefore, it cannot achieve the precise synthesis of short-chain DNA such as primers, probes, and adapters, and it cannot achieve the synthesis of long-chain DNA to meet the needs of information storage.

[0004] At present, the in vitro acquisition of single-stranded DNA sequences is based on the column synthesis technology of "phosphoramidite synthesis". This method is easy to automate, but it requires many chemical reaction steps to add each base, and the products and intermediates have many influences and complex interactions. How to ensure the success rate of the synthesis of the target base on the DNA chain is the key and difficulty of this process: Assuming that the success rate of adding bases in each step can reach 99.5%, that is, there is a 0.5% failure rate in each base addition cycle, when a section of DNA containing 100 bases is to be synthesized, the success rate is only 60.6%. Therefore, it is urgent to develop new DNA synthesis processes to reduce the error rate of each base addition cycle and increase the synthesis length of the DNA chain. Summary of the invention

[0005] The following is an overview of the subject matter described in detail herein. This overview is not intended to limit the scope of protection of this application.

[0006] The present application provides a DNA synthesis chip, which is a thin film transistor semiconductor array, and the thin film transistor semiconductor array includes: multiple reaction sites and multiple reaction site control units composed of thin film transistors, and each reaction site control unit includes at least one pixel circuit, and the pixel circuit is composed of the thin film transistor to form pixel addressing and control, and each reaction site is configured to be able to synthesize in parallel a DNA chain that is consistent with or different from other reaction sites.

[0007] In an embodiment of the present application, the thin film transistor semiconductor array may also include: a droplet-driven working electrode, a dielectric barrier layer, a hydrophobic functional layer, a droplet movement channel and a microbead accommodating hole, the droplet-driven working electrode is arranged on the pixel circuit, the dielectric barrier layer is arranged on the side of the droplet-driven working electrode away from the pixel circuit, the hydrophobic functional layer is arranged on the side of the dielectric barrier layer away from the pixel circuit, the microbead accommodating hole corresponds to the reaction sites one by one, and the multiple reaction sites are separated by the droplet movement channel.

[0008] In an embodiment of the present application, the droplet movement channel and the microbead accommodating hole can be defined by the dielectric barrier layer and the hydrophobic functional layer, the reaction site is arranged on the surface of the dielectric barrier layer away from the pixel circuit side and below the microbead accommodating hole, and the reaction site is located on the same planar layer as the droplet movement channel and the microbead accommodating hole.

[0009] In an embodiment of the present application, the thin film transistor semiconductor array may further include a functional layer arranged between the dielectric barrier layer and the hydrophobic functional layer, the droplet movement channel and the microbead accommodating hole are defined by the functional layer, the reaction site is arranged on the surface of the hydrophobic functional layer away from the pixel circuit side and above the microbead accommodating hole, the reaction site and the droplet movement channel and the microbead accommodating hole are located on different planar layers and are bridged by a flow channel.

[0010] In an embodiment of the present application, the thin film transistor semiconductor array may also include: a droplet-driven working electrode, a dielectric barrier layer, a hydrophobic functional layer, a droplet movement channel and a biochemical reaction working electrode, the droplet-driven working electrode is arranged on the pixel circuit, the dielectric barrier layer is arranged on the side of the droplet-driven working electrode away from the pixel circuit, the hydrophobic functional layer is arranged on the side of the dielectric barrier layer away from the pixel circuit, the droplet movement channel is defined by the dielectric barrier layer and the hydrophobic functional layer, the biochemical reaction working electrode corresponds one-to-one to the reaction site, and the biochemical reaction working electrode is configured to carry and limit the biochemical reaction.

[0011] In an embodiment of the present application, the biochemical reaction working electrode may be disposed on a side of the hydrophobic functional layer away from the pixel circuit, and the reaction site is located on a surface of the biochemical reaction working electrode away from the pixel circuit.

[0012] In an embodiment of the present application, a portion of the biochemical reaction working electrode may be covered by the dielectric barrier layer and / or the hydrophobic functional layer, and the remaining portion of the biochemical reaction working electrode is exposed, and the exposed portion of the biochemical reaction working electrode carries and limits the biochemical reaction.

[0013] In an embodiment of the present application, the thin film transistor semiconductor array may further include: a row drive signal line, a column drive signal line and a binding gold finger, and the pixel circuit is connected to the binding gold finger through at least one row drive signal line or column drive signal line.

[0014] In the embodiment of the present application, the thin film transistor semiconductor array may further include: a liquid inlet, a liquid outlet and an upper cover plate, and an upper cover plate common electrode is disposed on the upper cover plate.

[0015] In the embodiment of the present application, the thin film transistor may include a substrate, a bottom gate, a gate dielectric layer, an active layer, an active doping layer, a source electrode, a drain electrode and a passivation layer.

[0016] The present application also provides a DNA synthesis method, which is performed using the DNA synthesis chip as described above, and comprises:

[0017] Reaction substrates and reagents for DNA synthesis are injected into the thin film transistor semiconductor array. Under the control of the pixel circuit, the reaction substrates and reagents for DNA synthesis synthesize DNA chains at the reaction sites.

[0018] In an embodiment of the present application, the DNA synthesis method may be performed using a DNA synthesis chip having a thin film transistor semiconductor array including microbead receiving holes as described above, and the DNA synthesis method may include:

[0019] S1: dispersing microbeads as carriers for DNA chain synthesis into microbead receiving holes of the thin film transistor semiconductor array;

[0020] S2: injecting reaction substrates and reagents for DNA synthesis into the thin film transistor semiconductor array, wherein the reaction substrates and reagents are dispersed into droplets under the control of the pixel circuit, and the pixel circuit controls the droplet driving working electrode, and the droplet driving working electrode drives the droplets containing the reaction substrate having the first target base to reach the position of the microbeads through the droplet movement channel, contact the microbeads, and react on the surface of the microbeads, and the first target base of the reaction substrate having the first target base is connected to the microbeads;

[0021] S3: Introducing droplets containing reaction substrates having the second, third, fourth, ... Nth target bases into the location of the microbeads and performing a base extension process to obtain a desired DNA chain.

[0022] In the embodiment of the present application, the DNA synthesis method can be performed using a DNA synthesis chip having a thin film transistor semiconductor array including a biochemical reaction working electrode as described above, including:

[0023] S1': injecting reaction substrates and reagents for DNA synthesis into the thin film transistor semiconductor array, wherein the reaction substrates and reagents are dispersed into droplets under the control of the pixel circuit, and the pixel circuit controls the droplet driving working electrode, wherein the droplet driving working electrode drives the droplets containing the reaction substrate having the first target base to reach the position of the biochemical reaction working electrode via the droplet movement channel, contact the biochemical reaction working electrode, and react on the surface exposed by the biochemical reaction working electrode, wherein the first target base of the reaction substrate having the first target base is connected to the biochemical reaction working electrode;

[0024] S2': Introducing a droplet containing a reaction substrate having the second, third, fourth, ... Nth target base into the position where the biochemical reaction working electrode is located and performing a base extension process to obtain a desired DNA chain.

[0025] In the embodiments of the present application, the first, second, third, fourth, ... Nth target bases may all carry a dimethyl terephthalate protecting group;

[0026] The base extension process includes multiple cycle reactions, and each cycle reaction includes: a deprotection group reaction, a coupling reaction, and an oxidation reaction.

[0027] In the embodiments of the present application, in each cycle reaction, the reaction substrate and reagents pass through the channel where the microbeads connected with the target base or the biochemical reaction working electrode are located.

[0028] In the embodiment of the present application, after each cycle reaction, the reaction substrate and reagent do not pass through the channel where the microbeads connected to the target base or the biochemical reaction working electrode are located.

[0029] The DNA synthesis chip and DNA synthesis method of the present application are designed based on thin film transistors (TFTs). A large number of DNA chemical synthesis reaction sites are distributed on the surface of a thin film transistor array according to a specific rule, and then a suitable addressing and synthesis control mechanism is designed to automatically control the DNA chain synthesis pathway and synthesis process of tens of thousands of chemical synthesis reaction sites on the TFT array, so that the DNA synthesis reaction can be carried out in a specific area (pixel) as needed. This is a truly programmable, high-throughput DNA synthesis chip.

[0030] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0032] Figure 1 A schematic diagram of a DNA synthesis chip of one exemplary embodiment of the present application;

[0033] Figure 2 An enlarged view of a local pixel of a DNA synthesis chip of a structure of an exemplary embodiment of the present application;

[0034] Figure 3 A cross-sectional side view of a single pixel of a DNA synthesis chip of a structure of an exemplary embodiment of the present application;

[0035] Figure 4 A side cross-sectional view of a single pixel of a DNA synthesis chip of another structure according to an exemplary embodiment of the present application;

[0036] Figure 5 A schematic diagram of the reaction route of the chemical reaction corresponding to the extension of the base;

[0037] Figure 6 It is a schematic diagram of the structure of a specific probe synthesized by the DNA synthesis method of the present application.

[0038] The meanings of the symbols in the accompanying drawings are:

[0039] 101A / 101B-substrate; 102A / 102B-bottom gate; 103A / 103B-gate dielectric layer; 104A / 104B-active layer; 105A / 105B-active doping layer; 106A / 106B-source-drain electrode; 107A / 107B-passivation layer; 108 / 108A / 108B-droplet-driven working electrode; 109 / 109A / 109B-dielectric barrier layer; 10 10 / 1010A / 1010B-hydrophobic functional layer; 1011B-biochemical reaction working electrode; 401-binding gold finger; 204 / 204A-microbeads; 301-DNA barcode; 302-characteristic multiple index segment DNA; 303-synthesized target DNA chain; 304-DNA base to be connected; 305-capping protection group; 402-row drive signal line; 403-column drive signal line. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present application more clear, the embodiments of the present application will be described in detail below with reference to the accompanying drawings. It should be noted that the embodiments and features in the embodiments of the present application can be combined with each other arbitrarily without conflict.

[0041] An embodiment of the present application provides a DNA synthesis chip, which is a thin film transistor (TFT) semiconductor array, and the thin film transistor semiconductor array includes: multiple reaction sites and multiple reaction site control units composed of thin film transistors, and each reaction site control unit includes at least one pixel circuit, and the pixel circuit is composed of the thin film transistor to form pixel addressing and control, and each reaction site is configured to be able to synthesize in parallel a DNA chain that is consistent with or different from other reaction sites.

[0042] In an embodiment of the present application, the thin film transistor semiconductor array of the DNA synthesis chip of the first structure may also include: a droplet-driven working electrode, a dielectric barrier layer, a hydrophobic functional layer, a droplet movement channel and a microbead accommodating hole, wherein the droplet-driven working electrode is arranged on the pixel circuit, the dielectric barrier layer is arranged on the side of the droplet-driven working electrode away from the pixel circuit, the hydrophobic functional layer is arranged on the side of the dielectric barrier layer away from the pixel circuit, the microbead accommodating hole corresponds to the reaction sites one by one, and the multiple reaction sites are separated by the droplet movement channel.

[0043] In an embodiment of the present application, the droplet movement channel and the microbead accommodating hole can be defined by the dielectric barrier layer and the hydrophobic functional layer, and the reaction site is arranged on the surface of the dielectric barrier layer away from the pixel circuit side and below the microbead accommodating hole. At this time, the reaction site and the droplet movement channel and the microbead accommodating hole are located on the same planar layer.

[0044] In an embodiment of the present application, the thin film transistor semiconductor array may further include a functional layer arranged between the dielectric barrier layer and the hydrophobic functional layer, the droplet movement channel and the microbead accommodating hole are defined by the functional layer, and the reaction site is arranged on the surface of the hydrophobic functional layer away from the pixel circuit side and above the microbead accommodating hole. At this time, the reaction site and the droplet movement channel and the microbead accommodating hole are located on different planar layers, but at the position where connection is required, the reaction site and the droplet movement channel and the microbead accommodating hole are bridged by a flow channel.

[0045] In an embodiment of the present application, the thin film transistor semiconductor array of the DNA synthesis chip of the second structure may also include: a droplet-driven working electrode, a dielectric barrier layer, a hydrophobic functional layer, a droplet movement channel and a biochemical reaction working electrode, wherein the droplet-driven working electrode is arranged on the pixel circuit, the dielectric barrier layer is arranged on the side of the droplet-driven working electrode away from the pixel circuit, the hydrophobic functional layer is arranged on the side of the dielectric barrier layer away from the pixel circuit, the droplet movement channel is defined by the dielectric barrier layer and the hydrophobic functional layer, the biochemical reaction working electrode corresponds one-to-one to the reaction site, and the biochemical reaction working electrode is configured to carry and limit the biochemical reaction.

[0046] In an embodiment of the present application, the biochemical reaction working electrode may be disposed on a side of the hydrophobic functional layer away from the pixel circuit, and the reaction site is located on a surface of the biochemical reaction working electrode away from the pixel circuit.

[0047] In an embodiment of the present application, a portion of the biochemical reaction working electrode is covered by the dielectric barrier layer and / or the hydrophobic functional layer, and the remaining portion of the biochemical reaction working electrode is exposed, and the exposed portion of the biochemical reaction working electrode carries and defines the biochemical reaction. The reaction site is located on the surface of the exposed portion of the biochemical reaction working electrode.

[0048] In the description of the present application, “the droplet movement channel (and the microbead accommodating hole) are defined by…” means that the droplet movement channel (and the microbead accommodating hole) are defined by…, and the formation method is, for example, before the hydrophobic functional layer is arranged on the dielectric barrier layer, the droplet movement channel (and the microbead accommodating hole) is specially arranged on the front layer according to a certain three-dimensional shape, and then the dielectric barrier layer and the hydrophobic functional layer are arranged above these positions, so that the dielectric barrier layer and the hydrophobic functional layer define the droplet movement channel (and the microbead accommodating hole) on the front layer with a three-dimensional shape; it is also possible to specially arrange the droplet movement channel (and the microbead accommodating hole) on the dielectric barrier layer according to a certain three-dimensional shape before the hydrophobic functional layer is arranged. The resistive barrier layer is provided with a liquid drop movement channel (and a microbead accommodating hole), and then a hydrophobic functional layer is provided above these positions, so that the hydrophobic functional layer defines a liquid drop movement channel (and a microbead accommodating hole) on the three-dimensional dielectric resistive barrier layer; or when providing the dielectric resistive barrier layer and / or the hydrophobic functional layer, a part of the dielectric resistive barrier layer and / or the hydrophobic functional layer is specially provided according to a certain three-dimensional shape and / or surface characteristics, so that the part of the dielectric resistive barrier layer and / or the hydrophobic functional layer has the structure and surface characteristics of the liquid drop movement channel (and the microbead accommodating hole), so that the part of the dielectric resistive barrier layer and / or the hydrophobic functional layer can serve as the liquid drop movement channel and the microbead accommodating hole.

[0049] In the description of the present application, the term "supporting and limiting biochemical reactions" refers to providing a carrier for the biochemical reaction so that the biochemical reaction can proceed on the carrier in a desired reaction direction.

[0050] In the embodiment of the present application, the thin film transistor semiconductor array may further include: row / column drive signal lines and binding gold fingers, and the pixel circuit is connected to the binding gold fingers through at least one row / column drive signal line.

[0051] Figure 1 A schematic diagram of a DNA synthesis chip of one exemplary embodiment of the present application; Figure 2 An enlarged view of a local pixel of a DNA synthesis chip of a structure of an exemplary embodiment of the present application; Figure 3 A cross-sectional side view of a single pixel of a DNA synthesis chip according to one exemplary embodiment of the present application.

[0052] In the exemplary embodiment of the present application, Figures 1 to 3As shown, the DNA synthesis chip is a thin film transistor semiconductor array, which includes: multiple reaction sites, multiple reaction site control units composed of thin film transistors, droplet drive working electrodes 108 / 108A, dielectric barrier layers 109 / 109A, hydrophobic functional layers 1010 / 1010A, binding gold fingers 401, row drive signal lines 402 and column drive signal lines 403; wherein each reaction site control unit includes at least one pixel circuit, the pixel circuit is composed of the thin film transistor to form pixel addressing and control, and each reaction site is The invention is configured to be able to synthesize in parallel a DNA chain that is consistent with or different from other reaction sites; the droplet-driven working electrode 108 / 108A is arranged on the pixel circuit, the dielectric barrier layer 109 / 109A is arranged on the side of the droplet-driven working electrode 108 / 108A away from the pixel circuit, the hydrophobic functional layer 1010 / 1010A is arranged on the side of the dielectric barrier layer away from the pixel circuit, and the dielectric barrier layer 109 / 109A and the hydrophobic functional layer 1010 / 1010A define a droplet movement channel (for example, Figure 2 The A-A' channel, C-C' channel and D-D' channel in the dielectric barrier layer 109 / 109A) and a microbead accommodating hole capable of accommodating microbeads 204, the reaction site is arranged on the surface of the dielectric barrier layer 109 / 109A away from the pixel circuit side and is arranged below the microbead accommodating hole, and the reaction site is located on the same plane layer as the droplet movement channel and the microbead accommodating hole (such as Figure 3 As shown), the multiple reaction sites are separated by the droplet movement channel; the pixel circuit is connected to the binding gold finger 401 through at least one row drive signal line 402 or column drive signal line 403.

[0053] The thin film transistor may include a substrate 101A, a bottom gate 102A, a gate dielectric layer 103A, an active layer 104A, an active doping layer (also called an active injection layer) 105A, a source-drain electrode 106A and a passivation layer 107A.

[0054] Figure 4 A side cross-sectional view of a single pixel of a DNA synthesis chip of another structure according to an exemplary embodiment of the present application.

[0055] In the exemplary embodiment of the present application, Figure 4 As shown, the DNA synthesis chip is a thin film transistor semiconductor array, which includes: a plurality of reaction sites, a plurality of reaction site control units composed of thin film transistors, a droplet driving working electrode 108B, a dielectric barrier layer 109B, a hydrophobic functional layer 1010B, a biochemical reaction working electrode 1011B, a binding gold finger and a row / column driving signal line (with Figure 1The binding gold finger 401, the row drive signal line 402 and the column drive signal line 403 are the same); wherein each reaction site control unit includes at least one pixel circuit, the pixel circuit is composed of the thin film transistor to form pixel addressing and control, and each reaction site is configured to be able to synthesize in parallel a DNA chain that is consistent with or different from other reaction sites; the droplet drive working electrode 108B is arranged on the pixel circuit, the dielectric barrier layer 109B is arranged on the side of the droplet drive working electrode 108B away from the pixel circuit, the hydrophobic functional layer 1010B is arranged on the side of the dielectric barrier layer 109B away from the pixel circuit, and the dielectric barrier layer 109B and the hydrophobic functional layer 1010B define a droplet movement channel (with Figure 2 The A-A' channel, C-C' channel and D-D' channel in the same); the biochemical reaction working electrode 1011B is arranged on a side of the hydrophobic functional layer 1010B away from the pixel circuit, and the biochemical reaction working electrode 1011B is configured to carry and limit the biochemical reaction; the pixel circuit is connected to the binding gold finger through at least one row / column driving signal line.

[0056] And, in Figure 4 In the DNA synthesis chip shown, the reaction site is located on the surface of the biochemical reaction working electrode away from the pixel circuit, and the entire surface of the biochemical reaction working electrode is exposed, so the entire surface of the biochemical reaction working electrode can carry and limit the biochemical reaction.

[0057] The thin film transistor may include a substrate 101B, a bottom gate 102B, a gate dielectric layer 103B, an active layer 104B, an active doping layer (also called an active injection layer) 105B, a source-drain electrode 106B and a passivation layer 107B.

[0058] In an embodiment of the present application, the thin film transistor semiconductor array may further include: a liquid inlet, a liquid outlet and an upper cover plate (not shown in the figure); the liquid inlet and the liquid outlet connect the thin film transistor semiconductor array with the outside world, the liquid inlet is used to add reaction substrates and reagents into the thin film transistor semiconductor array, and the liquid outlet is used to discharge the reaction substrates, reagents and reaction products after the reaction from the thin film transistor semiconductor array; the liquid inlet and the liquid outlet may be physical holes, grooves, pools, etc., or may be electrodes having a size equal to or larger than that of the droplet-driven working electrode 108 / 108A / 108B, and may be arranged on one side of the thin film transistor semiconductor array or on the upper cover plate; an upper cover plate common electrode is arranged on the upper cover plate. When the upper cover plate common electrode is in a pixelated array, the upper cover plate common electrode corresponds one-to-one with the droplet driving working electrode 108 / 108A / 108B; when the upper cover plate common electrode is an unpatterned whole-surface electrode, the upper cover plate common electrode can be aligned as a whole with the droplet driving working electrode.

[0059] In an embodiment of the present application, the reaction site control unit may include a temperature control and temperature measurement functional unit to monitor the temperature of the reaction site so as to provide a suitable reaction temperature when a reaction is carried out at the reaction site.

[0060] In the embodiment of the present application, the pixel circuit refers to a specially designed circuit unit in each pixel of the thin film transistor semiconductor array, which can be used to control the droplet drive working electrode, and then perform pixel addressing and control, and can also control the dispersion and directional movement of reaction substrates and reagents, as well as control the changes in necessary parameters (such as voltage, current, charge, ion concentration, temperature, acidity and alkalinity, etc.) in the DNA reaction process of the pixel, and then accurately control the DNA synthesis reaction. In addition to the above-mentioned pixel circuit, the reaction site control unit can also include some other auxiliary circuit modules or components, such as auxiliary electrodes, temperature control and temperature measurement functional units, etc.

[0061] The disclosed embodiment also provides a DNA synthesis method, which is performed using the DNA synthesis chip as described above, and includes: injecting reaction substrates and reagents for DNA synthesis into the thin film transistor semiconductor array, and under the control of the pixel circuit, the reaction substrates and reagents for DNA synthesis synthesize DNA chains at the reaction sites.

[0062] In the embodiment of the present application, the DNA synthesis method can be performed using a DNA synthesis chip having a first structure, including:

[0063] S1: dispersing microbeads as carriers for DNA chain synthesis into microbead receiving holes of the thin film transistor semiconductor array;

[0064] S2: injecting reaction substrates and reagents for DNA synthesis into the thin film transistor semiconductor array, wherein the reaction substrates and reagents are dispersed into droplets under the control of the pixel circuit, and the pixel circuit controls the droplet driving working electrode, and the droplet driving working electrode drives the droplets containing the reaction substrate having the first target base to reach the position of the microbeads through the droplet movement channel, contact the microbeads, and react on the surface of the microbeads, and the first target base of the reaction substrate having the first target base is connected to the microbeads;

[0065] S3: Introducing droplets containing reaction substrates having the second, third, fourth, ... Nth target bases into the location of the microbeads and performing a base extension process to obtain a desired DNA chain.

[0066] In the examples of the present application, the first, second, third, fourth, ... Nth target bases all carry a dimethyl terephthalate protecting group;

[0067] The base extension process includes multiple cycle reactions, and each cycle reaction includes: a deprotection group reaction, a coupling reaction, and an oxidation reaction.

[0068] In the present embodiment, in each cycle reaction, the reaction substrate and reagent pass through the channel (such as Figure 2 A-A' channel in the figure).

[0069] In the present embodiment, after each cycle of reaction, the reaction substrate and reagent do not pass through the channel where the microbeads or biochemical reaction working electrodes connected to the target base are located (eg Figure 2 C-C' and D-D' channels in the

[0070] In an exemplary embodiment of the present application, the above-mentioned DNA synthesis method may include:

[0071] S1: synthesize a considerable number of DNA chains into carriers, i.e. microbeads (which can be gel microbeads used for sequencing, such as Figure 2 and Figure 3204, 204A), are mixed in a set ratio and dispersed on a thin film transistor semiconductor array, and can be dispersed by means of shaking, centrifugation, etc., so that the microbeads fall randomly but as evenly as possible into the microbead receiving holes of the thin film transistor semiconductor array; by detecting the change in capacitance between the droplet-driven working electrode in the microbead receiving hole on the DNA synthesis chip and the common electrode of the upper cover plate, it can be known whether there are microbeads embedded in the microbead receiving hole, as well as the distribution (mapping) of the microbeads on the entire panel array.

[0072] S2: The reaction substrates and reagents for DNA synthesis (for example, four base substrates A / C / G / T with protecting groups, sustained-release solution, acidic deprotection solution, etc.) are injected into the liquid inlet end of the thin-film transistor semiconductor array from the outside according to the designed probe sequence, and the reaction substrates and reagents are divided into sufficiently small droplets under the control of the pixel circuit; the droplets are separately and sequentially applied with external voltage / current driving signals according to a preset path and timing by an external signal programming method, and the pixel circuit controls the droplet driving working electrode, and the droplet driving working electrode drives the droplet to the location of the microbead through the droplet movement channel to fully contact the microbead (at least the bottom of the microbead), and a chemical reaction corresponding to the extension of the base occurs on the surface of the microbead.

[0073] Figure 5 The schematic diagram of the reaction route of the chemical reaction corresponding to the extension of the base. Figure 5 As shown, the chemical reaction corresponding to the extension of the base includes three main steps: "Deprotection" - "Coupling" - "Oxidation".

[0074] Specifically, firstly, a small droplet containing the first target base substrate is introduced to the location of the microbead and fully contacts the microbead, so that the 3' end of the base is connected to the microbead;

[0075] The following step S3 may include:

[0076] Then, a small droplet containing a second base substrate is introduced to allow coupling between the two bases.

[0077] Next, oxidation is performed, for example, oxidizing the phosphorous acid group into a phosphoric acid group;

[0078] Finally, an acidic slow-release solution containing hydrogen ions is introduced (or inducing conditions such as ultraviolet light are introduced) to remove the DMT protecting group connected to the 5' hydroxyl group of the second base, leaving a free 5' hydroxyl group.

[0079] By repeating the above process of "deprotection" - "coupling" - "oxidation", the target base will be connected to the previous 5' base end in a predetermined sequence, and the DNA chain will continue to extend. The positions of different pixels on the DNA synthesis chip will synthesize the desired DNA chain according to the design intention.

[0080] Figure 6 Schematic diagram of the structure of a specific probe synthesized by the DNA synthesis method of the present application. Figure 6 As shown, the specific probe is connected to a microbead 204, which contains a necessary DNA barcode (DNABarcode) 301, a characteristic multiple index segment DNA (NMI) 302, a synthesized target DNA chain 303, a DNA base to be connected 304, and a blocking protective group 305 that appears during the synthesis process.

[0081] In the embodiment of the present application, the DNA synthesis method can be performed using a DNA synthesis chip having a second structure, including:

[0082] S1': injecting reaction substrates and reagents for DNA synthesis into the thin film transistor semiconductor array, wherein the reaction substrates and reagents are dispersed into droplets under the control of the pixel circuit, and the pixel circuit controls the droplet driving working electrode, wherein the droplet driving working electrode drives the droplets containing the reaction substrate having the first target base to reach the position of the biochemical reaction working electrode via the droplet movement channel, contact the biochemical reaction working electrode, and react on the surface exposed by the biochemical reaction working electrode, wherein the first target base of the reaction substrate having the first target base is connected to the biochemical reaction working electrode;

[0083] S2': Introducing a droplet containing a reaction substrate having the second, third, fourth, ... Nth target base into the position where the biochemical reaction working electrode is located and performing a base extension process to obtain a desired DNA chain.

[0084] In the examples of the present application, the first, second, third, fourth, ... Nth target bases all carry a DMT protecting group.

[0085] In the present embodiment, in each cycle reaction, the reaction substrate and reagent pass through the channel (such as Figure 2 A-A' channel in the figure).

[0086] In the present embodiment, after each cycle of reaction, the reaction substrate and reagent do not pass through the channel where the microbeads or biochemical reaction working electrodes connected to the target base are located (eg Figure 2 C-C' and D-D' channels in the

[0087] In an exemplary embodiment of the present application, the above-mentioned DNA synthesis method may include:

[0088] S1': Four base substrates A / C / G / T with protective groups, slow-release solution, acidic deprotection solution, etc. are injected into the liquid inlet end of the thin film transistor semiconductor array from the outside respectively and sequentially according to the designed probe sequence, and are divided into sufficiently small droplets; the droplet containing the first target base substrate is introduced into the position of the biochemical reaction working electrode and is in full contact with the biochemical reaction working electrode, so that the 3' end of the base is connected to the biochemical reaction working electrode;

[0089] S2': A small droplet containing a second base substrate is introduced to allow coupling between the two bases, followed by oxidation and deprotection group reactions to achieve base extension and obtain the desired DNA chain.

[0090] In the description of the present application, it should be noted that the terms "upper", "lower", "one side", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the structure referred to has a specific orientation, is constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present application.

[0091] Although the embodiments disclosed in this application are as above, the contents described are only embodiments adopted to facilitate understanding of this application and are not intended to limit this application. Any technician in the field to which this application belongs can make any modifications and changes in the form and details of implementation without departing from the spirit and scope disclosed in this application, but the scope of patent protection of this application shall still be based on the scope defined in the attached claims.

Claims

1. A method for DNA synthesis, characterized in that: The DNA synthesis chip is carried out, and the DNA synthesis chip is a thin film transistor semiconductor array, and the thin film transistor semiconductor array includes: a plurality of reaction sites, a plurality of reaction site control units composed of thin film transistors, a droplet driving working electrode, a dielectric barrier layer, a hydrophobic functional layer, a droplet movement channel and a microbead receiving hole; Each reaction site control unit includes at least one pixel circuit, the pixel circuit is composed of the thin film transistor to form pixel addressing and control, and each reaction site is configured to be able to synthesize a DNA chain that is consistent with or different from other reaction sites in parallel; The droplet-driven working electrode is arranged on the pixel circuit, the dielectric barrier layer is arranged on the side of the droplet-driven working electrode away from the pixel circuit, the hydrophobic functional layer is arranged on the side of the dielectric barrier layer away from the pixel circuit, the microbead-accommodating holes correspond to the reaction sites one by one, and the multiple reaction sites are separated by the droplet movement channel; The DNA synthesis method comprises: S1: dispersing microbeads as carriers for DNA chain synthesis into microbead receiving holes of the thin film transistor semiconductor array; S2: injecting reaction substrates and reagents for DNA synthesis into the thin film transistor semiconductor array, wherein the reaction substrates and reagents are dispersed into droplets under the control of the pixel circuit, wherein the pixel circuit controls the droplet driving working electrode, wherein the droplet driving working electrode drives the droplets containing the reaction substrate having the first target base to reach the position of the microbeads via the droplet movement channel, contact the microbeads, and react on the surface of the microbeads, wherein the first target base of the reaction substrate having the first target base is connected to the microbeads; S3: Introducing droplets containing reaction substrates having the second, third, fourth, ... Nth target bases into the location of the microbeads and performing a base extension process to obtain a desired DNA chain.

2. The DNA synthesis method according to claim 1, characterized in that The first, second, third, fourth, ... Nth target bases all carry a dimethyl terephthalate protecting group; The base extension process includes multiple cycle reactions, and each cycle reaction includes: a deprotection group reaction, a coupling reaction, and an oxidation reaction.

3. The DNA synthesis method according to claim 2, characterized in that In each cycle reaction, the reaction substrate and reagents pass through the channel where the microbeads to which the target bases have been attached are located.

4. The DNA synthesis method according to claim 2, characterized in that After each cycle reaction, the reaction substrate and reagents do not pass through the channel where the microbeads to which the target bases have been linked are located.

5. The method for DNA synthesis according to any one of claims 1 to 4, characterized in that: The droplet movement channel and the microbead accommodating hole are defined by the dielectric barrier layer and the hydrophobic functional layer, the reaction site is arranged on the surface of the dielectric barrier layer away from the pixel circuit side and below the microbead accommodating hole, and the reaction site is located on the same planar layer as the droplet movement channel and the microbead accommodating hole.

6. The method for DNA synthesis according to any one of claims 1 to 4, characterized in that: The thin film transistor semiconductor array also includes a functional layer arranged between the dielectric barrier layer and the hydrophobic functional layer, the droplet movement channel and the microbead accommodating hole are defined by the functional layer, the reaction site is arranged on the surface of the hydrophobic functional layer away from the pixel circuit side and above the microbead accommodating hole, the reaction site and the droplet movement channel and the microbead accommodating hole are located on different planar layers and are bridged by a flow channel.

7. The method for DNA synthesis according to any one of claims 1 to 4, characterized in that: The thin film transistor semiconductor array further comprises: a row drive signal line, a column drive signal line and a binding gold finger, and the pixel circuit is connected to the binding gold finger through at least one row drive signal line or column drive signal line.

8. The method for DNA synthesis according to any one of claims 1 to 4, characterized in that: The thin film transistor semiconductor array further comprises: a liquid inlet, a liquid outlet and an upper cover plate, and an upper cover plate common electrode is arranged on the upper cover plate.

9. The method for DNA synthesis according to any one of claims 1 to 4, characterized in that: The thin film transistor includes a substrate, a bottom gate, a gate dielectric layer, an active layer, an active doping layer, a source electrode, a drain electrode and a passivation layer.

10. A method for DNA synthesis, characterized in that: The DNA synthesis chip is carried out, and the DNA synthesis chip is a thin film transistor semiconductor array, and the thin film transistor semiconductor array includes: a plurality of reaction sites, a plurality of reaction site control units composed of thin film transistors, a droplet driving working electrode, a dielectric barrier layer, a hydrophobic functional layer, a droplet movement channel and a biochemical reaction working electrode; Each reaction site control unit includes at least one pixel circuit, the pixel circuit is composed of the thin film transistor to form pixel addressing and control, and each reaction site is configured to be able to synthesize a DNA chain that is consistent with or different from other reaction sites in parallel; The droplet driving working electrode is arranged on the pixel circuit, the dielectric barrier layer is arranged on the side of the droplet driving working electrode away from the pixel circuit, the hydrophobic functional layer is arranged on the side of the dielectric barrier layer away from the pixel circuit, the droplet movement channel is defined by the dielectric barrier layer and the hydrophobic functional layer, the biochemical reaction working electrode corresponds to the reaction site one by one, and the biochemical reaction working electrode is configured to carry and define a biochemical reaction; The DNA synthesis method comprises: S1': injecting reaction substrates and reagents for DNA synthesis into the thin film transistor semiconductor array, wherein the reaction substrates and reagents are dispersed into droplets under the control of the pixel circuit, and the pixel circuit controls the droplet driving working electrode, wherein the droplet driving working electrode drives the droplets containing the reaction substrate having the first target base to reach the position of the biochemical reaction working electrode via the droplet movement channel, contact the biochemical reaction working electrode, and react on the surface exposed by the biochemical reaction working electrode, wherein the first target base of the reaction substrate having the first target base is connected to the biochemical reaction working electrode; S2': Introducing a droplet containing a reaction substrate having the second, third, fourth, ... Nth target base into the position where the biochemical reaction working electrode is located and performing a base extension process to obtain a desired DNA chain.

11. The method for DNA synthesis according to claim 10, characterized in that: The first, second, third, fourth, ... Nth target bases all carry a dimethyl terephthalate protecting group; The base extension process includes multiple cycle reactions, and each cycle reaction includes: a deprotection group reaction, a coupling reaction, and an oxidation reaction.

12. The DNA synthesis method according to claim 11, characterized in that: In each cycle reaction, the reaction substrate and reagent pass through the channel where the biochemical reaction working electrode connected to the target base is located.

13. The DNA synthesis method according to claim 11, characterized in that: After each cycle reaction, the reaction substrate and reagent do not pass through the channel where the biochemical reaction working electrode connected to the target base is located.

14. The method for DNA synthesis according to any one of claims 10 to 13, characterized in that: The biochemical reaction working electrode is arranged on a side of the hydrophobic functional layer away from the pixel circuit, and the reaction site is located on a surface of the biochemical reaction working electrode away from the pixel circuit.

15. The method for DNA synthesis according to any one of claims 10 to 13, characterized in that: A portion of the biochemical reaction working electrode is covered by the dielectric barrier layer and / or the hydrophobic functional layer, and the remaining portion of the biochemical reaction working electrode is exposed, and the exposed portion of the biochemical reaction working electrode carries and defines the biochemical reaction.

16. The method for DNA synthesis according to any one of claims 10 to 13, characterized in that: The thin film transistor semiconductor array further comprises: a row drive signal line, a column drive signal line and a binding gold finger, and the pixel circuit is connected to the binding gold finger through at least one row drive signal line or column drive signal line.

17. The method for DNA synthesis according to any one of claims 10 to 13, characterized in that: The thin film transistor semiconductor array further comprises: a liquid inlet, a liquid outlet and an upper cover plate, and an upper cover plate common electrode is arranged on the upper cover plate.

18. The method for DNA synthesis according to any one of claims 10 to 13, characterized in that: The thin film transistor includes a substrate, a bottom gate, a gate dielectric layer, an active layer, an active doping layer, a source electrode, a drain electrode and a passivation layer.

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