Gene sequencing method and gene sequencing system

Abstract

The embodiment of the invention discloses a gene sequencing method and a gene sequencing system. The gene sequencing method comprises the steps that a gene sequencing chip is provided, the gene sequencing chip comprises a plurality of micro-reaction chambers and a flow channel communicated with the micro-reaction chambers, one end of the flow channel is a sample inlet end, the other end of the flow channel is a sample outlet end, and the inner surface of the gene sequencing chip is subjected to hydrophobic modification; a sequencing buffer solution is introduced into the sample introduction end of the flow channel; introducing isolation gas into the sample introduction end of the flow channel to form an isolation gas column; introducing a water-phase reaction liquid into a sample introduction end of the flow channel; introducing sealing gas into the sample introduction end of the flow channel to form a sealing gas column; and carrying out a sequencing reaction through the gene sequencing chip. The gene sequencing method can at least partially solve the problem that in the prior art, the utilization rate of water-phase reaction liquid with high cost is low.

Description

Technical Field

[0001] The present disclosure relates to the field of gene sequencing technology, and in particular to a gene sequencing method and a gene sequencing system. Background Art

[0002] High-throughput microarray biochips are the core components of cutting-edge biological detection technologies such as digital PCR, digital ELISA and gene sequencing. High-throughput microarray biochips can be used to sequence hundreds of thousands, millions, or even hundreds of millions of DNA molecules at a time.

[0003] The high-throughput microarray biochip has a flow channel and multiple micro-pits connected to the flow channel. In a single gene sequencing cycle, it is necessary to sequentially introduce aqueous reaction liquid-oil phase sealing liquid-sequencing cleaning liquid-sequencing buffer into the flow channel. The oil phase sealing liquid can be used to seal the aqueous reaction liquid in each micro-pit of the high-throughput microarray biochip into an independent unit for biochemical reaction and signal detection.

[0004] The inventors found that when performing multiple gene sequencing cycles, after the previous cycle is completed, the flow channel is filled with sequencing buffer. The newly introduced aqueous reaction liquid in this cycle will be miscible with it and then diluted, and cannot meet the sequencing reaction requirements. Therefore, an excess liquid input volume (approximately 66% to 100% of the flow channel volume) is required to push the diluted aqueous reaction liquid out of the flow channel so that the aqueous reaction liquid retained in the flow channel can meet the sequencing reaction requirements. The cost of the aqueous reaction liquid is high. The above method will make the sequencing cost higher, and it may also aggravate the adsorption of proteins in the aqueous reaction liquid on the chip surface, resulting in changes in the surface characteristics of the chip, and ultimately affecting the sequencing quality. Summary of the invention

[0005] In view of this, the embodiments of the present disclosure provide a gene sequencing method and a gene sequencing system, which at least partially solve the problem of high cost and low utilization rate of aqueous phase reaction liquid in the prior art.

[0006] In a first aspect, the present disclosure provides a gene sequencing method, which adopts the following technical solution:

[0007] The gene sequencing method comprises:

[0008] A gene sequencing chip is provided, the gene sequencing chip comprising a plurality of micro-reaction chambers and a flow channel connected to the plurality of micro-reaction chambers, one end of the flow channel being a sample inlet end and the other end being a sample outlet end, and the inner surface of the gene sequencing chip being hydrophobically modified;

[0009] Introducing a sequencing buffer into the injection end of the flow channel;

[0010] Introducing an isolation gas into the injection end of the flow channel to form an isolation gas column;

[0011] introducing an aqueous reaction liquid into the injection end of the flow channel;

[0012] Introducing sealing gas into the injection end of the flow channel;

[0013] The gene sequencing chip is used to perform a sequencing reaction.

[0014] Optionally, the width of the flow channel is 1 mm to 20 mm, and the height is 30 μm to 200 μm; the injection flow rate of the isolation gas is 1 mm / s to 20 mm / s; and the injection flow rate of the sealing gas is 1 mm / s to 20 mm / s.

[0015] Optionally, the total volume of the isolation gas introduced is more than 10% of the volume of the flow channel; the total volume of the sealing gas introduced is more than 100% of the volume of the flow channel; the total volume of the aqueous reaction liquid introduced is 5% to 60% of the volume of the flow channel.

[0016] Optionally, the total volume of the sequencing buffer introduced is 3 to 15 times the volume of the flow channel.

[0017] Optionally, the width of the flow channel is 1 mm to 20 mm, and the height is 30 μm to 200 μm; the injection flow rate of the sequencing buffer is 5 mm / s to 50 mm / s.

[0018] Optionally, the isolation gas is air, nitrogen, oxygen, saturated water vapor or a rare gas; the sealing gas is air, nitrogen, oxygen, saturated water vapor or a rare gas.

[0019] The inner surface of the gene sequencing chip includes a portion inside the microreaction chamber and a portion of the surface between the microreaction chambers, and the entire inner surface of the gene sequencing chip has hydrophobic modification.

[0020] In a second aspect, the present disclosure provides a gene sequencing system, which adopts the following technical solutions:

[0021] The gene sequencing system comprises:

[0022] A gene sequencing chip, the gene sequencing chip comprising a plurality of micro-reaction chambers and a flow channel connected to the plurality of micro-reaction chambers, one end of the flow channel being a sample inlet end and the other end being a sample outlet end, the inner surface of the gene sequencing chip being hydrophobically modified;

[0023] A fluid supply device, comprising a buffer containing bottle, a reaction liquid containing bottle, a cleaning liquid containing bottle, a gas containing bottle, a control valve and a power source, wherein the buffer containing bottle, the reaction liquid containing bottle, the cleaning liquid containing bottle and the gas containing bottle are connected to the control valve through a pipeline, the control valve is connected to the sample inlet end of the flow channel through the pipeline, and the power source is connected to the sample outlet end of the flow channel through the pipeline;

[0024] Under the driving force provided by the power source and the control of the control valve, the fluid supply device sequentially supplies sequencing buffer, isolation gas, aqueous reaction liquid, sealing gas and sequencing cleaning liquid to the flow channel.

[0025] Optionally, the control valve is a rotary valve having at least four valve ports.

[0026] Optionally, the gene sequencing system further includes: a waste liquid collection bottle, the waste liquid collection bottle is connected to the power source through a pipeline, and the used fluid can be driven into the waste liquid collection bottle by the power source.

[0027] The disclosed embodiment provides a gene sequencing method and a gene sequencing system. In the process of applying the gene sequencing method to perform gene sequencing, a sequencing buffer is first introduced into the injection end of the flow channel, and then an isolation gas is introduced to form an isolation gas column. After the aqueous reaction liquid is introduced, a sealing gas is introduced to form a sealing gas column. The sealing gas is used to push out the aqueous reaction liquid in the flow channel, and each micro-reaction chamber is sealed into an independent unit, and then a sequencing reaction is performed. In the above process, on the one hand, the aqueous reaction liquid and the sequencing buffer are isolated by the isolation gas column, and the aqueous reaction liquid will not be diluted. On the other hand, the aqueous reaction liquid is sealed by the gas phase. When the gas phase is sealed, the aqueous reaction liquid is sandwiched between the isolation gas column and the sealing gas column, which can also ensure that the liquid column length of the aqueous reaction liquid remains stable during the dynamic liquid injection process, and the concentration of the effective ingredient is relatively uniform, so as to achieve a small volume of rapid injection of the aqueous reaction liquid, thereby improving the utilization efficiency of the aqueous reaction liquid and reducing the sequencing cost. In addition, the isolation gas column and the sealing gas column will not have any residue, and will not change the surface characteristics of the chip.

[0028] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following preferred embodiments are specifically cited and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 A flow chart of a gene sequencing method provided in an embodiment of the present disclosure;

[0031] Figure 2A schematic diagram of a gene sequencing process provided by an embodiment of the present disclosure;

[0032] Figure 3 A schematic diagram of a gene sequencing system provided in an embodiment of the present disclosure;

[0033] Figure 4 The embodiments of the present disclosure provide Figure 2 d is the actual picture of the state shown;

[0034] Figure 5 A comparison diagram of the fluorescence intensity when there is an "isolation gas column" and when there is no "isolation gas column" provided in the embodiment of the present disclosure;

[0035] Figure 6 A graph showing changes in fluorescence intensity of 200 tests of the gene sequencing method provided in an embodiment of the present disclosure;

[0036] Figure 7 This is a test graph of protein residue on the surface of a gene sequencing chip after 200 tests with and without an "isolation air column" provided in an embodiment of the present disclosure. DETAILED DESCRIPTION

[0037] The present disclosure is further described in detail below in conjunction with the accompanying drawings and implementations. It is understood that the specific implementations described herein are only used to explain the relevant content, rather than to limit the present disclosure. It should also be noted that, for ease of description, only the parts related to the present disclosure are shown in the accompanying drawings.

[0038] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in the present disclosure can be combined with each other. The technical solution of the present disclosure will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0039] Unless otherwise specified, the exemplary embodiments / embodiments shown will be understood as providing exemplary features of various details of some ways in which the technical concept of the present disclosure can be implemented in practice. Therefore, unless otherwise specified, the features of the various embodiments / embodiments can be combined, separated, interchanged and / or rearranged without departing from the technical concept of the present disclosure.

[0040] The terms used here are for the purpose of describing specific embodiments, and are not intended to be restrictive. As used here, unless the context clearly indicates otherwise, the singular forms "one (kind, person)" and "said (the)" are also intended to include plural forms. In addition, when the terms "comprise" and / or "include" and their variations are used in this specification, it is explained that there are stated features, integral bodies, steps, operations, parts, assemblies and / or their groups, but it is not excluded that there are or add one or more other features, integral bodies, steps, operations, parts, assemblies and / or their groups. It should also be noted that, as used here, the terms "substantially", "approximately" and other similar terms are used as approximate terms and not as degree terms, so that they are used to explain the inherent deviations of the measured values, calculated values ​​and / or the values ​​provided that will be recognized by those of ordinary skill in the art.

[0041] The present disclosure provides a gene sequencing method, specifically, Figure 1 As shown, the gene sequencing method includes:

[0042] Step S1, providing a gene sequencing chip, the gene sequencing chip comprising a plurality of micro-reaction chambers and a flow channel connected to the plurality of micro-reaction chambers, one end of the flow channel being an injection end and the other end being a sample output end, the inner surface of the gene sequencing chip being hydrophobically modified.

[0043] Among them, the micro-reaction chamber is the basic unit for chemical reactions. It is micro-nanoscale and can be cylindrical, truncated, grooved, quasi-truncated, quasi-hexagonal column structure, etc., or a combination thereof. Multiple micro-reaction chambers can be arranged in an array. For high-throughput sequencing, the number of micro-reaction chambers on a gene sequencing chip can be as high as 10 6 or 10 7 or 10 8 or 10 9 Or higher order of magnitude. The material of the gene sequencing chip can be glass, quartz, silicon-based, PMMA, PC and other plastic materials. The opening diameter of the micro-reaction chamber can be 0.2μm to 5μm, preferably 0.3μm to 3μm, and the period of the micro-reaction chamber is 0.4μm to 8μm, preferably 0.5μm to 5μm.

[0044] Optionally, the width of the flow channel of the gene sequencing chip is 1mm to 20mm, and the height is 30μm to 200μm. Its length can be 5cm to 10cm, which can be set specifically according to the length of the gene sequencing chip. For a gene sequencing chip, it may include only one flow channel, which is connected to all micro-reaction chambers, or it may include multiple (for example, 2 to 8) flow channels arranged in parallel, each flow channel is respectively connected to a part of the micro-reaction chambers, and each flow channel shares the injection end and the sample outlet end. The volume of the flow channel mentioned in the subsequent embodiments of the present disclosure refers to the volume of a single flow channel.

[0045] In one example, a gene sequencing chip includes a base plate and a cover plate including a plurality of micro-pits (which may be arranged in an array), the base plate and the cover plate having a side with micro-pits are bonded by an adhesive layer, the adhesive layer has a first groove extending along a first direction, the first direction is consistent with the length direction of the gene sequencing chip, and the first groove is connected to the plurality of micro-pits. The first groove is a flow channel, and each micro-pit corresponds to a micro-reaction chamber.

[0046] In another example, a gene sequencing chip includes a base plate and a cover plate including a plurality of micro-pits. The base plate has a second groove extending along a first direction, the first direction is consistent with the length direction of the gene sequencing chip, and a side of the base plate having the second groove is sealed and fixed to a side of the cover plate having the micro-pits, and the second groove is connected to the plurality of micro-pits. The second groove is a flow channel, and each micro-pit corresponds to a micro-reaction chamber. The sealing and fixing method can be UV adhesive bonding or laser bonding.

[0047] Optionally, the inner surface of the gene sequencing chip 1 includes a portion inside the micro-reaction chamber (including the surface of the side walls and the bottom), and a portion of the surface between the micro-reaction chambers. In the disclosed embodiment, the hydrophobic modification can be performed during the preparation of the gene sequencing chip, or after the preparation of the gene sequencing chip is completed. The above hydrophobic modified materials can be: fluorinated silanes or chlorosilanes, such as methylfluorosilane, trimethylfluorosilane, trichlorofluorosilane, etc.

[0048] In the disclosed embodiment, only the inner surface of the entire gene sequencing chip needs to be hydrophobically modified to meet the needs, and no differentiated modification is required, which greatly reduces the difficulty of chemical modification. In the prior art, when the oily fluid seals the aqueous reaction liquid in the micro-reaction chamber, in order to achieve a good sealing effect, it is necessary to differentiate and modify the inside and outside of the micro-reaction chamber. Specifically, the inside of the micro-reaction chamber (i.e., the bottom surface and the side wall) is hydrophilically modified, while the outside of the micro-reaction chamber (i.e., the surface between the micro-reaction chambers) is hydrophobically modified. This modification technology is difficult and the effect is unstable.

[0049] Step S2: introducing sequencing buffer into the injection end of the flow channel.

[0050] The sequencing buffer is mainly used to provide a suitable pH environment inside the gene sequencing chip for subsequent sequencing reactions.

[0051] The greater the amount of sequencing buffer introduced, the better the effect. Optionally, the total volume of sequencing buffer introduced is 3 to 15 times the volume of the flow channel.

[0052] When the sequencing buffer is drawn in by negative pressure, the injection flow rate of the sequencing buffer can be determined according to the negative pressure working pressure, and the flow resistance generated by the sequencing buffer flowing through the flow channel should be less than the negative pressure working pressure. For incompressible fluids, the calculation formula for the flow channel resistance R is R = 12QηL / h 3 w, where Q is the fluid flow rate, η is the fluid dynamic viscosity, L is the flow channel length, h is the flow channel height, and w is the flow channel width. For example, when the negative pressure working pressure is within 70kPa, taking the flow channel width as 1mm to 20mm and the height as 30μm to 200μm, the injection flow rate of the sequencing buffer is 5mm / s to 50mm / s. When providing positive pressure to drive the sequencing buffer to pass, the principle is the same and will not be repeated here.

[0053] Step S3, introducing isolation gas into the injection end of the flow channel to form an isolation gas column.

[0054] Exemplarily, when the negative pressure working pressure is within 70 kPa, taking the width of the flow channel as 1 mm to 20 mm and the height as 30 μm to 200 μm, the injection flow rate of the isolation gas is 1 mm / s to 20 mm / s.

[0055] The amount of the isolation gas introduced, i.e., the total volume of the isolation gas, should be sufficient to fit the side wall of the flow channel and separate the sequencing buffer. The total volume of the isolation gas introduced may be less than the volume of the flow channel. Preferably, the total volume of the isolation gas introduced is more than 10% of the volume of the flow channel, and more preferably 30% to 60%.

[0056] The isolation gas only needs to not affect the sequencing reaction. Optionally, the isolation gas is air, nitrogen, oxygen, saturated water vapor or a rare gas.

[0057] Step S4, introducing the aqueous reaction liquid into the injection end of the flow channel.

[0058] The amount of the aqueous phase reaction liquid introduced should be as low as possible to reduce costs, while fully filling the micro-reaction chamber and ensuring that the sequencing reaction is fully carried out. Preferably, the total volume of the aqueous phase reaction liquid introduced is 5% to 60% of the volume of the flow channel, and more preferably 20% to 30%.

[0059] For example, when the negative pressure working pressure is within 70 kPa, taking the width of the flow channel as 1 mm to 20 mm and the height as 30 μm to 200 μm, the injection flow rate of the aqueous reaction liquid is 5 mm / s to 50 mm / s.

[0060] Step S5, introducing sealing gas into the injection end of the flow channel.

[0061] Exemplarily, when the negative pressure working pressure is within 70 kPa, taking the width of the flow channel as 1 mm to 20 mm and the height as 30 μm to 200 μm, the injection flow rate of the sealing gas is 1 mm / s to 20 mm / s.

[0062] Among them, the amount of sealing gas introduced, that is, the total volume of the sealing gas, should be able to seal the aqueous reaction liquid in the micro-pit, so that the aqueous reaction liquid in each micro-reaction chamber is isolated from other micro-reaction chambers. The total volume of the sealing gas should be greater than the volume of the flow channel. Preferably, the total volume of the sealing gas introduced is more than 100% of the volume of the flow channel, and more preferably 120% to 300%.

[0063] The sealing gas can be any gas as long as it does not affect the sequencing reaction. Optionally, the sealing gas is air, nitrogen, oxygen, saturated water vapor or a rare gas. Preferably, the sealing gas is the same as the isolation gas.

[0064] Step S6: performing sequencing reaction using a gene sequencing chip.

[0065] The sequencing reaction of the nucleic acid molecules to be tested is carried out in multiple micro-reaction chambers on the gene sequencing chip. The nucleic acid molecules to be tested or their fragments can be fixed in the micro-reaction chambers. After providing an aqueous reaction liquid to each micro-reaction chamber, the nucleic acid molecules to be tested or their fragments in each micro-reaction chamber undergo a sequencing reaction with the aqueous reaction liquid.

[0066] The above sequencing reaction refers to a nucleic acid synthesis reaction catalyzed by nucleic acid polymerase. The nucleotides (or deoxynucleotides) used in the sequencing reaction can be natural or chemically modified. Among them, the 3' end of the chemically modified nucleotides (or deoxynucleotides) can be open or closed. Examples of sequencing methods using nucleotides (or deoxynucleotides) with open 3' ends include pyrophosphate sequencing, semiconductor sequencing, fluorescence sequencing, etc., and examples of sequencing methods using nucleotides (or deoxynucleotides) with closed 3' ends include reversible cycle termination method and dideoxynucleotide termination method, etc.

[0067] "High-throughput sequencing" or "next generation sequencing" includes sequence determination using methods that determine the sequences of many (typically thousands to billions) nucleic acids in an inherently parallel manner, i.e., where DNA templates are prepared for sequencing not one at a time, but rather are processed in batches, and preferably many sequences are read out in parallel, or using ultra-high-throughput serial processing that can itself be parallelized. Such methods include, but are not limited to, pyrosequencing (e.g., as commercialized by 454 Life Sciences, Inc., Branford, CT); sequencing by ligation (e.g., as commercialized in the SOLiD™ technology, Life Technologies, Inc., Carlsbad, CA); sequencing by synthesis using modified nucleotides (such as commercialized in the TruSeq™ and HiSeq™ technologies by Illumina, Inc., San Diego, CA; commercialized in the HeliScope™ by Helicos Biosciences Corporation, Cambridge, MA; and commercialized in the PacBio RS by Pacific Biosciences of California, Inc., Menlo Park, CA), sequencing by ion detection technology (such as the Ion Torrent™ technology, Life Technologies, Carlsbad, CA); sequencing by DNA nanoballs (Complete Genomics, Inc., Mountain View, CA); nanopore-based sequencing technology (e.g., as developed by Oxford Nanopore Technologies, LTD, Oxford, UK), and, for example, highly parallelized sequencing methods.

[0068] Optionally, the phosphoric acid of the nucleotide substrate is modified with a fluorophore, and an enzyme is used to release the fluorophore on the nucleotide substrate. Preferably, a DNA polymerase is first used to release the polyphosphate-substituted fluorophore, and then a phosphatase is used to remove the substituted polyphosphate, thereby releasing the fluorophore. Due to the sealed gas, each micro-reaction chamber is isolated from each other, and the released fluorescent group is confined in each micro-reaction chamber, successfully reflecting the sequence of the nucleic acid molecule to be tested.

[0069] Alternatively, the polyphosphate nucleotide substrate molecule may refer to a nucleotide having 4 to 9 phosphate molecules. The modified nucleotide substrate molecule having a fluorophore may be labeled with one fluorophore for single-color sequencing, or with different fluorophores for multi-color sequencing.

[0070] Alternatively, if Figure 2As shown, the gene sequencing method in the embodiment of the present disclosure further includes: after the sequencing reaction is completed, an appropriate amount of sequencing cleaning solution is introduced into the injection end of the flow channel to clean the gene sequencing chip. The sequencing cleaning solution is mainly used to clean and remove the aqueous reaction solution in the previous round of sequencing reaction. Figure 2 middle, Figure 2 a is the initial state of the gene sequencing chip filled with sequencing buffer. Figure 2 b is the state where a short isolation gas column is added after the sequencing buffer. Figure 2 c is the state of the aqueous phase reaction solution entering the gene sequencing chip. Figure 2 d is the state where a sealed gas column is added after the aqueous phase reaction liquid. Figure 2 e is the state where the gene sequencing chip is completely sealed by the sealed air column, and the sequencing reaction is performed in this state. Figure 2 f is the state where the chip is filled with the sequencing cleaning solution after the aqueous phase reaction solution has been washed away. Figure 2 a-2f is one sequencing cycle. If multiple sequencing cycles are required, just repeat the above process.

[0071] The greater the amount of sequencing cleaning solution introduced, the better the effect. Optionally, the total volume of the sequencing cleaning solution introduced is 3 to 15 times the volume of the flow channel.

[0072] Exemplarily, when the negative pressure working pressure is within 70 kPa, taking the width of the flow channel as 1 mm to 20 mm and the height as 30 μm to 200 μm, the injection flow rate of the sequencing cleaning solution is 5 mm / s to 50 mm / s.

[0073] The disclosed embodiment provides a gene sequencing method. In the process of applying the gene sequencing method to perform gene sequencing, a sequencing buffer is first introduced into the injection end of the flow channel, and then an isolation gas is introduced to form an isolation gas column. After the aqueous reaction liquid is introduced, a sealing gas is introduced to form a sealing gas column. The sealing gas is used to push the aqueous reaction liquid in the flow channel, and each micro-reaction chamber is sealed into an independent unit, and then a sequencing reaction is performed. In the above process, on the one hand, the aqueous reaction liquid and the sequencing buffer are isolated by the isolation gas column, and the aqueous reaction liquid will not be diluted. On the other hand, the aqueous reaction liquid is sealed by the gas phase. When the gas phase is sealed, the aqueous reaction liquid is sandwiched between the isolation gas column and the sealing gas column, which can also ensure that the liquid column length of the aqueous reaction liquid remains stable during the dynamic liquid injection process, and the concentration of the effective component is relatively uniform, so as to achieve a small volume of rapid injection of the aqueous reaction liquid, thereby improving the utilization efficiency of the aqueous reaction liquid and reducing the sequencing cost. Among them, the liquid injection volume of the aqueous reaction liquid is within 60% of the volume of the chip flow channel, and can be as low as 5%, which can meet the test needs.

[0074] In addition, in the prior art, proteins (such as enzymes, antigens, antibodies, etc.) and fluorescent group-modified nucleotides contained in the aqueous reaction liquid are often hydrophobic, and the oil phase sealing liquid will extract the effective ingredients in the microreaction chamber to a certain extent. When the aqueous reaction liquid is sealed by gas phase, the extraction of effective ingredients such as proteins and fluorescent group-modified nucleotides in the aqueous reaction reagent can also be avoided.

[0075] In addition, the isolation gas column and the sealing gas column are both in the gas phase and will not remain in the gene sequencing chip or change the surface properties of the chip, which is superior to the oil phase sealing in the prior art.

[0076] Through the above gene sequencing method, hundreds of times of alternating liquid injection of gas / liquid columns of different components in the gene sequencing chip can be automatically realized, showing potential commercial application prospects in the field of microfluidics, especially in the field of microarray gene sequencing. At the same time, this gene sequencing method can also be extended to fields such as digital ELISA and high-throughput drug screening, which helps to save the usage volume of rare samples.

[0077] In addition, the present disclosure provides a gene sequencing system, specifically, as Figure 3 As shown, the gene sequencing system includes:

[0078] A gene sequencing chip 1, comprising a plurality of micro-reaction chambers and a flow channel connected to the plurality of micro-reaction chambers, one end of the flow channel being a sample inlet end and the other end being a sample outlet end, and the inner surface of the gene sequencing chip 1 being hydrophobically modified;

[0079] The fluid supply device includes a plurality of liquid holding bottles 2 (including a buffer holding bottle, a reaction liquid holding bottle, and a cleaning liquid holding bottle), a gas holding bottle 3, a control valve 6, and a power source 5. The buffer holding bottle, the reaction liquid holding bottle, the cleaning liquid holding bottle, and the gas holding bottle 3 are connected through corresponding pipelines ( Figure 3 The control valve 6 is connected to the control valve 6 through the corresponding pipeline ( Figure 3 The power source 5 is connected to the injection end of the flow channel through the corresponding pipeline ( Figure 3 The sample outlet end of the flow channel is represented by pipeline 13);

[0080] Under the control of the negative pressure of the power source 5 and the control valve 6, the fluid supply device sequentially supplies sequencing buffer, isolation gas, aqueous reaction liquid, sealing gas and sequencing cleaning liquid to the flow channel.

[0081] Optionally, the width of the flow channel is 1 mm to 20 mm, and the height is 30 μm to 200 μm. The flow channel with the above dimensions can be applied to gene sequencing chips of various specifications. Optionally, the cross section of the flow channel is square, and the aspect ratio of the cross section of the flow channel is within 60. The flow channel with the above aspect ratio has lower process requirements and is easier to achieve when achieving the isolation effect of the isolation gas column and the sealing effect of the sealing gas column.

[0082] Optionally, the control valve 6 is a rotary valve having at least four valve ports, and the four valve ports correspond to the buffer solution holding bottle, the reaction solution holding bottle, the cleaning solution holding bottle and the gas holding bottle 3, respectively. Figure 3 In the example shown, the fluid supply device may further include other liquid holding bottles and / or gas holding bottles for holding other liquids or gases as required. For example, when the sealing gas and the isolation gas are different, the fluid supply device includes two gas holding bottles for holding the sealing gas and the isolation gas respectively. Then, the control valve 6 may further include a valve port corresponding to the other liquid holding bottle or the gas holding bottle, and the two are connected by a pipeline ( Figure 3 In the figure, it is shown as pipeline 12) connected.

[0083] Optionally, the power source 5 is a syringe pump, which includes an E port (injection port) and an O port (discharge port). The driving force provided by the power source 5 can be a driving force under positive pressure or an attractive force under negative pressure, which can be provided according to actual needs.

[0084] Optionally, all of the above pipelines are PTFE (polytetrafluoroethylene) pipelines, which can be used normally within -60°C to +260°C and have reliable and excellent corrosion resistance.

[0085] Alternatively, if Figure 3 As shown, the fluid supply device also includes: a waste liquid collection bottle 4, the waste liquid collection bottle 4 is connected through a pipeline ( Figure 3 The pipeline 14 (indicated in the figure) is connected to the power source 5, and the used fluid can be driven by the power source 5 to the waste liquid collection bottle 4.

[0086] In order to facilitate those skilled in the art to better understand and implement the gene sequencing method and gene sequencing system provided by the embodiments of the present disclosure, the embodiments of the present disclosure provide a specific embodiment and a corresponding comparative example below.

[0087] Embodiment 1:

[0088] The process of performing gene sequencing using the gene sequencing method and gene sequencing system provided by the embodiments of the present disclosure includes:

[0089] (1) Packaging of gene sequencing chips.

[0090] First, a glass plate with a length*width*thickness of 75mm*32mm*1mm and a micro-pit array quartz plate with a length*width*thickness of 65mm*25mm*0.7mm were selected as the bottom plate and cover plate of the gene sequencing chip respectively; then, the glass plate and the quartz plate were placed in a plasma cleaning machine (Plasma box) for cleaning for 7 minutes to clean the surface; then, a double-sided tape with a length*width*thickness of 65mm*25mm*0.05mm was selected as the bonding layer, and the first groove with a length*width*thickness of 60mm*3mm*0.05mm was cut out of the central symmetric film of the double-sided tape as the flow channel (width of 3mm); then, the double-sided tape was sandwiched between the glass plate and the quartz plate using a packaging tool; finally, the bonded gene sequencing chip was placed on a hot press, and hot-pressed at 70°C for 2 minutes under 120kgf to press and expel the air on both sides of the double-sided tape, so that the gene sequencing chip was tightly packaged.

[0091] (2) Hydrophobic modification of the inner surface of the microarray chip.

[0092] Use a pipette to fill the flow channel of the packaged gene sequencing chip with hydrophobic material (such as 3M electronic coating agent), let it stand for 6 minutes, and then slowly push the hydrophobic material out of the flow channel of the gene sequencing chip, leaving a hydrophobic coating on the inner surface of the gene sequencing chip. Place the gene sequencing chip on a heating plate and heat it at 135°C for 15 minutes to solidify the hydrophobic coating on the inner surface of the gene sequencing chip.

[0093] (3) Programmed fluid control and optical detection.

[0094] The implementation process for a single gene sequencing cycle is as follows:

[0095] Sequencing buffer injection stage: control valve 6 switches to the valve port connected to the buffer storage bottle, the syringe pump opens port E to form a pathway of "buffer storage bottle-control valve 6-gene sequencing chip 1-syringe pump", the syringe pump draws 100 μl of sequencing buffer at 20 μl / s to flow through the gene sequencing chip 1, the sequencing buffer is injected and fills the gene sequencing chip 1 under negative pressure, then the syringe pump closes port E and opens port O, the reagent in the syringe pump is emptied by positive pressure and collected in the waste liquid collection bottle 4.

[0096] In the “isolation gas column” injection stage: control valve 6 switches to the valve port corresponding to the gas holding bottle 3, and the injection pump opens the E port to form a “gas holding bottle 3-control valve 6-gene sequencing chip 1-injection pump” pathway. The injection pump draws 5 μl of gas into the gene sequencing chip 1 at 10 μl / s, and then the injection pump opens the O port to empty the reagent in the injection pump under positive pressure and collect it in the waste liquid collection bottle 4.

[0097] Aqueous reaction liquid injection stage: control valve 6 switches to the corresponding valve port of the reaction liquid holding bottle, and the injection pump opens port E to form a pathway of "reaction liquid holding bottle-control valve 6-gene sequencing chip 1-injection pump". The injection pump draws 2 μl of aqueous reaction liquid into the gene sequencing chip 1 at 10 μl / s, and then the injection pump opens port O to empty the reagent in the injection pump under positive pressure and collect it in the waste liquid collection bottle 4.

[0098] In the "sealed gas column" injection stage: the control valve 6 switches to the valve port corresponding to the gas holding bottle 3, and the syringe pump opens the E port to form a "gas holding bottle 3-control valve 6-gene sequencing chip 1-syringe pump" path. The syringe pump draws 20 μl of gas into the gene sequencing chip 1 at 4 μl / s. At this time, the gas volume is about twice the volume of the gene sequencing chip 1 cavity, so that the gas completely fills the gene sequencing chip 1 cavity to seal the micro-reaction chamber. Then the syringe pump opens the O port, and the reagent in the syringe pump is emptied by positive pressure and collected in the waste liquid collection bottle 4.

[0099] Sequencing reaction stage: After the micro-reaction chamber is sealed, the gene sequencing chip 1 is heated to 55° C. for 40 seconds to perform sequencing reaction, and the CCD camera scans the chip to detect the fluorescence signal of the sequencing reaction in the micro-reaction chamber.

[0100] Chip cleaning stage: control valve 6 switches to the valve port corresponding to the cleaning liquid holding bottle, and the injection pump opens port E to form a pathway of "cleaning liquid holding bottle-control valve 6-gene sequencing chip 1-injection pump". The injection pump draws 100 μl of sequencing cleaning liquid into and cleans the gene sequencing chip 1 at 20 μl / s. Then the injection pump opens port O to empty the reagent in the injection pump under positive pressure and collect it in the waste liquid collection bottle 4.

[0101] The above process is repeated according to the required number of sequencing reactions.

[0102] When the technical solution in the above embodiment 1 is adopted, the following technical effects are achieved:

[0103] like Figure 2 d and Figure 4 As shown, after completing the injection of sequencing buffer, "isolation gas column" injection, aqueous reaction liquid injection and "sealing gas column" injection, in the hydrophobic treated gene sequencing chip, the isolation gas column and the sealing gas column can fit tightly with the side wall of the gene sequencing chip, ensuring that the "isolation gas column" can well separate the sequencing buffer and the aqueous reaction liquid, avoiding the dilution of the aqueous reaction liquid by the sequencing buffer, improving the utilization efficiency of the aqueous reaction liquid, and reducing the sequencing cost. At the same time, the "sealing gas column" can also well seal the aqueous reaction liquid in each micro-reaction chamber to form an independent reaction space. Figure 4 The dark, light, dark and light parts arranged from left to right in the figure are: sequencing buffer, isolation gas column, aqueous reaction liquid and sealing gas column.

[0104] like Figure 5 As shown, a 10 μM PO (Peking Orange) fluorescent dye aqueous solution was used to simulate the aqueous phase reaction solution. Figure 5 The horizontal axis in a is the volume V of the aqueous phase reaction liquid inlet Reagent The flow channel volume V of the gene sequencing chip Chip The ordinate is the detected fluorescence intensity value. Figure 5 The vertical axis in b is the fluorescence intensity ratio when the "isolation gas column" is added to the corresponding volume of the aqueous phase reaction liquid inlet and when it is not added. Figure 5 It can be seen from a that as the volume of the aqueous phase reaction liquid increases, the fluorescence intensity when the "isolation gas column" is added is higher than when it is not added. Figure 5 b It can be seen that when the volume of the aqueous reaction liquid inlet is 20% of the flow channel volume of the gene sequencing chip, the aqueous reaction liquid is not diluted due to the effect of the "isolation gas column", and the fluorescence intensity in the microreaction chamber is 3 times that when the "isolation gas column" is not added, indicating that the gene sequencing method and gene sequencing system provided in the embodiments of the present disclosure can achieve the effect of saving the volume of sequencing reagents, etc.

[0105] In the disclosed embodiment, the fluorescence intensity in the micro-reaction chamber was tested after 200 injections of sequencing buffer, "isolation gas column", aqueous reaction solution, and "sealed gas column". Figure 6 It is a statistical graph of fluorescence intensity distribution of 200 rounds in the same field of view. The average value of fluorescence intensity of each round is about 800, and the CV value is about 5%, which shows that the gene sequencing method and gene sequencing system provided by the embodiments of the present disclosure have good stability and good applicability in the field of gene sequencing.

[0106] In addition, the protein residues were tested in the embodiments of the present disclosure with or without the addition of an isolation gas column. Figure 7 As shown in a, in the technical solution provided by the embodiment of the present disclosure, after 200 injections of sequencing buffer, "isolation gas column" injection, 20% of the chip's flow channel volume aqueous phase reaction liquid injection, and "sealed gas column" injection, no protein residue was seen on the surface of the gene sequencing chip, while Figure 7 As shown in b, after 200 injections of sequencing buffer, 100% of the chip's flow channel volume aqueous reaction solution, and "sealed gas column" injection, there is obvious protein residue on the surface of the gene sequencing chip ( Figure 7 The bright spots in b are the proteins in the aqueous reaction solution remaining on the chip surface. Figure 7 It can be explained that the small volume aqueous reaction liquid injection method of adding an "isolation gas column" in the embodiment of the present disclosure not only saves the volume of the aqueous reaction liquid, but also effectively alleviates protein adsorption and accumulation, thereby ensuring good sequencing quality.

[0107] In the description of this specification, the description with reference to the terms "one embodiment / method", "some embodiments / methods", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment / method or example are included in at least one embodiment / method or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment / method or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments / methods or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments / methods or examples described in this specification and the features of the different embodiments / methods or examples, unless they are contradictory.

[0108] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0109] Those skilled in the art should understand that the above embodiments are only for the purpose of clearly illustrating the present disclosure, and are not intended to limit the scope of the present disclosure. For those skilled in the art, other changes or modifications may be made based on the above disclosure, and these changes or modifications are still within the scope of the present disclosure.

Claims

1. A gene sequencing method, characterized in that: include: A gene sequencing chip is provided, the gene sequencing chip comprising a plurality of micro-reaction chambers and a flow channel connected to the plurality of micro-reaction chambers, one end of the flow channel being a sample inlet end and the other end being a sample outlet end, and the inner surface of the gene sequencing chip being hydrophobically modified; Introducing a sequencing buffer into the injection end of the flow channel; Introducing an isolation gas into the injection end of the flow channel to form an isolation gas column; introducing an aqueous reaction liquid into the injection end of the flow channel; Introducing sealing gas into the injection end of the flow channel to form a sealing gas column; The gene sequencing chip is used to perform a sequencing reaction.

2. The gene sequencing method according to claim 1, characterized in that: The width of the flow channel is 1 mm to 20 mm, and the height is 30 μm to 200 μm; the injection flow rate of the isolation gas is 1 mm / s to 20 mm / s; and the injection flow rate of the sealing gas is 1 mm / s to 20 mm / s.

3. The gene sequencing method according to claim 2, characterized in that: The total volume of the insulating gas introduced is more than 10% of the volume of the flow channel; the total volume of the sealing gas introduced is more than 100% of the volume of the flow channel; the total volume of the aqueous reaction liquid introduced is 5% to 60% of the volume of the flow channel.

4. The gene sequencing method according to claim 1, characterized in that: The total volume of the sequencing buffer introduced is 3 to 15 times the volume of the flow channel.

5. The gene sequencing method according to claim 4, characterized in that: The width of the flow channel is 1 mm to 20 mm, and the height is 30 μm to 200 μm; the injection flow rate of the sequencing buffer is 5 mm / s to 50 mm / s.

6. The gene sequencing method according to claim 1, characterized in that: The isolation gas is air, nitrogen, oxygen, saturated water vapor or a rare gas; the sealing gas is air, nitrogen, oxygen, saturated water vapor or a rare gas.

7. The gene sequencing method according to claim 1, characterized in that: The inner surface of the gene sequencing chip includes a portion inside the microreaction chamber and a portion of the surface between the microreaction chambers, and the entire inner surface of the gene sequencing chip has hydrophobic modification.

8. A gene sequencing system, characterized in that: include: A gene sequencing chip, the gene sequencing chip comprising a plurality of micro-reaction chambers and a flow channel connected to the plurality of micro-reaction chambers, one end of the flow channel being a sample inlet end and the other end being a sample outlet end, the inner surface of the gene sequencing chip being hydrophobically modified; A fluid supply device, comprising a buffer containing bottle, a reaction liquid containing bottle, a cleaning liquid containing bottle, a gas containing bottle, a control valve and a power source, wherein the buffer containing bottle, the reaction liquid containing bottle, the cleaning liquid containing bottle and the gas containing bottle are connected to the control valve through a pipeline, the control valve is connected to the sample inlet end of the flow channel through the pipeline, and the power source is connected to the sample outlet end of the flow channel through the pipeline; Under the driving force provided by the power source and the control of the control valve, the fluid supply device sequentially supplies sequencing buffer, isolation gas, aqueous reaction liquid, sealing gas and sequencing cleaning liquid to the flow channel.

9. The gene sequencing system according to claim 8, characterized in that: The control valve is a rotary valve having at least four valve ports.

10. The gene sequencing system according to claim 8, characterized in that: Also includes: A waste liquid collection bottle is connected to the power source through a pipeline, and the used fluid can be driven into the waste liquid collection bottle by the power source.

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