A recyclable sealed oil temperature-controlled nucleic acid sequencing system

Abstract

The present invention provides a recyclable sealed oil temperature-controlled nucleic acid sequencing system. The temperature of the chip is controlled by the sealed oil, and at the same time, the recycling of the sealed oil can be achieved. This method avoids the traditional complex heating structure and can control multiple gene sequencing chips simultaneously, reducing the consumption of the sealed oil.

Description

Technical Field

[0001] The present invention relates to a recyclable sealed oil temperature-controlled nucleic acid sequencing system, belonging to the field of gene sequencing. Background Art

[0002] The principle of nucleic acid sequencing is generally relatively simple. However, due to its very small signal and very small amount of reactants, it is extremely difficult to control the precision of the reaction. Generally, precisely controlling the sequencing reaction is a difficulty in the sequencing field, mainly requiring control of (1) a relatively high degree of reaction completion. This also belongs to a difficulty in the chemical field, and the unpredictability of the reaction increases the difficulty of this control. (2) The reaction conditions need to be perfectly controlled. This includes temperature control. Only by well controlling the reaction conditions can the influence of the environment be reduced as much as possible. The traditional temperature control method for sequencers is to heat the platform and the chip on which the chip is placed by heating devices such as Peltier, and then heat the reaction solution in the chip chamber through heat transfer on the chip panel. For a micro-pit array chip, the volume of the solution in the micro-pits is usually at the femtoliter (fL, 10 -9 L) level, and its heat transfer is very fast, while the indirect heat transfer through the chip panel greatly affects the temperature control efficiency. Many efforts have been made to improve the heating and cooling rates of the reaction solution in the micro-pit chip. For example, a layer of gold is plated on the surface of the micro-pits, and the plasma effect occurs on the metal surface under light irradiation at a certain wavelength to heat the liquid in the micro-pits. These methods are complex and expensive. The present invention provides a temperature control method for an oil-sealed chip, which uses sealed oil to control the temperature of the chip and can simultaneously recycle the sealed oil. This method avoids the traditional complex heating structure and can simultaneously control multiple gene sequencing chips, reducing the consumption of sealed oil. Summary of the Invention

[0003] The present invention provides a recyclable sealed oil temperature-controlled nucleic acid sequencing system, which is characterized by including,

[0004] (a) A first temperature control system, including the following reaction solution and its storage device,

[0005] (1) A sequencing reagent configured at a first temperature and its storage device,

[0006] (2) Sealed oil configured at a first temperature and its storage device;

[0007] (b) A first fluid switching device and a second fluid switching device;

[0008] (c) A second temperature control system, including a reagent at a second temperature and its storage device;

[0009] (d) A gene sequencing chip;

[0010] (e) An optical system;

[0011] (f) Waste liquid recovery device;

[0012] Among them, the reagents in the first temperature control system and the reagents in the second temperature control system are connected to the first fluid switching device through a fluid pipeline, and then connected to the inlet of the gene sequencing chip; the second fluid switching device is connected to the outlet of the gene sequencing chip; the second fluid switching device is also connected to at least one of the sealed oil storage device at the first temperature and the sealed oil storage device at the second temperature; the temperature of the gene sequencing chip is controlled by the first temperature control system fluid and the second temperature control system fluid flowing through the gene sequencing chip.

[0013] According to a preferred embodiment, the first fluid switching device and the second fluid switching device are rotary valves.

[0014] According to a preferred embodiment, the temperature of the first temperature control system is controlled at 0-25 °C, preferably 4-20 °C; more preferably 4-15 °C.

[0015] According to a preferred embodiment, the temperature of the second temperature control system is controlled at 60-95 °C; preferably 65-90 °C.

[0016] According to a preferred embodiment, it further includes a normal temperature storage device, and the normal temperature storage device includes a normal temperature washing solution storage device.

[0017] According to a preferred embodiment, the gene sequencing chip includes a first gene sequencing chip and a second gene sequencing chip.

[0018] The present invention provides a recyclable sealed oil temperature-controlled nucleic acid sequencing system, which is characterized in that it includes,

[0019] (a) A first temperature control system, including the following reagents and their storage devices,

[0020] (1) A sequencing reagent storage device configured at a first temperature,

[0021] (2) A sealed oil storage device configured at a first temperature;

[0022] (b) A first fluid switching device and a second fluid switching device;

[0023] (c) A second temperature control system, including a sealed oil storage device configured at a second temperature;

[0024] (d) A gene sequencing chip;

[0025] (e) An optical system

[0026] Among them, the reagents in the first temperature control system and the reagents in the second temperature control system are connected to a first fluid switching device through a fluid pipeline, and then connected to the inlet of the gene sequencing chip; the sequencing reagents and the sealing oil in the first temperature control system, and the sealing oil in the second temperature control system can be sequentially added to the sequencing chip by the fluid switching device; a second fluid switching device is connected to the outlet of the gene sequencing chip; the second fluid switching device is also connected to at least one of the sealing oil storage devices at the first temperature and the sealing oil storage device at the second temperature; wherein the gene sequencing chip includes a first sequencing chip and a second sequencing chip; the optical system includes an objective lens for taking pictures to obtain sequencing signals; the first sequencing chip and the second sequencing chip share a set of optical systems; the temperature of the gene sequencing chip is controlled by the fluids in the first temperature control system and the second temperature control system flowing through the gene sequencing chip.

[0027] The present invention provides a recyclable sealing oil temperature-controlled nucleic acid sequencing method, characterized in that

[0028] it includes sealing oil at a first temperature and sealing oil at a second temperature;

[0029] it includes a gene sequencing chip;

[0030] wherein at least one surface of the reaction chamber of the gene sequencing chip has pre-processed micro-pits; after adding the sequencing reagents, the temperature of the gene sequencing chip is controlled by the sealing oil at the first temperature and the sealing oil at the second temperature.

[0031] According to a preferred embodiment, the sequencing reagents include sequencing reagent one and sequencing reagent two; each sequencing reagent contains two of the nucleotide molecules A, G, C, T, or each sequencing reagent contains two of the nucleotide molecules A, G, C, U; the nucleotide molecules in the two sequencing reagents are base complementary; the nucleotide molecules are nucleotide substrate molecules with a fluorophore having fluorescence switching properties modified at the 5'-polyphosphate end; the fluorescence switching property means that there is no fluorescence signal before sequencing and there is an obvious fluorescence signal after sequencing.

[0032] According to a preferred embodiment, the first temperature is 0 - 25 °C; the second temperature is 60 - 95 °C.

[0033] According to a preferred embodiment, the sequencing includes the following steps: introducing sequencing reagent one stored at the first temperature into the sequencing chip; introducing the sealing oil stored at the first temperature; introducing the sealing oil stored at the second temperature; taking pictures to record the sequencing signals after the sequencing reaction occurs; introducing sequencing reagent two stored at the first temperature; introducing the sealing oil stored at the first temperature; introducing the sealing oil stored at the second temperature; taking pictures to record the sequencing signals after the sequencing reaction occurs.

[0034] In this application, a special recyclable oil seal chip temperature control technology is adopted. There are many advantages to heating with hot and cold oil: (1) It improves the prior art where the previous oil was only used for sealing. It can not only heat the chip but also recycle the sealing oil. (2) The sequencing reaction itself requires oil for the technology to be used effectively. If the reaction does not require oil inherently, adding hot and cold oil may cause the reaction to proceed smoothly. (3) It replaces the original heating plate structure, saving the complex heating structure. The benefits brought by the streamlined structure are obvious. Firstly, a complex temperature control system is not needed, and secondly, there is no need for the hot and cold temperature cycling of the heating plate. This can probably shorten the heating control time by about 20%. Since gene sequencing is not a short-time reaction, for example, 10 minutes or 1 hour, the changes brought by this structure are significant. (4) It saves the heating and cooling structure. In a conventional electric heating structure, rapid cooling requires a cooling device, generally a water cooling device, which has a relatively large structure. In this regard, a lot of space in the sequencer can be saved, which is beneficial to the miniaturization of the sequencer. (5) It improves efficiency. By using the method of heating with hot and cold oil, multiple chips, such as 2, 3, 4 or more, can be controlled simultaneously. When multiple chips are controlled simultaneously, only different hot and cold oil fluids need to be added to the chips. If a conventional heating plate structure is used, when different chips require different temperatures, each chip needs to be equipped with a set of heating and cooling modules. Such a huge configuration is impossible or unrealistic to achieve. The hot and cold oil mechanism provides a completely new setting. By only controlling the fluids of each chip (including sequencing reagents, hot and cold oil, cleaning solution, etc., added according to the set program), the process of each chip can be precisely controlled. The complexity of multiple sets of chips is only reflected in the improvement of the fluid system. This improvement reflects a huge integration advantage. Brief Description of the Drawings

[0035] Figure 1 Diagram of the inventive device; labeled as: 1 - micro-pit array chip, 2 - chip fluid inlet, 3 - signal acquisition device, 4 - valve, 5 - pump, 6 - reagent temperature control table, 7 - bottle of washing solution, 8 - bottle of sequencing reagent, 9 - second bottle of sequencing reagent, 10 - bottle of cold oil, 11 - bottle of hot oil, 12 - second bottle of washing solution, 13 - waste liquid bottle, 14 - second rotary valve;

[0036] Figure 2 Fluid experiment, the chip is filled with an aqueous solution containing black dye;

[0037] Figure 3 Fluid experiment, the aqueous solution in the chip is discharged from the chip through the sealing oil;

[0038] Figure 4 Curve of temperature rise and fall for hot and cold oil temperature control and curve of temperature rise and fall for Peltier temperature control;

[0039] Figure 5Schematic diagram of oil seal

[0040] Figure 6 Chip structure diagram Detailed implementation manners

[0041] To further illustrate the core content of the present invention, the present invention will now be described by the following examples. The examples are for further explaining the content of the invention and do not impose any limitations on the present invention.

[0042] The field of gene sequencing belongs to a special field of biochemical applications. Generally, the temperature is controlled by electric heating or refrigeration, similar to a conventional PCR instrument. However, the difference is that during the gene sequencing process, a longer time is required, and a complex gene sequencing chip system and matching components are needed.

[0043] Gene sequencing belongs to a special application field, with small volume, long cycle, extremely high signal sensitivity requirements, and serious process influence. During the gene sequencing process, temperature control has always been a relatively difficult topic. Common temperature control is by means of electric heating, such as Peltier. Conventional heating methods have extremely high requirements for the chip. The smoothness, flatness, and heat transfer efficiency of the part where the bottom of the chip is in contact with the heating device will have a serious impact on the actual temperature of the sequencing.

[0044] The present invention provides a gene sequencing system with temperature control by cold and hot oil, comprising a first sequencing chip, a cold oil control system, a hot oil control system, a fluid switching system, and an optical system; wherein, the cold oil control system is used to generate cold oil at 0 - 20 degrees Celsius; the hot oil control system is used to generate hot oil at 60 - 95 degrees Celsius; the cold and hot oil can enter the sequencing chip controllably through the fluid switching system; wherein, the sequencing chip includes a fluid chamber, a fluid inlet, and a fluid outlet, and at least one inner surface of the sequencing chip has a micro reaction chamber prepared in advance; the cold oil is used to seal the micro reaction chamber in the sequencing chip and control the chip in a relatively low temperature range; the hot oil is used to provide the reaction temperature; wherein the optical system includes an objective lens for collecting the reaction signal in the micro reaction chamber.

[0045] The present invention provides a recyclable sealed oil temperature-controlled nucleic acid sequencing system, characterized in comprising

[0046] (a) A first temperature control system, comprising the following devices

[0047] (3) A sequencing reagent storage device configured at a first temperature

[0048] (4) A sealed oil storage device configured at a first temperature;

[0049] (b) A first fluid switching device and a second fluid switching device;

[0050] (c) A second temperature control system, including a storage device for sealed oil configured at a second temperature;

[0051] (d) A gene sequencing chip;

[0052] (e) An optical system

[0053] Wherein, the storage device in the first temperature control system and the storage device in the second temperature control system are connected to a first fluid switching device in the form of a pipeline, and then connected to the inlet of the gene sequencing chip; the sequencing reagent and sealed oil in the first temperature control system, and the sealed oil in the second temperature control system can be sequentially added to the sequencing chip by the fluid switching device; a second fluid switching device is connected to the outlet of the gene sequencing chip; the second fluid switching device is also connected to at least one of the storage device for sealed oil at a first temperature and the storage device for sealed oil at a second temperature; wherein the gene sequencing chip includes a first sequencing chip and a second sequencing chip; the optical system includes an objective lens for taking pictures to obtain sequencing signals; the first sequencing chip and the second sequencing chip share a set of optical systems; the temperature of the gene sequencing chip is controlled by the fluids in the first temperature control system and the second temperature control system flowing through the gene sequencing chip.

[0054] According to a preferred embodiment, the first sequencing chip and the second sequencing chip share an optical system and collect signals cyclically.

[0055] According to a preferred embodiment, the fluid in the first temperature control system includes a sequencing reaction solution and sealed oil; the fluid in the second temperature control system includes sealed oil. The first temperature control system provides a low-temperature reaction solution to the sequencing chip and provides sealed oil for sealing the reaction solution. The second temperature control system provides high-temperature sealed oil to the sequencing chip. The gene sequencing reaction is a process of cyclic temperature change, and sealed oils at different temperatures can enable the chip to maintain different temperatures.

[0056] According to a preferred embodiment, it further includes a washing solution storage device.

[0057] According to a preferred embodiment, the washing solution storage device is a room-temperature storage device.

[0058] According to a preferred embodiment, the washing solution storage device is located in the first temperature control system. The washing solution is in a low-temperature state.

[0059] According to a preferred embodiment, the hot and cold oil temperatures of the first sequencing chip and the second sequencing chip are different. That is to say, when the first sequencing chip enters the hot oil to keep it at the sequencing reaction temperature, the second sequencing chip can enter the cold oil. In this way, the operation of the two sequencing chips basically does not interfere with each other, and a set of fluid systems can be used to make the two sequencing chips work simultaneously. This greatly improves the application efficiency. Of course, since sequencing is divided into multiple steps and the time for each step is different, during the dual-chip sequencing process, it is inevitable that the first sequencing chip needs to enter the hot oil, but the second sequencing chip has not completed taking pictures. A simple solution is to adjust the order of fluid addition, such as delaying the addition order of the hot oil to the first sequencing chip. Generally speaking, the sequencing efficiency is improved. This advantage is significant. In existing sequencers, if multiple chips perform sequencing without interference, multiple heating platforms with different temperature functions are required, which is a complex project. Even more, each time a reaction chip is added, a heating platform needs to be added. The method provided by the present invention only needs to adjust the order of fluid addition to achieve the purpose of controlling the temperature of each chip, only increasing the complexity of the fluid system. To a certain extent, the number of chips on the same sequencer can be quickly increased without interference. In this regard, it is a qualitative improvement for the sequencer.

[0060] According to a preferred embodiment, the sequencing reagent is at the same temperature as the cold oil.

[0061] According to a preferred embodiment, the sequencing reagent and the cold oil are at the same temperature during the sequencing operation.

[0062] According to a preferred embodiment, it further includes a sequencing reagent storage device and a washing solution storage device.

[0063] According to a preferred embodiment, it includes Test Reagent 1, stored in a sequencing reagent bottle 1; includes Sequencing Reagent 2, stored in a sequencing reagent bottle 2; includes Sequencing Reagent 3, stored in a sequencing reagent bottle 3.

[0064] According to a preferred embodiment, there are two kinds of sequencing reagents, namely Sequencing Reagent 1 and Sequencing Reagent 2.

[0065] According to a preferred embodiment, there are three kinds of sequencing reagents.

[0066] According to a preferred embodiment, there is one kind of cleaning solution.

[0067] According to a preferred embodiment, there are two kinds of cleaning solutions.

[0068] According to a preferred embodiment, the fluid switching system includes a rotary valve.

[0069] According to a preferred embodiment, the sequencing is performed using a gene sequencer.

[0070] According to a preferred embodiment, the sequencer used for the sequencing includes a fluid system, an optical system, and a chip stage.

[0071] According to a preferred embodiment, the storage temperature of the reagent storage device is 0 - 20 degrees Celsius.

[0072] According to a preferred embodiment, the storage temperature of the cold oil is 0 - 20 degrees Celsius.

[0073] According to a preferred embodiment, the storage temperature of the hot oil is 60 - 95 degrees Celsius, preferably 65 - 75 degrees Celsius.

[0074] According to a preferred embodiment, the function of the rotary valve is to form a passage between two or more inlets and outlets on the rotary valve. This belongs to the common function of a rotary valve.

[0075] According to a preferred embodiment, the reagent storage device is used to store sequencing reagents.

[0076] According to a preferred embodiment, the hot oil storage device is used to store hot oil.

[0077] According to a preferred embodiment, the cold oil storage device is used to store cold oil.

[0078] According to a preferred embodiment, the reagent storage device, the hot oil storage device, and the cold oil storage device are connected to a first rotary valve through pipelines.

[0079] According to a preferred embodiment, the first rotary valve is connected to a sequencing chip. The first rotary valve transports sequencing reagents, hot and cold oils, and other necessary reagents into the sequencing chip.

[0080] According to a preferred embodiment, the first rotary valve is connected to the chip inlet of the sequencing chip.

[0081] According to a preferred embodiment, the syringe pump is connected to the chip outlet of the sequencing chip.

[0082] The outlet of the gene sequencing chip is connected to at least one of the storage devices for sealing oil at a first temperature and sealing oil at a second temperature. When the sequencing reaction solution and the sealing oil are recovered into the sealing oil storage device, since the water and oil do not mix, and the water is clearly on the upper layer, it will not affect the recycling of the oil, and no sequencing reaction solution will enter the chip to cause contamination.

[0083] According to a preferred embodiment, the sealing oil is FC40 or FC70. Common fluorinated sealing oils are extremely difficult to dissolve in water and have a density greater than that of water. The sealing oil and water are recovered simultaneously without causing cross - contamination.

[0084] The gene sequencing chip belongs to conventional technology. Commonly, CN2017105741742, CNCN2017105741441, CN201710630287X, 201811643917.8, 201910156547.3; the content of these patents can be incorporated into this patent by reference.

[0085] Generally, a rotary valve is used as the fluid control structure. The fluid components are connected by plastic tubes. Generally, polytetrafluoroethylene tubes are used for connection. For example, common ones are 0.3mm, 0.6mm, etc. The liquid path control method has been introduced in the applicant's previous patents, such as CN2017211569462; the content of this patent can be incorporated into this patent by reference.

[0086] Generally, a heating plate is used to control the temperature of the reagent. A temperature probe can be pre-placed inside the reagent bottle, or a temperature probe can be set on the structure of the reagent needle. Commonly, the reagent bottle is replaceable, and it is more convenient to use the reagent needle to carry the temperature probe. The constant temperature control of the reagent is not a complicated technology. Generally, the temperature drop of the reagent after passing through the connecting pipeline and the rotary valve will also be considered, and the temperature of the oil flowing out of the chip can be detected by measuring the temperature of the outlet reagent. Usually, the temperature of the high-temperature oil is 0.5 - 5 degrees Celsius higher than the actual required temperature of the chip, preferably 0.5 - 2 degrees Celsius. Usually, the temperature of the low-temperature oil is 0.5 - 5 degrees Celsius lower than the actual required temperature of the chip, preferably 1 - 3 degrees Celsius. The temperature control is empirical, and fine-tuning can be carried out before the equipment actually works.

[0087] The function of the washing liquid is to clean the oil. The washing liquid is generally small molecule organic reagents such as ethanol and acetone.

[0088] Generally, the hot and cold oils use the same component oil. Generally, fluorine oil is used. The oil plays a sealing role. Commonly, it is introduced in the applicant's patents CN201710630287X and CN2017211569462; part of the content of these two patents can be incorporated into this patent by reference.

[0089] The specific embodiments in the specific implementation manners of the present invention are only further descriptions of the present invention and do not constitute limiting factors of the present invention.

[0090] Example 1

[0091] See the invention device Figure 1 . Figure 11 is a micro-pit array chip; 2 is the chip fluid inlet; 3 is the signal acquisition device; 4 is the rotary valve; 5 is the pump; 6 is the reagent temperature control table; 7 is a bottle of washing solution; 8 is a bottle of sequencing reagent; 9 is a second bottle of sequencing reagent; 10 is the cold oil bottle; 11 is the hot oil bottle; 12 is a second bottle of washing solution; 13 is the waste liquid bottle; 14 is the second rotary valve. Among them, the micro-pit array chip has been described in Patent CN2017105741742. The fluid system can refer to CN2017211569301. The valve in component 4 is a rotary valve with multiple inlets and outlets. The pump in component 5 is a syringe pump for providing power.

[0092] The sequencing chip is shown in Example 5

[0093] The sequencing process is as follows:

[0094] 1. Inject 10 mL of washing solution to rinse the chip, and the rinsing waste liquid enters the waste liquid bottle.

[0095] 2. Inject 100 μL of sequencing reagent one (8), and the excess liquid enters the waste liquid bottle.

[0096] 3. Inject cooling oil (10), and the excess oil is passed through the second rotary valve 14 into the hot oil storage device 11.

[0097] 4. Inject hot oil (11), and the excess oil is passed through the second rotary valve 14 into the hot oil storage device 11.

[0098] 5. Wait for 1 min.

[0099] 6. Excite with 473 nm laser and take a fluorescence image.

[0100] 7. Inject 10 mL of washing solution (7) to rinse the chip, and the rinsing waste liquid enters the waste liquid bottle.

[0101] 8. Inject 100 μL of sequencing reagent two (9), and the excess liquid enters the waste liquid bottle.

[0102] 9. Inject cooling oil (10), and the excess oil is passed through the second rotary valve 14 into the hot oil storage device 11.

[0103] 10. Inject hot oil (11), and the excess oil is passed through the second rotary valve 14 into the hot oil storage device 11.

[0104] 11. Wait for 1 min.

[0105] 12. Excite with 473 nm laser and take a fluorescence image.

[0106] Repeat steps 1 - 13 fifty times to obtain 100 fluorescence signals.

[0107] The excess oil will not be wasted. Compared with directly introducing the oil into the waste liquid bottle, more than 200 ml of oil can be saved for each sequencing. Commonly used sealing fluorinated oils, such as FC40, etc., are denser than water. The excess aqueous liquid is located above the oil and will not contaminate the oil or cause liquid crosstalk. During this process, the first rotary valve 4 and the second rotary valve 14 need to be comprehensively controlled. Only when the oil enters the chip, the liquid discharged from the chip will be connected to the oil storage device through the second rotary valve; in other states, the discharged liquid will be connected to the waste liquid bottle.

[0108] Example 2

[0109] Hot and cold oils are used for the sequencing reaction. The sequencing device includes: a reaction chamber with the DNA fragments to be tested planted on its surface, a pump valve system for fluid control, an optical system for signal acquisition, a central control system, and a temperature control system for controlling the temperature of the reaction reagents. See Figure 1 . The test process includes: (1) Experiment preparation: Place the chip on the chip table, place the sequencing reaction reagents and the cold oil bottle on the 15°C temperature control table, place the hot oil bottle on the 65°C temperature control table, and place the first washing solution and the second washing solution at room temperature; (2) The pump valve system first controls the first washing solution to enter the reaction chamber to clean the reaction channel; (3) The pump valve system then controls the corresponding reaction solution to enter the reaction chamber; (4) The pump valve system controls the 15°C cold oil to enter the chip; (5) Turn on the light source, and the camera collects signals to record the background value; (6) The pump valve system controls the 65°C hot oil to enter the chip, and the polymerase extension reaction proceeds, maintaining the reaction time for 45 seconds; (7) Draw the 15°C cold oil into the chip again to collect signals. (8) The pump valve system pumps the second washing solution into the reaction chamber. Steps (2)-(8) are one sequencing reaction cycle. Sequencing reaction solutions are cycled in this way until the set number of cycles.

[0110] 2+2 sequencing, single color: Configure 3 sets of reaction solutions, two bottles in each set, and each bottle contains two bases labeled with fluorescent groups, and the fluorescent groups are all X. The two bottles in one set exactly contain the complete 4 kinds of bases. The 6 bottles of solutions do not repeat each other.

[0111] The first bottle The second bottle The first set AX + CX GX + TX The second set AX + GX CX + TX The third set AX + TX CX + GX

[0112] The first bottle of reaction solution is sequencing reagent one; the second bottle of reaction solution is sequencing reagent two.

[0113] The complete sequencing process includes three rounds, and the three rounds are carried out in sequence. The sequencing processes of each round respectively use the above three sets of reagents. Otherwise, they are exactly the same (using the same sequencing primers and the reaction conditions are exactly the same).

[0114] Each round of sequencing includes:

[0115] 1. Hybridize the sequencing primer on the prepared DNA array

[0116] 2. Start the sequencing process. Repeat the process of 2.1 - 2.4 a limited number of times.

[0117] 2.1 Introduce the first bottle of reagent. React and collect fluorescence signals.

[0118] 2.2 Wash all the residual reaction solutions and the generated fluorescent molecules in the flowcell.

[0119] 2.3 Introduce the second bottle of reagent. React and collect fluorescence signals.

[0120] 2.2 Wash all the residual reaction solutions and the generated fluorescent molecules in the flowcell.

[0121] 3. Unwind the extended sequencing primer.

[0122] At this point, the next round of experiment can be carried out.

[0123] Prepare the reaction solution:

[0124] Prepare the washing solution for the sequencing reaction solution, abbreviated as the washing solution, containing:

[0125] 20 mM Tris-HCl pH 8.8

[0126] 10 mM (NH4)2SO4

[0127] 50 mM KCl

[0128] 2 mM MgSO4

[0129] 0.1% 20

[0130] Place the sequencing chip on the sequencer.

[0131] Use the first set of reaction solutions for sequencing. Corresponding to Figure 1 , follow the sequencing process described in Example 1.

[0132] Example 3

[0133] Through the fluid experiment pictures, the sequencing oil seal effect can be directly seen. As Figure 2 , Figure 3 shown, when the objective lens is focused on the micropit plane, after the oil seal, it is found that there is basically no water (sequencing reagent) residue. And the results of a large number of experiments confirm that there is no serious water residue in multiple rounds of reactions. That is to confirm that the oil seal with low-temperature oil or the oil seal with alternating hot and cold will not affect the experimental results.

[0134] Example 4

[0135] The method according to Embodiment 1. Set the temperature of the hot oil to 70 degrees Celsius and the temperature of the cold oil to 4 degrees Celsius. Through multiple rounds of experiments, it is found that the heating efficiency of the hot and cold oils is good, and the temperature changes are shown in Figure 4 the dotted line in. Turn off the hot oil and cold oil temperature control. Place the same reagent on the heating plate and set the temperature to cycle between 70 degrees Celsius and 4 degrees Celsius. The heating and cooling times used in the two groups of experiments are the same. The chip test temperature of the heating plate is shown in Figure 4 the solid line in. This experiment can illustrate that (1) the heating and cooling rates of the oil heating and the electric heating chip are basically the same. The difference in the maximum temperature is because the oil heating directly heats the chip, with higher efficiency. According to the specific situation, the required specific temperature is controllable. When changing the material supporting the chip below, the oil heating and cooling will become faster. Simply put, when changing the material at the bottom of the chip, since only the chip needs to be heated without considering the heating of large auxiliary equipment, the efficiency is increased by more than 20%.

[0136] Generally, during electric heating, the bottom of the chip is in direct contact with the metal hot plate, which has good thermal conductivity. While during oil heating, it is not required that the bottom material has good thermal conductivity. This also gives more choices for the bottom material. This selectivity has great advantages for industrial design. The processing and maintenance of the flatness of the hot plate surface are not easy tasks. While for the oil heating method, there is no need to consider the problems of the hot plate material and processing.

[0137] It should be specifically noted that the direction of the liquid discharged from the chip does not affect the temperature change of the chip.

[0138] Embodiment 5

[0139] The device according to Embodiment 1. The chip is mainly divided into three layers. From top to bottom, they are the micro-reaction chamber chip layer 103, the middle glue layer 102, and the lower bottom plate layer 101. Punch holes in the upper micro-reaction chamber chip layer. The middle glue layer has a hollow reaction chamber structure, and the bottom plate layer below is a piece of glass. The three layers are assembled together to form the chip. The external fluid enters through the holes in the micro-reaction chamber chip layer, then enters the reaction chamber formed by the middle glue layer, and finally exits the chip through the holes at the other end of the micro-reaction chamber.

[0140] As Figure 5 described, first fill the internal fluid channel with the aqueous fluid 1200, and the micro-pit part is also filled with the aqueous fluid. Then introduce the oil-phase fluid 1200, as Figure 5 (A) shows. During the process of the oil-phase fluid discharging the aqueous fluid from the reaction chamber, the aqueous fluid will be sealed inside the micro-pits, thus forming independent reaction units with each micro-pit as a unit. Corresponding to this embodiment, the aqueous fluid can be a sequencing reagent. The oil-phase fluid can be cold oil and hot oil.

[0141] corresponding to Figure 5 the described fluid structure. The cross-sectional view of its chip structure can be as Figure 6 shown. The bottom plate glass layer is 101, the middle layer is the die-cut double-sided adhesive 102, and the upper layer is the microchannel plate 103. Among them, in 102, the die-cut mechanism forms a cavity-type reaction chamber. On the lower surface of 103, that is, the surface in contact with 102, an array of micro reaction chambers is etched; it is not shown in the figure. Holes are drilled at appropriate positions on the micro reaction chamber chip layer of 103 as channels for external fluid to enter and exit the reaction chamber. The holes are directly connected to the reaction chamber. Among them, the thickness of layer 101 is 1 mm, the thickness of layer 102 is 0.1 mm, and the thickness of 103 is 1 mm.

[0142] The embodiments of the present invention are further explanations of the present invention and do not affect the protection scope of the invention.

Claims

1. A recyclable sealed oil temperature-controlled nucleic acid sequencing system, characterized in that it includes (a) A first temperature control system, comprising the following reagents and their storage devices, (1) Sequencing reagents configured at a first temperature and their storage devices, (2) Sealed oil configured at a first temperature and its storage device; (b) A first fluid switching device and a second fluid switching device; (c) A second temperature control system, comprising sealed oil at a second temperature and its storage device; (d) A gene sequencing chip, and the gene sequencing chip is a micro-pit array chip; (e) An optical system; (f) A waste liquid recovery device; wherein, the sequencing reagents, sealed oil in the first temperature control system, and the sealed oil in the second temperature control system are respectively connected to the first fluid switching device through fluid pipelines, and then connected to the inlet of the gene sequencing chip; the sequencing reagents and sealed oil in the first temperature control system, and the sealed oil in the second temperature control system can be sequentially added to the sequencing chip by the fluid switching device; the second fluid switching device is connected to the outlet of the gene sequencing chip; the second fluid switching device is also connected to at least one of the sealed oil storage device at the first temperature and the sealed oil storage device at the second temperature; the temperature of the gene sequencing chip is controlled by the sealed oil of the first temperature control system and the sealed oil of the second temperature control system flowing through the gene sequencing chip.

2. The recyclable sealed oil temperature-controlled nucleic acid sequencing system according to claim 1, wherein the first fluid switching device and the second fluid switching device are rotary valves.

3. The recyclable sealed oil temperature-controlled nucleic acid sequencing system according to claim 1, characterized in that the temperature of the first temperature control system is controlled at 0-25 degrees Celsius; the temperature of the second temperature control system is controlled at 60-95 degrees Celsius.

4. The recyclable sealed oil temperature-controlled nucleic acid sequencing system according to claim 1, characterized in that it further includes a normal temperature storage device, and the normal temperature storage device includes a normal temperature washing solution storage device.

5. The recyclable sealed oil temperature-controlled nucleic acid sequencing system according to claim 1, characterized in that the gene sequencing chip includes a first gene sequencing chip and a second gene sequencing chip; the first gene sequencing chip and the second gene sequencing chip share a set of optical systems.

6. A recyclable sealed oil temperature-controlled nucleic acid sequencing system, characterized in that it includes (a) A first temperature control system, comprising the following reagents and their storage devices, (1) Sequencing reagents configured at a first temperature and their storage devices, (2) Sealed oil configured at a first temperature and its storage device; (b) A first fluid switching device and a second fluid switching device; (c) A second temperature control system, comprising sealed oil configured at a second temperature and its storage device; (d) A gene sequencing chip, and the gene sequencing chip is a micro-pit array chip; (e) An optical system Among them, the sequencing reagent, the sealing oil in the first temperature control system, and the sealing oil in the second temperature control system are respectively connected to a first fluid switching device through fluid pipelines, and then connected to the inlet of the gene sequencing chip; the sequencing reagent and the sealing oil in the first temperature control system, and the sealing oil in the second temperature control system can be sequentially added to the sequencing chip by the fluid switching device; a second fluid switching device is connected to the outlet of the gene sequencing chip; the second fluid switching device is also connected to at least one of the sealing oil storage devices at the first temperature and the sealing oil storage devices at the second temperature; wherein the gene sequencing chip includes a first sequencing chip and a second sequencing chip; the optical system includes an objective lens for taking pictures to obtain sequencing signals; the first sequencing chip and the second sequencing chip share a set of optical systems; the temperature of the gene sequencing chip is controlled by the sealing oil in the first temperature control system and the sealing oil in the second temperature control system flowing through the gene sequencing chip.

7. A method for controlling temperature in nucleic acid sequencing using the nucleic acid sequencing system according to any one of claims 1-6, characterized in that, it includes sealing oil at a first temperature and sealing oil at a second temperature; it includes a gene sequencing chip; wherein at least one surface of the reaction chamber of the gene sequencing chip has pre-processed micro-pits; after adding the sequencing reagent, the temperature of the gene sequencing chip is controlled by the sealing oil at the first temperature and the sealing oil at the second temperature.

8. The method according to claim 7, characterized in that, the sequencing reagent includes sequencing reagent one and sequencing reagent two; each sequencing reagent contains two of the nucleotide molecules A, G, C, T, or each sequencing reagent contains two of the nucleotide molecules A, G, C, U; the nucleotide molecules in the two sequencing reagents are base complementary; the nucleotide molecule is a nucleotide substrate molecule with a fluorophore having fluorescence switching properties modified at the 5'-polyphosphate end; the fluorescence switching property means that there is no fluorescence signal before sequencing and there is an obvious fluorescence signal after sequencing.

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