A nucleic acid sequencing system with reagent temperature control

Abstract

The application provides a nucleic acid sequencing system for controlling temperature of reagents. The sealed oil with different temperatures is used as a fluid, which not only provides a sealing function for the nucleic acid sequencing chip, but also controls the temperature of the nucleic acid sequencing chip simultaneously. The problems of complex device, complex control and large space occupation of the traditional heating mode are avoided. Multiple chips can be controlled simultaneously, and the complexity of the sequencer is not increased greatly.

Description

Technical Field

[0001] The present invention relates to a nucleic acid sequencing system with reagent temperature control, belonging to the field of gene sequencing. Background Art

[0002] Gene sequencing is an emerging industry in recent years. The current mainstream of gene sequencing is second-generation sequencing. The core idea of ​​the second-generation sequencing technology is sequencing by synthesis, that is, determining the sequence of nucleic acids by capturing the markers at the newly synthesized ends. Generally, second-generation sequencing technologies require periodic heating and cooling processes. Not only second-generation sequencing, but also similar PCR requires periodic heating and cooling. The accuracy of temperature control affects whether the biological reaction can occur correctly, and the heating and cooling rate directly restricts the duration and cycle of the test. The conventional temperature control method relies on heating devices such as Peltier to heat the platform where the chip is placed and the chip, and then heat the reaction solution in the chip chamber through heat transfer from the chip panel. For micropit array chips, the volume of the solution in the micropit is usually femtoliters (fL, 10 -9 L) level, its heat transfer is very fast, while the indirect heat transfer through the chip panel greatly affects the efficiency of temperature control. A lot of work is devoted to improving the heating and cooling rate of the reaction liquid in the micro-pit chip. For example, a layer of gold is plated on the surface of the micro-pit, and a plasma effect occurs on the metal surface under a certain wavelength of light, thereby heating the liquid in the micro-pit. These methods are complex and expensive. The present invention discloses a system for controlling the temperature of a gene sequencing chip using reagents, which heats and cools the reaction liquid by using hot and cold fluids; it is fast, simple and accurate. Summary of the Invention

[0003] The present invention provides a reagent temperature-controlled nucleic acid sequencing system, characterized in that it includes a first temperature control system, including reagents at a first temperature and storage devices thereof; a reagent switching system; a second temperature control system, including reagents configured at a second temperature and storage devices thereof; a gene sequencing chip; wherein the gene sequencing chip includes a first reagent layer and a second reagent layer; the reagents stored in the storage device in the first temperature control system and the reagents stored in the storage device in the second temperature control system are connected to the reagent switching system via a fluid pipeline; and one of the inner surfaces of the gene sequencing chip has pre-prepared micro-pits.

[0004] According to a preferred embodiment, the temperature of the first temperature control system is controlled at 0-25 degrees Celsius, preferably 4-20 degrees Celsius, and more preferably 4-15 degrees Celsius; the temperature of the second temperature control system is controlled at 60-95 degrees Celsius, preferably 65-90 degrees Celsius.

[0005] According to a preferred embodiment, it further comprises a normal temperature storage device, wherein the normal temperature storage device comprises a normal temperature lotion storage device.

[0006] According to a preferred embodiment, the gene sequencing chip includes a first gene sequencing chip and a second gene sequencing chip.

[0007] According to a preferred embodiment, the first and second sequencing chips share an optical system and cyclically collect signals. A nucleic acid sequencing system with sealed oil temperature control is characterized by comprising: a first temperature control system containing reagents and storage devices at a first temperature; a reagent switching system; a second temperature control system containing reagents configured to a second temperature and storage devices; and a gene sequencing chip; wherein the reagents stored in the storage device of the first temperature control system and the reagents stored in the storage device of the second temperature control system are connected to the reagent switching system via a pipeline and can be selectively added to the gene sequencing chip; wherein the gene sequencing chip includes a first sequencing chip and a second sequencing chip; the two sequencing chips are connected to the reagent switching system via a fluid pipeline and can be selectively added with reagents; the reagents at the first temperature include sequencing reagents and sealing oil.

[0008] A gene sequencing method using sealing oil temperature control is characterized by comprising: a first temperature control system comprising a reagent at a first temperature and a storage device thereof; a reagent switching system; a second temperature control system comprising a reagent configured at a second temperature and a storage device thereof; a gene sequencing chip; wherein at least one surface of a reaction chamber of the gene sequencing chip has pre-processed micro-pits; the reagent at the first temperature comprises a sequencing reagent and sealing oil; the reagent at the second temperature is sealing oil; during sequencing, the sequencing reagent at the first temperature is added to the gene sequencing chip through the reagent switching system; the sealing oil at the first temperature is added to the gene sequencing chip; the sealing oil at the second temperature is added to the gene sequencing chip; and detection is performed to obtain a sequencing signal.

[0009] According to a preferred embodiment, the sealing oil at the first temperature and the sealing oil at the second temperature have the same composition.

[0010] According to a preferred embodiment, it is characterized in that the sequencing reagents include sequencing reagent one and sequencing reagent two; each sequencing reagent contains two of A, G, C, and T nucleotide molecules, or each reaction solution contains two of A, G, C, and U nucleotide molecules; the bases of the nucleotide molecules in the two sequencing reagents are complementary.

[0011] According to a preferred embodiment, the method comprises the following steps:

[0012] introducing sequencing reagent 1 stored at a first temperature into the sequencing chip;

[0013] introducing sealing oil stored at a first temperature;

[0014] introducing sealing oil stored at a second temperature;

[0015] After the sequencing reaction occurs, take a photo to record the sequencing signal;

[0016] introducing sequencing reagent 2 stored at the first temperature;

[0017] introducing sealing oil stored at a first temperature;

[0018] introducing sealing oil stored at a second temperature;

[0019] After the sequencing reaction occurs, take a photo to record the sequencing signal.

[0020] This application uses a special oil-sealed chip technology. As far as the applicant knows, this technology has not been applied by others in the field of gene sequencing. The present invention has the following advantages: (1) Chip layering; the first layer of the chip can also be called the reaction chamber layer, and the second layer of the chip can also be called the heating layer; the double-layer chip ensures that the heating and cooling fluids and the reaction liquid enter different chip layers. The fluid heating is carried out in a manner closest to the reaction chamber. (2) The original heating plate structure is replaced. The complex heating structure is saved. The benefits of the streamlined structure are obvious. First, there is no need for a complex temperature control system, and there is no need for the heating plate to cycle between hot and cold temperatures. This can shorten the heating control time by about 20%. Since gene sequencing is not a short-term reaction, such as 10 minutes, such as 1 hour; generally, it can reach 15-50 hours, the change brought about by this structure is significant. (3) The heating and cooling structure is saved. In conventional electric heating structures, rapid cooling requires a cooling device. Generally, a water cooling device is used. The structure of this device is relatively large. From this aspect, a lot of sequencer space can be saved, which is conducive to the miniaturization of the sequencer. (4) Improved efficiency. By using hot and cold oil heating, the Nth chip can be controlled simultaneously. When multiple chips are controlled at the same time, you only need to add different hot and cold oil fluids 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. This huge configuration is impossible to achieve, or it is unrealistic. The hot and cold oil mechanism, by contrast, proposes a completely new setting. It is only necessary to control the fluid of each chip (including sequencing reagents, hot and cold oil, cleaning fluid, etc., added according to the set program) to accurately control the process of each chip. The complexity of multiple sets of chips is only reflected in the improvement of the fluid system. This improvement reflects the huge advantages of integration. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Diagram of the invention device; labeled as: 1-micropit array chip, 2-chip fluid inlet, 3-signal acquisition device, 4-valve, 5-pump, 6-reagent temperature control station, 7-one bottle of wash solution, 8-one bottle of sequencing reagent, 9-two bottles of sequencing reagent, 10-cold oil bottle, 11-hot oil bottle, 12-two bottles of wash solution, 13-waste liquid bottle.

[0022] Figure 2 These are the temperature rise and fall curves for hot and cold oil temperature control and the temperature rise and fall curves for Peltier temperature control.

[0023] Figure 3 Chip structure diagram. DETAILED DESCRIPTION

[0024] In order to further illustrate the core content of the present invention, the present invention is now illustrated by the following examples. The examples are for further explaining the invention content and do not limit the present invention.

[0025] Gene sequencing is a specialized field of biochemical applications. Generally, temperature control is achieved through electric heating and cooling. This specialized application requires small size, long cycles, extremely high signal sensitivity, and significant process impact. Temperature control has always been a challenging issue during the sequencing process. Common temperature control methods utilize electric heating, such as Peltier technology. Conventional heating methods place extremely high demands on the chip. The smoothness, flatness, and heat transfer efficiency of the chip bottom where the heating device meets the heating device significantly impact the actual sequencing temperature.

[0026] The present invention provides a reagent temperature-controlled nucleic acid sequencing system, characterized in that it includes a first temperature control system, including reagents at a first temperature and storage devices thereof; a reagent switching system; a second temperature control system, including reagents configured at a second temperature and storage devices thereof; a gene sequencing chip; wherein the gene sequencing chip includes a first reagent layer and a second reagent layer; the reagents stored in the storage device in the first temperature control system and the reagents stored in the storage device in the second temperature control system are connected to the reagent switching system via a fluid pipeline; and one of the inner surfaces of the gene sequencing chip has pre-prepared micro-pits.

[0027] Generally, hot and cold oils aren't the only heating and cooling fluid options. The chip is divided into two layers: the first layer is used for the entry of sequencing reagents and the reaction, while the second layer maintains the required temperature for sequencing. Therefore, there are no strict restrictions on the fluid in the second layer. Common non-volatile fluids such as water, aqueous salt solutions, glycerol solutions in water, and fluorinated oils can serve as heating and cooling fluids. Since the heating and cooling fluids don't come into contact with the sequencing reagents, their selection is broad.

[0028] According to a preferred embodiment, the heating and cooling fluid is selected from water, fluorinated oil, and a mixture of aqueous solutions.

[0029] The micro-reactors of gene sequencing chips are oriented in a certain direction. Advantageously, they are typically placed on the inner surface, closest to the outer surface of the chip. The chip is structured in multiple layers, and imaging is most convenient when the micro-reactors are closest to the outer surface, eliminating the need to image through the second layer of the chip.

[0030] There's no specific limit on the thickness of each layer of a gene sequencing chip. Generally, a thin layer of glass separates the first and second layers of a gene sequencing chip. For example, 0.3mm thick BF33. Generally, no more than 0.5mm is appropriate. Excessively thick glass, for example, exceeding 1mm, can affect heating and cooling rates.

[0031] The other structural parts of the sequencing chip involved in the present invention have been described in the aforementioned patents and will not be repeated in the present invention.

[0032] According to a preferred embodiment, a second sequencing chip is included.

[0033] According to a preferred embodiment, the first sequencing chip and the second sequencing chip share an optical system and collect signals in a cycle.

[0034] According to a preferred embodiment, the hot and cold fluid temperatures of the first and second sequencing chips are different. That is, while the first sequencing chip is exposed to the hot fluid to maintain its sequencing reaction temperature, the second sequencing chip can be exposed to the cold fluid. Oil can be used as the hot and cold fluids. This ensures that the two sequencing chips operate with minimal interference, allowing both to operate simultaneously using a single fluid system. This significantly improves application efficiency. Of course, since sequencing is divided into multiple steps, each with different timings, it's inevitable that during dual-chip sequencing, the first sequencing chip will need to be exposed to the hot fluid before the second sequencing chip has finished capturing its image. A simple solution is to adjust the order in which the fluids are added, for example, by delaying the addition of the hot fluid to the first sequencing chip. Overall, this improves sequencing efficiency. This benefit is significant. In existing sequencers, sequencing multiple chips without interfering with each other requires multiple heating platforms with different temperature capabilities, a complex process. Furthermore, each time a reaction chip is added, an additional heating platform is required. The method provided by the present invention achieves the goal of controlling the temperature of each chip by simply adjusting the order in which the fluids are added, which only increases the complexity of the fluid system. To a certain extent, it can quickly increase the number of chips on a single sequencer without interfering with each other. In this sense, it represents a qualitative improvement for sequencers.

[0035] According to a preferred embodiment, the sequencing reagents are kept at the same temperature as the cooling oil.

[0036] According to a preferred embodiment, the temperatures of the sequencing reagents and the cooling oil are the same during the sequencing run.

[0037] According to a preferred embodiment, the system further comprises a sequencing reagent storage device and a washing solution storage device.

[0038] According to a preferred embodiment, the test reagent 1 is included and contained in a sequencing reagent bottle 1; the sequencing reagent 2 is included and contained in a sequencing reagent bottle 2; and the sequencing reagent 3 is included and contained in a sequencing reagent bottle 3.

[0039] According to a preferred embodiment, there are two sequencing reagents, namely sequencing reagent one and sequencing reagent two.

[0040] According to a preferred embodiment, there are three sequencing reagents.

[0041] According to a preferred embodiment, the cleaning liquid is one kind.

[0042] According to a preferred embodiment, there are two types of cleaning liquids.

[0043] According to a preferred embodiment, the fluid switching system comprises a rotary valve.

[0044] According to a preferred embodiment, the sequencing is performed using a gene sequencer.

[0045] According to a preferred embodiment, the sequencer used for sequencing includes a fluid system, an optical system, and a chip stage.

[0046] According to a preferred embodiment, the storage temperature of the reagent storage device is 0-20 degrees Celsius.

[0047] According to a preferred embodiment, the storage temperature of the cold oil is 0-20 degrees Celsius.

[0048] According to a preferred embodiment, the storage temperature of the hot oil is 60-95 degrees Celsius, preferably 65-75 degrees Celsius.

[0049] According to a preferred embodiment, the storage temperature of the hot oil or the second temperature reagent is 30-45 degrees Celsius. When different catalytic enzymes are selected, the temperature used by the gene sequencing chip is different.

[0050] 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, which is a common function of a rotary valve.

[0051] According to a preferred embodiment, the reagent storage device is used to store sequencing reagents.

[0052] According to a preferred embodiment, the thermal oil storage device is used to store thermal oil.

[0053] According to a preferred embodiment, the cold oil storage device is used to store cold oil.

[0054] According to the preferred embodiment, the reagent storage device, the hot oil storage device, and the cold oil storage device are connected to the first rotary valve through pipelines.

[0055] According to the preferred embodiment, the first rotary valve is connected to the sequencing chip. The first rotary valve delivers the sequencing reagent, the hot / cold oil, and other necessary reagents into the sequencing chip.

[0056] According to the preferred embodiment, the first rotary valve is connected to the chip inlet of the sequencing chip.

[0057] According to the preferred embodiment, the syringe pump is connected to the chip outlet of the sequencing chip.

[0058] The gene sequencing chip belongs to the conventional process. Commonly, CN2017105741742, CN CN2017105741441, CN201710630287X, 201811643917.8, 201910156547.3; the contents of these patents can be incorporated by reference in this patent.

[0059] Generally, the rotary valve is used as the fluid control structure. The fluid components are connected through plastic pipes. Generally, polytetrafluoroethylene pipes are used for connection. For example, commonly used 0.3mm, 0.6mm, etc. The way of liquid path control has been introduced in the applicant's previous patents, such as CN2017211569462; the contents of this patent can be incorporated by reference in this patent.

[0060] Generally, the heating plate is used to control the temperature of the reagent. The temperature probe can be placed inside the reagent bottle in advance, or the temperature probe can be set on the structure of the reagent needle. Commonly, the reagent bottle is replaceable, and the use of reagent needle with temperature probe is more convenient. The constant temperature control of the reagent is not a complex technology. Generally, the temperature reduction of the reagent after passing through the connecting pipeline and the rotary valve can be detected by testing the outlet reagent temperature to detect the temperature of the oil flowing out of the chip. Generally, the temperature of the high-temperature oil is 0.5-5 degrees Celsius higher than the actual temperature required by the chip, preferably 0.5-2 degrees Celsius. Generally, the temperature of the low-temperature oil is 0.5-5 degrees Celsius lower than the actual temperature required by the chip, preferably 1-3 degrees Celsius. The temperature control is empirical, and the equipment can be fine-tuned before actual work.

[0061] The function of the washing solution is to clean the oil. The washing solution is generally small-molecule organic reagents such as ethanol and acetone.

[0062] Generally, hot and cold oils use oils with the same composition. Fluorine oil is generally used. The oil acts as a seal. Common examples of this method are described in the applicant's patents CN201710630287X and CN2017211569462; portions of these two patents are incorporated herein by reference.

[0063] Using hot and cold oil to control temperature is rare in microfluidic chips. Passing hot and cold oil into the chip to directly heat it and provide an oil seal is not a common technology.

[0064] The specific embodiments of the present invention are merely further explanations of the present invention and are not intended to be limiting factors of the present invention.

[0065] Example 1

[0066] Hot and cold oil is used for sequencing reaction. The sequencing device includes: a reaction chamber with DNA fragments to be tested planted on the surface, a pump and valve system for fluid control, an optical system for collecting signals and a central control system, and a temperature control system for controlling the temperature of the reaction reagents. Figure 1 The test process includes: (1) Experimental preparation: Place the chip on the chip table, place the sequencing reaction reagents and cold oil bottle on the 15℃ temperature control table, place the hot oil bottle on the 65℃ temperature control table, and place the wash solution 1 and wash solution 2 at room temperature; (2) The pump valve system first controls the wash solution 1 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℃ cold oil to enter the chip; (5) Turn on the light source, and the camera collects signals and records background values; (6) The pump valve system controls the 65℃ hot oil to enter the chip, and the polymerase extension reaction is carried out, keeping the reaction time at 45 seconds; (7) Pump 15℃ cold oil into the chip again and collect signals. (8) The pump valve system pumps wash solution 2 into the reaction chamber. Steps (2)-(8) are a sequencing reaction cycle. The sequencing reaction solution is cycled in this way until the set number of cycles is reached.

[0067] 2+2 sequencing, single-color: Prepare three sets of two reaction solutions, each containing two fluorescently labeled bases (X). Both reaction solutions in one set contain exactly four bases. All six solutions are unique.

[0068] First bottle Second bottle The first set AX+CX GX+TX Second set AX+GX CX+TX The third set AX+TX CX+GX

[0069] The first bottle of reaction solution is sequencing reagent 1; the second bottle of reaction solution is sequencing reagent 2.

[0070] The complete sequencing process consists of three rounds, which are performed sequentially. Each round of sequencing uses the three sets of reagents described above. Otherwise, the three rounds are identical (using the same sequencing primers and reaction conditions).

[0071] The sequencing chip structure is described in Example 4.

[0072] Each cycle of sequencing comprises:

[0073] 1. Hybridize the sequencing primer to the DNA array that has been prepared

[0074] 2. Start the sequencing process. Repeat 2.1-2.4 for a limited number of times.

[0075] 2.1. Add the first reagent. React and collect the fluorescent signal.

[0076] 2.2. Wash the flowcell to remove all residual reagents and fluorescent molecules produced

[0077] 2.3. Add the second reagent. React and collect the fluorescent signal.

[0078] 2.2. Wash the flowcell to remove all residual reagents and fluorescent molecules produced

[0079] 3. Unwind the extended sequencing primer.

[0080] At this point, the next cycle of sequencing can be performed.

[0081] Prepare the reagents:

[0082] Prepare the sequencing reagent solution, referred to as the wash solution, containing:

[0083] 20 mM Tris-HCl pH 8.8

[0084] 10 mM (NH4)2SO4

[0085] 50 mM KCl

[0086] 2 mM MgSO4

[0087] 0.1% Tween 20 20

[0088] The sequencing chip is described in Example 4.

[0089] Place the sequencing chip in the sequencer.

[0090] Use the first set of reagents to sequence. Corresponding to Figure 1 , follow the procedure below.

[0091] 1. Flush the chip with 10 mL of the wash solution

[0092] 2. Add 100 uL of sequencing reagent one (8) to the first layer of the chip

[0093] 3. Add the cooling oil (10) to the second layer of the chip

[0094] 4. Pass hot oil (11) into the second layer of the chip

[0095] 5. Wait 1 minute

[0096] 6. Use 473nm laser excitation to capture fluorescence images.

[0097] 7. Pass 10 mL of washing solution (7) to rinse the chip

[0098] 8. Add 100uL sequencing reagent 2 (9)

[0099] 9. Introduce cooling oil (10) into the second layer of the chip

[0100] 10. Pass hot oil (11) into the second layer of the chip

[0101] 11. Wait 1 minute

[0102] 12. Use 473 nm laser excitation to capture fluorescence images.

[0103] Repeat steps 1-13 50 times to obtain 100 fluorescence signals.

[0104] Example 2

[0105] According to the method of Example 1, the temperature of the hot oil is set to 70 degrees Celsius and the temperature of the cold oil is set to 4 degrees Celsius. Multiple rounds of experiments found that the heating efficiency of the hot and cold oils is good, and the temperature changes are shown in Table 1. Figure 2 The hot oil and cold oil temperature control is turned off. The same reagent is placed on the heating plate and the temperature is set to 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 2 The solid line shows that (1) the heating and cooling rates of the oil-heated and electrically heated chips are essentially the same. The difference in maximum temperature is because oil heating directly heats the chip, which is more efficient. Depending on the specific situation, a specific temperature can be controlled. When the material supporting the chip is changed, the oil heating and cooling rates become even faster. Simply changing the material at the bottom of the chip improves efficiency by more than 20% because only the chip needs to be heated, without having to worry about heating the bulky auxiliary equipment.

[0106] Typically, electric heating places the chip bottom in direct contact with the metal heating plate, which provides excellent thermal conductivity. Oil heating, however, does not require the bottom material to have excellent thermal conductivity. This allows for greater choice in base material, a significant advantage for industrial design. Maintaining and maintaining the flatness of the hot plate surface is challenging. Oil heating, however, eliminates the need to consider hot plate material and processing issues.

[0107] Example 3

[0108] The apparatus according to embodiment 1. Figure 1 1 is the micro-well array chip; 2 is the chip fluid inlet; 3 is the signal acquisition device; 4 is the valve; 5 is the pump; 6 is the reagent temperature control station; 7 is a bottle of wash solution; 8 is a bottle of sequencing reagent; 9 is two bottles of sequencing reagent; 10 is a bottle of cold oil; 11 is a bottle of hot oil; 12 is two bottles of wash solution. The detailed structure of the micro-well array chip is described in Example 4; other functional structures are described in patent CN2017105741742. The fluid system can be found in CN2017211569301. The valve in component 4 is a rotary valve with multiple inlets and outlets. The pump in component 5 is a syringe pump, which provides power.

[0109] The number of inlets and outlets on the microwell array chip can be adjusted based on specific needs. During the actual sequencing process, after the sequencing chip is placed on the sequencer, cooling oil is introduced in step 3, requiring approximately 50-100 ml, and hot oil is introduced in step 4, requiring 50-100 ml. The same applies to steps 9 and 10.

[0110] According to this feature, this embodiment improves the Figure 1 The cooling and heating liquid used in the second layer of the sequencing chip is connected to the cooling or heating liquid. This achieves the recycling of the cooling or heating liquid. Of course, a more complex device can also be designed to determine the direction of recycling of the cooling and heating liquids based on the remaining amount. For example, when the cooling liquid is low, all the liquid in the second layer of the chip is recycled into the cooling liquid 10; when the heating liquid is low, all the liquid in the second layer of the chip is recycled into the heating liquid 11.

[0111] 1. Add 10 mL of washing solution to rinse the chip, and the waste solution is discharged into the waste bottle;

[0112] 2. Add 100uL sequencing reagent 1 (8), and the excess liquid will enter the waste bottle;

[0113] 3. Cooling oil (10) is introduced, and the excess oil enters the cold oil bottle 10;

[0114] 4. Add hot oil (11) and the excess oil goes into the cold oil bottle 10;

[0115] 5. Wait for 1 minute;

[0116] 6. Use 473nm laser excitation to capture fluorescence images;

[0117] 7. Pass 10 mL of washing solution (7) to rinse the chip, and the waste solution is discharged into the waste liquid bottle;

[0118] 8. Add 100uL sequencing reagent 2 (9), and the excess liquid enters the waste bottle;

[0119] 9. Cooling oil (10) is introduced, and the excess oil enters the cold oil bottle 10;

[0120] 10, hot oil (11) is introduced, and the excess oil enters the cold oil bottle 10;

[0121] 11. Wait 1 minute

[0122] 12. Use 473 nm laser excitation to capture fluorescence images.

[0123] Repeat steps 1-13 50 times to obtain 100 fluorescence signals.

[0124] With this improvement, excess oil will not be wasted. Compared with Example 1, more than 200 ml of oil can be saved per sequencing.

[0125] Example 4

[0126] The device described in Example 1. The chip is mainly divided into three layers, see Figure 3 From top to bottom, they are the base layer 101, the middle spacer layer 103, and the second base layer 105. The three layers are assembled together to form a chip, with the different chip layers bonded together with double-sided tape. External fluid enters through the holes in the micro-reactor chip layer.

[0127] The cross-section of the chip structure can be shown as Figure 3 As shown, the bottom glass layer is 101, the fluid layer of the heating and cooling liquid is 102, the middle layer 103 with micro-pits on one side, the reaction liquid layer is 104, and the second bottom glass layer is 105. In 102, the die-cutting mechanism forms a cavity-type reaction chamber. The lower surface of 103, that is, the surface in contact with 102, is etched with an array of micro-reaction chambers. Among them, 101, 103, and 105 are physical layers. 101 and 105 are made of BF33 glass with a thickness of 1mm. 103 can be made of dry-etched glass or microchannel plate with a thickness of 0.3-0.5mm. The thickness used in this reaction is 0.3mm. In order to save fluid, the thickness of layer 102 is 0.05mm, and the thickness of layer 104 is 0.08mm. The 102 layer and the 104 layer are separated and formed by die-cut double-sided tape with pre-prepared patterns.

[0128] The embodiments of this patent are further explanations of the present invention and do not affect the scope of protection of the patent.

Claims

1. A nucleic acid sequencing system with reagent temperature control, characterized in that The invention comprises a first temperature control system including a reagent configured to be at a first temperature and a storage device thereof; a reagent switching system; A second temperature control system includes a reagent configured to maintain a second temperature and a storage device therefor; a gene sequencing chip; wherein the gene sequencing chip includes a first reagent layer and a second reagent layer; the reagent stored in the storage device of the first temperature control system and the reagent stored in the storage device of the second temperature control system are connected to a reagent switching system via a fluid pipeline; one inner surface of the gene sequencing chip has pre-prepared micro-pits; The first temperature control system includes a sequencing reaction solution and a storage device thereof, and also includes a cooling liquid and a storage device thereof; the sequencing reaction solution is added to the first reagent layer of the gene sequencing chip through the reagent switching device; and the cooling liquid is added to the second reagent layer of the gene sequencing chip through the reagent switching device; The reagent in the second temperature control system is a heating liquid, which is added to the second reagent layer of the gene sequencing chip through a reagent switching device; the cooling liquid and the heating liquid jointly control the temperature of the gene sequencing chip.

2. The system according to claim 1, wherein: 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.

3. The system according to claim 1, wherein: The gene sequencing chip includes a first gene sequencing chip and a second gene sequencing chip.

4. A gene sequencing method for sealing oil temperature control, characterized in that include: A first temperature control system, comprising a reagent at a first temperature and a storage device thereof; Reagent switching system; A second temperature control system comprising a reagent configured to be at a second temperature and a storage device thereof; a gene sequencing chip; Wherein, at least one surface of the reaction chamber of the gene sequencing chip has pre-processed micro-pits; the reagents at the first temperature include sequencing reagents and sealing oil; the reagents at the second temperature are sealing oil; The gene sequencing chip includes a first reagent layer and a second reagent layer; The first temperature control system includes sequencing reagents and storage devices thereof, and also includes cooling liquid and storage devices thereof; the sequencing reagents are added to the first reagent layer of the gene sequencing chip through the reagent switching device; and the cooling liquid is added to the second reagent layer of the gene sequencing chip through the reagent switching device; The reagent in the second temperature control system is a heating liquid, which is added to the second reagent layer of the gene sequencing chip through the reagent switching device; the cooling liquid and the heating liquid jointly control the temperature of the gene sequencing chip; During sequencing, a sequencing reagent at a first temperature is added to the gene sequencing chip through a reagent switching system; sealing oil at a first temperature is added to the gene sequencing chip; sealing oil at a second temperature is added to the gene sequencing chip; and detection is performed to obtain a sequencing signal.

5. The method according to claim 4, characterized in that The sequencing reagents include sequencing reagent one and sequencing reagent two; each sequencing reagent contains two of A, G, C, and T nucleotide molecules, or each reaction solution contains two of A, G, C, and U nucleotide molecules; the bases of the nucleotide molecules in the two sequencing reagents are complementary.

6. The method according to claim 4, characterized in that The following steps are included: introducing sequencing reagent 1 stored at a first temperature into the sequencing chip; introducing sealing oil stored at a first temperature; introducing sealing oil stored at a second temperature; After the sequencing reaction occurs, take a photo to obtain the sequencing signal; introducing sequencing reagent 2 stored at the first temperature; introducing sealing oil stored at a first temperature; introducing sealing oil stored at a second temperature; After the sequencing reaction occurs, take a photo to obtain the sequencing signal.

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