CN112969527A - Micro-fluidic chip, preparation method thereof and DNA (deoxyribonucleic acid) synthesis method - Google Patents
Micro-fluidic chip, preparation method thereof and DNA (deoxyribonucleic acid) synthesis method Download PDFInfo
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Abstract
一种微流体芯片及其制备方法和DNA合成方法。微流控芯片包括片体，片体内设置有输入流道(21、22)、输出流道(31、32)以及反应腔室(40)，反应腔室(40)的入口设有第一栅栏式筛阀(51)，反应腔室(40)的出口设有第二栅栏式筛阀(52)。第一栅栏式筛阀(51)的口径大于固相载体的尺寸，第二栅栏式筛阀(52)的口径小于固相载体的尺寸。由于在反应腔室(40)的入口和出口分别设置有第一栅栏式筛阀(51)和第二栅栏式筛阀(52)，第一栅栏式筛阀(51)的口径大于固相载体的尺寸，第二栅栏式筛阀(52)的口径小于固相载体的尺寸，能够有效控制固相载体进入反应腔室(40)内部后被限制在反应腔室(40)内部进行基于合成反应，提高了反应的正确率；并且反应腔室(40)内可容纳大量的固相载体进行合成反应，从而合成通量高。
A microfluidic chip and its preparation method and DNA synthesis method. The microfluidic chip includes a chip body, and the chip body is provided with input flow channels (21, 22), output flow channels (31, 32) and a reaction chamber (40), and the inlet of the reaction chamber (40) is provided with a first fence Type sieve valve (51), the outlet of the reaction chamber (40) is provided with a second grid type sieve valve (52). The diameter of the first fence type sieve valve (51) is larger than the size of the solid phase carrier, and the diameter of the second fence type sieve valve (52) is smaller than the size of the solid phase carrier. Since the inlet and outlet of the reaction chamber (40) are respectively provided with the first grid-type sieve valve (51) and the second grid-type sieve valve (52), the diameter of the first grid-type sieve valve (51) is larger than that of the solid phase carrier The size of the second fence-type sieve valve (52) is smaller than the size of the solid phase carrier, which can effectively control the solid phase carrier to enter the reaction chamber (40) and be confined inside the reaction chamber (40) to carry out the synthesis-based reaction. , the correct rate of the reaction is improved; and the reaction chamber (40) can accommodate a large number of solid-phase carriers for the synthesis reaction, so that the synthesis throughput is high.
Description
The application relates to a DNA synthesis technology, in particular to a micro-fluidic chip, a preparation method thereof and a DNA synthesis method.
DNA is the basic genetic material in a living body. Artificially synthesized in vitro DNA can replicate any naturally occurring DNA function or create new DNA functions as required for research and application. With the development of genomics, molecular biology, system biology and synthetic biology, artificially synthesized DNA has wide application value in the fields of cell engineering modification, gene editing, disease diagnosis and treatment, new material development and the like. Since Todd, Khorana and co-workers in the fifties of the twentieth century reported for the first time DNA synthesis, the synthetic methods have undergone a long-term development, and the current classical methods include column synthesis developed in the eighties, and microarray-based high-throughput synthesis developed in the nineties. A column synthesizer, for example, dr. oligo192, controls the addition of a reagent by an electromagnetic valve, and performs a solid phase synthesis reaction on a porous reaction column having a size of the order of centimeters, and this reaction has a low error rate, but the synthesis flux is not high and a large amount of raw materials are required. Microarray synthesizers, such as the CustomAlray synthesizer, reduce the synthesis reaction into reaction wells of micron order, and the synthesis pool of one chip has tens of thousands of reaction wells, thus improving the synthesis flux and reducing the consumption of raw materials, but the reaction is not easy to control and the error rate is high. Further advances in DNA synthesis require more efficient engineering techniques to achieve. From the viewpoint of chemical reaction, in order to improve the reaction efficiency, it is necessary to maintain the concentration of the reagent at a certain level as much as possible and to remove the residual reagent as soon as possible after the reaction; in order to reduce by-products and reduce the error rate, it is necessary to shorten the reaction time while ensuring the reaction is sufficient, and for this reason, four-step reactions in the reaction cycle of DNA synthesis need to be controlled as precisely as possible. From the perspective of synthesis cost, in order to reduce the consumption of raw materials, the use of raw materials needs to be strictly controlled under the condition of ensuring reasonable output; in order to shorten the time of the whole target synthesis under the condition of ensuring the flux, the flow of the synthesis needs to be optimally designed, such as the sequence of adding the combination monomers and the reagents is optimized, and the target product is obtained as soon as possible. In summary, there is no technology that can realize DNA synthesis at low cost and high throughput while ensuring low error rate. How to realize efficient and low-cost DNA synthesis by high-level technical means is a problem to be solved urgently.
Microfluidic technology is a technology that enables precise operational control of microscale fluids. Due to its advantages of precise controllability, high throughput and low cost, microfluidic technology has been widely used in various fields of life sciences. Some of the existing DNA synthesis methods have been tried using some microfluidic technologies, such as etching technology of electrochemical synthesis chip and mask illumination technology of program control in photochemical synthesis, but the methods and applications for synthesizing DNA using microfluidic chip are relatively few. DNA synthesis is the most fundamental ring in the fields of molecular biology, synthetic biology and the like, and has placed high demands on scale and cost. From theory and previous practice, it can be found that the microfluidic technology has great potential and wide application prospect in the field of DNA synthesis.
The existing single-base DNA synthesis methods, including chemical synthesis, electrochemical synthesis and photochemical synthesis of porous glass, are based on phosphoramidite chemical synthesis four-step methods, and DNA monomers are added one by one according to a predetermined sequence. Some commercial synthesizers have entered the market based on single base DNA synthesis methods. These synthesizers can be broadly divided into two categories: column synthesizers and microarray synthesizers. Oligo192, is a solid phase synthesis reaction on a porous reaction column with a size of centimeter grade by controlling the addition of reagents through an electromagnetic valve, and has high reaction accuracy, but the synthesis flux is not high and the required raw materials are large. Microarray synthesizers, such as the CustomAlray synthesizer, reduce the synthesis reaction into reaction wells of micron order, and one chip has tens of thousands of reaction wells on the synthesis pool, thus improving the synthesis flux and reducing the consumption of raw materials, but the reaction is not easy to control and the accuracy is not high.
Disclosure of Invention
The embodiment provides a micro-fluidic chip, including the lamellar body, be equipped with input flow channel in the lamellar body, output flow channel and reaction chamber, the lamellar body is extended out to input flow channel's one end, the other end extends to the entry linkage with reaction chamber, the lamellar body is extended out to output flow channel's one end, the other end extends to the exit linkage with reaction chamber, reaction chamber's entry is equipped with first barrier formula sieve valve, reaction chamber's export is equipped with second barrier formula sieve valve, the bore of first barrier formula sieve valve is greater than the size of solid carrier, the bore of second barrier formula sieve valve is less than the size of solid carrier.
In one embodiment, a method for manufacturing a microfluidic chip is provided, which includes the steps of:
preparing an upper chip with a first input flow channel and a first output flow channel;
preparing a lower chip with a second input flow channel, a second output flow channel, a reaction chamber, a first fence type sieve valve and a second fence type sieve valve, wherein the second input flow channel is connected with an inlet of the reaction chamber through the first fence type sieve valve, the second output flow channel is connected with an outlet of the reaction chamber through the second fence type sieve valve, the caliber of the first fence type sieve valve is larger than the size of a solid-phase carrier, and the caliber of the second fence type sieve valve is smaller than the size of the solid-phase carrier;
and compounding the upper chip and the lower chip into the microfluidic chip, wherein one end of the first input flow channel extends to be in butt joint with the second input flow channel, the other end of the first input flow channel extends to be in butt joint with the upper surface of the upper chip, one end of the first output flow channel extends to be in butt joint with the second output flow channel, and the other end of the first output flow channel extends to be in butt joint with the upper surface of the upper chip.
In one embodiment, a method for DNA synthesis is provided, comprising the steps of:
preparing a micro-fluidic chip with a corresponding flow channel and a reaction chamber according to a target DNA or RNA sequence to be synthesized;
designing a solid phase carrier modified for synthesis according to a target DNA or RNA sequence to be synthesized;
inputting the modified solid phase carrier into the reaction chamber from the input flow channel of the microfluidic chip of the embodiment;
then inputting the reagent into the reaction chamber from the input flow channel of the microfluidic chip for synthetic reaction;
after the synthesis reaction is finished, outputting the solid phase carrier in the reaction chamber to the outside of the chip from the output flow channel and collecting the solid phase carrier;
and carrying out ammonolysis, purification and gene assembly on the output and collected solid phase carrier to prepare the target DNA or RNA.
According to the microfluidic chip, the preparation method thereof and the DNA synthesis method of the embodiment, the inlet and the outlet of the reaction chamber are respectively provided with the first fence type sieve valve and the second fence type sieve valve, the caliber of the first fence type sieve valve is larger than the size of the solid phase carrier, and the first fence type sieve valve is smaller than the size of the solid phase carrier, so that the solid phase carrier can be effectively controlled to be limited in the reaction chamber to carry out synthesis reaction after entering the reaction chamber, the reaction accuracy is improved, and a large amount of solid phase carriers can be accommodated in the reaction chamber to carry out synthesis reaction, so that the synthesis flux is high.
FIG. 1 is a schematic structural diagram of a microfluidic chip according to an embodiment;
FIG. 2 is a schematic diagram of a reaction chamber according to an embodiment;
FIG. 3 is a flow chart of a method of fabricating a microfluidic chip according to an embodiment;
FIG. 4 is a flow chart of a method of fabricating an upper chip in one embodiment;
FIG. 5 is a flow chart of a method for manufacturing a lower chip in one embodiment;
FIG. 6 is a schematic diagram of a method of fabricating a lower chip in one embodiment;
FIG. 7 is a schematic view of a reaction chamber template in one embodiment;
FIG. 8 is a flowchart of a DNA synthesis method in one embodiment;
FIG. 9 shows the result of detecting fluorescent monomers in DNA synthesis in one example.
The embodiment of the invention provides a micro-fluidic chip which can reduce the material cost through the reaction control of micron scale and can also ensure the accuracy of the synthesized product by utilizing a solid phase synthesis method. In addition, the microfluidic chip also has the characteristics of expansion and easy integration, and the flux can be flexibly adjusted according to the requirement.
Example 1:
as shown in fig. 1, the present embodiment provides a microfluidic chip, where the microfluidic chip includes a sheet body, the sheet body 10 includes an upper chip 11 and a lower chip 12, the upper chip 11 and the lower chip 12 are made of glass, silicon wafer or polydimethylsiloxane, and the upper chip 11 and the lower chip 12 are bonded and compounded together. In other embodiments, the upper chip 11 and the lower chip 12 may be a unitary structure.
In this embodiment, the lower surface of the upper chip 11 is provided with a first input flow channel 21 and a first output flow channel 31, the upper surface of the lower chip 12 is provided with a second input flow channel 22, a second output flow channel 32 and a reaction chamber 40, the reaction chamber 40 has an inlet and an outlet, the inlet of the reaction chamber 40 is provided with a first barrier type sieve valve 51, the outlet of the reaction chamber 40 is provided with a second barrier type sieve valve 52, the second input flow channel 22 is connected with the inlet of the reaction chamber 40 through the first barrier type sieve valve 51, and the second outflow flow channel 32 is connected with the outlet of the reaction chamber 40 through the second barrier type sieve valve 52.
The first input flow channel 21 and the first output flow channel 31 on the upper chip 11, and the second input flow channel 22, the second output flow channel 32, the reaction chamber 40, the first barrier type sieve valve 51 and the second barrier type sieve valve 52 on the lower chip 12 are all open grooves, the first input flow channel 21 and the first output flow channel 31 enclose a complete flow channel with the lower chip 12, and the second input flow channel 22, the second output flow channel 32, the reaction chamber 40, the first barrier type sieve valve 51 and the second barrier type sieve valve 52 enclose a complete flow channel, cavity and valve with the upper chip 11.
After the upper chip 11 and the lower chip 12 are combined together, one end of the first input flow channel 21 extends to be in butt joint with the second input flow channel 22, the other end of the first input flow channel 21 is provided with a guide hole extending out of the upper surface of the upper chip 11, and the first input flow channel 21 and the second input flow channel 22 are in butt joint to form the input flow channel 20. The first inlet flow channel 21 is a control flow channel, a valve is attached to the first inlet flow channel 21, and the first inlet flow channel 21 is narrower than the second inlet flow channel 22.
Similarly, one end of the first output flow channel 31 extends to be in butt joint with the second output flow channel 32, the other end of the first output flow channel 31 is provided with a guide hole extending out of the upper surface of the upper chip 11, and the first output flow channel 31 and the second output flow channel 32 are in butt joint to form the output flow channel 30. The first output flow path 31 is a control flow path, a valve is attached to the first output flow path 31, and the first output flow path 31 is narrower than the second output flow path 32. The first input flow channel 21 and the first output flow channel 31 are provided with valves for precisely controlling the introduction and the derivation, improving the efficiency of the synthesis.
In this embodiment, the input flow channels 20 and the output flow channels 30 may be provided in a plurality, the reaction chambers 40 may be provided in a plurality, the plurality of input flow channels 20 are collected into one flow channel, and then the plurality of flow channels are branched to be respectively butted with inlets of the plurality of reaction chambers 40, that is, each input flow channel 20 is butted with all the reaction chambers 40. Similarly, the plurality of output channels 30 are collected into one channel, and then branched into a plurality of channels to be respectively connected to the outlets of the plurality of reaction chambers 40. In other embodiments, a plurality of input flow channels 20 and a plurality of output flow channels 30 may be separately disposed, and finally, a plurality of flow channels are divided from each of the input flow channels 20 and the output flow channels 30 to be connected to the reaction chamber 40.
In this embodiment, the plurality of second input runners 22 and the plurality of second output runners 23 are respectively gathered together and then branched to be connected with the reaction chamber 40, the plurality of first input runners 21 and the plurality of first output runners 31 are independent from each other, the first input runners 21 correspond to the second input runners 22 one by one, and the first output runners 31 correspond to the second output runners 32 one by one.
The number of the input flow channels 20 and the output flow channels 30 and the number of the reaction chambers 40 can be set according to specific requirements, for example, four single-base monomers are required for the synthesis of a single base, at least four reagents are required for the chemical synthesis reaction, and at least 8 input flow channels 20 are required. The number of the second output flow channels 32 corresponds to the number of the reaction chambers 40.
As shown in fig. 1 and 2, in the present embodiment, the reaction chamber 40 has two, preferably pie-shaped structures, and the reaction chamber 40 may also have a square-shaped structure. Two reaction chambers 40 are arranged side by side, the reaction chamber 40 has an inlet and three outlets, the three outlets are respectively provided with a second barrier type sieve valve 52, and the branched outflow channels of the output flow channel 30 are respectively connected with the outlets of the reaction chambers 40.
The first barrier screen valve 51 and the second barrier screen valve 52 are composed of several long thin pipes side by side. The aperture of the first barrier type sieve valve 51 is slightly larger than the size of the solid phase carrier for synthesizing the gene, and the aperture of the second barrier type sieve valve 52 is slightly smaller than the size of the solid phase carrier, so that the solid phase carrier can be input into the reaction chamber 40 and is limited in the reaction chamber 40 by the second barrier type sieve valve 52. The specific size of the first barrier sieve valve 51 and the second barrier sieve valve 52 can be designed according to the size of the actual solid carrier, for example, the diameter and the length of the second barrier sieve valve 52 can be designed to be 4 micrometers, 5 micrometers, 6 micrometers, etc., the height of the pipe section of the second barrier sieve valve 52 is 20 micrometers, the width is 5 micrometers, and the solid carrier can be effectively blocked.
The micro-fluidic chip of this embodiment is further provided with an auxiliary flow channel 60, the auxiliary flow channel 60 is disposed on the lower surface of the upper chip 11 and is also an open-type groove, the auxiliary flow channel 60 and the lower chip 12 enclose to form a complete flow channel structure, the auxiliary flow channel 60 has one, the auxiliary flow channel 60 extends across the outlets of the reaction chambers 40, the auxiliary flow channel 60 is connected to six outlets of the two reaction chambers 40, and one end of the auxiliary flow channel 60 has a guide hole extending to the upper surface of the upper chip 11. The auxiliary flow channel 60 can be used not only for adding reactants but also for more convenient collection and back flushing of the substances in the reaction chamber 40 and for checking malfunction during operation of the chip.
The microfluidic chip of this embodiment is used to connect with an automatic control device for controlling the introduction and removal of reactants and reagents.
According to the micro-fluidic chip provided by the embodiment, the first fence type sieve valve 51 and the second fence type sieve valve 52 are respectively arranged at the inlet and the outlet of the reaction chamber 40, the caliber of the first fence type sieve valve 51 is larger than the size of a solid phase carrier, and the first fence type sieve valve 52 is smaller than the size of the solid phase carrier, so that the solid phase carrier can be effectively controlled to be limited in the reaction chamber 40 after entering the reaction chamber 40 to perform a synthetic reaction, the reaction accuracy is improved, a large amount of solid phase carriers can be accommodated in the reaction chamber 40 to perform the synthetic reaction, and the synthetic flux is high. And, there are many input and output pipelines, and many reaction chambers 40, have further improved flux and efficiency that is synthesized.
Example 2:
the embodiment provides a method for preparing a microfluidic chip, which is used for preparing the microfluidic chip of the embodiment. The preparation method mainly prepares the upper chip and the lower chip through photoetching, dry etching or reverse mould processes, and the upper chip and the lower chip are compounded together, and the preparation method can adopt the combined preparation of photoetching and reverse mould, also can adopt the combined preparation of dry etching and reverse mould, or adopt the combined preparation of photoetching, dry etching and reverse mould. The present embodiment is described by taking a combination of photolithography, dry etching, and reverse molding as an example.
As shown in fig. 3, the method for preparing the microfluidic chip of this embodiment mainly includes the following steps:
s100: preparing an upper chip;
as shown in fig. 4, the preparation of the upper chip mainly includes the following sub-steps:
s101: initial preparation;
the method comprises the steps of initially preparing to place a substrate on a platform, coating a layer of photoresist on the upper surface of the substrate, and attaching a mask with a first input runner, a first output runner and an auxiliary runner pattern on the photoresist.
S102: UV illumination;
UV (ultraviolet) is vertically irradiated on the photoresist attached with the mask, the part of the photoresist which is not blocked by the mask is subjected to chain reaction, the part of the photoresist after reaction is dissolved in a dissolving agent, and the blocked part is not dissolved in the dissolving agent.
S103: stripping the photoresist;
the parts of the photoresist which are illuminated are dissolved away by the dissolving agent, and only the shielded parts of the photoresist remain, wherein the parts are the patterns of the first input flow channel, the first output flow channel and the auxiliary flow channel.
S104: etching by reactive ions;
and etching the substrate into a pattern same as the photoresist by adopting an ion etching technology to prepare an upper chip template with a structure opposite to that of the upper chip.
S105: preparing a reverse mold;
and (4) performing reverse molding to obtain an upper chip according to the upper chip template by adopting a reverse molding technology, and punching the upper chip to prepare the final upper chip.
S200: preparing a lower chip;
as shown in fig. 5 and 6, the preparation of the lower chip mainly includes the following sub-steps:
s201: initial preparation;
initially, a substrate 2 is placed on a stage 1, a photoresist 3 is coated on an upper surface of the substrate 2, and a mask 4 having a reaction chamber pattern is attached on the photoresist 3.
S202: first UV illumination;
UV (ultraviolet) is vertically irradiated on the photoresist 3 attached with the mask 4, the part of the photoresist 3 which is not shielded by the mask 4 is subjected to chain reaction, the part of the photoresist 3 after reaction is dissolved in a dissolving agent, and the shielded part is not dissolved in the dissolving agent.
S203: stripping the photoresist for the first time;
the light-irradiated portion of the photoresist 3 is dissolved away by the solvent, and only the shielded portion of the photoresist 3 remains, which is the pattern of the reaction chamber.
S204: etching by reactive ions;
the substrate 2 will be etched into the same pattern as the photoresist 3 using ion etching techniques.
S205: coating the photoresist again;
the photoresist 3 is coated on the platform 1 with the substrate 2, the substrate 2 is covered by the photoresist 3, and a mask 4 with a second input flow channel, a second output flow channel, a first barrier type sieve valve and a second barrier type sieve valve pattern is attached to the region outside the reaction chamber region.
S206: performing secondary UV illumination;
the stage 1 with the mask 4 is vertically irradiated with UV (ultraviolet rays), and the irradiated photoresist 3 undergoes a chain reaction.
S207: stripping the photoresist for the second time;
and dissolving the exposed photoresist 3 by using a dissolving agent, and preparing a lower chip template corresponding to a lower chip by using the photoresist 3 with patterns corresponding to the second input flow channel, the second output flow channel, the first barrier type sieve valve and the second barrier type sieve valve, as shown in fig. 7.
S208: and (4) preparing a reverse mold.
And (5) performing reverse molding on the lower chip according to the lower chip template by adopting a reverse molding technology to obtain the lower chip. And a second input flow channel in the prepared lower chip is connected with an inlet of the reaction chamber through a first fence type sieve valve, a second output flow channel is connected with an outlet of the reaction chamber through a second fence type sieve valve, the caliber of the first fence type sieve valve is slightly larger than the size of the solid-phase carrier, and the caliber of the second fence type sieve valve is slightly smaller than the size of the solid-phase carrier.
S300: and compounding the upper chip and the lower chip together.
And bonding the prepared upper chip and the prepared lower chip together to prepare the microfluidic chip with the flow channel and the reaction chamber, wherein valves are arranged on the first input flow channel, the first output flow channel and the auxiliary flow channel.
Specifically, in the prepared micro-fluidic chip, one end of a first input flow channel extends to be in butt joint with a second input flow channel, the other end of the first input flow channel extends to the upper surface of the upper chip, one end of a first output flow channel extends to be in butt joint with a second output flow channel, and the other end of the first output flow channel extends to the upper surface of the upper chip. One end of the auxiliary flow passage extends to be in butt joint with the outlet of the reaction chamber, and the other end of the auxiliary flow passage extends to be in butt joint with the upper surface of the upper chip.
The preparation method of the microfluidic chip provided by the embodiment adopts the photoetching, dry etching and reverse mold processes, and the preparation precision and efficiency are high.
Example 3:
this example provides a method for gene synthesis based on the microfluidic chip of the above example and suitable for DNA synthesis and RNA synthesis. This example illustrates the synthesis of DNA.
As shown in FIG. 8, the DNA synthesis method of this example includes the following steps:
s10: preparing a micro-fluidic chip;
designing and preparing a microfluidic chip having sufficient flow channels and reaction chambers according to target DNA to be synthesized, for example, four single-base monomers are required for single-base synthesis, and at least four reagents are required for chemical synthesis reaction, thus at least 8 input flow channels are required; in order to meet the requirements of synthesis throughput and yield, a plurality of reaction chambers with different sizes and shapes can be designed. The preparation method is as described in the above examples.
S20: designing a solid phase carrier;
designing and modifying a solid phase carrier for synthesis according to a target DNA or RNA sequence to be synthesized; the immobilization carrier is CPG with active functional groups modified by amino, and the diameter of the immobilization carrier is any one of 5 μm, 25 μm, 50 μm, 100 μm, 200 μm and 500 μm. The pore diameter of the fixing carrier is
Any one of them. The connecting molecule of the fixed carrier is a compound with any one or more functional groups of ester group, lipid group, thioester group, o-nitrobenzyl group, coumarin group, hydroxyl group, sulfhydryl group, thiolether group, carboxyl group, aldehyde group, amino group, amide group, alkenyl group and alkynyl group.
S30: inputting a solid phase carrier;
and inputting the modified solid phase carrier into the reaction chamber from the input flow channel of the microfluidic chip.
S40: inputting a reagent;
after the solid phase carrier is input into the reaction chamber, the positive pressure or negative pressure drive and the valve control are carried out, and the reagent is added into the reaction chamber in which the solid phase carrier is stored from the input flow channel for carrying out the synthetic reaction.
The steps of the synthesis reaction, reagents, solvents and times used are shown in the following table:
TABLE 1 synthetic reaction step scheme
Wherein, if the chain length of the DNA sequence to be synthesized is short (the number of cycles is less than or equal to 25), the capping step in the experimental operation can be omitted, for example, if a longer chain length DNA sequence is to be synthesized (the number of cycles is more than 25), the capping step is needed to reduce the error rate and obtain enough target DNA.
S50: outputting the solid phase carrier;
after the synthesis reaction is finished, the solid phase carrier in the reaction chamber is independently output to a container outside the chip from the output flow channel through positive pressure or negative pressure driving and valve control.
S60: ammonolysis, purification and gene assembly.
And (3) carrying out ammonolysis, purification and gene assembly on the output and collected solid phase carrier in sequence to prepare the target DNA. As shown in FIG. 9, the detection result of the fluorescent monomer of the target DNA was synthesized.
Wherein, the reagent adopted by the ammonolysis is any one of ammonia water, ammonia gas and methylamine, the temperature of the ammonolysis is any one of 25 ℃, 60 ℃ and 90 ℃, the time of the ammonolysis is any one of 2h, 5h, 10h, 18h and 24h, and the purification mode is any one of desalination, MOP, PAGE Plus and HPLC.
The synthesis method of the embodiment can be accurately and automatically controlled by a controller, and the controller controls driving and valves to accurately input and output reactants and reagents so as to realize high-flux high-accuracy synthesis reaction.
The embodiment provides a gene synthesis method, which adopts multi-channel, multi-reaction chamber and valve control, can reduce material cost through micron-scale reaction control, and can also ensure the accuracy of a synthesized product by using a solid-phase synthesis method. In addition, the chip used for synthesis also has the characteristics of expandability and easy integration, and the flux can be flexibly adjusted according to the requirement. The DNA synthesis method based on microfluidics provided by the invention improves the synthesis efficiency, technically avoids the defects of the current commercial synthesizer, and provides a new path with higher feasibility for the development of the future synthesizer.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. Variations of the above-described embodiments may be made by those skilled in the art, consistent with the principles of the invention.
Claims (26)
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| CN115323019A (en) * | 2022-08-31 | 2022-11-11 | 深圳市曙芯生物科技有限公司 | High-flux gene synthesis method |
| CN117732525A (en) * | 2024-02-19 | 2024-03-22 | 杭州联川生物技术股份有限公司 | A kind of microfluidic chip manufacturing method |
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| CN115323019A (en) * | 2022-08-31 | 2022-11-11 | 深圳市曙芯生物科技有限公司 | High-flux gene synthesis method |
| CN117732525A (en) * | 2024-02-19 | 2024-03-22 | 杭州联川生物技术股份有限公司 | A kind of microfluidic chip manufacturing method |
| CN117732525B (en) * | 2024-02-19 | 2024-05-14 | 杭州联川生物技术股份有限公司 | A method for manufacturing a microfluidic chip |
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| WO2020093322A1 (en) | 2020-05-14 |
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