TWI732404B - Apparatus and method for preparing nucleic acid sequences using enzyme - Google Patents

Abstract

An apparatus for preparing nucleic acid sequences using an enzyme, including a reactor, a plurality of nucleotide material bottles, a deblocking material bottle, and a liquid delivering device. The reactor includes a reaction substrate having a pretreated surface. Each of the nucleotide material bottles is adapted to contain a first reaction solution, and the first reaction solution includes a reaction enzyme and a nucleotide having a terminal protecting group. The deblocking material bottle is adapted to contain a deblocking solution. The liquid delivering device is connected to the reactor, the nucleotide material bottles and the deblocking material bottle, and is adapted to deliver the first reaction solution and the deblocking solution to the pretreated surface. The reaction enzyme is adapted to dispose the nucleotide having the terminal protecting group on the pretreated surface. The deblocking solution is adapted to remove the terminal protecting group of the nucleotide. The reactor has an operating temperature of 45℃ - 105℃. A method for preparing nucleic acid sequences using an enzyme of the invention is provided.

Description

Apparatus and method for preparing nucleic acid sequence using enzyme

The present invention relates to a device and method for preparing biomolecular structures, and more particularly to a device and method for preparing nucleic acid sequences using enzymes.

In the middle of the 20th century, several breakthroughs in genetic and biochemical research led to the development of today's medical field. The origin of these series of events was the X-ray-induced gene knockout study in 1941. From the results, it was known that genes were directly involved in the function of enzymes. Then it was discovered that genes are composed of nucleic acid (DNA), and the double-stranded helix is a structure of nucleic acid with genetic information and can be accurately replicated by DNA polymerase.

Nucleic acid synthesis is essential to modern biotechnology. The scientific community's ability to artificially synthesize DNA, RNA and protein has enabled the rapid development of the field of biotechnology. Artificial DNA synthesis, a growing market with unlimited business opportunities, enables biotech and pharmaceutical companies to develop a series of peptide therapies, such as insulin for the treatment of diabetes. It allows researchers to characterize cellular proteins to develop new small molecule therapies to treat diseases faced by today's aging population, such as heart disease and cancer.

However, the current DNA synthesis technology cannot meet the needs of the biotechnology industry. Despite the many benefits of DNA synthesis, there are still bottlenecks in the development of the artificial DNA synthesis industry, which hinders the development of the field of biotechnology. Although DNA synthesis is a mature technology, it is actually very difficult to synthesize DNA strands longer than 200 nucleotides, and most DNA synthesis companies can only provide up to 120 nucleotides. In comparison, the average protein-coding gene is on the order of 2000-3000 nucleotides, while the average eukaryotic genome is billions of nucleotides. Therefore, all major gene synthesis companies nowadays use the "synthesis and bonding" technology. About 40-60 overlapping sequence fragments are synthesized by PCR and bonded together (Young, L. et al. (2004) Nucleic Acid Res . 32, e59). Conventional methods provided by gene synthesis companies usually allow lengths of up to 3000 base pairs for routine production.

There are two main types of nucleic acid synthesis methods today: chemical synthesis and enzymatic synthesis. The most common nucleic acid chemical synthesis method is the phosphoramidite method described by Adams et al. (1983, J. Amer, Chem Soc. , 105: 661) and Froehler et al. (1983, Tetrahedron Lett 24: 3171). . In this method, each nucleotide to be added is equipped with a protective group on the 5'-OH group to avoid uncontrolled polymerization of the same type of nucleotide, usually 5'-OH group A trityl group can be used as the protecting group of the group. In order to avoid the possible degradation caused by the use of strong reactants, the nitrogen-containing bases carried by the nucleotides can also be further protected. The protective groups usually used include isobutyryl groups (Reddy et al. 1997, Nucleosides & Nucleotides 16: 1589 ). Every time a new nucleotide is added, the 5'-OH group of the last nucleotide in the chain undergoes a deprotection reaction to make it available for the next polymerization step. The nitrogen-containing bases carried by the nucleotides are only deprotected after all polymerization reactions are completed.

The chemical synthesis method described above requires a large amount of unstable, dangerous and expensive reactants, which can affect the environment and health. In addition, the equipment used in this chemical synthesis method is very complicated, requires a lot of investment, and must be operated by professional technicians. One of the main disadvantages of this chemical synthesis method is the lower yield. In each cycle, the success rate of the coupling reaction is only 98% to 99.5%, so that the nucleic acid sequence in preparation may not have the correct sequence. As the synthesis process progresses, the reaction medium will be filled with incorrect sequence fragments. This type of deletion error will therefore have a serious impact and cause the reading frame of the nucleic acid fragment to change.

For example, in order to make the coupling reaction have a 99% correct rate, the synthesis yield of a 70-nucleotide nucleic acid will be lower than 50%. This means that after 70 cycles of adding the nucleotide step, the reaction medium will contain more error sequence fragments than correct sequence fragments, which makes the synthesis reaction unsuitable for continuing.

Therefore, the methods of chemically synthesizing nucleic acids are not efficient for the synthesis of longer fragments, because they generate a large number of fragments with wrong sequences. In fact, the length of the fragments that can be effectively produced by chemical synthesis methods is about 50 to 100 nucleotides.

On the other hand, the difference between enzyme synthesis and chemical synthesis is that enzyme synthesis uses enzymes to carry out the nucleotide coupling step.

US patent US 7060440B1 discloses a method for DNA synthesis using a template-dependent polymerase. However, this technique is not suitable for de novo synthesis of nucleic acids due to the need for existing nucleic acid strands as templates.

Therefore, there is a need for enzymes that can perform coupling reactions between nucleotides without template strands. DNA polymerase is one of the eligible enzymes. DNA polymerases are divided into seven evolutionary families based on their amino acid sequences: A, B, C, D, X, Y, and RT. There is no correlation between the families of DNA polymerases, that is, members of one family are not homologous to members of any other family. Through the homology of DNA polymerase and the prototype member of the family, it can be determined as a member of the family. For example, members of family A are homologous to E. coli DNA polymerase I; members of family B are homologous to E. coli DNA polymerase II; members of family C are homologous to E. coli DNA polymerase III; members of family D Homologous to Pyrococcus furiosus DNA polymerase; members of X family are homologous to eukaryotic DNA polymerase β; members of Y family are homologous to eukaryotic RAD30; and members of RT family are homologous to reverse transcriptase .

Many documents (Ud-Dean et al. (2009) Syst. Synth. Boil. 2, 67-73, US 5763594 and US 8808989) have disclosed the use of terminal deoxynucleotidyl transferase (TdT) for acceptance. Controlled de novo single-stranded DNA synthesis. On the other hand, uncontrolled de novo single-stranded DNA synthesis utilizes the deoxynucleotide triphosphate (dNTP) 3'tailing property of terminal deoxynucleotidyl transferase on single-stranded DNA to create, for example, the next generation Homopolymer adaptor sequences prepared by sequencing libraries (Roychoudhur R. et al. (1976) Nucleic Acids Res. 3, 10-116 and WO 2003/050242).

However, since the main purpose of terminal deoxynucleotidyl transferase is to increase the diversity of antigen receptors, it may be difficult for it to exhibit normal and predictable behavior like other polymerases. This means that terminal deoxynucleotidyl transferases still have to face many challenges to achieve high-yield automation in the nucleotide synthesis cycle. Even though many documents (WO 2016128731A1, WO 2018217689A1, WO 2019135007A1) have disclosed the use of modified terminal deoxynucleotidyl transferase for enzyme synthesis, for terminal deoxynucleotidyl transferase, there is still a limit to the reaction rate itself. Moreover, when a large number of nucleic acid sequences are synthesized, the probability of error will still be increased.

To sum up, the chemical synthesis of nucleic acids can synthesize a large amount of nucleic acids, but the cost of the reagents used is too high and will pollute the environment, and the probability of errors in the synthesis process is high. The enzymatic synthesis of nucleic acids is inexpensive and has a high degree of accuracy of nucleic acid sequences. However, due to the reaction rate of enzymes, large-scale synthesis cannot be performed. Therefore, there is still no better nucleic acid synthesis method that can solve the problem of correct and large-scale nucleic acid synthesis.

The invention provides a device for preparing nucleic acid sequences using enzymes, which can improve the efficiency of preparing nucleic acid sequences.

The present invention provides a method for preparing nucleic acid sequences using enzymes, which can improve the efficiency of preparing nucleic acid sequences.

The device for preparing nucleic acid sequences using enzymes provided by an embodiment of the present invention includes a reactor, a plurality of nucleotide material bottles, deprotection material bottles, and a liquid delivery device. The reactor includes a reaction substrate, and the reaction substrate has a pretreated surface. Each nucleotide material bottle is suitable for accommodating a first reaction solution, and the first reaction solution includes a reaction enzyme and a nucleotide with a terminal protective group. The deprotection material bottle is suitable for containing the deprotection solution. The liquid delivery device is connected to the reactor, the nucleotide material bottles and the deprotection material bottles, and is suitable for delivering the first reaction liquid and the deprotection solution to the pretreatment surface. The reaction enzyme is suitable for arranging nucleotides with terminal protecting groups on the pretreated surface, and the deprotection solution is suitable for removing the terminal protecting groups of nucleotides. The working temperature of the reactor is 45℃~105℃.

In an embodiment of the present invention, the working temperature of the above-mentioned reactor is 50°C to 85°C.

In an embodiment of the present invention, the working temperature of the above-mentioned reactor is 55°C to 75°C.

In an embodiment of the present invention, the above-mentioned reaction enzyme is DNA polymerase.

In an embodiment of the present invention, the above-mentioned reaction enzyme is A family DNA polymerase.

In an embodiment of the present invention, the above-mentioned reaction enzyme is a family B DNA polymerase.

In an embodiment of the present invention, the above-mentioned reaction enzyme is X family DNA polymerase.

In an embodiment of the present invention, the above-mentioned liquid delivery device includes an extraction device, which is suitable for extracting the first reaction liquid and the deprotection solution to the pretreatment surface.

In an embodiment of the present invention, the above-mentioned extraction device is an injection pump or a pneumatic valve.

In an embodiment of the present invention, the above-mentioned pretreated surface has a plurality of primers, which are suitable for connecting nucleotides with terminal protecting groups.

In an embodiment of the present invention, the aforementioned deprotection solution includes a reducing agent or a deprotection enzyme.

In an embodiment of the present invention, the aforementioned device for preparing nucleic acid sequences using enzymes further includes a restriction enzyme material bottle suitable for containing a second reaction solution, the second reaction solution includes restriction enzyme, and the liquid delivery device is further connected to the restriction enzyme material Bottle to deliver the second reaction liquid to the pretreatment surface.

In an embodiment of the present invention, the above-mentioned apparatus for preparing nucleic acid sequences using enzymes further includes a heater, which is connected to the reactor and is suitable for heating the reactor.

In an embodiment of the present invention, the above-mentioned liquid delivery device includes a first switching valve and a plurality of first delivery tubes, and the first delivery tubes are connected between the nucleotide material bottles and the first switching valve and deprotected Between the material bottle and the first switching valve, the first switching valve is connected to the reactor and is suitable for switching between these first delivery pipes.

In an embodiment of the present invention, the above-mentioned device for preparing nucleic acid sequences using enzymes further includes a waste liquid bottle and a product bottle, wherein the liquid conveying device includes a second switching valve and a plurality of second conveying pipes, and the second conveying pipes are connected Between the waste liquid bottle and the second switching valve and between the product bottle and the second switching valve, the second switching valve is connected to the reactor and is suitable for switching between these second delivery pipes.

In an embodiment of the present invention, the above-mentioned device for preparing nucleic acid sequences using enzymes further includes a power supply element, which is suitable for supplying power to the liquid delivery device.

The method for preparing a nucleic acid sequence using enzymes provided in an embodiment of the present invention includes: (1) providing a reaction substrate, and the reaction substrate has a pretreated surface. (2) The nucleotides with terminal protecting groups are arranged on the pretreatment surface by reaction enzyme, and the working temperature is 45℃~105℃. (3) Remove the terminal protective group of the nucleotide by using a deprotection solution. (4) Connect another nucleotide with a terminal protecting group to the nucleotide by a reaction enzyme, and the working temperature is 45°C~105°C. (5) Determine whether the nucleic acid sequence is complete, if so, obtain the nucleic acid sequence, if not, repeat steps (3) and (4).

In an embodiment of the present invention, the above-mentioned method for disposing nucleotides with terminal protecting groups on the pretreatment surface includes disposing a first reaction solution on the pretreatment surface, and the first reaction solution includes a reaction enzyme and a terminal protection. Base nucleotides.

In an embodiment of the present invention, the above-mentioned method of disposing the first reaction liquid on the pretreatment surface includes transporting the first reaction liquid to the pretreatment surface by a liquid conveying device.

In an embodiment of the present invention, the above-mentioned method of adjusting the working temperature to 45°C to 105°C includes heating the reaction substrate by a heater.

In an embodiment of the present invention, the above-mentioned method for preparing a nucleic acid sequence using an enzyme further includes: (6) cutting the nucleic acid sequence from the pretreated surface by restriction enzymes.

In the apparatus and method for preparing nucleic acid sequences using enzymes in the embodiments of the present invention, since enzymes are used to synthesize nucleic acid sequences, compared with chemical synthesis, it is less likely to pollute the environment and reduce costs, and the working temperature of the reactor is set at 45°C~105°C, compared with the conventional method of using enzymes at 37°C, the embodiments of the present invention can show better activity when using enzymes above 45°C, and cooperate with the device of the present invention for preparing nucleic acid sequences using enzymes. The synthesis of multiple nucleic acid sequences can be performed at the same time, so the efficiency of preparing nucleic acid sequences can be improved.

In order to make the above and other objects, features and advantages of the present invention more comprehensible, the following specific examples are given in conjunction with the accompanying drawings, which are described in detail as follows.

Fig. 1 is a block diagram of an apparatus for preparing nucleic acid sequences using enzymes according to an embodiment of the present invention. Please refer to FIG. 1, the apparatus 100 for preparing a nucleic acid sequence using enzymes in this embodiment includes a reactor 110, a plurality of nucleotide material bottles 120, a deprotection material bottle 130, and a liquid delivery device 140. The reactor 110 includes a reaction substrate 111, and the reaction substrate 111 has a pretreatment surface 1111. Each nucleotide material bottle 120 is suitable for accommodating a first reaction solution 121. The first reaction solution 121 includes, for example, a reaction enzyme and nucleotides with terminal protective groups. The deprotection material bottle 130 is suitable for containing the deprotection solution 131. The liquid delivery device 140 is connected to the reactor 110, the nucleotide material bottles 120 and the deprotection material bottles 130, and is suitable for delivering the first reaction solution 121 and the deprotection solution 131 to the pretreatment surface 1111.

The material of the reaction substrate 111 includes, for example, silicon, glass (silicon dioxide), metal or polymer, such as polycarbonate or polymethyl methacrylate, but not limited thereto. The reaction substrate 111 is, for example, a plate-like structure or a three-dimensional structure, and the pretreatment surface 1111 is a planar or three-dimensional structure of the plate-like structure as a whole. For example, the pretreatment surface 1111 of the three-dimensional structure may be a spherical surface of superparamagnetic beads (SPMB), or a porous surface containing controlled pore glass (CPG).

In FIG. 1, four nucleotide material bottles 120 are taken as an example, but the number is not limited to this. Taking the synthetic single-stranded deoxyribonucleic acid (ssDNA) sequence as an example, the nitrogen-containing bases of the nucleotides required for its synthesis can be divided into four categories: adenine (A), thymine (T), and cytosine ( C), guanine (G), so the first reaction solution 121 in the four nucleotide material bottles 120 corresponds to the four different types of nucleotides (dAMP, dTMP, dCMP, dGMP), which are divided into The first reaction liquid 121a, 121b, 121c, 121d. When synthesizing ribonucleic acid (RNA) sequences, it is also necessary to use a nucleotide (UMP) material whose nitrogenous base is uracil (U). According to different design requirements, more than one nucleotide material bottle 120 can be used for each type of nucleotide, or only one, two or three of the four different types of nucleotides can be used. Therefore, the present invention does not Limit the number of nucleotide material bottles 120.

The "reactive enzyme", "nucleotide with terminal protecting group", "nucleotide", "restriction enzyme", etc. mentioned in the full text of the present invention should be regarded as a general term for these substances, rather than representing their actual quantity. For example, there are multiple reaction enzymes and nucleotides with terminal protecting groups in the first reaction solution.

The apparatus 100 for preparing nucleic acid sequences using enzymes, for example, further includes a heater 150 connected to the reactor 110 and suitable for heating the reactor 110. The working temperature of the reactor 110 is, for example, 45° C. to 105° C., preferably 50° C. to 85° C. ℃, more preferably 55℃~75℃. The heater 150 includes, for example, a heating element 151 and a heating control element 152. The heating element 151 is, for example, arranged in the reactor 110 to heat the reactor 110, and the heating control element 152 is connected to the heating element 151, but it is not limited thereto.

The apparatus 100 for preparing nucleic acid sequences using enzymes, for example, further includes a power supply element 160 and a control module 170. The power supply element 160 is suitable for providing power to the liquid conveying device 140 and the heater 150. The control module 170 is electrically connected to the liquid delivery device 140 and the heater 150, and is adapted to enable the liquid delivery device 140 to select different nucleotide material bottles 120 or deprotection material bottles 130, and to control the heating temperature of the heater 150. The control module 170 is, for example, an electronic device capable of achieving control functions, such as a tablet computer or a desktop computer. The specific implementation aspects of the apparatus 100 for preparing nucleic acid sequences using enzymes will be further described below in conjunction with the drawings, but the specific architecture of the apparatus 100 for preparing nucleic acid sequences using enzymes of the present invention is not limited to the examples listed below.

Fig. 2 is a schematic diagram of an apparatus for preparing nucleic acid sequences using enzymes according to an embodiment of the present invention. Please refer to FIG. 2, the apparatus 200 for preparing nucleic acid sequences using enzymes in this embodiment includes a reactor 210, a plurality of nucleotide material bottles 220, a deprotection material bottle 230, a liquid delivery device 240, a heater 250, a power supply element 260, and Control module 270. The reactor 210 includes a reaction substrate 211, and the reaction substrate 211 has a pretreatment surface 2111. Each nucleotide material bottle 220 is suitable for containing the first reaction solution 221. The deprotection material bottle 230 is suitable for containing the deprotection solution 231. The heater 250 includes, for example, a heating element 251 and a heating control element 252. The apparatus 200 for preparing a nucleic acid sequence using an enzyme in this embodiment is similar to the above-mentioned apparatus 100 for preparing a nucleic acid sequence using an enzyme, so the description of each element is not repeated here. The apparatus 200 for preparing nucleic acid sequences using enzymes of this embodiment, for example, further includes a nucleotide material bottle 220 containing a first reaction solution 221e of a nucleotide material using uracil, a restriction enzyme material bottle 280, a cleaning bottle 290, and waste For the liquid bottle 2000 and the product bottle 2001, the liquid conveying device 240 includes, for example, an extraction device 241, a first switching valve 242, a plurality of first conveying tubes 243, a second switching valve 244, a plurality of second conveying tubes 245, and a plurality of thirds. Transport pipe 246. The restriction enzyme material bottle 280 is suitable for containing the second reaction solution 281, and the second reaction solution 281 includes restriction enzymes. The cleaning bottle 290 is adapted to contain a cleaning solution 291, and the cleaning solution 291 is used to clean the pretreatment surface 2111 after the reaction, so as to avoid affecting the next reaction. The waste liquid bottle 2000 is suitable for containing the first reaction solution 221, the deprotection solution 231, the second reaction solution 281, and the cleaning solution 291 that have been used. The product bottle 2001 is suitable for containing the prepared nucleic acid sequence.

In the liquid delivery device 240, a plurality of first delivery tubes 243 are connected between the plurality of nucleotide material bottles 220 and the first switching valve 242, and between the deprotection material bottle 230 and the first switching valve 242, the enzyme material is restricted. Between the bottle 280 and the first switching valve 242, and between the cleaning bottle 290 and the first switching valve 242, the first switching valve 242 is connected to the pretreatment surface 2111 via a third delivery pipe 246, and is suitable for the first delivery Switch between tubes 243. A plurality of second delivery tubes 245 are connected between the waste liquid bottle 2000 and the second switching valve 244, and between the product bottle 2001 and the second switching valve 244. The second switching valve 244 is connected to the pretreatment via the third delivery tube 246 The surface 2111 is suitable for switching between these second conveying tubes 245. The extraction device 241 is, for example, an injection pump or a pneumatic valve, but not limited thereto. In FIG. 2, the injection pump is taken as an example, and the extraction device 241 is connected to the second switching valve 244.

Specifically, taking the delivery of the first reaction solution 221 as an example, when the extraction device 241 performs extraction, first the first reaction solution 221 of one of the plurality of nucleotide material bottles 220 (that is, the first reaction solution One of 221a, 221b, 221c, 221d, and 221e) is delivered to the pretreatment surface 2111 through the first delivery pipe 243, the first switching valve 242, and the third delivery pipe 246 in order to undergo reaction. After the reaction is completed, the extraction device 241 will continue to extract, so that the first reaction solution 221 on the pretreatment surface 2111 is delivered to the extraction device 241 through the third delivery pipe 246 and the second switching valve 244 in sequence. Then, the second switching valve 244 switches the valve path to the waste liquid bottle 2000, and the extraction device 241 pushes and transports the first reaction liquid 221 to the waste liquid bottle 2000 via the second delivery pipe 245. If the deprotection solution 231 needs to be reacted next, the first switching valve 242 will switch the valve path to the deprotection material bottle 230, and the second switching valve 244 will switch the valve path back to the pretreatment surface 2111, and the extraction device 241 starts to extract After the deprotection solution 231 reacts on the pretreatment surface 2111, the deprotection solution 231 is delivered to the waste liquid bottle 2000. After the preparation of the nucleic acid sequence is completed, the second switching valve 244 switches the valve path to the pretreatment surface 2111, and the extraction device 241 will extract the nucleic acid sequence of the pretreatment surface 2111 through the third delivery pipe 246 and the second switching valve 244 in sequence. Then, the second switching valve 244 switches the valve path to the product bottle 2001, and the extraction device 241 pushes the nucleic acid sequence to the product bottle 2001 via the second delivery tube 245. The above method of using the liquid delivery device 240 is applicable to multiple nucleotide material bottles 220, deprotection material bottles 230, restriction enzyme material bottles 280, and cleaning bottles 290. It should be noted that the above is only a specific implementation of the device 200 for preparing nucleic acid sequences using enzymes. Depending on the design architecture, the liquid delivery device 240 may include different elements and delivery methods.

Fig. 3 is a schematic diagram of the combination of a reactor and a heating element according to an embodiment of the present invention. Please refer to FIGS. 2 and 3. In FIG. 2, the heating element 251 of the heater 250 is arranged in the reactor 210. Specifically, the reactor 210 includes, for example, a reaction substrate 211 and an upper cover 212, and the reaction substrate 211 and the upper cover 212 is configured on the heating element 251. The reaction substrate 211 and the upper cover 212 are combined to form a cavity 213, and the surface of the reaction substrate 211 facing the cavity 213 is the pretreatment surface 2111. The upper cover 212 has a plurality of through holes 212a, and the through holes 212a are suitable for passing the third conveying pipe 246 to convey the first reaction solution 221, the deprotection solution 231, the second reaction solution 281 and the cleaning solution 291 to the pretreatment On the surface 2111, after the reaction is completed, the first reaction solution 221, the deprotection solution 231, the second reaction solution 281, and the cleaning solution 291 are transferred to the waste liquid bottle 2000. In other words, the cavity 213 is used as a flow channel. The heating element 251 of the heater 250 directly heats the reaction substrate 211 during the reaction process. This embodiment is only an implementation aspect of heating, and the present invention does not particularly limit the configuration of the heating element 251.

The apparatus 200 for preparing nucleic acid sequences using enzymes of this embodiment uses the pretreatment surface 2111 of the reaction substrate 211 to prepare nucleic acid sequences, which can simultaneously synthesize multiple nucleic acid sequences and improve the efficiency of nucleic acid sequence preparation.

Fig. 4 is a schematic diagram of the combination of a reactor and a heating element according to another embodiment of the present invention. Please refer to FIG. 2 and FIG. 4, this embodiment is another way of combining the reactor 310 with the heating element 351 of the heater 350. The reactor 310 includes, for example, a reaction substrate 311 and a shell 314. The housing 314 has a cavity 313 inside, and the reaction substrate 311 is disposed in the cavity 313. The surface of the housing 314 has a plurality of through holes 314a, and the through holes 314a are suitable for passing the third conveying pipe 246. The function of the through holes 314a is the same as that described above, and will not be repeated here. The material of the housing 314 is, for example, glass, but it is not limited thereto. The reaction substrate 311 in this embodiment has a three-dimensional structure, such as a spherical shape, and the number can be multiple, and the pretreatment surface 3111 is a spherical surface of the reaction substrate 311. The material of the reaction substrate 311 is, for example, superparamagnetic particles, but it is not limited thereto. The heating element 351 of the heater 350 covers the reactor 310 to heat the reactor 310.

Fig. 5 is a schematic diagram of an apparatus for preparing nucleic acid sequences using enzymes according to another embodiment of the present invention. 5, the apparatus 400 for preparing a nucleic acid sequence using enzymes in this embodiment includes a reactor 410, a plurality of nucleotide material bottles 420, a deprotection material bottle 430, a liquid delivery device 440, a heater 450, a power supply element 460, The control module 470, the restriction enzyme material bottle 480, the cleaning bottle 490, the waste liquid bottle 4000, and the product bottle 4001. The reactor 410 includes a reaction substrate 411, and the reaction substrate 411 has a pretreatment surface 4111. Each nucleotide material bottle 420 is suitable for containing the first reaction solution 421. The deprotection material bottle 430 is suitable for containing the deprotection solution 431. The heater 450 includes a heating element 451 and a heating control element 452, for example. The liquid delivery device 440 includes, for example, an extraction device 441, a first switching valve 442, a plurality of first delivery pipes 443, a second switching valve 444, a plurality of second delivery pipes 445, and a plurality of third delivery pipes 446. The restriction enzyme material bottle 480 is suitable for containing the second reaction solution 481, and the second reaction solution 481 includes restriction enzymes. The cleaning bottle 490 is suitable for containing the cleaning solution 491. The apparatus 400 for preparing nucleic acid sequences using enzymes in this embodiment has similar structure and advantages to the above-mentioned apparatus 200 for preparing nucleic acid sequences using enzymes. Therefore, the functions of each element will not be repeated here. The only difference lies in the use of enzymes in this embodiment. In the device 400 for preparing a nucleic acid sequence, the liquid delivery device 440 further includes a plurality of fourth delivery tubes 447, for example. These fourth delivery tubes 447 are respectively connected between the plurality of nucleotide material bottles 420 containing the first reaction solutions 421a, 421b, 421c, and 421d and the second switching valve 444. With these fourth delivery pipes 447, the first reaction solution 421a, 421b, 421c, and 421d after the reaction can be recycled back to a plurality of nucleotide material bottles 420, which can be reused to achieve cost-saving effects.

Hereinafter, the method for preparing the nucleic acid sequence will be described in detail. In the embodiment, the synthesis of a single-stranded deoxyribonucleic acid (ssDNA) sequence is taken as an example. Fig. 6 is a schematic flow chart of a method for preparing a nucleic acid sequence using an enzyme according to an embodiment of the present invention. 7A to 7E are schematic diagrams of a reaction for synthesizing a nucleic acid sequence according to an embodiment of the present invention. Please refer to FIG. 6 and FIG. 7A first, the method for preparing a nucleic acid sequence using enzymes in this embodiment includes the following steps: Step S101 is performed: a reaction substrate 511 is provided, and the reaction substrate 511 has a pretreatment surface 5111. The pretreatment surface 5111 has, for example, a plurality of primers 5112. In this embodiment, the primers 5112 are, for example, single-stranded DNA, but it is not limited thereto. In this embodiment, for example, a uracil nucleotide U is first configured on the primer 5112, but it is not limited to this. Next, proceed to step S102: arranging nucleotides with terminal protecting groups on the pretreatment surface 5111 by reaction enzymes, and the working temperature is 45°C~105°C, preferably 50°C~85°C, more preferably 55°C ~75°C, specifically, the working temperature for preparing the nucleic acid sequence is, for example, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C , 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃ or 75℃, but not limited to this.

The method of arranging nucleotides with terminal protecting groups on the pretreatment surface includes, for example, arranging a first reaction solution on the pretreatment surface 5111. The first reaction solution includes reaction enzymes and nucleotides with terminal protecting groups. The method of disposing on the pretreatment surface 5111 includes, for example, conveying the first reaction liquid to the pretreatment surface 5111 by a liquid conveying device. The first reaction liquid is, for example, the first reaction liquid 121, 221, 421 of any of the above-mentioned embodiments. The liquid delivery device is, for example, the liquid delivery device 140, 240, 440 of any of the above embodiments.

Please refer to FIG. 7B, the method for arranging the nucleotide 522 with the terminal protecting group PG on the pretreated surface 5111 includes the reaction enzyme 523 connecting the nucleotide 522 with the terminal protecting group PG to the uracil on the primers 5112 Nucleotide U. The reaction enzyme 523 in this embodiment is, for example, a DNA polymerase, especially a heat-resistant DNA polymerase, but it is not limited to this. DNA polymerases include, for example, family A DNA polymerases, family B DNA polymerases, and family X DNA polymerases, examples of which include Taq polymerase, archaeal DNA polymerase (archaeal DNA polymerase), heat-resistant reverse transcriptase, and the like. When the temperature of these DNA polymerases is above 45°C, compared with the conventional terminal deoxynucleotidyl transferase (TdT) used at 37°C, they can have better activity and improve the efficiency of nucleic acid sequence synthesis. Examples of the terminal protecting group PG include methyl, 2-nitrobenzyl, 3'-O-(2-cyanoethyl), allyl, amine, azido, tert-butoxyethoxy and the like.

After step S102 is completed, the reaction enzyme 523 and the unreacted nucleotide 522 with the terminal protective group PG can be washed away by the washing solution to avoid affecting subsequent reactions. In this embodiment, the cleaning solution is, for example, deionized water, but it is not limited to this. The cleaning solution is, for example, the cleaning solutions 291 and 491 of any of the foregoing embodiments. Next, proceed to step S103: the terminal protecting group PG of nucleotide 522 is removed by the deprotection solution 531. Referring to FIG. 7C, the deprotection solution 531 may be delivered to the pretreatment surface 5111 by the liquid delivery device 140, 240, 440, for example. The function of the deprotection solution 531 is to remove the terminal protecting group PG of the nucleotide 522, so that the nucleotide 522 can be connected to other nucleotides 522 to synthesize the nucleic acid sequence. The deprotection solution 531 is classified into a chemical type and an enzyme type according to different types, for example. Examples of chemical types include reducing agents such as dithiothreitol (DTT) or dithioerythritol (DTE), etc.; examples of enzyme types include deprotection enzymes such as alkaline phosphatase, pyrophosphatase, Cattle small intestine alkaline phosphatase and so on.

After step S103 is completed, the deprotection solution 531 and the terminal protecting group PG are washed away with a cleaning solution. Then, step S104 is performed: another nucleotide 522 with a terminal protecting group PG is connected to the nucleotide 522 arranged on the pretreatment surface 5111 by the reaction enzyme 523. Please refer to FIG. 7D. The reaction of step S104 is similar to that of step S102. The difference is that the nucleotide 522 with the terminal protecting group PG is linked to the nucleotide 522 previously linked to the primer 5112. According to the length of the designed nucleic acid sequence, proceed to step S105: determine whether the designed nucleic acid sequence is complete, if so, obtain the nucleic acid sequence, if not, repeat steps S103 and S104, and continue to extend the length of the nucleic acid sequence until the design is completed The nucleic acid sequence.

After the nucleic acid sequence synthesis is completed, the method for preparing the nucleic acid sequence using enzymes, for example, further includes step S106: cutting the nucleic acid sequence from the pretreated surface by restriction enzymes. 2 and 7E, the restriction enzyme material bottle 280 in the apparatus 200 for preparing nucleic acid sequences using enzymes is suitable for containing the second reaction solution 281, and the second reaction solution 281 includes the restriction enzyme 581a. After the liquid delivery device 240 delivers the second reaction solution 281 to the pretreatment surface 5111, the restriction enzyme 581a will cut the synthesized nucleic acid sequence S from the pretreatment surface 5111. After that, the nucleic acid sequence S is collected and transported to the product bottle 2001 to complete the preparation process of the nucleic acid sequence S. Examples of restriction enzyme 581a include uracil DNA glycosylase (UDG), endonuclease VIII, USER enzyme (NEB #M5508), etc., and combinations thereof. In this embodiment, for example, the restriction enzyme 581a that cuts the uracil nucleotide U is used. When the nucleic acid sequence is completed, since it is a synthetic deoxyribonucleic acid sequence, the uracil nucleotide U is not included in the sequence. For restriction enzyme 581a, there is only a previously configured single uracil nucleotide U cut position, which can then correctly cleave the nucleic acid sequence S. Fig. 7E is a simple schematic diagram of the cut. Different restriction enzymes 581a will have different cutting positions, and the present invention is not particularly limited. The above-mentioned method can also be applied to the apparatus 400 for preparing nucleic acid sequences using enzymes.

To sum up, in the apparatus and method for preparing nucleic acid sequences using enzymes in the embodiments of the present invention, since enzymes are used to synthesize nucleic acid sequences, compared with chemical synthesis, it is less likely to pollute the environment and reduce costs, while the reactor The working temperature is set at 45°C~105°C. Compared with the conventional method of using enzymes at 37°C, the embodiment of the present invention can show better activity when using enzymes above 45°C, and it can be used in conjunction with the enzyme preparation of the present invention. The nucleic acid sequence device can simultaneously synthesize multiple nucleic acid sequences, so the efficiency of preparing nucleic acid sequences can be improved.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to those defined by the attached patent application scope.

100, 200, 400: devices for preparing nucleic acid sequences using enzymes 110, 210, 310, 410: reactor 111, 211, 311, 411, 511: reaction substrate 212: Upper cover 212a: Through hole 213, 313: Cavity 314: Shell 314 a: Through hole 1111, 2111, 3111, 4111, 5111: Pretreatment surface 5112: Introduction 120, 220, 420: Nucleotide material bottle 121, 121a, 121b, 121c, 121d, 221, 221a, 221b, 221c, 221d, 221e, 421, 421a, 421b, 421c, 421d, 421e: first reaction liquid 522: Nucleotide 523: Reaction enzyme 130, 230, 430: To protect the material bottle 131, 231, 431, 531: Deprotection solution 140, 240, 440: Liquid delivery device 241, 441: extraction device 242, 442: the first switching valve 243, 443: The first conveying pipe 244, 444: the second switching valve 245, 445: the second conveying pipe 246, 446: The third conveying pipe 447: The fourth conveying pipe 150, 250, 350, 450: heater 151, 251, 351, 451: heating elements 152, 252, 452: heating control elements 160, 260, 460: power supply components 170, 270, 470: control module 280, 480: Restricted enzyme material bottle 281, 481: second reaction liquid 581a: Restriction enzyme 290, 490: cleaning bottle 291, 491: cleaning solution 2000, 4000: waste liquid bottle 2001, 4001: Product bottle PG: terminal protecting group S: Nucleic acid sequence U: Uracil Nucleotide S101, S102, S103, S104, S105, S106: steps

Fig. 1 is a block diagram of an apparatus for preparing nucleic acid sequences using enzymes according to an embodiment of the present invention. Fig. 2 is a schematic diagram of an apparatus for preparing nucleic acid sequences using enzymes according to an embodiment of the present invention. Fig. 3 is a schematic diagram of the combination of a reactor and a heater according to an embodiment of the present invention. Fig. 4 is a schematic diagram of the combination of a reactor and a heater according to another embodiment of the present invention. Fig. 5 is a schematic diagram of an apparatus for preparing nucleic acid sequences using enzymes according to another embodiment of the present invention. Fig. 6 is a schematic flow chart of a method for preparing a nucleic acid sequence using an enzyme according to an embodiment of the present invention. 7A to 7E are schematic diagrams of a reaction for synthesizing a nucleic acid sequence according to an embodiment of the present invention.

200: Device for preparing nucleic acid sequence using enzyme 210: Reactor 211: Reactive substrate 2111: Pretreatment surface 220: Nucleotide material bottle 221, 221a, 221b, 221c, 221d, 221e: the first reaction liquid 230: To protect the material bottle 231: Deprotection solution 240: Liquid delivery device 241: Extraction device 242: The first switching valve 243: The first delivery pipe 244: The second switching valve 245: The second conveying pipe 246: The third delivery pipe 250: heater 251: Heating element 252: Heating control element 260: power supply element 270: control module 280: Restriction enzyme material bottle 281: The second reaction solution 290: cleaning bottle 291: Cleaning solution 2000: Waste bottle 2001: Product bottle

Claims (24)

A device for preparing nucleic acid sequences using enzymes, comprising: a reactor, including a reaction substrate, the reaction substrate having a pretreatment surface; a plurality of nucleotide material bottles, wherein each of the nucleotide material bottles is suitable for containing A first reaction solution, the first reaction solution comprising a DNA polymerase and a nucleotide with a terminal protecting group; a deprotection material bottle suitable for containing a deprotection solution; a liquid delivery device connected to the Reactor, the nucleotide material bottles, and the deprotection material bottle, and are suitable for delivering the first reaction solution and the deprotection solution to the pretreatment surface, and the DNA polymerase is suitable for having the terminal protecting group The nucleotides are arranged on the pretreatment surface, the deprotection solution is suitable for removing the terminal protecting group of the nucleotides; and a restriction enzyme material bottle is suitable for containing a second reaction solution, the second reaction solution A restriction enzyme is included, and the liquid delivery device is further connected to the restriction enzyme material bottle to deliver the second reaction liquid to the pretreatment surface. The working temperature of the reactor is 45℃~105℃. The device for preparing nucleic acid sequences using enzymes as described in claim 1, wherein the working temperature of the reactor is 50°C to 85°C. The device for preparing nucleic acid sequences using enzymes as described in claim 2, wherein the working temperature of the reactor is 55°C to 75°C. The device for preparing a nucleic acid sequence using an enzyme as described in claim 1, wherein the DNA polymerase is an A family DNA polymerase. The device for preparing a nucleic acid sequence using an enzyme as described in claim 1, wherein the DNA polymerase is a family B DNA polymerase. The device for preparing a nucleic acid sequence using an enzyme as described in claim 1, wherein the DNA polymerase is an X family DNA polymerase. The device for preparing a nucleic acid sequence using an enzyme according to claim 1, wherein the liquid delivery device includes an extraction device adapted to extract the first reaction solution and the deprotection solution to the pretreatment surface. The device for preparing a nucleic acid sequence using an enzyme according to claim 7, wherein the extraction device is an injection pump or a pneumatic valve. The device for preparing a nucleic acid sequence using an enzyme as described in claim 1, wherein the pretreated surface has a plurality of primers suitable for connecting the nucleotide with the terminal protecting group. The device for preparing a nucleic acid sequence using an enzyme as described in claim 1, wherein the deprotection solution includes a reducing agent or a deprotection enzyme. The apparatus for preparing nucleic acid sequences using enzymes as described in claim 1, further comprising a heater connected to the reactor and suitable for heating the reactor. The device for preparing a nucleic acid sequence using an enzyme according to claim 1, wherein the liquid delivery device includes a first switching valve and a plurality of first delivery tubes, and the first delivery tubes are connected to the nucleotide material bottles and Between the first switching valve and between the deprotection material bottle and the first switching valve, the first switching valve is connected to the reactor and is suitable for switching between the first delivery pipes. The device for preparing nucleic acid sequences using enzymes as described in claim 1, further comprising a waste liquid bottle and a product bottle, wherein the liquid conveying device comprises a second switching valve and a plurality of second conveying pipes, the second conveying A tube is connected between the waste liquid bottle and the second switching valve and between the product bottle and the second switching valve. The second switching valve is connected to the reactor and is suitable for connecting between the second delivery pipes. Switch. The device for preparing nucleic acid sequences using enzymes as described in claim 1, further comprising a power supply element suitable for supplying power to the liquid delivery device. A method for preparing nucleic acid sequences using enzymes includes: (1) providing a reaction substrate with a pretreatment surface; (2) using a DNA polymerase to convert a nucleotide with a terminal protecting group It is arranged on the pretreatment surface, and the working temperature is 45°C~105°C; (3) The terminal protecting group of the nucleotide is removed by a deprotection solution; (4) The DNA polymerase will have the The other nucleotide of the terminal protecting group is connected to the nucleotide, and the working temperature is 45℃~105℃; (5) Determine whether the nucleic acid sequence is completed, if it is, obtain a nucleic acid sequence, if not, repeat step (3) ) And (4); and (6) cutting the nucleic acid sequence from the pretreated surface by a restriction enzyme. The method for preparing a nucleic acid sequence using an enzyme as described in claim 15, wherein the working temperature is 50°C to 85°C. The method for preparing a nucleic acid sequence using an enzyme as described in claim 15, wherein the working temperature is 55°C to 75°C. The method for preparing a nucleic acid sequence using an enzyme as described in claim 15, wherein the method for disposing the nucleotide with the terminal protecting group on the pretreatment surface includes disposing a first reaction solution on the pretreatment surface, and The first reaction solution includes the DNA polymerase and the nucleotide with the terminal protecting group. The method for preparing a nucleic acid sequence using an enzyme according to claim 15, wherein the method of disposing the first reaction solution on the pretreatment surface includes transporting the first reaction solution to the pretreatment surface by a liquid conveying device. The method for preparing a nucleic acid sequence using an enzyme according to claim 15, wherein the method of adjusting the working temperature to 45°C to 105°C includes heating the reaction substrate by a heater. The method for preparing a nucleic acid sequence using an enzyme according to claim 15, wherein the pretreatment surface has a plurality of primers, and the method for arranging the nucleotide with the terminal protecting group on the pretreatment surface includes polymerizing by the DNA The enzyme connects the nucleotide with the terminal protecting group to the primers. The method for preparing a nucleic acid sequence using an enzyme as described in claim 16, wherein the DNA polymerase is an A family DNA polymerase. The method for preparing a nucleic acid sequence using an enzyme as described in claim 16, wherein the DNA polymerase is a B family DNA polymerase. The method for preparing a nucleic acid sequence using an enzyme as described in claim 16, wherein the DNA polymerase is an X family DNA polymerase.

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