TWI732405B - Method for preparing nucleic acid sequences using enzyme - Google Patents

Abstract

A method for preparing nucleic acid sequences using an enzyme, including: (1) providing a reaction substrate having a pretreated surface. (2) Disposing a nucleotide having a terminal protecting group on the pretreated surface by a reaction enzyme, and a reaction temperature is 45℃ - 105℃. (3) Removing the terminal protecting group of the nucleotide by irradiation or heating. (4) Coupling another nucleotide having the terminal protecting group to the nucleotide by the reaction enzyme, and a reaction temperature is 45℃ - 105℃. (5) Determining whether nucleic acid sequence is completed, and if so, obtaining the nucleic acid sequence, if otherwise repeating steps (3) and (4). The method for preparing nucleic acid sequences using an enzyme of the invention may increase the efficiency of preparing nucleic acid sequences.

Description

Method for preparing nucleic acid sequence using enzyme

The present invention relates to a method for preparing biomolecular structures, and in particular to a 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 knock-out 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.

WO 95/00530 A1 discloses a method for preparing a nucleotide array by synthesizing nucleotide probes on a substrate using photolithography. The nucleotides are immobilized on the substrate, and the photosensitive protective group on the 5'terminal hydroxyl group is used for base synthesis in the 3'to 5'direction. In each synthesis cycle, the protective group is selectively removed by irradiating the surface with a photolithography mask. The deprotected hydroxyl group is coupled to the selected 5'-photoprotected nucleotide phosphite, while the nucleic acid sequence in the unirradiated area of the surface remains protected And can't respond. According to the need, different nucleotides are used to repeat a round of illumination and coupling until the desired nucleic acid sequence is obtained.

US Patent US 20160184788A1 discloses a method of selectively masking one or more sites on a surface and a method of synthesizing a molecular array. It fixes nucleotides on a substrate, and uses a heat-sensitive protective group on the 5'terminal hydroxyl group to base synthesis in the 3'to 5'direction. By heating at different positions, the thermosensitive protective group of the nucleotide located at the position can be removed, and the deprotected hydroxyl group is coupled with the selected 5'-photoprotected nucleotide phosphite, so it can Prepare a large number of different nucleic acid sequences.

However, 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. Conventionally, DNA polymerase is often used to synthesize DNA. 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 It is 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 accepting 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, for 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.

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 enzyme synthesis method of nucleic acid is cheap and the accuracy of the nucleic acid sequence is high. However, due to It is impossible to carry out large-scale synthesis due to the reaction rate of the enzyme. Therefore, there is still no better nucleic acid synthesis method that can solve the problem of correct and large-scale nucleic acid synthesis.

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

The method for preparing nucleic acid sequences using enzymes provided in an embodiment of the present invention includes: (1) providing a reaction substrate with a pretreated surface. (2) The nucleotides with terminal protective groups are arranged on the pretreatment surface by reaction enzymes, and the reaction temperature is 45°C~105°C. (3) The terminal protective group of the nucleotide is removed by irradiation or heating. (4) Connect another nucleotide with a terminal protecting group to the nucleotide by a reaction enzyme, and the reaction 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 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 method of removing the terminal protective groups of nucleotides by illumination or heating includes partial removal in a patterned manner.

In an embodiment of the present invention, the above-mentioned method for removing the terminal protective groups of nucleotides by illumination includes using ultraviolet digital light processing (DLP) wafers for illumination.

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

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

In an embodiment of the present invention, the above-mentioned pretreated surface has a plurality of primers, and the method for arranging nucleotides with terminal protecting groups on the pretreated surface includes linking the nucleotides with terminal protecting groups by a reaction enzyme To these primers.

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 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 reaction temperature 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, thus improving the efficiency of preparing nucleic acid sequences.

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.

100, 300: light-emitting element

310: Reflective light valve

200, 400, 600, 700: pretreatment surface

210, 410, 610, 710: primer

220, 420, 620, 720a, 720c, 720g, 720t: nucleotides

I, II, III, IV: area

PG: terminal protecting group

S101, S102, S103, S104, S105, S106: steps

Figure 1 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.

2A and 2B are schematic diagrams of removing the terminal protecting group of nucleotides according to an embodiment of the present invention.

3A and 3B are schematic diagrams of removing the terminal protective group of a nucleotide according to another embodiment of the present invention.

4A and 4B are schematic diagrams of removing the terminal protective group of a nucleotide according to another embodiment of the present invention.

5A to FIG. 5I are schematic diagrams of steps of a method for preparing a nucleic acid sequence using an enzyme according to an embodiment of the present invention.

Figure 1 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. 1, the method for preparing a nucleic acid sequence using an enzyme in this embodiment includes the following steps: Step S101: Provide a reaction substrate, the reaction substrate has a pre-treated surface, and then proceed to Step S102: The reaction enzyme will have an end The nucleotide of the protecting group is arranged on the pretreatment surface, and the reaction temperature is 45°C~105°C, preferably 50°C~85°C, more preferably 55°C~75°C, specifically, it is used for preparing nucleic acid sequence The reaction temperature 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°C, 67°C, 68°C, 69°C, 70°C ℃, 71℃, 72℃, 73℃, 74℃ or 75℃, but not limited to this.

The material of the reaction substrate includes, for example, silicon, glass (silicon dioxide), metal or polymer, such as polycarbonate or polymethyl methacrylate, but not limited to this. Specifically, the reaction substrate is, for example, a plate-like structure such as a wafer or a flat plate, and the pretreatment surface is a plane of the plate-like structure.

In this embodiment, for example, the synthesis of deoxyribonucleic acid (DNA) sequences is taken as an example. Specifically, the nitrogen-containing bases of nucleotides required for synthesis can be further divided into four categories: adenine (A) , Thymine (T), cytosine (C), guanine (G), so four different types of nucleotides (dAMP, dTMP, dCMP, dGMP) are used. When synthesizing ribonucleic acid (RNA) sequences, it is also necessary to use nucleotides (UMP) whose nitrogen-containing base is uracil (U). The reaction enzyme is, for example, a DNA polymerase, especially a heat-resistant DNA polymerase, but it is not limited thereto. 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, 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.

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, the number of reaction enzymes and nucleotides with terminal protecting groups are both multiple.

The method of arranging nucleotides with terminal protecting groups on the pretreatment surface by reaction enzymes includes, for example, an impregnation method or a liquid flow method. Specifically, a reaction enzyme and a nucleotide having a terminal protecting group are prepared as a formulation solution. In the impregnation method, the reaction substrate is immersed in the formulation solution, and the temperature of the solution is maintained at 45°C to 105°C, waiting for the completion of the reaction. The way of liquid flow is to flow the formulation solution over the pretreatment surface of the reaction substrate and maintain the temperature of the reaction substrate at 45°C to 105°C. The foregoing manners are only implementation aspects of the present invention, and are not intended to limit the scope of the present invention.

The pretreatment surface has, for example, multiple primers, but it is not limited to this. The method of arranging nucleotides with terminal protecting groups on the pretreated surface includes linking nucleotides with terminal protecting groups to these primers by a reaction enzyme. In this example, when synthesizing deoxyribonucleic acid sequences, with the assistance of primers, it is easier for DNA polymerase (reaction enzyme) to arrange nucleotides with terminal protecting groups on the pretreatment surface. These primers are, for example, single Strands of DNA, but not limited to this. In another embodiment, the pretreatment surface may not have primers, for example.

Next, proceed to step S103: removing the terminal protective group of the nucleotide by irradiation or heating. For example, terminal protecting groups are classified into photosensitive type and heat-sensitive type. Examples of terminal protecting groups include methyl, 2-nitrobenzyl, 3'-O-(2-cyanoethyl), allyl, amine, and Nitrogen, tert-butoxyethoxy, etc., but not limited to this. The light-sensitive terminal protective group can be removed by light, and the heat-sensitive terminal protective group can be removed by heating. The nucleotide after the terminal protection group is removed can be connected to the next nucleotide to extend the sequence. The following will further illustrate the specific implementation of removing the terminal protection group of nucleotides in conjunction with the diagram, but the specific method of removing the terminal protection group of nucleotides by light or heating of the present invention is not limited to the examples listed below. .

2A and 2B are schematic diagrams of removing the terminal protecting group of nucleotides according to an embodiment of the present invention. 2A and 2B, this embodiment uses a photosensitive terminal protective group PG, so the pretreatment surface 200 can be illuminated by the light-emitting element 100, and the nucleotide 220 connected to the primer 210 is illuminated by the light L Then the terminal protective group PG will decompose, as shown in Figure 2B. The light-emitting element 100 is, for example, a light-emitting diode or a laser diode, but it is not limited thereto. The lighting treatment is not limited to the use of the light-emitting element 100, and for example, natural light may be used. Since the pretreatment surface 200 can be configured with multiple nucleotides 220 at the same time, multiple nucleic acid sequences can be prepared at one time, which improves the preparation efficiency.

In addition, the pretreatment surface 200 can also be divided into a plurality of regions, each region includes a nucleic acid sequence in preparation, and the terminal protective group PG is partially removed in a patterned manner to achieve the effect of simultaneously preparing a plurality of different nucleic acid sequences. 3A and 3B are schematic diagrams of removing the terminal protective group of a nucleotide according to another embodiment of the present invention. Please refer to FIG. 3A and FIG. 3B. This embodiment also uses a photosensitive end protecting group PG, but the difference is that this embodiment uses ultraviolet digital light processing (DLP) wafers for illumination. Specifically, the ultraviolet digital light processing chip includes a light-emitting element 300 and a reflective light valve 310. In FIGS. 3A and 3B, multiple regions are separated by dotted lines. After the light L emitted from the light-emitting element 300 is transmitted to the reflective light valve 310, the reflective light valve 310 is patterned to partially illuminate the pretreatment surface 400. There are several regions, so that the terminal protective group PG of the nucleotide 420 connected to the primer 410 in these regions will be decomposed after being irradiated by the light L, and the next nucleotide 420 can be connected, as shown in FIG. 3B. By designing a patterning method, different nucleic acid sequences can be prepared in each region at the same time.

4A and 4B are schematic diagrams of removing the terminal protective group of a nucleotide according to another embodiment of the present invention. Please refer to FIGS. 4A and 4B. The method of this embodiment is similar to the above-mentioned method with similar advantages. The only difference is that this embodiment uses a heat-sensitive terminal protective group PG, so the pretreatment surface 600 is processed by the heating element 500. Heating (heat energy transfer is indicated by a curved arrow), connected to the primer The terminal protecting group PG of 610 nucleotide 620 will be decomposed after heating, as shown in Figure 4B. The heat treatment can also locally remove the terminal protective group PG in a patterned manner, that is, in the form of zone heating. For example, when a silicon wafer is used as the reaction substrate, the circuit can be distributed in each area, and the current passing through can be controlled to achieve the effect of area heating.

Please refer to FIG. 1 again. After the terminal protecting group of the nucleotide is removed, step S104 is followed: another nucleotide with a terminal protecting group is connected to the nucleotide disposed on the pretreatment surface by a reaction enzyme. The reaction of step S104 is similar to that of step S102, except that the nucleotide with a terminal protecting group is linked to the nucleotide previously linked to the primer. According to the length of the nucleic acid sequence to be synthesized, 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 Complete the designed nucleic acid sequence. When the steps are repeated, if step S103 is to partially remove the terminal protection groups in different regions in a patterned manner, the lengths of the nucleic acid sequences in different regions may be different. The specific implementation of the method for preparing a nucleic acid sequence using an enzyme will be further described below in conjunction with a diagram, but the specific manner of the method for preparing a nucleic acid sequence using an enzyme of the present invention is not limited to the examples listed below.

5A to FIG. 5I are schematic diagrams of steps of a method for preparing a nucleic acid sequence using an enzyme according to an embodiment of the present invention. Please refer to FIG. 1 and FIG. 5A to FIG. 5I. In FIG. 5A, step S101 is performed: a reaction substrate with a pretreatment surface 700 is provided. The pretreatment surface 700 has a plurality of primers 710. In this embodiment, the primers 710 are, for example, It is a single strand of DNA, but not limited to this. The pretreatment surface 700 here is divided into 4 areas: I, II, III, and IV. Each region has multiple primers 710, and regions I, II, III, and IV are respectively designed to prepare different nucleic acid sequences.

Step S102 is performed in FIG. 5B: the nucleotide 720c with the terminal protecting group PG is connected to the plurality of primers 710 on the pretreatment surface 700 by the reaction enzyme, and the reaction temperature is 45°C to 105°C. The nucleotide 720 used in this embodiment includes nucleotides 720a, 720c, 720g, and 720t. It corresponds to adenine nucleotides (dAMP), cytosine nucleotides (dCMP), guanine purine nucleotides (dGMP), thymine nucleotides (dTMP). The nucleotide 720c configured in FIG. 5B is a cytosine nucleotide.

Step S103 is performed in FIG. 5C: the terminal protecting group PG of nucleotide 720 is removed by irradiation or heating. Here, the terminal protective group PG in different regions is partially removed in a patterned manner, and the implementation of illumination or heating has been described in detail, and will not be repeated here. In Figure 5C, the terminal protecting group PG of nucleotide 720c in regions II and III is removed.

Step S104 is performed in FIG. 5D: another nucleotide 720 having a terminal protecting group PG is connected to the nucleotide 720 arranged on the pretreatment surface 700 by a reaction enzyme. In Fig. 5D, only the nucleotide 720c of region II and III is additionally ligated with another nucleotide 720g by the reaction enzyme.

Then proceed to step S105: determine whether the designed nucleic acid sequence is completed. When it is determined that the nucleic acid sequence is not completed, steps S103 and S104 are repeated. In Figure 5E, step S103 is performed again to remove the terminal protecting group of nucleotide 720c in region I. PG and the terminal protecting group PG of the 720g nucleotide of region III. Next, in FIG. 5F, step S104 is performed again, and another nucleotide 720t having a terminal protecting group PG is connected to nucleotide 720c of region I and nucleotide 720g of region III by a reaction enzyme.

After that, step S105 is performed again to determine whether the nucleic acid sequence is complete. If it is not completed, steps S103 and S104 are repeated. Figure 5G removes the terminal protection group PG of nucleotide 720t in region I and the end of nucleotide 720c in region IV Protecting group PG. Fig. 5H uses a reaction enzyme to link another nucleotide 720a with a terminal protecting group PG to nucleotide 720t in region I and nucleotide 720c in region IV.

Steps S103 and S104 will be repeated until it is determined that the different nucleic acid sequences S in the regions I, II, III, and IV are respectively synthesized and completed, as shown in FIG. 5I. The above is the implementation of the patterned method of partially removing the terminal protective group PG in the different regions I, II, III, and IV by illumination or heating. Through this method, a large number of and different types can be prepared in a short time. Nucleic acid sequence S. When these are different When the nucleic acid sequence S belongs to the sequence fragments of the same gene, by bonding the subsequent sequence fragments, a nucleic acid sequence with a longer length than the conventional technology can be produced in quantity.

After the synthesis of the nucleic acid sequence S is completed, the method for preparing the nucleic acid sequence using enzymes, for example, further includes step S106: cutting the nucleic acid sequence S from the pre-treated surface 700 by restriction enzymes. When the restriction enzyme is configured on the pretreatment surface, the restriction enzyme will cut the synthesized nucleic acid sequence S from the pretreatment surface 700. After that, the nucleic acid sequence S is collected, and the preparation process of the nucleic acid sequence S is completed. Examples of restriction enzymes include uracil DNA glycosylase (UDG), endonuclease VIII, USER enzyme (NEB #M5508), etc., and combinations thereof. For example, when a restriction enzyme that cuts uracil nucleotides (UMP) is used, before the reaction enzyme arranges the nucleotide 720 with the terminal protecting group PG on the primer 710, it will first place a urine on the primer 710. Pyrimidine nucleotides, when the nucleic acid sequence is completed, since it is a synthetic deoxyribonucleic acid sequence, the sequence does not contain uracil nucleotides. For restriction enzymes, only the previously configured single uracil nucleotide cut Position, and then the nucleic acid sequence can be cleaved correctly. Different restriction enzymes will have different cutting positions, and the present invention is not particularly limited.

The above-mentioned pretreatment surfaces 200, 400, 600, 700 are only used to distinguish different embodiments and use different numbers, which can be used interchangeably in each embodiment, primers 210, 410, 610, 710 and nucleotide 220, 420, 620, 720a, 720c, 720g, 720t can be used in the same way.

In summary, in the 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 to chemical synthesis, it is less likely to pollute the environment and reduce costs, and the reaction temperature is set at 45°C to 105°C, compared to 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, thus improving the efficiency of preparing nucleic acid sequences.

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.

S101, S102, S103, S104, S105, S106: steps

Claims (9)

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 reaction temperature is 45°C~105°C; (3) The terminal protecting group of the nucleotide is removed by light or heating; (4) The terminal is possessed by the DNA polymerase The other nucleotide of the protecting group is connected to the nucleotide, and the reaction temperature is 45°C~105°C; (5) Determine whether the nucleic acid sequence is complete, if so, 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 1, 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 1, wherein the DNA polymerase is a family B DNA polymerase. The method 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 method for preparing a nucleic acid sequence using an enzyme as described in claim 1, wherein the method of removing the terminal protective group of the nucleotide by light or heating includes partial removal in a patterned manner. The method for preparing a nucleic acid sequence using an enzyme as described in claim 1, wherein the method of removing the terminal protective group of the nucleotide by irradiation includes irradiating the wafer with an ultraviolet digital light treatment. The method for preparing a nucleic acid sequence using an enzyme as described in claim 1, wherein the reaction temperature is 50°C to 85°C. The method for preparing a nucleic acid sequence using an enzyme as described in claim 1, wherein the reaction temperature is 55°C to 75°C. The method for preparing a nucleic acid sequence using an enzyme according to claim 1, 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 using the reaction enzyme The nucleotide with the terminal protecting group is connected to the primers.

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