Double-stranded DNA synthesis method

Abstract

To develop methods for synthesizing target double-stranded DNA fragments accurately, easily, efficiently and quickly, regardless of their sequences, in the method for synthesizing double-stranded DNA fragments using PCR.SOLUTION: Provided is a method for synthesizing double-stranded DNA by ligating short double-stranded DNA with overlap extension PCR to obtain a target double-stranded DNA fragment, characterized by comprising a multi-stage annealing temperature setting in the PCR cycle of the overlap extension PCR.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel method for synthesizing double-stranded DNA. [Background technology]

[0002] In the field of genetic engineering, DNA synthesis to construct long-chain DNA having a novel sequence is carried out for various purposes. Methods for synthesizing such long-chain DNA consist of roughly two steps. The first step is to synthesize double-stranded DNA fragments from a large number of single-stranded oligo DNAs. The second step is to construct long-chain DNA by assembling a large number of double-stranded DNA fragments obtained in the first step. Known methods for constructing such long-chain DNA include the yeast enrichment method (see Non-Patent Document 1), the Gibson assembly method (see Non-Patent Document 2), the Golden gate method (see Non-Patent Document 3), the LCR method (see Non-Patent Document 4), and the OGAB method (see Non-Patent Document 5).

[0003] Several methods for synthesizing double-stranded DNA fragments are known, including, for example, synthesizing double-stranded DNA fragments by simultaneously joining and assembling single-stranded oligo DNAs containing multiple homologous regions in a PCR reaction solution (see Patent Document 1 and Non-Patent Documents 6-8). In these methods, multiple single-stranded oligo DNAs are mixed in a single PCR reaction solution, and joining these single-stranded oligo DNAs by PCR can lead to undesirable situations such as mismatches between the single-stranded oligo DNAs and the formation of primer dimers. For these reasons, these synthesis methods are not necessarily capable of synthesizing double-stranded DNA fragments for any DNA sequence. In particular, synthesis may be difficult or impossible when the target DNA sequence is a complex DNA sequence containing long repetitive sequences, consecutive identical base sequences, etc.

[0004] Furthermore, in these synthesis methods, single-stranded oligo DNAs serving as starting materials must be designed to synthesize double-stranded DNAs having a desired DNA sequence. When designing these single-stranded oligo DNAs, the length of the homologous regions for joining multiple single-stranded oligo DNAs must be adjusted to set the annealing temperature of the PCR reaction. Additionally, to prevent hairpin formation and other problems in the single-stranded oligo DNAs during the synthesis process, the length, number, and GC content of each single-stranded oligo DNA must be adjusted. Specifically, in conventional synthesis methods, the design of single-stranded oligo DNAs corresponding to the desired DNA sequence to be synthesized is complex. Furthermore, even when synthesizing a large number of double-stranded DNA fragments (several to several hundred), single-stranded oligo DNAs must be designed for each DNA sequence, resulting in the inconvenience of time-consuming and labor-intensive design of single-stranded oligo DNAs.

[0005] In the second step, synthesizing long-chain DNA, the numerous double-stranded DNA fragments obtained in the first step are used as accumulation materials. When accumulating double-stranded DNA fragments in the second step, if even one double-stranded DNA fragment is missing, it becomes impossible to synthesize the desired long-chain DNA. However, conventional methods for synthesizing double-stranded DNA fragments have the disadvantage of requiring time and effort to synthesize a large number of double-stranded DNA fragments (several to hundreds of fragments). Furthermore, depending on the complexity of the target DNA sequence, synthesis can be difficult or impossible. Therefore, it is difficult to quickly obtain all of the double-stranded DNA fragments that serve as accumulation materials. In other words, conventional methods for synthesizing double-stranded DNA fragments are unable to quickly supply the double-stranded DNA fragments that serve as accumulation materials for long-chain DNA synthesis, creating a bottleneck. Therefore, there is a strong demand for a new method that can efficiently and quickly synthesize double-stranded DNA fragments as an alternative to conventional synthesis methods. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent Publication No. 20080182296 [Non-Patent Document]

[0007] [Non-Patent Document 1] Gibson, D. G., et al. Science, 5867, 1215 - 1220., 2008 [Non-Patent Document 2] Gibson, D. G., et al. Nat. Methods, 6, 343 - 345., 2009 [Non-Patent Document 3] Engler, C., et al. PLoS ONE, 4, e5553., 2009 [Non-Patent Document 4] Stefan de Kok, S., et al. ACS Synth. Biol., 3, 97 - 106., 2014 [Non-Patent Document 5] Tsuge, K., et al. Nucleic Acids Res., 31, e133., 2003、Tsuge, K., et al. Sci. Rep., 5, 10655., 2015 [Non-Patent Document 6] ​​​​​​​​​​​​​​​​​​

[0008] Under these circumstances, the present inventors have undertaken this research with the objective of solving the above-mentioned problems and developing a method for accurately, easily, efficiently, and quickly synthesizing double-stranded DNA fragments that can be used as accumulation materials for long-chain DNA synthesis. That is, the objective of the present invention is to develop a method for synthesizing double-stranded DNA fragments using PCR that can accurately, easily, efficiently, and quickly synthesize a desired double-stranded DNA fragment regardless of its sequence. [Means for solving the problem]

[0009] As a result of intensive research to solve the above problems, the present inventors have discovered that in a method for synthesizing double-stranded DNA in which short double-stranded DNAs are ligated by overlap extension PCR to obtain a desired double-stranded DNA fragment, by setting the annealing temperature in multiple stages in the PCR cycle, the desired double-stranded DNA fragment can be synthesized accurately, easily, efficiently, and quickly, and have completed the present invention. That is, the gist of the present invention is as follows.

[0010] [1] A method for synthesizing double-stranded DNA, in which short double-stranded DNAs are ligated by overlap extension PCR to obtain a desired double-stranded DNA fragment, comprising: A method for synthesizing double-stranded DNA, characterized in that the PCR cycle of the overlap extension PCR has multiple stages of annealing temperature settings. [2] The method for synthesizing double-stranded DNA according to [1], wherein the annealing temperature is set in 2 to 20 stages. [3] The method for synthesizing double-stranded DNA according to [1] or [2], wherein the annealing temperature is set to 65°C to 85°C. [4] The method for synthesizing double-stranded DNA according to any one of [1] to [3], wherein the temperature holding time at each stage of the annealing temperature setting is 10 seconds to 2 minutes. [5] The method for synthesizing double-stranded DNA according to any one of [1] to [4], wherein the overlap region of DNA in the overlap extension PCR is 10 to 40 bases. [6] The method for synthesizing double-stranded DNA according to any one of [1] to [5], wherein 3 to 20 types of short double-stranded DNA are ligated in the overlap extension PCR. [7] The method for synthesizing double-stranded DNA according to any one of [1] to [6], wherein the short double-stranded DNA used in the overlap extension PCR is synthesized by primer extension PCR. [8] The method for synthesizing double-stranded DNA according to [7], wherein the PCR cycle of the primer extension PCR has multiple annealing temperature settings. [9] The method for synthesizing double-stranded DNA according to [7] or [8], wherein the short double-stranded DNA synthesized by the primer extension PCR has a size of 100 to 300 bases.

[10] A method for synthesizing double-stranded DNA, comprising: a primer extension PCR step of synthesizing short double-stranded DNA by primer extension PCR; and an overlap extension PCR step in which the double-stranded DNA synthesized in the primer extension PCR step is ligated to synthesize a double-stranded DNA fragment of interest by overlap extension PCR; wherein the PCR cycle of the overlap extension PCR has multiple annealing temperature settings. [Effects of the Invention]

[0011] According to the present invention, by setting multiple annealing temperatures in the PCR cycle, it is possible to combine multiple DNA fragments with various Tm values, thereby enabling easy, accurate, efficient, and rapid assembly of single-stranded oligo DNA fragments containing numerous homologous regions. Furthermore, because the method for synthesizing double-stranded DNA of the present invention is applicable to combining DNA fragments with various Tm values, it is unnecessary to optimize the sequence of the single-stranded oligo DNA fragments used as raw materials, and it is not necessary to carefully adjust the temperature setting for each target double-stranded DNA fragment. Furthermore, according to the present invention, it is possible to easily, accurately, efficiently, and rapidly synthesize a double-stranded DNA fragment of interest regardless of its sequence, thereby enabling the synthesis of double-stranded DNA fragments that have previously been difficult or impossible to synthesize due to their complex sequences (e.g., long repetitive sequences, consecutive identical base sequences, AT-rich or GC-rich sequences, etc.). [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram schematically illustrating the method for synthesizing double-stranded DNA of the present invention. [Figure 2] FIG. 1 shows the results of examining PCR conditions for the method for synthesizing double-stranded DNA of the present invention. [Figure 3] FIG. 1 shows the results of examining PCR conditions for the method for synthesizing double-stranded DNA of the present invention. [Figure 4] FIG. 1 shows the results of comparing double-stranded DNA fragments obtained by a conventional synthesis method and the synthesis method of the present invention. [Figure 5] FIG. 1 shows the results of electrophoresis of double-stranded DNAs with different sequences simultaneously synthesized by the method for synthesizing double-stranded DNA of the present invention. [Figure 6] FIG. 1 shows the results of comparing the quality of chemically synthesized single-stranded DNA. [Figure 7] FIG. 1 shows the results of comparing double-stranded DNA fragments obtained by a conventional synthesis method and the synthesis method of the present invention. [Figure 8] FIG. 1 shows the results of comparing double-stranded DNA fragments obtained by a conventional synthesis method and the synthesis method of the present invention. [Figure 9] FIG. 1 shows the results of comparing double-stranded DNA fragments obtained by a conventional synthesis method and the synthesis method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0013] The method for synthesizing double-stranded DNA of the present invention will be described in detail below. Unless otherwise specified, molecular biological techniques in this specification can be performed by methods described in general experimental manuals known to those skilled in the art, or methods based thereon. Furthermore, unless otherwise specified, the terms used in this specification should be interpreted as having the meanings commonly used in the relevant technical field.

[0014] <Double-stranded DNA synthesis method> The method for synthesizing double-stranded DNA of the present invention is a method for obtaining a double-stranded DNA fragment of interest by ligating short double-stranded DNAs by overlap extension PCR, characterized in that the PCR cycle of the overlap extension PCR has multiple annealing temperature settings. Because the method for synthesizing double-stranded DNA of the present invention has multiple annealing temperature settings in the PCR cycle, it can be applied to the joining of multiple DNAs with various Tm values, thereby enabling the easy, accurate, efficient, and rapid assembly of single-stranded oligo DNAs containing multiple homologous regions. Furthermore, because the method for synthesizing double-stranded DNA of the present invention can be applied to the joining of DNAs with various Tm values, it is unnecessary to optimize the sequence of the single-stranded oligo DNA used as the material, and it is not necessary to carefully adjust the temperature setting for each double-stranded DNA fragment of interest. Furthermore, according to the present invention, it is possible to easily, accurately, efficiently, and quickly synthesize a target double-stranded DNA fragment regardless of its sequence, and therefore it is possible to synthesize double-stranded DNA fragments that have previously been difficult or impossible to synthesize due to their complex sequences (e.g., long repetitive sequences, consecutive identical base sequences, AT-rich or GC-rich sequences, etc.).

[0015] Furthermore, the short double-stranded DNA used in the overlap extension PCR is preferably synthesized by primer extension PCR. Therefore, the present invention can also be described as a method for synthesizing double-stranded DNA, comprising a primer extension PCR step for synthesizing short double-stranded DNA by primer extension PCR, and an overlap extension PCR step for synthesizing a desired double-stranded DNA fragment by overlap extension PCR by ligating the double-stranded DNA synthesized in the primer extension PCR step, and characterized by having multiple annealing temperature settings in the PCR cycle of the overlap extension PCR. Each step of the method for synthesizing double-stranded DNA of the present invention will be described in detail below. Figure 1 shows a schematic diagram of the method for synthesizing double-stranded DNA of the present invention.

[0016] [Primer extension PCR process] This step involves synthesizing short double-stranded DNAs by primer extension PCR. The short double-stranded DNAs obtained in this step are ligated in the overlap extension PCR step described below to form double-stranded DNA fragments that can be used as building blocks for long-chain DNA synthesis. Therefore, the type of double-stranded DNA fragments required as building blocks are determined based on the sequence of the long double-stranded DNA to be synthesized. These double-stranded DNA fragments are then divided into several segments with designed sequences, which become the short double-stranded DNAs synthesized in this step.

[0017] Here, in the present invention, primer extension PCR refers to a reaction in which a pair of single-stranded oligo DNAs, each having a region that complementarily binds to the other's end, are joined together and each strand is extended with a polymerase to synthesize double-stranded DNA.

[0018] (i) Sequence design of single-stranded oligo DNA The full-length sequence of a double-stranded DNA fragment obtained by the method of synthesizing double-stranded DNA of the present invention is divided into any number of short double-stranded DNAs. The size of these short double-stranded DNAs is typically 100 to 300 bases, preferably 120 to 250 bases, more preferably 140 to 180 bases, and even more preferably around 150 bases. The number of short double-stranded DNAs to be divided can be determined appropriately depending on the length of the full-length sequence, but is typically 3 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably around 3.

[0019] The divided short double-stranded DNAs are designed to contain overlapping regions of any length at the 5'- or 3'-end. The length of this overlapping region is typically 5 to 50 bases, preferably 10 to 40 bases, more preferably 15 to 40 bases, and even more preferably approximately 30 bases. The pair of single-stranded oligo DNAs used to synthesize the short double-stranded DNAs designed as above are 60 to 300 bases long, including the overlapping region, preferably 100 to 200 bases, and more preferably 150 to 200 bases. In this process, using relatively long single-stranded oligo DNAs reduces the number of single-stranded oligo DNAs used in double-stranded DNA synthesis and eliminates bias in the GC content ratio in the single-stranded oligo DNA sequences, thereby preventing the formation of DNA secondary structures such as hairpin structures. These single-stranded oligo DNAs can be prepared by chemical synthesis.

[0020] These single-stranded oligo DNAs may contain incomplete single-stranded oligo DNAs generated during the chemical synthesis process, which are different from full-length single-stranded oligo DNAs. In this process, as long as full-length single-stranded oligo DNAs account for 15% or more of the single-stranded oligo DNAs used, a DNA amplification product of the desired synthetic double-stranded DNA can be obtained, and double-stranded DNA with the correct sequence can be obtained by cloning. In other words, even if the single-stranded oligo DNAs used contain approximately 85% incomplete single-stranded oligo DNAs containing unreacted material, a DNA amplification product of the desired synthetic double-stranded DNA can be obtained.

[0021] (ii) PCR cycle The number of pairs of single-stranded oligo DNAs corresponding to the number of divisions is adjusted separately to an appropriate concentration using sterile water or TE buffer. The concentration is typically 0.1 to 10 μM, preferably 0.25 to 5 μM, and more preferably around 1 μM. A PCR reaction solution (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase; NEB, etc.) is prepared, and the above pairs of single-stranded oligo DNAs are added to each solution to adjust the total volume to equal amounts. A PCR reaction solution is prepared for each synthesis of short double-stranded DNA, and only the two single-stranded oligo DNAs that make up a pair are mixed into each PCR reaction solution. This prevents mismatching between single-stranded oligo DNAs and the formation of primer dimers during the joining of the single-stranded oligo DNAs.

[0022] Next, a short double-stranded DNA is synthesized by a primer extension PCR reaction using a thermal cycler. The PCR reaction temperature conditions are 98°C for 2 minutes, followed by a thermal denaturation step, an annealing step, and an extension step (each cycle) for any number of cycles. The number of cycles is usually 5 or more, preferably 10 or more. Although there is no problem with more than 10 cycles, a sufficient amount of synthesis can be obtained in this process with about 10 cycles.

[0023] The conditions for the heat denaturation and extension steps can be the same as those for conventional PCR, for example, 98°C for 30 seconds for the heat denaturation step, and 72°C for 50 seconds for the extension step, although the conditions vary depending on the polymerase used.

[0024] The present invention differs significantly from conventional methods in that the annealing step has multiple annealing temperature settings. The annealing temperature settings are in the range of 50°C to 90°C, preferably 60°C to 85°C, with 2 to 20 temperature settings, preferably 2 to 15 temperature settings, more preferably 2 to 10 temperature settings, and even more preferably 2 to 8 temperature settings. The annealing temperature retention time at each step is typically 10 seconds or longer, preferably 20 seconds or longer, more preferably 30 seconds or longer, even more preferably 40 seconds or longer, and particularly preferably 50 seconds or longer. Reaction times longer than 50 seconds are not problematic, and may be up to about 2 minutes, although approximately 50 seconds may be sufficient. After the primer extension PCR step is complete, overnight incubation is also acceptable. The temperature during this time is preferably in the range of 4°C to 72°C.

[0025] By setting multiple annealing temperatures in the PCR of this step, it is possible to compensate for the difference in Tm value between each pair of single-stranded oligo DNA combinations, and therefore PCR for synthesizing all double-stranded DNA in this step can be performed at once.

[0026] [Overlap extension PCR process] This step involves ligating the short double-stranded DNA fragments synthesized in the primer extension PCR step by overlap extension PCR to obtain the desired double-stranded DNA fragment. This PCR cycle is characterized by having multiple annealing temperature settings.

[0027] Here, overlap extension PCR refers to a reaction in which, during amplification of target DNA in a PCR reaction, a new sequence is added to the 5' end of a target DNA-specific primer, thereby adding a new sequence to the PCR product. This new sequence is designed to be complementary between multiple target DNAs, thereby fusing the ends of multiple target DNAs during annealing and synthesizing a single PCR product through a subsequent extension reaction using DNA polymerase. In the present invention, this reaction is performed to ligate multiple short double-stranded DNAs synthesized in the primer extension PCR step to create a single double-stranded DNA fragment. The short double-stranded DNAs have homologous regions at the junction. When thermally denatured into single strands, the homologous regions complementarily bind during the annealing step, allowing new sequences at the 5' ends of each strand to be added to the PCR product. Because this new sequence is designed to be complementary to another double-stranded DNA, the DNA ends are fused during annealing, allowing a single PCR product to be synthesized through a subsequent extension reaction using DNA polymerase.

[0028] Specifically, the procedure is as follows: Equal amounts of the short double-stranded DNA synthesized in the primer extension PCR step are added to a PCR reaction solution (1 x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase; NEB, etc.) for synthesizing full-length double-stranded DNA. This PCR reaction solution contains primers for amplifying the full-length double-stranded DNA.

[0029] Next, a full-length double-stranded DNA fragment is synthesized by PCR using a thermal cycler. The PCR reaction is performed at 98°C for 2 minutes, followed by a desired number of cycles, each cycle consisting of a thermal denaturation step, an annealing step, and an extension step. The number of cycles is usually 10 or more, preferably 20 or more. Although there is no problem with more than 20 cycles, approximately 20 cycles is sufficient to obtain a sufficient amount of synthesis for this process.

[0030] The conditions for the heat denaturation and extension steps can be the same as those for conventional PCR, for example, 98°C for 30 seconds for the heat denaturation step, and 72°C for 50 seconds for the extension step, although the conditions vary depending on the polymerase used.

[0031] The present invention differs significantly from conventional methods in that the annealing step has multiple annealing temperature settings. The annealing temperature is set in the range of 50°C to 90°C, preferably 60°C to 85°C, with 2 to 20 temperature settings, preferably 2 to 15 temperature settings, more preferably 2 to 10 temperature settings, and even more preferably 2 to 8 temperature settings. The annealing temperature is held for each temperature setting step typically for 10 seconds or more, preferably 20 seconds or more, more preferably 30 seconds or more, even more preferably 40 seconds or more, and particularly preferably 50 seconds or more. Reaction times longer than 50 seconds are not problematic, and may be up to about 2 minutes, although about 50 seconds may be sufficient.

[0032] The multiple annealing temperature settings in this PCR process compensate for differences in Tm values ​​that arise when assembling short double-stranded DNA or splicing individual single-stranded DNAs together, eliminating the need to optimize the Tm values ​​of the homologous regions of the single-stranded oligo DNA required for splicing. This allows the length of the homologous region of the single-stranded oligo DNA to be fixed at 30 bases, simplifying the design of single-stranded oligo DNA when synthesizing double-stranded DNAs with numerous different sequences. It also allows the temperature conditions for the primer extension PCR reaction and the overlap extension PCR reaction to be standardized.

[0033] Furthermore, the method of the present invention allows multiple double-stranded DNA fragments with different sequences to be synthesized simultaneously in parallel. While there is no particular limit to the number of double-stranded DNA fragments that can be synthesized simultaneously, simultaneous synthesis of tens to hundreds of fragments is possible, resulting in significantly high throughput. Furthermore, by applying the synthesis method of the present invention to a program for a liquid dispensing robot, the simultaneous synthesis of multiple double-stranded DNA fragments can be automated.

[0034] The double-stranded DNA fragments that can be synthesized by the synthesis method of the present invention are not particularly limited in sequence or length, but a length of up to 5,000 base pairs is preferred in consideration of the accuracy of the thermostable DNA polymerase used in PCR. Therefore, the number of short double-stranded DNA fragments ligated by overlap extension PCR is usually about 3 to 20 fragments, preferably 2 to 10 fragments, more preferably 2 to 5 fragments, and even more preferably about 3 fragments.

[0035] Furthermore, the synthesis method of the present invention also makes it possible to synthesize double-stranded DNA fragments containing DNA sequences that are generally considered to be complex sequences, such as long repetitive sequences, consecutive identical base sequences, AT-rich sequences, and GC-rich sequences.

[0036] The double-stranded DNA fragments obtained by the synthesis method of the present invention can be accumulated in large numbers to construct long-chain DNA by using a method for synthesizing long-chain DNA, such as the yeast accumulation method (see Non-Patent Document 1), the Gibson assembly method (see Non-Patent Document 2), the Golden gate method (see Non-Patent Document 3), the LCR method (see Non-Patent Document 4), or the OGAB method (see Non-Patent Document 5).

[0037] In the synthesis of long-chain DNA, double-stranded DNA fragments are used as accumulation materials, but if even one of these double-stranded DNA fragments is missing, it becomes impossible to synthesize the desired long-chain DNA. The synthesis method of the present invention can simultaneously synthesize multiple double-stranded DNA fragments regardless of the complexity of the target DNA sequence to be synthesized, and therefore can rapidly synthesize double-stranded DNA fragments that serve as accumulation materials for the synthesis of long-chain DNA. Therefore, the synthesis method of the present invention can rapidly supply the double-stranded DNA fragments necessary for the synthesis of long-chain DNA, thereby significantly improving the throughput of the method for synthesizing long-chain DNA.

[0038] The PCR product of the double-stranded DNA fragment obtained by the synthesis method of the present invention is mixed with a vector and subjected to DNA ligation reaction to perform DNA cloning, followed by transformation of E. coli competent cells by introducing the plasmid. DNA cloning of the DNA amplification product of the synthetic double-stranded DNA fragment can be performed by any of the following methods, without particular limitation: restriction enzyme cloning, TA cloning, In-Fusion cloning, blunt-end cloning, etc. The vector DNA used can be any of the following: restriction enzyme cloning vector, In-Fusion cloning vector, TA cloning vector, blunt-end cloning vector, etc., without particular limitation.

[0039] The DNA amplification products of double-stranded DNA fragments obtained by the synthesis method of the present invention can suppress the appearance of non-specific DNA amplification products shorter than the length of the target sequence, which are generated by PCR reactions. Therefore, the synthesized double-stranded DNA fragments obtained by the synthesis method of the present invention can be subjected to DNA cloning methods such as TA cloning and blunt-end cloning without undergoing steps such as gel excision and purification to isolate only the target double-stranded DNA fragment, and cloned DNA containing the target double-stranded DNA fragment can be quickly obtained.

[0040] <Program> The present invention also includes a PCR program for the method for synthesizing double-stranded DNA of the present invention described above. The program for PCR conditions of the present invention can be used in a device used in conjunction with PCR, such as a thermal cycler. The program of the present invention specifies the temperature conditions and the like of PCR in the method for synthesizing double-stranded DNA of the present invention, and the details of the program can be applied to the explanation in the section "Method for synthesizing double-stranded DNA."

[0041] <Device> The present invention also includes an apparatus capable of realizing the above-described method for synthesizing double-stranded DNA of the present invention. The apparatus of the present invention is also an apparatus incorporating the above-described program of the present invention. Specifically, it is an apparatus used in connection with PCR, such as a thermal cycler, incorporating the above-described program of the present invention. The program specifies the PCR temperature conditions and the like in the DNA synthesis method of the present invention, and the details of the program are applicable to the explanation in the section "Method for synthesizing double-stranded DNA."

[0042] <Automatic double-stranded DNA synthesis system> The present invention also includes an automated double-stranded DNA synthesis system characterized by using the above-described method for synthesizing double-stranded DNA of the present invention. In the above-described method for synthesizing double-stranded DNA of the present invention, PCR can be performed under the same conditions regardless of the target DNA sequence to synthesize double-stranded DNA, enabling a high-throughput synthesis system using a liquid dispensing robot or the like, or an automated synthesis system that can automate a series of these steps. Such an automated double-stranded DNA synthesis system of the present invention may use the above-described program of the present invention or the device of the present invention. [Example]

[0043] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples.

[0044] The reagents and test methods used in the examples are as follows: Single-stranded oligo DNA, which was used as the material for double-stranded DNA synthesis, was manufactured by Nippon Techno Service Co., Ltd. and FASMAC Co., Ltd. DNA amplification by PCR reaction used for double-stranded DNA synthesis was performed using Phusion High-Fidelity DNA polymerase manufactured by NEB according to the attached instructions. DNA cloning of the DNA amplification product was performed using 10x A-attachment Mix manufactured by Toyobo Co., Ltd., T-Vector pMD19 (Simple) and DNA Ligation kit manufactured by TaKaRa Co., Ltd. E. coli JM109 competent cells manufactured by TaKaRa Co., Ltd., and transformation using a plasmid vector was performed according to the attached instructions. The medium components and agar of LB medium were manufactured by Becton Dickinson Co., Ltd. TM Tryptone, Bacto TM Yeast Extract, Bacto TMAgar). Ampicillin and carbenicillin antibiotics were purchased from Nacalai Tesque. Preparation of PCR reaction templates from E. coli colonies was performed using Kanto Chemical's Cica Geneus DNA Extraction Reagent according to the attached instructions. DNA amplification from E. coli colonies by colony direct PCR was performed using TaKaRa Ex-Taq Hot Start kit according to the attached instructions. The temperature conditions for the colony direct PCR reaction were as follows: 95°C for 2 minutes, followed by 30 cycles of the following cycles: 95°C for 20 seconds, 58°C for 30 seconds, and 72°C for 1 minute per 1 kb of amplification length. DNA purification was performed using Qiagen's MinElute PCR Purification kit according to the attached instructions. DNA sequencing was performed using Thermo Fisher Scientific's BigDye Terminator v3.1 Cycle Sequencing Kit according to the attached instructions. The temperature conditions for the DNA sequencing reaction were as follows. After 2 minutes at 95°C, 30 cycles of the following temperature cycles were performed: 95°C for 5 seconds; 50°C for 10 seconds; and 60°C for 2 minutes and 30 seconds. Purification of the DNA sequencing reaction product was performed using a BigDye Terminator Purification kit (Thermo Fisher Scientific) according to the accompanying instructions. DNA sequencing was performed using an Applied Biosystems 3500xL Genetic analyzer (Thermo Fisher Scientific) according to the accompanying instructions. Agarose gel electrophoresis of the PCR reaction product was performed using an agarose gel electrophoresis apparatus (i-MyRun.NC) (Cosmo Bio) according to the accompanying instructions. DNA staining after electrophoresis was performed using Biotium's GelRed nucleic acid fluorescent staining reagent according to the accompanying instructions. Electrophoresis of single-stranded oligo DNA was performed using an XCell SureLock MiniCell electrophoresis apparatus manufactured by Thermo Fisher Scientific and 10% Novex TBE-Urea gel according to the attached instructions. After electrophoresis, the single-stranded oligo DNA was stained using SYBR Green II nucleic acid fluorescent staining reagent manufactured by TaKaRa according to the attached instructions.Analysis of single-stranded oligo DNA was performed using a Bio-Rad Gel Doc EZ system gel imaging analyzer according to the manufacturer's instructions. All other biochemical reagents were from Thermo Fisher Scientific and Nacalai Tesque.

[0045] (Example 1) Investigation of PCR reaction conditions in the synthesis method of the present invention (investigation of temperature retention time for each multi-stage temperature gradient) (1) Sequence design of single-stranded oligo DNA The desired full-length double-stranded DNA sequence was divided into three short double-stranded DNA fragments. These short double-stranded DNA fragments were designed to contain a 30-base-pair overlap region at the 5'- or 3'-end. Specifically, the six single-stranded oligo DNAs used to synthesize the three short double-stranded DNA fragments were designed to be approximately 150 bases long, including a 30-base overlap region (SEQ ID NOs: 1 to 6). A forward primer (SEQ ID NO: 7) and a reverse primer (SEQ ID NO: 8) were also designed for overlap extension PCR.

[0046] (2) Two-step double-stranded DNA synthesis The six single-stranded oligo DNAs required for double-stranded DNA synthesis were prepared at a concentration of 1 μM in sterile water or TE buffer (Nacalai Tesque). Three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared to synthesize short double-stranded DNA fragments by primer extension PCR. To the first PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 1) and single-stranded oligo DNA (SEQ ID NO: 2) was added. To the second PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 3) and single-stranded oligo DNA (SEQ ID NO: 4) was added. To the third PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 5) and single-stranded oligo DNA (SEQ ID NO: 6) was added. The total volume was adjusted to 25 μL. PCR reaction solution B (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase, 0.1 μM forward primer (SEQ ID NO: 7), 0.1 μM reverse primer (SEQ ID NO: 8) was prepared to a total volume of 25 μL for synthesizing full-length double-stranded DNA by overlap extension PCR.

[0047] Three short double-stranded DNA fragments were synthesized by primer extension PCR using a thermal cycler manufactured by TaKaRa Co., Ltd. The temperature conditions for the PCR reaction were as follows:

[0048] After 2 minutes at 98°C, 10 cycles of the following temperature cycle were performed.

[0049] (Thermal denaturation stage) 98℃, 30 seconds; (Annealing stage) 77.5℃, 10 seconds; 75℃, 10 seconds; 72.5℃, 10 seconds; 70℃, 10 seconds; 67.5℃, 10 seconds; 65℃, 10 seconds; 62.5℃, 10 seconds (elongation stage) 72℃, 50 seconds

[0050] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0051] Next, the three short double-stranded DNA fragments prepared above were ligated in a TaKaRa thermal cycler by overlap extension PCR to synthesize a full-length double-stranded DNA under the following temperature conditions:

[0052] After 2 minutes at 98°C, the following temperature cycle was repeated 20 times. (Thermal denaturation stage) 98℃, 30 seconds; (Annealing stage) 77.5℃, 10 seconds; 75℃, 10 seconds; 72.5℃, 10 seconds; 70℃, 10 seconds; 67.5℃, 10 seconds; 65℃, 10 seconds; 62.5℃, 10 seconds (elongation stage) 72℃, 50 seconds.

[0053] Then, the mixture was treated at 72°C for 3 minutes.

[0054] Full-length double-stranded DNA was synthesized under the same PCR conditions as above, except that all temperature holding times in the annealing step were 20 seconds, 30 seconds, 40 seconds, 50 seconds, or 2 minutes. Subsequently, a portion of the DNA amplification products of the double-stranded DNA fragments obtained under the six conditions of the synthesis method of the present invention (temperature holding times in the annealing step were 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, and 2 minutes) was electrophoresed on a 1% agarose gel using an agarose gel electrophoresis apparatus (Cosmo Bio). The results are shown in Figure 2.

[0055] As a result, the DNA amplification products of double-stranded DNA fragments obtained when the temperature holding time of the PCR reaction conditions in the synthesis method of the present invention was 50 seconds or longer showed reduced appearance of nonspecific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence) compared to the DNA amplification products of double-stranded DNA fragments obtained when the temperature holding time of the PCR reaction conditions was less than 50 seconds. That is, a temperature holding time of 50 seconds or longer is particularly preferred for the PCR reaction conditions in the synthesis method of the present invention. On the other hand, although the DNA amplification products of double-stranded DNA fragments obtained when the temperature holding time of the PCR reaction conditions was 10, 20, 30, or 40 seconds contained nonspecific DNA amplification products, it was still possible to obtain the target double-stranded DNA fragment. Furthermore, since the appearance of nonspecific DNA amplification products can be suppressed when the temperature holding time of the PCR reaction conditions in the synthesis method of the present invention was 50 seconds or longer, it was possible to quickly obtain cloned DNA containing the target double-stranded DNA fragment.

[0056] (Example 2) Investigation of PCR reaction conditions for the synthesis method of the present invention (investigation of the number of temperature gradient steps) (1) Sequence design of single-stranded oligo DNA The desired full-length double-stranded DNA sequence was divided into three short double-stranded DNA fragments. These short double-stranded DNA fragments were designed to contain a 30-base-pair overlap region at the 5'- or 3'-end. Specifically, the six single-stranded oligo DNAs used to synthesize the three short double-stranded DNA fragments were designed to be approximately 150 bases long, including a 30-base overlap region (SEQ ID NOs: 9 to 14). Additionally, a forward primer (SEQ ID NO: 15) and a reverse primer (SEQ ID NO: 16) were designed for the overlap extension PCR reaction.

[0057] (2) Two-step double-stranded DNA synthesis The six single-stranded oligo DNAs required for double-stranded DNA synthesis were prepared at a concentration of 1 μM in sterile water or TE buffer (Nacalai Tesque). Three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase) for synthesizing short double-stranded DNA fragments were prepared. To the first PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 9) and single-stranded oligo DNA (SEQ ID NO: 10) was added. To the second PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 11) and single-stranded oligo DNA (SEQ ID NO: 12) was added. To the third PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 13) and single-stranded oligo DNA (SEQ ID NO: 14) was added. The total volume was adjusted to 25 μL. PCR reaction solution B for synthesizing full-length double-stranded DNA (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase, 0.1 μM forward primer (SEQ ID NO: 15), 0.1 μM reverse primer (SEQ ID NO: 16) was prepared in a total volume of 25 μL.

[0058] A full-length double-stranded DNA fragment was synthesized under the following conditions (i) to (vii) (different numbers of annealing temperature settings).

[0059] (i) 0 level Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0060] 98℃, 30 seconds; 72℃, 50 seconds

[0061] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0062] Next, a full-length double-stranded DNA was synthesized by multi-stage ligation of three short double-stranded DNA fragments using overlap extension PCR in a TaKaRa thermal cycler. The PCR reaction conditions were 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0063] 98℃, 30 seconds; 72℃, 50 seconds

[0064] Then, it was treated at 72°C for 3 minutes.

[0065] (ii) 1st stage Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0066] 98℃, 30 seconds; 77.5℃, 50 seconds; 72℃, 50 seconds

[0067] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0068] Next, a full-length double-stranded DNA was synthesized by multi-stage ligation of three short double-stranded DNA fragments using overlap extension PCR in a TaKaRa thermal cycler. The PCR reaction conditions were 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0069] 98℃, 30 seconds; 77.5℃, 50 seconds; 72℃, 50 seconds

[0070] Then, it was treated at 72°C for 3 minutes.

[0071] (iii) Two-stage Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0072] 98℃, 30 seconds; 77.5℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0073] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0074] Next, a full-length double-stranded DNA was synthesized by multi-stage ligation of three short double-stranded DNA fragments using overlap extension PCR in a TaKaRa thermal cycler. The PCR reaction conditions were 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0075] 98℃, 30 seconds; 77.5℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0076] Then, it was treated at 72°C for 3 minutes.

[0077] (iv) Three-stage Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0078] 98℃, 30 seconds; 77.5℃, 50 seconds; 70℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0079] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0080] Next, using a TaKaRa thermal cycler, overlap extension PCR was performed to synthesize full-length double-stranded DNA by multistage ligation of three short double-stranded DNA fragments. The PCR reaction conditions were 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles: 98°C for 30 seconds, 77.5°C for 50 seconds, 70°C for 50 seconds, 62.5°C for 50 seconds, and 72°C for 50 seconds. Then, it was treated at 72°C for 3 minutes.

[0081] (v) 7 levels Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0082] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0083] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0084] Next, a full-length double-stranded DNA was synthesized by multi-stage ligation of three short double-stranded DNA fragments using overlap extension PCR in a TaKaRa thermal cycler. The PCR reaction conditions were 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0085] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0086] Then, it was treated at 72°C for 3 minutes.

[0087] (vi) 8 stages Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0088] 98℃, 30 seconds; 77.5℃, 50 seconds; 75.4℃, 50 seconds; 73.2℃, 50 seconds; 71.1℃, 50 seconds; 68.9℃, 50 seconds; 66.8℃, 50 seconds; 64.6℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0089] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0090] Next, a full-length double-stranded DNA was synthesized by multi-stage ligation of three short double-stranded DNA fragments using overlap extension PCR in a TaKaRa thermal cycler. The PCR reaction conditions were 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0091] 98℃, 30 seconds; 77.5℃, 50 seconds; 75.4℃, 50 seconds; 73.2℃, 50 seconds; 71.1℃, 50 seconds; 68.9℃, 50 seconds; 66.8℃, 50 seconds; 64.6℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0092] Then, it was treated at 72°C for 3 minutes.

[0093] (vii) Continuous temperature gradient Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0094] 98°C, 30 seconds; 77.5°C to 62.5°C, 0.04°C / second; 72°C, 50 seconds

[0095] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0096] Next, a full-length double-stranded DNA was synthesized by multi-stage ligation of three short double-stranded DNA fragments using overlap extension PCR in a TaKaRa thermal cycler. The PCR reaction conditions were 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0097] 98°C, 30 seconds; 77.5°C to 62.5°C, 0.04°C / second; 72°C, 50 seconds

[0098] Then, it was treated at 72°C for 3 minutes.

[0099] Then, a portion of the DNA amplification products of the double-stranded DNA fragments obtained under the seven conditions of the synthesis method of the present invention was electrophoresed on a 1% agarose gel using an agarose gel electrophoresis apparatus (Cosmo Bio). The results are shown in Figure 3.

[0100] As shown in Figure 3, the number of temperature gradient steps, which is a PCR reaction condition in the synthesis method of the present invention, is preferably 2 to 8 steps. There is no problem with the number of temperature gradient steps being 8 or more, and the reaction may be carried out under PCR reaction conditions with 8 or more temperature gradient steps, but it can also be said that 2 to 8 steps is sufficient. On the other hand, it has been confirmed that if the PCR reaction conditions in the synthesis method of the present invention do not include a temperature holding time for each temperature gradient (continuous temperature gradient) or if the temperature holding time is very short, it is difficult to obtain a DNA amplification product of the desired double-stranded DNA fragment even if the number of temperature gradient steps is increased (data not shown).

[0101] (Example 3) Comparison of double-stranded DNA fragments obtained by conventional synthesis methods and the synthesis method of the present invention - 1 (Comparison of double-stranded DNA fragments obtained by the synthesis method of a contract synthesis company and the synthesis method of the present invention) (1) Sequence design of single-stranded oligo DNA The desired full-length double-stranded DNA sequence was divided into three short double-stranded DNA fragments. These short double-stranded DNA fragments were designed to contain a 30-base-pair overlap region at the 5'- or 3'-end. Specifically, the six single-stranded oligo DNAs used to synthesize the three short double-stranded DNA fragments were designed to be approximately 150 bases long, including a 30-base overlap region (SEQ ID NOs: 17 to 22). Additionally, a forward primer (SEQ ID NO: 23) and a reverse primer (SEQ ID NO: 24) were designed for the overlap extension PCR reaction.

[0102] (2) Two-step double-stranded DNA synthesis The six single-stranded oligo DNAs required for double-stranded DNA synthesis were prepared at a concentration of 1 μM in sterile water or TE buffer (Nacalai Tesque). Three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared to synthesize short double-stranded DNA fragments. To the first PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 17) and single-stranded oligo DNA (SEQ ID NO: 18) was added. To the second PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 19) and single-stranded oligo DNA (SEQ ID NO: 20) was added. To the third PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 21) and single-stranded oligo DNA (SEQ ID NO: 22) was added. The total volume was adjusted to 25 μL. PCR reaction solution B for synthesizing full-length double-stranded DNA (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / µL Phusion High-Fidelity DNA polymerase, 0.1 µM forward primer (SEQ ID NO: 23), 0.1 µM reverse primer (SEQ ID NO: 24) was prepared in a total volume of 25 µL.

[0103] Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0104] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0105] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0106] Furthermore, a full-length double-stranded DNA was synthesized by multi-stage overlap extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature set at 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0107] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0108] Then, it was treated at 72°C for 3 minutes.

[0109] A portion of the DNA amplification product of the synthetic double-stranded DNA obtained by the method of the present invention and a portion of the solution of synthetic double-stranded DNA synthesized by a contract synthesis company (using a conventional synthesis method) were electrophoresed on a 1% agarose gel using an agarose gel electrophoresis apparatus (Cosmo Bio). The results are shown in Figure 4.

[0110] As shown in Figure 4, the DNA amplification products of double-stranded DNA fragments obtained by the synthesis method of the present invention were able to significantly reduce the occurrence of non-specific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence) compared to the DNA amplification products of double-stranded DNA fragments obtained by the synthesis method of the contract synthesis company. The DNA amplification products of double-stranded DNA fragments obtained by the contract synthesis company, in which a significant amount of non-specific DNA amplification products appeared, require steps such as gel excision and purification to extract only the desired double-stranded DNA fragment, so it is not preferable to apply DNA cloning methods such as TA cloning and blunt-end cloning, and it is difficult to quickly obtain cloned DNA.

[0111] On the other hand, the DNA amplification products of double-stranded DNA fragments obtained by the synthesis method of the present invention were able to significantly reduce the appearance of non-specific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence). Therefore, the DNA amplification products of double-stranded DNA fragments obtained under the PCR reaction conditions of the synthesis method of the present invention can be subjected to DNA cloning methods such as TA cloning and blunt-end cloning without undergoing steps such as gel excision and purification to isolate only the desired double-stranded DNA fragment, making it possible to quickly obtain cloned DNA containing the desired double-stranded DNA fragment. As a result, it can be said that the synthesis method of the present invention is superior to conventional DNA synthesis methods in that it increases the efficiency of DNA cloning and allows the desired cloned DNA to be quickly obtained.

[0112] (Example 4) Simultaneous synthesis of multiple different double-stranded DNA sequences and analysis of chemically synthesized single-stranded oligo DNA (1) Sequence design of single-stranded oligo DNA Each of the four target full-length double-stranded DNA sequences was divided into three short double-stranded DNA fragments. These short double-stranded DNA fragments were designed to contain a 30-base-pair overlap region at the 5'- or 3'-end. The six single-stranded oligo DNAs used to synthesize the three short double-stranded DNA fragments corresponding to the first full-length double-stranded DNA were designed to be approximately 150 bases long and contain a 30-base overlap region (SEQ ID NOS: 25-30). The six single-stranded oligo DNAs used to synthesize the three short double-stranded DNA fragments corresponding to the second full-length double-stranded DNA were designed to be approximately 150 bases long and contain a 30-base overlap region (SEQ ID NOS: 31-36). The six single-stranded oligo DNAs used to synthesize the three short double-stranded DNA fragments corresponding to the third full-length double-stranded DNA were designed to be approximately 150 bases long and contain a 30-base overlap region (SEQ ID NOS: 37-42). Six single-stranded oligo DNAs (SEQ ID NOs: 43 to 48) were designed to be approximately 150 bases long, including a 30-base overlap region, for synthesis of three short double-stranded DNA fragments corresponding to the fourth full-length double-stranded DNA. Additionally, a forward primer (SEQ ID NO: 49) and a reverse primer (SEQ ID NO: 50) were designed for overlap extension PCR.

[0113] (2) Two-step double-stranded DNA synthesis The 24 single-stranded oligo DNAs required for synthesizing double-stranded DNAs of the four different target sequences were prepared to a concentration of 1 μM in sterile water or TE buffer (Nacalai Tesque).

[0114] Three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared to synthesize the short double-stranded DNA fragments required for the synthesis of the first double-stranded DNA. To the first PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 25) and single-stranded oligo DNA (SEQ ID NO: 26) was added. To the second PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 27) and single-stranded oligo DNA (SEQ ID NO: 28) was added. To the third PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 29) and single-stranded oligo DNA (SEQ ID NO: 30) was added. The total volume was adjusted to 25 μL. To synthesize the first full-length double-stranded DNA, PCR reaction solution B (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase, 0.1 μM forward primer (SEQ ID NO: 49), 0.1 μM reverse primer (SEQ ID NO: 50) was prepared in a total volume of 25 μL.

[0115] To synthesize the short double-stranded DNA fragments required for the synthesis of the second double-stranded DNA, three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared. Then, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 31) and single-stranded oligo DNA (SEQ ID NO: 32) was added to the first PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 33) and single-stranded oligo DNA (SEQ ID NO: 34) was added to the second PCR reaction solution A, and 1 μL each of single-stranded oligo DNA (SEQ ID NO: 35) and single-stranded oligo DNA (SEQ ID NO: 36) was added to the third PCR reaction solution A. The total volume was adjusted to 25 μL. PCR reaction solution B for synthesizing the second full-length double-stranded DNA (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / µL Phusion High-Fidelity DNA polymerase, 0.1 µM forward primer (SEQ ID NO: 49), 0.1 µM reverse primer (SEQ ID NO: 50) was prepared in a total volume of 25 µL.

[0116] To synthesize the short double-stranded DNA fragments required for the synthesis of the third double-stranded DNA, three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared. Then, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 37) and single-stranded oligo DNA (SEQ ID NO: 38) was added to the first PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 39) and single-stranded oligo DNA (SEQ ID NO: 40) was added to the second PCR reaction solution A, and 1 μL each of single-stranded oligo DNA (SEQ ID NO: 41) and single-stranded oligo DNA (SEQ ID NO: 42) was added to the third PCR reaction solution A. The total volume was adjusted to 25 μL. To synthesize the third full-length double-stranded DNA, PCR reaction solution B (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase, 0.1 μM forward primer (SEQ ID NO: 49), 0.1 μM reverse primer (SEQ ID NO: 50) was prepared in a total volume of 25 μL.

[0117] To synthesize the short double-stranded DNA fragments required for the synthesis of the fourth double-stranded DNA, three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared. Then, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 43) and single-stranded oligo DNA (SEQ ID NO: 44) was added to the first PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 45) and single-stranded oligo DNA (SEQ ID NO: 46) was added to the second PCR reaction solution A, and 1 μL each of single-stranded oligo DNA (SEQ ID NO: 47) and single-stranded oligo DNA (SEQ ID NO: 48) was added to the third PCR reaction solution A. The total volume was adjusted to 25 μL. PCR reaction solution B for synthesizing the fourth full-length double-stranded DNA (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / µL Phusion High-Fidelity DNA polymerase, 0.1 µM forward primer (SEQ ID NO: 49), 0.1 µM reverse primer (SEQ ID NO: 50) was prepared in a total volume of 25 µL.

[0118] Each short double-stranded DNA fragment was synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0119] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0120] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0121] Furthermore, a full-length double-stranded DNA was synthesized by multi-stage ligation of three short double-stranded DNA fragments using an overlap extension PCR reaction in a TaKaRa thermal cycler, where the PCR reaction was carried out at 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0122] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0123] Then, it was treated at 72°C for 3 minutes.

[0124] Thereafter, a portion of the DNA amplification product of the double-stranded DNA synthesis was subjected to electrophoresis on a 1% agarose gel using an agarose gel electrophoresis apparatus (Cosmo Bio). The results are shown in Figure 5.

[0125] As shown in Figure 5, the synthesis method of the present invention was able to simultaneously obtain DNA amplification products of double-stranded DNA fragments of four target DNA sequences. This result is due to the fact that the Tm value can be compensated for by providing a temperature holding time for each of the multiple temperature gradients in the PCR reaction conditions. In other words, compared to conventional synthesis methods, the synthesis method of the present invention can simultaneously obtain multiple double-stranded DNA fragments of target DNA sequences without the need for sequence design.

[0126] The synthesis method of the present invention can obtain double-stranded DNA fragments even from complex DNA sequences such as long repetitive sequences, consecutive identical base sequences, AT-rich sequences, and GC-rich sequences, so there is no particular limitation on the complexity of the target DNA sequences to be simultaneously synthesized. The synthesis method of the present invention can simultaneously synthesize a large number of double-stranded DNA fragments, in the order of several tens to several hundreds, resulting in extremely high throughput. Furthermore, by applying the synthesis method of the present invention to the program of a liquid dispensing robot, the simultaneous synthesis of multiple double-stranded DNA fragments can also be automated.

[0127] The DNA amplification products of multiple double-stranded DNA fragments obtained by the synthesis method of the present invention were able to significantly reduce the appearance of non-specific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence). Therefore, the DNA amplification products of the double-stranded DNA fragments obtained in this manner can be subjected to DNA cloning methods such as TA cloning and blunt-end cloning without undergoing steps such as gel excision and purification to isolate only the desired double-stranded DNA fragments, and cloned DNA containing the desired double-stranded DNA fragments can be quickly obtained.

[0128] The method for synthesizing long-chain DNA accumulates double-stranded DNA fragments by using a large number of double-stranded DNA fragments obtained by the method for synthesizing double-stranded DNA fragments as accumulation materials. Therefore, if even one of the double-stranded DNA fragments is missing, it becomes impossible to synthesize long-chain DNA. However, conventional synthesis methods have the disadvantage of requiring time and effort to synthesize a large number of double-stranded DNA fragments. Furthermore, depending on the complexity of the target DNA sequence, synthesis may be difficult or impossible, making it difficult to quickly obtain all of the double-stranded DNA fragments that serve as accumulation materials. In other words, conventional methods for synthesizing double-stranded DNA fragments are unable to quickly supply double-stranded DNA fragments that serve as accumulation materials for long-chain DNA synthesis, creating a bottleneck. In contrast, the synthesis method of the present invention can simultaneously synthesize multiple double-stranded DNA fragments regardless of the complexity of the target DNA sequence to be synthesized, thereby enabling the rapid synthesis of a large number of double-stranded DNA fragments that serve as accumulation materials for long-chain DNA synthesis. In other words, the synthesis method of the present invention can rapidly supply double-stranded DNA fragments necessary for synthesizing long-chain DNA, thereby significantly improving the throughput of the method for synthesizing long-chain DNA.

[0129] (3) Analysis of chemically synthesized single-stranded oligo DNA used in double-stranded DNA synthesis To analyze the chemically synthesized single-stranded oligo DNA used in the double-stranded DNA synthesis of the present invention, the single-stranded oligo DNA was subjected to gel electrophoresis. Electrophoresis of the single-stranded oligo DNA was performed using an XCell SureLock MiniCell Electrophoresis System (Thermo Fisher Scientific) and 10% Novex TBE-Urea Gel (Thermo Fisher Scientific) according to the attached instructions. After electrophoresis, the single-stranded oligo DNA was stained with SYBR Green II nucleic acid fluorescent staining reagent (TaKaRa) according to the attached instructions. Gel analysis of the single-stranded oligo DNA was performed using a gel imaging analyzer, Gel Doc EZ System (Bio-Rad), according to the attached instructions. The results are shown in Figure 6.

[0130] As shown in Figure 6, the content of single-stranded oligo DNA used in the synthesis method of the present invention was 60% to 15% for full-length single-stranded oligo DNA, while the content of incomplete single-stranded oligo DNA was 40% to 85%. Incomplete single-stranded oligo DNA is a reaction intermediate product such as an unreacted substance generated during the chemical synthesis of single-stranded oligo DNA.

[0131] In the synthesis of double-stranded DNA fragments by the synthesis method of the present invention, single-stranded oligo DNA containing 85% incomplete single-stranded oligo DNA (full-length single-stranded oligo DNA of 15% or more) was used. Therefore, even when using very low-quality single-stranded oligo DNA containing approximately 85% incomplete single-stranded oligo DNA, the synthesis method of the present invention was able to obtain a DNA amplification product of the desired double-stranded DNA fragment. Because the DNA amplification product of the obtained double-stranded DNA fragment suppresses the appearance of nonspecific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence), DNA cloning methods such as TA cloning and blunt-end cloning can be applied without the need for gel excision and purification steps to isolate only the desired double-stranded DNA fragment, allowing for the rapid isolation of cloned DNA containing the desired double-stranded DNA fragment.

[0132] (Example 5) Cloning and sequence analysis of double-stranded DNA fragments obtained by the synthesis method of the present invention (1) DNA ligation and transformation of the DNA amplification products of double-stranded DNA fragments obtained by the synthesis method of the present invention To clone the DNA amplification product of the double-stranded DNA synthesized in Example 4, a dA overhang was added to the 3'-end of the DNA amplification product using 10x A-attachment Mix (TOYOBO). The DNA amplification product was then purified using a MinElute PCR Purification kit (QIAgen). The purified synthetic DNA was then mixed with T-Vector pMD19 (Simple) (TaKaRa), and a DNA Ligation kit (TaKaRa) was added at a 1:1 ratio to the DNA mixture. The DNA ligation reaction solution was incubated at 16°C in an incubator (TAITEC) for 1 hour to overnight. After the DNA ligation reaction, E. coli JM109 competent cells (TaKaRa) were transformed according to the manufacturer's instructions. The cells were then spread on LB agar medium containing 100 μg / mL kanamycin (Nacalai Tesque) and cultured overnight at 37°C.

[0133] (2) DNA sequencing analysis DNA extracts were prepared from E. coli colonies using Cica Genesis DNA Extraction Reagent (Kanto Chemical) according to the attached instructions. The DNA extract was used as a template for PCR using TaKaRa Ex-Taq Hot Start (TaKaRa) with M13 forward primer (SEQ ID NO: 51) and M13 reverse primer (SEQ ID NO: 52) according to the attached instructions. The target DNA sequence was amplified using the DNA extract as a template for PCR using TaKaRa Ex-Taq Hot Start (TaKaRa) with M13 forward primer (SEQ ID NO: 51) and M13 reverse primer (SEQ ID NO: 52) according to the attached instructions. The PCR reaction was performed at 95°C for 2 minutes, followed by 30 cycles of the following temperature cycles: 95°C for 20 seconds; 58°C for 30 seconds; and 72°C for 60 seconds. A portion of the resulting PCR product solution was electrophoresed on a 1% agarose gel (Thermo Fisher Scientific). The remaining PCR product solution was then purified using the MinElute PCR Purification kit (Qiagen) according to the attached instructions.

[0134] The purified DNA product obtained above was sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) according to the attached instructions, using the M13 forward primer (SEQ ID NO: 51) and the M13 reverse primer (SEQ ID NO: 52). The DNA sequencing reaction conditions were 95°C for 2 minutes, followed by 30 cycles of the following temperature cycles: 95°C for 5 seconds; 50°C for 10 seconds; and 60°C for 2 minutes and 30 seconds. The resulting DNA sequencing reaction product was purified using the BigDye Terminator Purification kit (Thermo Fisher Scientific) according to the attached instructions. The target DNA sequence was then analyzed using an Applied Biosystems 3500xL Genetic analyzer (Thermo Fisher Scientific) according to the attached instructions.

[0135] As a result of DNA sequence analysis, it was possible to obtain a cloned DNA with the exact sequence of the synthetic double-stranded DNA obtained by the synthesis method of the present invention.

[0136] (Example 6) Comparison of double-stranded DNA fragments obtained by conventional synthesis methods and the synthesis method of the present invention - 2 (Comparison of PCR reaction conditions for the double-stranded DNA synthesis method described in U.S. Patent Application Publication US20080182296A1 with the double-stranded DNA synthesis method of the present invention) (1) Sequence design of single-stranded oligo DNA The desired full-length double-stranded DNA sequence was divided into three short double-stranded DNA fragments. These short double-stranded DNA fragments were designed to contain a 30-base-pair overlap region at the 5'- or 3'-end. The six single-stranded oligo DNAs used to synthesize these three short double-stranded DNA fragments were designed to be approximately 150 bases long, including a 30-base overlap region (SEQ ID NOs: 53 to 58). Additionally, a forward primer (SEQ ID NO: 59) and a reverse primer (SEQ ID NO: 60) were designed for the overlap extension PCR reaction.

[0137] (2) Two-step double-stranded DNA synthesis The six single-stranded oligo DNAs required for double-stranded DNA synthesis were prepared at a concentration of 1 μM in sterile water or TE buffer (Nacalai Tesque). Three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared to synthesize short double-stranded DNA fragments. To the first PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 53) and single-stranded oligo DNA (SEQ ID NO: 54) was added. To the second PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 55) and single-stranded oligo DNA (SEQ ID NO: 56) was added. To the third PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 57) and single-stranded oligo DNA (SEQ ID NO: 58) was added. The total volume was adjusted to 25 μL. PCR reaction solution B for synthesizing full-length double-stranded DNA (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / µL Phusion High-Fidelity DNA polymerase, 0.1 µM forward primer (SEQ ID NO: 59), 0.1 µM reverse primer (SEQ ID NO: 60) was prepared in a total volume of 25 µL.

[0138] (i) Conventional synthesis method (method described in U.S. Patent Application Publication US20080182296A1; PCR-directed gene synthesis from a large number of overlapping oligodeoxyribonucleotides); Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler. The PCR reaction was performed at 95°C for 4 minutes, followed by 10 cycles of the following temperature cycles:

[0139] 98℃, 30 seconds; 50℃, 30 seconds; 72℃, 30 seconds

[0140] Thereafter, the mixture was treated at 72°C for 5 minutes, and 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to the PCR reaction solution B while maintaining the temperature at 72°C.

[0141] Next, a full-length double-stranded DNA was synthesized by multi-stage ligation of three short double-stranded DNA fragments using overlap extension PCR in a TaKaRa thermal cycler. The PCR reaction was performed at 95°C for 4 minutes, followed by 20 cycles of the following temperature cycles:

[0142] 98℃, 30 seconds; 50℃, 30 seconds; 72℃, 30 seconds

[0143] Then, the mixture was treated at 72°C for 5 minutes.

[0144] (ii) The synthesis method of the present invention Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0145] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0146] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0147] Next, a full-length double-stranded DNA was synthesized by multi-stage ligation of three short double-stranded DNA fragments using overlap extension PCR in a TaKaRa thermal cycler. The PCR reaction was performed at 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0148] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0149] Then, it was treated at 72°C for 3 minutes.

[0150] A portion of the DNA amplification products of the double-stranded DNA fragments obtained by PCR reaction conditions (i) of the conventional synthesis method and (ii) of the synthesis method of the present invention was electrophoresed on a 1% agarose gel using an agarose gel electrophoresis apparatus (Cosmo Bio). The results are shown in Figure 7.

[0151] As shown in Figure 7, under the PCR reaction conditions of the conventional synthesis method, a DNA amplification product of a double-stranded DNA fragment with the length of the target DNA sequence was not obtained. In contrast, the synthesis method of the present invention was able to obtain a DNA amplification product of a double-stranded DNA fragment with the length of the target DNA sequence. Since the DNA amplification product of the double-stranded DNA fragment obtained in this case can suppress the appearance of non-specific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence), DNA cloning methods such as TA cloning and blunt-end cloning can be applied without going through steps such as gel excision and purification to isolate only the target double-stranded DNA fragment, and cloned DNA containing the target double-stranded DNA fragment can be quickly obtained.

[0152] Even if it is difficult to synthesize a target DNA sequence under PCR reaction conditions of a conventional synthesis method (the method described in U.S. Patent Application Publication US20080182296A1), a double-stranded DNA fragment of the target DNA sequence can be obtained by applying the synthesis method of the present invention. Therefore, the synthesis method of the present invention is superior to conventional synthesis methods because it can synthesize a double-stranded DNA fragment of the target DNA sequence regardless of the complexity of the target DNA sequence.

[0153] (Example 7) Comparison of double-stranded DNA fragments obtained by conventional synthesis methods and the synthesis method of the present invention - 3 (Comparison between the two-step double-stranded DNA synthesis method in which all single-stranded oligo DNAs are assembled in the first-step PCR reaction described in Non-Patent Document 6 and the double-stranded DNA synthesis method of the present invention) (1) Sequence design of single-stranded oligo DNA The desired full-length double-stranded DNA sequence was divided into three short double-stranded DNA fragments. These short double-stranded DNA fragments were designed to contain a 30-base-pair overlap region at the 5'- or 3'-end. The six single-stranded oligo DNAs used to synthesize these three short double-stranded DNA fragments were designed to be approximately 150 bases long, including a 30-base overlap region (SEQ ID NOs: 61 to 66). Additionally, a forward primer (SEQ ID NO: 67) and a reverse primer (SEQ ID NO: 68) were designed for the overlap extension PCR reaction.

[0154] (2) Double-stranded DNA synthesis Six single-stranded oligo DNAs required for double-stranded DNA synthesis were prepared to a concentration of 1 μM in sterile water or TE buffer (Nacalai Tesque).

[0155] (i) Conventional synthesis method (a two-step double-stranded DNA synthesis method in which all single-stranded oligo DNAs are assembled in the first PCR reaction; Non-Patent Document 6) In the first step, to assemble all the single-stranded oligo DNAs, one PCR reaction solution A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase) was prepared, and 1 μL of each single-stranded oligo DNA (SEQ ID NOs: 61 to 66) was added. This was adjusted to a total volume of 25 μL. Next, PCR reaction solution B (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase, 0.1 μM forward primer (SEQ ID NO: 67), 0.1 μM reverse primer (SEQ ID NO: 68) was prepared to synthesize full-length double-stranded DNA. This was adjusted to a total volume of 25 μL.

[0156] All single-stranded oligo DNAs were assembled by the first PCR reaction using a thermal cycler manufactured by TaKaRa Co. The PCR reaction was carried out at 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0157] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0158] Thereafter, 1 μL of PCR reaction solution A containing the assembled single-stranded oligo DNA was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0159] Using a TaKaRa thermal cycler, full-length double-stranded DNA was synthesized from the single-stranded oligo DNA assembled in the second PCR reaction. The PCR reaction temperature conditions were 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0160] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0161] Then, it was treated at 72°C for 3 minutes.

[0162] (ii) The synthesis method of the present invention Three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared to synthesize short double-stranded DNA fragments. To the first PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 61) and single-stranded oligo DNA (SEQ ID NO: 62) was added. To the second PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 63) and single-stranded oligo DNA (SEQ ID NO: 64) was added. To the third PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 65) and single-stranded oligo DNA (SEQ ID NO: 66) was added. The total volume was adjusted to 25 μL. PCR reaction solution B for synthesizing full-length double-stranded DNA (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / µL Phusion High-Fidelity DNA polymerase, 0.1 µM forward primer (SEQ ID NO: 67), 0.1 µM reverse primer (SEQ ID NO: 68) was prepared in a total volume of 25 µL.

[0163] Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0164] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0165] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0166] A full-length double-stranded DNA was synthesized by multi-stage overlap extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature set at 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0167] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0168] Then, it was treated at 72°C for 3 minutes.

[0169] Portions of the DNA amplification products of the double-stranded DNA fragments obtained by the conventional synthesis method (i) and the synthesis method of the present invention (ii) were electrophoresed on a 1% agarose gel using an agarose gel electrophoresis apparatus (Cosmo Bio). The results are shown in Figure 8.

[0170] As shown in Figure 8, the conventional synthesis method did not produce a DNA amplification product of a double-stranded DNA fragment of the desired DNA sequence length. On the other hand, the synthesis method of the present invention was able to obtain a DNA amplification product of a double-stranded DNA fragment of the desired DNA sequence. As a result, it can be said that the synthesis method of the present invention was able to accurately synthesize a short double-stranded DNA by applying a primer extension PCR reaction in the first step of the synthesis method, and therefore was able to synthesize a double-stranded DNA fragment of the desired DNA sequence.

[0171] The DNA amplification product of the double-stranded DNA fragment obtained in this way can suppress the appearance of non-specific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence). Therefore, DNA cloning methods such as TA cloning and blunt-end cloning can be applied without going through steps such as gel excision and purification to extract only the target double-stranded DNA fragment, and cloned DNA containing the target double-stranded DNA fragment can be quickly obtained.

[0172] Even if the target DNA sequence is difficult to synthesize using conventional synthesis methods, the synthesis method of the present invention can be used to obtain a double-stranded DNA fragment of the target DNA sequence. Therefore, the synthesis method of the present invention is superior to conventional synthesis methods because it can synthesize a double-stranded DNA fragment of the target DNA sequence regardless of the complexity of the target DNA sequence.

[0173] (Example 8) Comparison of double-stranded DNA fragments obtained by conventional synthesis methods and the synthesis method of the present invention - 4 (Comparison of the single-step double-stranded DNA synthesis method described in Non-Patent Document 9 with the double-stranded DNA synthesis method of the present invention) (1) Sequence design of single-stranded oligo DNA The desired full-length double-stranded DNA sequence was divided into three short double-stranded DNA fragments. These short double-stranded DNA fragments were designed to contain a 30-base-pair overlap region at the 5'- or 3'-end. The six single-stranded oligo DNAs used to synthesize these three short double-stranded DNA fragments were designed to be approximately 150 bases long, including a 30-base overlap region (SEQ ID NOs: 69 to 74). Additionally, a forward primer (SEQ ID NO: 75) and a reverse primer (SEQ ID NO: 76) were designed for the overlap extension PCR reaction.

[0174] (2) Double-stranded DNA synthesis Six single-stranded oligo DNAs required for double-stranded DNA synthesis were prepared to a concentration of 1 μM using sterile water or TE buffer (Nacalai Tesque). (i) Conventional synthesis method (one-step double-stranded DNA synthesis method; Non-Patent Document 9) To synthesize double-stranded DNA from all single-stranded oligo DNAs, one PCR reaction solution A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase, 0.1 μM forward primer (SEQ ID NO: 75), 0.1 μM reverse primer (SEQ ID NO: 76) was prepared, and 1 μL of each single-stranded oligo DNA (SEQ ID NOs: 69 to 74) was added to the reaction solution. The total volume was adjusted to 25 μL.

[0175] Single-stranded oligo DNA was assembled by PCR using a TaKaRa thermal cycler to synthesize full-length double-stranded DNA. The PCR reaction was performed at 98°C for 2 minutes, followed by 30 cycles of the following temperature cycles:

[0176] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0177] Then, it was treated at 72°C for 3 minutes.

[0178] (ii) The synthesis method of the present invention Three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTPs, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared to synthesize short double-stranded DNA fragments. To the first PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 69) and single-stranded oligo DNA (SEQ ID NO: 70) was added. To the second PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 71) and single-stranded oligo DNA (SEQ ID NO: 72) was added. To the third PCR reaction solution A, 1 μL each of single-stranded oligo DNA (SEQ ID NO: 73) and single-stranded oligo DNA (SEQ ID NO: 74) was added. The total volume was adjusted to 25 μL. PCR reaction solution B for synthesizing full-length double-stranded DNA (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / µL Phusion High-Fidelity DNA polymerase, 0.1 µM forward primer (SEQ ID NO: 75), 0.1 µM reverse primer (SEQ ID NO: 76) was prepared in a total volume of 25 µL.

[0179] Three short double-stranded DNA fragments were synthesized by primer extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature conditions being 98°C for 2 minutes, followed by 10 cycles of the following temperature cycles:

[0180] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0181] Then, 1 μL of each of the PCR reaction solutions A containing the three short double-stranded DNAs was added to PCR reaction solution B while maintaining the temperature at 72°C.

[0182] A full-length double-stranded DNA was synthesized by multi-stage overlap extension PCR using a TaKaRa thermal cycler, with the PCR reaction temperature set at 98°C for 2 minutes, followed by 20 cycles of the following temperature cycles:

[0183] 98℃, 30 seconds; 77.5℃, 50 seconds; 75℃, 50 seconds; 72.5℃, 50 seconds; 70℃, 50 seconds; 67.5℃, 50 seconds; 65℃, 50 seconds; 62.5℃, 50 seconds; 72℃, 50 seconds

[0184] Then, it was treated at 72°C for 3 minutes.

[0185] Portions of the DNA amplification products of the double-stranded DNA fragments obtained by the conventional synthesis method (i) and the synthesis method of the present invention (ii) were electrophoresed on a 1% agarose gel using an agarose gel electrophoresis apparatus (Cosmo Bio). The results are shown in Figure 9.

[0186] As shown in Figure 9, the conventional synthesis method did not produce a DNA amplification product of a double-stranded DNA fragment of the length of the target DNA sequence. On the other hand, the synthesis method of the present invention was able to obtain a DNA amplification product of a double-stranded DNA fragment of the target DNA sequence. As a result, it can be said that the synthesis method of the present invention was able to synthesize a double-stranded DNA fragment of the target DNA sequence by dividing the process into a primer extension PCR reaction and a second overlap extension PCR reaction.

[0187] The DNA amplification product of the double-stranded DNA fragment obtained in this way can suppress the appearance of non-specific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence). Therefore, DNA cloning methods such as TA cloning and blunt-end cloning can be applied without going through steps such as gel excision and purification to extract only the target double-stranded DNA fragment, and cloned DNA containing the target double-stranded DNA fragment can be quickly obtained.

[0188] The DNA sequence synthesized in this example is generally considered to be a complex sequence, containing long repetitive sequences, consecutive identical base sequences, and AT-rich regions. Therefore, it was found that the two-step double-stranded DNA synthesis method of the present invention can accurately synthesize double-stranded DNA of the target DNA sequence even from the above complex DNA sequence.

[0189] Even if the target DNA sequence is difficult or impossible to synthesize by conventional synthesis methods, the synthesis method of the present invention can be used to obtain a double-stranded DNA fragment of the target DNA sequence. Therefore, the synthesis method of the present invention is superior to conventional synthesis methods because it can synthesize a double-stranded DNA fragment of the target DNA sequence regardless of the complexity of the target DNA sequence. [Industrial Applicability]

[0190] According to the present invention, by setting multiple annealing temperatures in the PCR cycle, it is possible to combine multiple DNA fragments with various Tm values, thereby enabling easy, accurate, efficient, and rapid assembly of single-stranded oligo DNA fragments containing numerous homologous regions. Furthermore, because the method for synthesizing double-stranded DNA of the present invention is applicable to combining DNA fragments with various Tm values, it is unnecessary to optimize the sequence of the single-stranded oligo DNA fragments used as raw materials, and it is not necessary to carefully adjust the temperature setting for each target double-stranded DNA fragment. Furthermore, according to the present invention, it is possible to easily, accurately, efficiently, and rapidly synthesize a double-stranded DNA fragment of interest regardless of its sequence, thereby enabling the synthesis of double-stranded DNA fragments that have previously been difficult or impossible to synthesize due to their complex sequences (e.g., long repetitive sequences, consecutive identical base sequences, AT-rich or GC-rich sequences, etc.).

Claims

[Claim 1] The invention as shown in the drawings.

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