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 Art

[0002] DNA synthesis for constructing long-chain DNA having a novel sequence in the field of genetic engineering is carried out for various purposes. The method for synthesizing this long-chain DNA generally consists of two major steps. The first step is a step of synthesizing double-stranded DNA fragments from a large number of single-stranded oligo DNAs. The second step is a step of constructing long-chain DNA by integrating a large number of double-stranded DNA fragments obtained in the first step. As methods for constructing this long-chain DNA, a yeast integration method (see Non-Patent Document 1), a Gibson assembly method (see Non-Patent Document 2), a Golden gate method (see Non-Patent Document 3), an LCR method (see Non-Patent Document 4), an OGAB method (see Non-Patent Document 5), etc. are known.

[0003] As a method for synthesizing the above double-stranded DNA fragments, for example, there are several known methods for synthesizing double-stranded DNA fragments by simultaneously bonding and assembling single-stranded oligo DNAs containing a large number of homologous regions in a PCR reaction solution (see Patent Document 1, Non-Patent Documents 6 to 8). In these methods, since a large number of single-stranded oligo DNAs are mixed in one PCR reaction solution, when these single-stranded oligo DNAs are bonded by a PCR reaction, an inconvenient situation such as mismatches between single-stranded oligo DNAs and the formation of primer dimers occurs. For the above reasons, in these synthesis methods, it is not always possible to synthesize double-stranded DNA fragments for any DNA sequence to be synthesized. In particular, when the target DNA sequence to be synthesized is a complex DNA sequence containing a long repetitive sequence, a continuous identical base sequence, etc., synthesis may be difficult or impossible.

[0004] In addition, in these synthesis methods, in order to synthesize double-stranded DNA having the target DNA sequence, single-stranded oligo DNA serving as the starting material must be designed. When designing these single-stranded oligo DNAs, the length of the homologous region for sticking together a large number of single-stranded oligo DNAs must be adjusted for setting the annealing temperature of the PCR reaction. In addition, in order to prevent hairpin formation and the like of single-stranded oligo DNA in the synthesis process, it is necessary to adjust the length, number, and GC content composition of each single-stranded oligo DNA. That is, in the conventional synthesis methods, the design of single-stranded oligo DNA for the target DNA sequence to be synthesized is complicated, and even when attempting to synthesize a large number of double-stranded DNA fragments in units of several to several hundreds, single-stranded oligo DNA must be designed for each of those DNA sequences, resulting in the disadvantage that it takes time and effort to design single-stranded oligo DNA.

[0005] In the synthesis of long-chain DNA, which is the second step, a large number of double-stranded DNA fragments obtained in the first step are used as the assembly material. When assembling double-stranded DNA fragments in the synthesis of long-chain DNA in the second step, if even one of the double-stranded DNA fragments is missing, it becomes impossible to synthesize the target long-chain DNA. However, in the conventional methods for synthesizing double-stranded DNA fragments, there is the disadvantage that it takes time and effort to synthesize a large number of double-stranded DNA fragments in units of several to several hundreds, and depending on the complexity of the target DNA sequence to be synthesized, synthesis is difficult or impossible, so it is difficult to quickly obtain all the double-stranded DNA fragments serving as the assembly material. That is, in the conventional methods for synthesizing double-stranded DNA fragments, it is impossible to quickly supply the double-stranded DNA fragments serving as the assembly material for the synthesis of long-chain DNA, and the actual situation is that it has become a bottleneck. Therefore, there has been a strong demand for a new method that can efficiently and rapidly synthesize double-stranded DNA fragments to replace the conventional synthesis methods.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Document

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0008] Under such circumstances, the inventors of the present invention aimed to develop a method that can accurately, easily, efficiently, and rapidly synthesize double-stranded DNA fragments that can be used as integrated materials for long-chain DNA synthesis to solve the above-mentioned disadvantages. That is, the present invention aims to develop a method for synthesizing double-stranded DNA fragments using the PCR method, which can accurately, easily, efficiently, and rapidly synthesize the target double-stranded DNA fragments regardless of their sequences.

Means for Solving the Problems

[0009] As a result of intensive research to solve the above problems, the inventors of the present invention have found that in a method for synthesizing double-stranded DNA in which short double-stranded DNA is ligated by overlap extension PCR to obtain a target double-stranded DNA fragment, by setting the annealing temperature of the PCR cycle in multiple stages, the target double-stranded DNA fragment can be accurately, easily, efficiently, and rapidly synthesized, and thus the present invention has been completed. 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 DNA is ligated by overlap extension PCR to obtain a target double-stranded DNA fragment, characterized in that the PCR cycle of the overlap extension PCR has multiple-stage annealing temperature settings. [2] The method for synthesizing double-stranded DNA according to [1], wherein the annealing temperature setting is from 2 to 20 stages. [3] The method for synthesizing double-stranded DNA according to [1] or [2], wherein the annealing temperature setting is from 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 from 10 seconds to 2 minutes. [5] The method for synthesizing double-stranded DNA according to any one of [1] to [4], wherein the overlapping region of DNA in the overlap extension PCR is 10 bases to 40 bases. [6] The method for synthesizing double-stranded DNA according to any one of [1] to [5], wherein 3 to 20 short double-stranded DNAs 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-stage annealing temperature settings. [9] The method for synthesizing double-stranded DNA according to [7] or [8], wherein the size of the short double-stranded DNA synthesized by the primer extension PCR is 100 bases to 300 bases.

[10] A method for synthesizing double-stranded DNA, A primer extension PCR step of synthesizing a short double-stranded DNA by primer extension PCR, and An overlap extension PCR step of synthesizing a target double-stranded DNA fragment by ligating the double-stranded DNA synthesized in the primer extension PCR step by overlap extension PCR comprising, and having multiple-stage annealing temperature settings in the PCR cycle of the overlap extension PCR, a method for synthesizing double-stranded DNA.

Effect of the Invention

[0011] According to the present invention, by having a multi-stage annealing temperature setting in the PCR cycle, it becomes applicable to the ligation of a plurality of DNAs having various Tm values. Therefore, the assembly of single-stranded oligo DNAs containing a large number of homologous regions can be performed easily, accurately, efficiently, and rapidly. Further, since the double-stranded DNA synthesis method of the present invention is applicable to the ligation of DNAs having various Tm values, there is no need to optimize the single-stranded oligo DNA sequence as a material, and it is not necessary to finely re-set the temperature for each target double-stranded DNA fragment. Furthermore, according to the present invention, regardless of the sequence of the target double-stranded DNA fragment, it is possible to synthesize easily, accurately, efficiently, and rapidly. Thus, double-stranded DNA fragments that were difficult or impossible to synthesize due to conventional complex sequences (for example, long repetitive sequences, continuous identical base sequences, AT-rich, GC-rich sequences, etc.) can also be synthesized.

Brief Description of Drawings

[0012]

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Embodiments for Carrying Out the Invention

[0013] Hereinafter, the double-stranded DNA synthesis method of the present invention will be described in detail. In this specification, molecular biological techniques can be carried out by the methods described in general experimental manuals known to those skilled in the art or methods equivalent thereto, unless otherwise specified. Also, the terms used in this specification are interpreted in the meanings commonly used in the technical field, unless otherwise mentioned.

[0014] <Double-stranded DNA Synthesis Method> The double-stranded DNA synthesis method of the present invention is a method for obtaining a target double-stranded DNA fragment by ligating short double-stranded DNAs by overlap extension PCR, and is characterized in that, in the PCR cycle of the overlap extension PCR, it has annealing temperature settings in multiple stages. By having annealing temperature settings in multiple stages in the PCR cycle in this way, the double-stranded DNA synthesis method of the present invention can be applied to the ligation of multiple DNAs having various Tm values, so that the assembly of single-stranded oligo DNAs containing a large number of homologous regions can be carried out easily, accurately, efficiently, and rapidly. Further, since the double-stranded DNA synthesis method of the present invention can be applied to the ligation of DNAs having various Tm values, there is no need to optimize the sequence of the single-stranded oligo DNA used as a material, and it is not necessary to re-set the temperature finely for each target double-stranded DNA fragment. Furthermore, according to the present invention, it is possible to synthesize easily, accurately, efficiently, and rapidly regardless of the sequence of the target double-stranded DNA fragment, so that double-stranded DNA fragments that were difficult or impossible to synthesize due to being a conventional complex sequence (for example, a long repetitive sequence, a continuous identical base sequence, an AT-rich, GC-rich sequence, etc.) can also be synthesized.

[0015] In addition, the short double-stranded DNA used in the above overlap extension PCR is preferably synthesized by primer extension PCR. Therefore, the present invention includes a primer extension PCR step of synthesizing short double-stranded DNA by primer extension PCR, and an overlap extension PCR step of ligating the double-stranded DNA synthesized in the primer extension PCR step by overlap extension PCR to synthesize a target double-stranded DNA fragment. It can also be described as a double-stranded DNA synthesis method characterized by having multiple-stage annealing temperature settings in the PCR cycle of the overlap extension PCR. The double-stranded DNA synthesis method of the present invention will be described in detail for each step below. FIG. 1 schematically shows the steps of the double-stranded DNA synthesis method of the present invention.

[0016] [Primer Extension PCR Step] This step is a step of synthesizing short double-stranded DNA by primer extension PCR. The short double-stranded DNA obtained in this step is ligated in the overlap extension PCR step described later and becomes a double-stranded DNA fragment that can be used as an integration material for long-chain DNA synthesis. Therefore, from the sequence of the long double-stranded DNA to be finally synthesized, it is determined what kind of double-stranded DNA fragments are required as integration materials, and the short double-stranded DNA synthesized in this step is the one that is divided into several pieces and designed for the sequence.

[0017] Here, in the present invention, primer extension PCR refers to a reaction of synthesizing double-stranded DNA by bonding a pair of single-stranded oligo DNAs having regions that complementarily bind to each other's terminal portions and extending each strand with a polymerase.

[0018] (i) Sequence Design of Single-Stranded Oligo DNA The full-length sequence of the double-stranded DNA fragment obtained by the double-stranded DNA synthesis method of the present invention is divided into any number of short double-stranded DNAs. The size of this short double-stranded DNA is usually 100 bases to 300 bases, preferably 120 bases to 250 bases, more preferably 140 bases to 180 bases, and even more preferably around 150 bases. The number of short double-stranded DNAs to be divided can be appropriately determined according to the length of the full-length sequence, but is usually 3 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably about 3.

[0019] The above-mentioned divided short double-stranded DNAs are designed to include regions of arbitrary length that overlap with each other at their 5'- or 3'-ends. The length of this overlap region is usually 5 bases to 50 bases, preferably 10 bases to 40 bases, more preferably 15 bases to 40 bases, and even more preferably about 30 bases. The pair of single-stranded oligo DNAs used for the synthesis of the short double-stranded DNAs designed as described above are 60 bases to 300 bases including the above overlap region, preferably 100 bases to 200 bases, and more preferably 150 bases to 200 bases. In this step, by using relatively long single-stranded oligo DNAs, the number of single-stranded oligo DNAs used for double-stranded DNA synthesis can be reduced, and the bias in the GC content ratio in the single-stranded oligo DNA sequence can be eliminated. Therefore, the formation of DNA secondary structures such as hairpin structures can also be prevented. These single-stranded oligo DNAs can be prepared by chemical synthesis.

[0020] These single-stranded oligo DNAs may include single-stranded oligo DNAs of incomplete length generated during the chemical synthesis process, which are different from the full-length single-stranded oligo DNAs. In this step, if the full-length single-stranded oligo DNAs account for 15% or more in the single-stranded oligo DNAs used, a DNA amplification product of the target synthetic double-stranded DNA can be obtained, and a double-stranded DNA with an accurate sequence can be obtained by cloning. That is, even in a state where about 85% of the single-stranded oligo DNAs of incomplete length including unreacted substances are contained in the single-stranded oligo DNAs used, a DNA amplification product of the target synthetic double-stranded DNA can be obtained.

[0021] (ii) PCR cycle Pairs of single-stranded oligo DNAs in the number of sets corresponding to the above-mentioned number of divisions are each separately adjusted to an arbitrary concentration with sterilized water, TE buffer, or the like. The above concentration is usually 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 dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase; NEB or the like) is prepared, and the above pair of single-stranded oligo DNAs are added respectively so that the total amount becomes equal. At this time, a PCR reaction solution is prepared for each synthesis of a short double-stranded DNA, and only two single-stranded oligo DNAs that form a pair are mixed in one PCR reaction solution. Therefore, mismatching between single-stranded oligo DNAs and formation of primer dimers caused during the ligation of single-stranded oligo DNAs can be prevented.

[0022] Next, using a thermal cycler, short double-stranded DNAs are synthesized by primer extension PCR reaction. As the temperature conditions for the PCR reaction at this time, after 98 °C for 2 minutes, a heat denaturation stage / annealing stage / extension stage (extension) is defined as one cycle, and an arbitrary number of cycles are performed. The number of cycles is usually 5 cycles or more, preferably 10 cycles or more. There is no problem even if it exceeds 10 cycles, but about 10 cycles can provide a sufficient amount of synthesis in this step.

[0023] In the above heat denaturation stage and extension stage, conditions similar to those of the conventional PCR method can be selected. For example, in the heat denaturation stage, it is 98°C for 30 seconds, and in the extension stage, the conditions vary depending on the polymerase used. For example, it is 72°C for 50 seconds.

[0024] In the present invention, in the above annealing stage, the fact that it has a multi-stage annealing temperature setting is significantly different from the conventional method. As the annealing temperature setting, it is in the range of 50°C to 90°C, preferably in the range of 60°C to 85°C, and has a temperature setting of 2 to 20 stages, preferably 2 to 15 stages, more preferably 2 to 10 stages, and even more preferably 2 to 8 stages. The holding time of the annealing temperature at each stage is usually 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. There is no problem even if the reaction is carried out for a long time exceeding 50 seconds and it can be reacted up to about 2 minutes, but it can be said that about 50 seconds is sufficient. Incidentally, there is no problem even if it is incubated overnight after the primer extension PCR step is completed. The temperature at that time is preferably in the range of 4°C to 72°C.

[0025] By having a multi-stage annealing temperature setting in the PCR of this step, the difference in Tm values for each combination of pairs of single-stranded oligo DNAs can be complemented, so that PCR for all double-stranded DNA synthesis in this step can be carried out at once.

[0026] [Overlap Extension PCR Step] This step is a step of ligating the short double-stranded DNA synthesized in the above primer extension PCR step by overlap extension PCR to obtain the target double-stranded DNA fragment. This PCR cycle is characterized by having a multi-stage annealing temperature setting.

[0027] Here, overlap extension PCR refers to a reaction in which, when amplifying target DNA in a PCR reaction, by adding another sequence to the 5'-end side of a target DNA-specific primer, a new sequence is added to the PCR product, and this new sequence is designed to be complementary between multiple target DNAs, so that the ends of multiple target DNAs are fused during annealing, and then a single PCR product is synthesized in the subsequent extension reaction by DNA polymerase. In the present invention, it is a reaction performed to ligate a plurality of types of short double-stranded DNAs synthesized in the above primer extension PCR step to create a single double-stranded DNA fragment. The above short double-stranded DNAs have homologous regions at the ligation portions. When they become single-stranded due to heat denaturation, at the annealing stage, the homologous region portions bind complementarily, and the new sequences on the 5'-end sides of each other can be added to the PCR product. Since this new sequence is designed to be complementary to another double-stranded DNA, the ends of the DNA are fused during annealing, and a single PCR product can be synthesized in the subsequent extension reaction by DNA polymerase.

[0028] Specifically, it is carried out as follows. The short double-stranded DNAs synthesized in the above primer extension PCR step are added in equal amounts to a PCR reaction solution (1 x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase; manufactured by NEB or the like) for synthesizing full-length double-stranded DNA. This PCR reaction solution contains primers for amplifying full-length double-stranded DNA.

[0029] Next, using a thermal cycler, a full-length double-stranded DNA fragment is synthesized by PCR reaction. As the temperature conditions for the PCR reaction at this time, after 98°C for 2 minutes, the heat denaturation stage / annealing stage / extension stage (extension) is taken as one cycle, and an arbitrary number of cycles are performed. The number of cycles is usually 10 cycles or more, and preferably 20 cycles or more. There is no problem even if it exceeds 20 cycles, but about 20 cycles can provide a sufficient synthesis amount in this step.

[0030] In the above heat denaturation stage and extension stage, conditions similar to those of the conventional PCR method can be selected. For example, in the heat denaturation stage, it is 98°C for 30 seconds, and in the extension stage, the conditions vary depending on the polymerase used. For example, it is 72°C for 50 seconds.

[0031] In the present invention, in the above annealing stage, having a multi-stage annealing temperature setting is significantly different from the conventional method. As the annealing temperature setting, it is in the range of 50°C to 90°C, preferably in the range of 60°C to 85°C, with 2 to 20 stages, preferably 2 to 15 stages, more preferably 2 to 10 stages, and even more preferably 2 to 8 stages of temperature settings. The holding time of the annealing temperature for each stage is usually 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. There is no problem even if the reaction is carried out for a long time exceeding 50 seconds, and it may be carried out up to about 2 minutes, but it can be said that about 50 seconds is sufficient.

[0032] By having a multi-stage annealing temperature setting in the PCR of this step, it is possible to complement the assembly of short double-stranded DNAs and the difference in Tm values that occurs when bonding each single-stranded DNA together. Therefore, it is not necessary to optimize the Tm value for the homologous region of the single-stranded oligo DNA required for bonding. For this reason, the length of the homologous region of the single-stranded oligo DNA can be designed while fixed at 30 bases, and the design of the single-stranded oligo DNA when synthesizing many double-stranded DNAs with different sequences can be simplified. Also, the respective temperature conditions of the primer extension PCR reaction and the overlap extension PCR reaction can be unified.

[0033] Furthermore, according to the method of the present invention, double-stranded DNA fragments of different sequences can be synthesized simultaneously in parallel. The number of these double-stranded DNA fragments that can be synthesized simultaneously is not particularly limited, but since simultaneous synthesis in units of several tens to several hundreds is also possible, the throughput is extremely high. Also, by applying the synthesis method of the present invention to the program of a liquid dispensing robot, the synthesis of multiple double-stranded DNA fragments can be automated simultaneously.

[0034] The double-stranded DNA fragments that can be synthesized by the synthesis method of the present invention are not particularly limited in terms of sequence or length, but considering the accuracy of the heat-resistant DNA polymerase used in PCR, a length of up to 5,000 base pairs is preferred. Therefore, the number of short double-stranded DNA fragments ligated by the overlap extension PCR reaction 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] Also, according to the synthesis method of the present invention, it is possible to synthesize double-stranded DNA fragments containing DNA sequences generally regarded as complex sequences such as long repetitive sequences, continuous identical base sequences, AT-rich, and GC-rich.

[0036] The double-stranded DNA fragments obtained by the synthesis method of the present invention can be used to construct long-chain DNA by integrating a large number using synthesis methods for long-chain DNA such as the yeast integration 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).

[0037] In the synthesis of long-chain DNA, double-stranded DNA fragments are used as the assembly material. However, if even one of the double-stranded DNA fragments is missing, it becomes impossible to synthesize the target long-chain DNA. Since the synthesis method of the present invention can simultaneously synthesize multiple double-stranded DNA fragments regardless of the complexity of the DNA sequence of the target to be synthesized, it can rapidly synthesize the double-stranded DNA fragments that serve as the assembly material for the synthesis of long-chain DNA. Therefore, the synthesis method of the present invention can rapidly supply the double-stranded DNA fragments required for the synthesis of long-chain DNA, thus significantly improving the throughput of the long-chain DNA synthesis method.

[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 for DNA cloning, and then transformation is carried out by introducing a plasmid using E. coli competent cells. The DNA cloning of the DNA amplification product of the synthetic double-stranded DNA fragment may be any of the restriction enzyme cloning method, TA cloning method, In-Fusion cloning method, blunt-end cloning method, etc., and is not particularly limited. The vector DNA to be used may be any of the vector for restriction enzyme cloning, the vector for In-Fusion cloning, the vector for TA cloning, the vector for blunt-end cloning, etc., and is not particularly limited.

[0039] The DNA amplification product of the double-stranded DNA fragment obtained by the synthesis method of the present invention can suppress the appearance of non-specific DNA amplification products that are shorter than the length of the target sequence generated by the PCR reaction. Therefore, the synthetic double-stranded DNA fragment obtained by the synthesis method of the present invention can apply DNA cloning methods such as the TA cloning method and the blunt-end cloning method without going through steps such as gel excision purification for extracting only the target double-stranded DNA fragment, and can rapidly obtain cloned DNA containing the target double-stranded DNA fragment.

[0040] <Program> The present invention also includes the PCR program in the double-stranded DNA synthesis method of the present invention described above. The program for the PCR conditions of the present invention can be used in an apparatus used for PCR such as a thermal cycler. The program of the present invention defines the temperature conditions and the like of PCR in the double-stranded DNA synthesis method of the present invention, and the detailed content can apply the description in the section of "double-stranded DNA synthesis method".

[0041] <Apparatus> The present invention also includes an apparatus capable of realizing the double-stranded DNA synthesis method of the present invention described above. Further, the apparatus of the present invention is an apparatus in which the program of the present invention is incorporated. Specifically, it is an apparatus used for PCR such as a thermal cycler in which the program of the present invention is incorporated. The above program defines the temperature conditions and the like of PCR in the DNA synthesis method of the present invention, and the detailed content can apply the description in the section of "double-stranded DNA synthesis method".

[0042] <Double-stranded DNA automatic synthesis system> The present invention also includes a double-stranded DNA automatic synthesis system characterized by using the double-stranded DNA synthesis method of the present invention described above. In the double-stranded DNA synthesis method of the present invention described above, PCR can be performed under the same conditions regardless of the sequence of the target DNA, and a high-throughput synthesis system using a liquid dispensing robot or the like, or an automatic synthesis system capable of automatically performing a series of these steps becomes possible. Such a double-stranded DNA automatic synthesis system of the present invention may use the program of the present invention and the apparatus of the present invention described above.

Example

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

[0044] The reagents and the like used in the examples, and the common test methods and the like are as follows. Single-stranded oligo DNA used as a material for double-stranded DNA synthesis was purchased from Nippon Techno Service Co., Ltd. and Fasmac Co., Ltd. DNA amplification by PCR reaction used for double-stranded DNA synthesis was carried out using NEB's Phusion High-Fidelity DNA polymerase according to the attached instructions. For DNA cloning of the DNA amplification product, Toyobo's 10x A-attachment Mix, TaKaRa's T-Vector pMD19(Simple), and DNA Ligation kit were used. Escherichia coli competent cells were TaKaRa's Escherichia coli JM109 competent cells, and the transformation method using the plasmid vector was carried out according to the attached instructions. The medium components and agar of the LB medium were purchased from Becton Dickinson and Company (Bacto TM Tryptone, Bacto TM Yeast Extract, Bacto TMAgar). Antibiotics of ampicillin and carbenicillin were purchased from Nacalai Tesque. Preparation of the template for PCR reaction from E. coli colonies was performed using the Shikagene DNA extraction reagent manufactured by Kanto Chemical Co., Inc. according to the attached instructions. DNA amplification by colony direct PCR reaction from E. coli colonies was performed using TaKaRa Ex-Taq Hot Start manufactured by TaKaRa according to the attached instructions. The temperature conditions for the colony direct PCR reaction were as follows. After 95°C for 2 minutes, the following temperature cycle: 95°C for 20 seconds; 58°C for 30 seconds; 72°C for 1 minute per 1 kb of amplification length; was performed for 30 cycles. DNA purification was performed using the MinElute PCR Purification kit manufactured by Qiagen according to the attached instructions. The DNA sequencing reaction was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit manufactured by Thermo Fisher Scientific according to the attached instructions. The temperature conditions for the DNA sequencing reaction were as follows. After 95°C for 2 minutes, the following temperature cycle: 95°C for 5 seconds; 50°C for 10 seconds; 60°C for 2 minutes and 30 seconds; was performed for 30 cycles. Purification of the DNA sequencing reaction products was performed using the BigDye Terminator Purification kit manufactured by Thermo Fisher Scientific according to the attached instructions. DNA sequencing was performed using the Applied Biosystems 3500xL Genetic analyzer manufactured by Thermo Fisher Scientific according to the attached instructions. Agarose gel electrophoresis of the PCR reaction products was performed using the agarose gel electrophoresis apparatus (i-MyRun.NC) manufactured by Cosmo Bio Co., Ltd. according to the attached instructions. DNA staining after electrophoresis was performed using the GelRed nucleic acid fluorescent staining reagent manufactured by Biotium according to the attached instructions. Electrophoresis of single-stranded oligo DNA was performed using the XCell SureLock mini-cell electrophoresis apparatus and 10% Novex TBE-Urea gel manufactured by Thermo Fisher Scientific according to the attached instructions. Staining of single-stranded oligo DNA after electrophoresis was performed using the 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 gel imaging analysis device Gel Doc EZ System manufactured by Bio-Rad Laboratories, Inc. according to the attached instruction manual. As for all other biochemical reagents, those manufactured by Thermo Fisher Scientific and Nacalai Tesque were used.

[0045] (Example 1) Examination of PCR reaction conditions in the synthesis method of the present invention (examination of temperature holding time for each multi-step temperature gradient) (1) Sequence design of single-stranded oligo DNA The sequence of the target full-length double-stranded DNA was divided into three short double-stranded DNA fragments. Those short double-stranded DNA fragments were designed to contain a 30-base pair overlap region at the 5'- or 3'-end. That is, the six single-stranded oligo DNAs used for the synthesis of the three short double-stranded DNA fragments were designed to be about 150 bases in length including a 30-base overlap region (SEQ ID NOs: 1 to 6). Also, a forward primer (SEQ ID NO: 7) and a reverse primer (SEQ ID NO: 8) used for the overlap extension PCR reaction were designed.

[0046] (2) Two-step type 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 (manufactured by Nacalai Tesque). After preparing three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase) for synthesizing short double-stranded DNA fragments by primer extension PCR, 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 first 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 second PCR reaction solution A, and 1 μL each of single-stranded oligo DNA (SEQ ID NO: 5) and single-stranded oligo DNA (SEQ ID NO: 6) was added to the third PCR reaction solution A. At this time, the total volume was adjusted to 25 μL. A 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) for synthesizing full-length double-stranded DNA by overlap extension PCR was prepared. At this time, the total volume was adjusted to 25 μL.

[0047] Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions of the PCR reaction at this time were as follows.

[0048] After 98°C for 2 minutes, the following temperature cycle was performed 10 cycles.

[0049] (Denaturation stage) 98°C, 30 seconds; (Annealing stage) 77.5°C, 10 seconds; 75°C, 10 seconds; 72.5°C, 10 seconds; 70°C, 10 seconds; 67.5°C, 10 seconds; 65°C, 10 seconds; 62.5°C, 10 seconds (Extension stage) 72°C, 50 seconds

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

[0051] Next, using a thermal cycler manufactured by TaKaRa, the three short double-stranded DNA fragments prepared above were multi-stage ligated by overlap extension PCR reaction to synthesize a full-length double-stranded DNA. The temperature conditions of the PCR reaction at this time are as follows.

[0052] After 98°C for 2 minutes, the following temperature cycle was performed 20 times. (Denaturation stage) 98°C, 30 seconds; (Annealing stage) 77.5°C, 10 seconds; 75°C, 10 seconds; 72.5°C, 10 seconds; 70°C, 10 seconds; 67.5°C, 10 seconds; 65°C, 10 seconds; 62.5°C, 10 seconds (Extension stage) 72°C, 50 seconds.

[0053] Thereafter, it was treated at 72°C for 3 minutes.

[0054] Full-length double-stranded DNAs were synthesized under the same PCR conditions as above except that all the temperature holding times in the annealing stage were set to 20 seconds, 30 seconds, 40 seconds, 50 seconds, or 2 minutes. Thereafter, a part of the DNA amplification products of the double-stranded DNA fragments obtained from the six conditions (temperature holding times in the annealing stage are 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 2 minutes) 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 2.

[0055] As a result, when the temperature holding time of the PCR reaction conditions of the synthesis method of the present invention is 50 seconds or more, the DNA amplification product of the double-stranded DNA fragment obtained can suppress the appearance of non-specific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence) compared to the DNA amplification product of the double-stranded DNA fragment obtained when the temperature holding time of the PCR reaction conditions is less than 50 seconds. That is, the temperature holding time of the PCR reaction conditions of the synthesis method of the present invention is particularly preferably 50 seconds or more. On the other hand, although the DNA amplification products of the double-stranded DNA fragments obtained when the temperature holding time of the PCR reaction conditions is 10 seconds, 20 seconds, 30 seconds, or 40 seconds contain non-specific DNA amplification products, it is possible to obtain the target double-stranded DNA fragment. When the temperature holding time of the PCR reaction conditions of the synthesis method of the present invention is 50 seconds or more, as described above, it is possible to suppress the appearance of non-specific DNA amplification products. Therefore, DNA cloning such as the TA cloning method and the blunt-end cloning method can be applied, and it is possible to quickly obtain cloned DNA containing the target double-stranded DNA fragment.

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

[0057] (2) Two-step type 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 (manufactured by Nacalai Tesque). After preparing three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase) for synthesizing short double-stranded DNA fragments, 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 first 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 second PCR reaction solution A, and 1 μL each of single-stranded oligo DNA (SEQ ID NO: 13) and single-stranded oligo DNA (SEQ ID NO: 14) was added to the third PCR reaction solution A. At this time, the total volume was adjusted to 25 μL. A 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: 15, 0.1 μM reverse primer: SEQ ID NO: 16) for synthesizing full-length double-stranded DNA was prepared. At this time, the total volume was adjusted to 25 μL.

[0058] Full-length double-stranded DNA fragments were synthesized under the following conditions (i) to (vii) with different numbers of annealing temperature setting steps.

[0059] (i) 0 steps Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions of the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 times.

[0060] 98°C, 30 seconds; 72°C, 50 seconds

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

[0062] Next, using a thermal cycler manufactured by TaKaRa, full-length double-stranded DNA was synthesized by multi-step ligation of three short double-stranded DNA fragments by overlap extension PCR. The PCR reaction conditions at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 times.

[0063] 98°C, 30 seconds; 72°C, 50 seconds

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

[0065] (ii) One-step Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions of the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 times.

[0066] 98°C, 30 seconds; 77.5°C, 50 seconds; 72°C, 50 seconds

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

[0068] Next, using a thermal cycler manufactured by TaKaRa, full-length double-stranded DNA was synthesized by multi-step ligation of three short double-stranded DNA fragments by overlap extension PCR. The PCR reaction conditions at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 times.

[0069] 98°C, 30 seconds; 77.5°C, 50 seconds; 72°C, 50 seconds

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

[0071] (iii) Two-step Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions for the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 cycles.

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

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

[0074] Next, using a thermal cycler manufactured by TaKaRa, the full-length double-stranded DNA was synthesized by overlapping extension PCR to multi-stage ligate three short double-stranded DNA fragments. The reaction conditions for the PCR at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 cycles.

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

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

[0077] (iv) Three steps Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions for the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 cycles.

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

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

[0080] Next, using a thermal cycler manufactured by TaKaRa, full-length double-stranded DNA was synthesized by multi-step ligation of three short double-stranded DNA fragments by overlap extension PCR. The reaction conditions for PCR at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 times. 98°C for 30 seconds; 77.5°C for 50 seconds; 70°C for 50 seconds; 62.5°C for 50 seconds; 72°C for 50 seconds. Thereafter, it was treated at 72°C for 3 minutes.

[0081] (v) Seven steps Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions for the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 times.

[0082] 98°C for 30 seconds; 77.5°C for 50 seconds; 75°C for 50 seconds; 72.5°C for 50 seconds; 70°C for 50 seconds; 67.5°C for 50 seconds; 65°C for 50 seconds; 62.5°C for 50 seconds; 72°C for 50 seconds

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

[0084] Next, using a thermal cycler manufactured by TaKaRa, full-length double-stranded DNA was synthesized by multi-step ligation of three short double-stranded DNA fragments by overlap extension PCR. The reaction conditions for PCR at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 times.

[0085] 98°C for 30 seconds; 77.5°C for 50 seconds; 75°C for 50 seconds; 72.5°C for 50 seconds; 70°C for 50 seconds; 67.5°C for 50 seconds; 65°C for 50 seconds; 62.5°C for 50 seconds; 72°C for 50 seconds

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

[0087] (vi) Eight steps Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions for the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 cycles.

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

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

[0090] Next, using a thermal cycler manufactured by TaKaRa, the full-length double-stranded DNA was synthesized by overlapping extension PCR to multi-stage ligate the three short double-stranded DNA fragments. The reaction conditions for the PCR at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 cycles.

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

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

[0093] (vii) Continuous temperature gradient Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions for the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 cycles.

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

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

[0096] Next, using a thermal cycler manufactured by TaKaRa, the full-length double-stranded DNA was synthesized by multi-step ligation of the three short double-stranded DNA fragments by overlap extension PCR. The PCR reaction conditions at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 times.

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

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

[0099] Subsequently, a part of the DNA amplification product of the double-stranded DNA fragment obtained from 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 steps of the temperature gradient, which is the PCR reaction condition of the synthesis method of the present invention, is preferably 2 to 8 steps. There is no problem even if the number of steps of the temperature gradient is 8 steps or more, and the reaction may be carried out under the PCR reaction conditions of the temperature gradient with 8 steps or more, but it can be said that about 2 to 8 steps are sufficient. On the other hand, when there is no temperature holding time for each temperature gradient of the PCR reaction condition of the synthesis method of the present invention (continuous temperature gradient), or when the temperature holding time is very short, it has been confirmed that it is difficult to obtain the DNA amplification product of the target double-stranded DNA fragment even if the number of steps of the temperature gradient is increased (data not shown).

[0101] (Example 3) Comparison of double-stranded DNA fragments obtained by the conventional synthesis method and the synthesis method of the present invention - 1 (Comparison of double-stranded DNA fragments obtained by the synthesis method of the contract synthesis company and the synthesis method of the present invention) (1) Sequence design of single-stranded oligo DNA The sequence of the target full-length double-stranded DNA 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. That is, the six single-stranded oligo DNAs used for the synthesis of the three short double-stranded DNA fragments were designed to be about 150 bases in length, including a 30-base overlap region (SEQ ID NOs: 17-22). Also, a forward primer (SEQ ID NO: 23) and a reverse primer (SEQ ID NO: 24) used for the overlap extension PCR reaction were designed.

[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 (manufactured by Nacalai Tesque). After preparing three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase) for synthesizing short double-stranded DNA fragments, 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 first 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 second PCR reaction solution A, and 1 μL each of single-stranded oligo DNA (SEQ ID NO: 21) and single-stranded oligo DNA (SEQ ID NO: 22) was added to the third PCR reaction solution A. At this time, the total volume was adjusted to 25 μL. A 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: 23, 0.1 μM reverse primer: SEQ ID NO: 24) for synthesizing the full-length double-stranded DNA was prepared. At this time, the total volume was adjusted to 25 μL.

[0103] Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions for the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 times.

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

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

[0106] Furthermore, using a thermal cycler manufactured by TaKaRa, a full-length double-stranded DNA was synthesized by overlapping and ligating three short double-stranded DNA fragments by overlap extension PCR reaction. The temperature conditions for the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 times.

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

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

[0109] A part of the DNA amplification product of the synthetic double-stranded DNA obtained by the method of the present invention and a part of the solution of the synthetic double-stranded DNA (by the conventional synthesis method) synthesized by commissioning a synthetic company 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 Fig. 4, the DNA amplification product of the double-stranded DNA fragment obtained by the synthesis method of the present invention can significantly suppress the appearance of non-specific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence) compared to the DNA amplification product of the double-stranded DNA fragment obtained by the synthesis method of the contract synthesis company. The DNA amplification product of the double-stranded DNA fragment of the contract synthesis company with a significant appearance of non-specific DNA amplification products needs to go through processes such as gel excision purification to extract only the target double-stranded DNA fragment. Therefore, it is not preferable to apply DNA cloning such as the TA cloning method and the blunt-end cloning method, and it is difficult to quickly obtain cloned DNA.

[0111] On the other hand, the DNA amplification product of the double-stranded DNA fragment obtained by the synthesis method of the present invention can very effectively suppress the appearance of non-specific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence). Therefore, the DNA amplification product of the double-stranded DNA fragment obtained under the PCR reaction conditions of the synthesis method of the present invention can apply DNA cloning methods such as the TA cloning method and the blunt-end cloning method without going through processes such as gel excision purification to extract only the target double-stranded DNA fragment, and can quickly obtain cloned DNA containing the target double-stranded DNA fragment. From the above results, it can be said that compared with the conventional DNA synthesis method, the synthesis method of the present invention is also excellent in terms of increasing the efficiency of DNA cloning and being able to quickly obtain the target cloned DNA.

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

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

[0114] To synthesize short double-stranded DNA fragments necessary for the synthesis of the first double-stranded DNA, three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared. 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 first 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 second PCR reaction solution A, and 1 μL each of single-stranded oligo DNA (SEQ ID NO: 29) and single-stranded oligo DNA (SEQ ID NO: 30) was added to the third PCR reaction solution A. At this time, the total volume was adjusted to 25 μL. To synthesize the first full-length double-stranded DNA, a 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. At this time, the total volume was adjusted to 25 μL.

[0115] To synthesize short double-stranded DNA fragments necessary for the synthesis of the second double-stranded DNA, three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTP, 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. At this time, the total volume was adjusted to 25 μL. A 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) for synthesizing the second full-length double-stranded DNA was prepared. At this time, the total volume was adjusted to 25 μL.

[0116] To synthesize 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 dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared. Subsequently, 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. At this time, the total volume was adjusted to 25 μL. A 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) for synthesizing the third full-length double-stranded DNA was prepared. At this time, the total volume was adjusted to 25 μL.

[0117] To synthesize short double-stranded DNA fragments necessary for the synthesis of the fourth double-stranded DNA, three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase) were prepared. Subsequently, 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. At this time, the total volume was adjusted to 25 μL. A 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) for synthesizing the fourth full-length double-stranded DNA was prepared. At this time, the total volume was adjusted to 25 μL.

[0118] Using a thermal cycler manufactured by TaKaRa, each short double-stranded DNA fragment was synthesized by primer extension PCR reaction. The temperature conditions of the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 times.

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

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

[0121] Furthermore, using a thermal cycler manufactured by TaKaRa, a full-length double-stranded DNA was synthesized by multi-step ligation of three short double-stranded DNA fragments through an overlap extension PCR reaction. The temperature conditions for the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 times.

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

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

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

[0125] As shown in Fig. 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 because the Tm value can be compensated by giving the temperature holding time for each multi-step temperature gradient of the PCR reaction conditions. That is, compared with the conventional synthesis method, the synthesis method of the present invention can simultaneously obtain a plurality of double-stranded DNA fragments of the target DNA sequence without sequence design.

[0126] The synthesis method of the present invention can also be used to obtain double-stranded DNA fragments of DNA sequences that are long repetitive sequences, continuous identical base sequences, AT-rich, GC-rich, etc. Therefore, the complexity of the target DNA sequences to be synthesized simultaneously is not particularly limited. According to the synthesis method of the present invention, a large number of double-stranded DNA fragments in units of several tens to several hundreds can be synthesized simultaneously, so the throughput is extremely high. In addition, by applying the synthesis method of the present invention to the program of a liquid dispensing robot, the 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 very effectively suppress 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 at this time can be applied with DNA cloning methods such as the TA cloning method and the blunt-end cloning method without going through steps such as gel excision purification for extracting only the target double-stranded DNA fragment, and cloned DNA containing the target double-stranded DNA fragment can be quickly obtained.

[0128] The method for synthesizing long-chain DNA uses a large number of double-stranded DNA fragments obtained by the method for synthesizing double-stranded DNA fragments as an integration material to integrate the double-stranded DNA fragments. Therefore, if even one of the double-stranded DNA fragments is missing, it is impossible to synthesize long-chain DNA. However, the conventional synthesis method has the disadvantages that it takes time and effort to synthesize a large number of double-stranded DNA fragments, and depending on the complexity of the target DNA sequence to be synthesized, the synthesis is difficult or impossible. Therefore, it is difficult to quickly obtain all the double-stranded DNA fragments that serve as the integration material. That is, with the conventional method for synthesizing double-stranded DNA fragments, it is not possible to quickly supply the double-stranded DNA fragments that serve as the integration material for the synthesis of long-chain DNA, which is the actual bottleneck. On the other hand, the synthesis method of the present invention can simultaneously synthesize a plurality of double-stranded DNA fragments regardless of the complexity of the target DNA sequence to be synthesized. Therefore, a large number of double-stranded DNA fragments that serve as the integration material for the synthesis of long-chain DNA can be quickly synthesized. That is, the synthesis method of the present invention can quickly supply the double-stranded DNA fragments required for the synthesis of long-chain DNA, so that the throughput of the method for synthesizing long-chain DNA can be greatly improved.

[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. The electrophoresis of the single-stranded oligo DNA was carried out using an XCell SureLock mini-cell electrophoresis apparatus (manufactured by Thermo Fisher Scientific) and a 10% Novex TBE-Urea gel (manufactured by Thermo Fisher Scientific) according to the attached instructions. The gel staining of the single-stranded oligo DNA after electrophoresis was carried out using SYBR Green II nucleic acid fluorescent staining reagent (manufactured by TaKaRa) according to the attached instructions. For the gel analysis of the single-stranded oligo DNA, a gel imaging analysis apparatus Gel Doc EZ system (manufactured by Bio-Rad) was used according to the attached instructions. The results are shown in Fig. 6.

[0130] As shown in Fig. 6, the content ratio of the single-stranded oligo DNA used in the synthesis method of the present invention was such that the ratio of the full-length single-stranded oligo DNA was contained from 60% to 15%, while the ratio of the non-full-length single-stranded oligo DNA was contained from 40% to 85%. The non-full-length single-stranded oligo DNA is a reaction intermediate product such as unreacted substances generated during the chemical synthesis of the single-stranded oligo DNA.

[0131] In the synthesis of the double-stranded DNA fragment by the synthesis method of the present invention, a single-stranded oligo DNA containing 85% of the non-full-length single-stranded oligo DNA (15% or more of the full-length single-stranded oligo DNA) was used. Therefore, even when the synthesis method of the present invention uses a very low-quality single-stranded oligo DNA containing about 85% of the non-full-length single-stranded oligo DNA in the single-stranded oligo DNA for the synthesis of the double-stranded DNA fragment, the DNA amplification product of the target double-stranded DNA fragment can be obtained. At this time, the DNA amplification product of the double-stranded DNA fragment obtained can suppress the appearance of non-specific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence). Therefore, without going through steps such as gel excision purification for extracting only the target double-stranded DNA fragment, DNA cloning methods such as the TA cloning method and the blunt-end cloning method can be applied, and a cloned DNA containing the target double-stranded DNA fragment can be obtained quickly.

[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 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 above, a dA overhang was added to the 3'-terminal site of the DNA amplification product using 10xA-attachment Mix (manufactured by TOYOBO). Subsequently, the DNA amplification product was purified using the MinElute PCR Purification kit (manufactured by QIAgen). Then, the purified synthetic DNA and T-Vector pMD19 (Simple) (manufactured by TaKaRa) were mixed, and DNA Ligation kit (manufactured by TaKaRa) was added so that the mixed DNA solution became 1:1. The DNA ligation reaction solution was left standing at 16°C for 1 hour to overnight using a thermostat (manufactured by TAITEC). After the DNA ligation reaction, transformation was performed using E. coli JM109 competent cells (manufactured by TaKaRa) according to the attached instructions, and spread on an LB agar medium containing 100 μg / mL of kanamycin (manufactured by Nacalai Tesque), and cultured overnight at 37°C.

[0133] (2) DNA Sequencing Analysis Using a Shikazinias DNA extraction reagent (manufactured by Kanto Chemical Co., Inc.), a DNA extract was prepared from E. coli colonies according to the attached instructions. Using the DNA extract as a template for the PCR reaction, with TaKaRa Ex-Taq Hot Start (manufactured by TaKaRa), and according to the attached instructions, the target DNA sequence was amplified by using the M13 forward primer (SEQ ID NO: 51) and the M13 reverse primer (SEQ ID NO: 52). The temperature conditions for the PCR reaction at this time were as follows: after 95°C for 2 minutes, the following temperature cycle was performed 30 times. 95°C for 20 seconds; 58°C for 30 seconds; 72°C for 60 seconds. A part of the obtained PCR reaction product solution was electrophoresed using a 1% agarose gel (manufactured by Thermo Fisher). Then, the remaining PCR reaction product solution was purified using a MinElute PCR Purification kit (manufactured by Qiagen) according to the attached instructions.

[0134] The purified DNA product obtained above was subjected to a sequencing reaction using the BigDye Terminator v3.1 Cycle Sequencing Kit (manufactured by Thermo Fisher Scientific), and according to the attached instructions, by using the M13 forward primer (SEQ ID NO: 51) and the M13 reverse primer (SEQ ID NO: 52). The reaction conditions for the DNA sequencing at this time were as follows: after 95°C for 2 minutes, the following temperature cycle was performed 30 times. 95°C for 5 seconds; 50°C for 10 seconds; 60°C for 2 minutes and 30 seconds. The obtained DNA sequencing reaction product was purified using a BigDye Terminator Purification kit (manufactured by Thermo Fisher Scientific) according to the attached instructions. Then, using an Applied Biosystems 3500xL Genetic analyzer (manufactured by Thermo Fisher Scientific), according to the attached instructions, the target DNA sequence was analyzed.

[0135] As a result of the DNA sequence analysis, a cloned DNA with the exact sequence of the synthetic double-stranded DNA obtained by the synthesis method of the present invention could be obtained.

[0136] (Example 6) Comparison between double-stranded DNA fragments obtained by the conventional synthesis method and the synthesis method of the present invention - 2 (Comparison between the PCR reaction conditions of the double-stranded DNA synthesis method described in US Patent Application Publication US20080182296A1 and the double-stranded DNA synthesis method of the present invention) (1) Sequence design of single-stranded oligo DNA The sequence of the target full-length double-stranded DNA 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 for the synthesis of these three short double-stranded DNA fragments were designed to be about 150 bases in length including a 30-base overlap region (SEQ ID NOs: 53 to 58). Also, a forward primer (SEQ ID NO: 59) and a reverse primer (SEQ ID NO: 60) used for the overlap extension PCR reaction were designed.

[0137] (2) Two-step double-stranded DNA synthesis 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 (manufactured by Nacalai Tesque). After preparing three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase) for synthesizing short double-stranded DNA fragments, 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 first 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 second PCR reaction solution A, and 1 μL each of single-stranded oligo DNA (SEQ ID NO: 57) and single-stranded oligo DNA (SEQ ID NO: 58) was added to the third PCR reaction solution A. At this time, the total volume was adjusted to 25 μL. A 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: 59, 0.1 μM reverse primer: SEQ ID NO: 60) for synthesizing full-length double-stranded DNA was prepared. At this time, the total volume was adjusted to 25 μL.

[0138] (i) Conventional synthesis method (the method described in US Patent Application Publication US20080182296A1; Pcr-directed gene synthesis from large number of overlapping oligodeoxyribonucleotides) Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions of the PCR reaction at this time were as follows: after 95°C for 4 minutes, the following temperature cycle was performed 10 times.

[0139] 98°C, 30 seconds; 50°C, 30 seconds; 72°C, 30 seconds

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

[0141] Next, using a thermal cycler manufactured by TaKaRa, full-length double-stranded DNA was synthesized by multi-step ligation of three short double-stranded DNA fragments by overlap extension PCR. The temperature conditions for the PCR reaction at this time were as follows: after 95°C for 4 minutes, the following temperature cycle was performed 20 times.

[0142] 98°C, 30 seconds; 50°C, 30 seconds; 72°C, 30 seconds

[0143] Thereafter, it was treated at 72°C for 5 minutes.

[0144] (ii) Synthesis method of the present invention Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions for the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 times.

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

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

[0147] Next, using a thermal cycler manufactured by TaKaRa, full-length double-stranded DNA was synthesized by multi-step ligation of three short double-stranded DNA fragments by overlap extension PCR. The temperature conditions for the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 times.

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

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

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

[0151] As shown in Fig. 7, under the PCR reaction conditions of the conventional synthesis method, a DNA amplification product of a double-stranded DNA fragment having the length of the target DNA sequence could not be obtained. 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. Since the DNA amplification product of the double-stranded DNA fragment obtained at this time 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 the TA cloning method and the blunt-end cloning method can be applied without going through steps such as gel excision purification for extracting only the target double-stranded DNA fragment, and cloned DNA containing the target double-stranded DNA fragment can be obtained quickly.

[0152] Under the PCR reaction conditions of the conventional synthesis method (the method described in US Patent Application Publication US20080182296A1), even when the target DNA sequence is difficult to synthesize, by applying the synthesis method of the present invention, a double-stranded DNA fragment of the target DNA sequence can be obtained. Therefore, the synthesis method of the present invention is superior to the conventional synthesis method 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 the conventional synthesis method 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 sequence of the target full-length double-stranded DNA 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'-terminus. The six single-stranded oligo DNAs used for the synthesis of these three short double-stranded DNA fragments were designed to be about 150 bases in length, including a 30-base overlap region (SEQ ID NOs: 61-66). Also, a forward primer (SEQ ID NO: 67) and a reverse primer (SEQ ID NO: 68) used for the overlap extension PCR reaction were designed.

[0154] (2) 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 (manufactured by Nacalai Tesque).

[0155] (i) Conventional synthesis method (two-step double-stranded DNA synthesis method in which all single-stranded oligo DNAs are assembled in the first-step 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 dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase) was prepared, and then 1 μL of each single-stranded oligo DNA (SEQ ID NOs: 61 to 66) was added. At this time, the total volume was adjusted to 25 μL. Then, a 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: 67, 0.1 μM reverse primer: SEQ ID NO: 68) for synthesizing the full-length double-stranded DNA was prepared. At this time, the total volume was adjusted to 25 μL.

[0156] Using a thermal cycler manufactured by TaKaRa, all the single-stranded oligo DNAs were assembled by the first-step PCR reaction. The temperature conditions of the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 times.

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

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

[0159] Using a thermal cycler manufactured by TaKaRa, full-length double-stranded DNA was synthesized from the assembled single-stranded oligo DNA by the second-stage PCR reaction. The temperature conditions for the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 times.

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

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

[0162] (ii) Synthesis method of the present invention After preparing three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase) for synthesizing short double-stranded DNA fragments, 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 first 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 second PCR reaction solution A, and 1 μL each of single-stranded oligo DNA (SEQ ID NO: 65) and single-stranded oligo DNA (SEQ ID NO: 66) was added to the third PCR reaction solution A. At this time, the total volume was adjusted to 25 μL. A 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: 67, 0.1 μM reverse primer: SEQ ID NO: 68) for synthesizing full-length double-stranded DNA was prepared. At this time, the total volume was adjusted to 25 μL.

[0163] Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions of the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 10 times.

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

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

[0166] Using a thermal cycler manufactured by TaKaRa, a full-length double-stranded DNA was synthesized by multi-step ligation of three short double-stranded DNA fragments by overlap extension PCR reaction. The temperature conditions of the PCR reaction at this time were as follows: after 98°C for 2 minutes, the following temperature cycle was performed 20 times.

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

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

[0169] A part of the DNA amplification product of the double-stranded DNA fragment obtained by the conventional synthesis method (i) and the synthesis method (ii) 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 8.

[0170] As shown in Figure 8, in the conventional synthesis method, a DNA amplification product of a double-stranded DNA fragment having the length of the target DNA sequence could not be obtained. 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, in the synthesis method of the present invention, short double-stranded DNA could be accurately synthesized by applying a primer extension PCR reaction in the first stage of the synthesis method, so it can be said that a double-stranded DNA fragment of the target DNA sequence could be synthesized.

[0171] Since the DNA amplification product of the double-stranded DNA fragment obtained at this time 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 the TA cloning method and the blunt-end cloning method can be applied without going through steps such as gel excision purification for extracting only the target double-stranded DNA fragment, and cloned DNA containing the target double-stranded DNA fragment can be obtained quickly.

[0172] In the conventional synthesis method, even when the target DNA sequence is difficult to synthesize, by applying the synthesis method of the present invention, a double-stranded DNA fragment of the target DNA sequence can be obtained. Therefore, the synthesis method of the present invention is not limited to the complexity of the target DNA sequence and can synthesize a double-stranded DNA fragment of the target DNA sequence, so it is superior to the conventional synthesis method.

[0173] (Example 8) Comparison of double-stranded DNA fragments obtained by the conventional synthesis method and the synthesis method of the present invention - 4 (Comparison between the one-step type double-stranded DNA synthesis method described in Non-Patent Document 9 and the double-stranded DNA synthesis method of the present invention) (1) Sequence design of single-stranded oligo DNA The sequence of the target full-length double-stranded DNA 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 for the synthesis of these three short double-stranded DNA fragments were designed to be about 150 bases in length including a 30-base overlap region (SEQ ID NOs: 69 to 74). Also, a forward primer (SEQ ID NO: 75) and a reverse primer (SEQ ID NO: 76) used for the overlap extension PCR reaction were designed.

[0174] (2) Double-stranded DNA synthesis The six single-stranded oligo DNAs required for double-stranded DNA synthesis were adjusted to a concentration of 1 μM in sterile water or TE buffer (manufactured by 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, after preparing one PCR reaction solution A (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), 1 μL of each single-stranded oligo DNA (SEQ ID NOs: 69 to 74) was added. At this time, the total volume was adjusted to 25 μL.

[0175] Using a thermal cycler manufactured by TaKaRa, single-stranded oligo DNA was assembled by PCR reaction to synthesize full-length double-stranded DNA. The temperature conditions for the PCR reaction at this time were 98°C for 2 minutes, followed by 30 cycles of the following temperature cycles.

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

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

[0178] (ii) Synthesis method of the present invention After preparing three PCR reaction solutions A (1x Phusion HF Buffer, 0.2 mM dNTP, 0.016 U / μL Phusion High-Fidelity DNA polymerase) for synthesizing short double-stranded DNA fragments, 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 first 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 second PCR reaction solution A, and 1 μL each of single-stranded oligo DNA (SEQ ID NO: 73) and single-stranded oligo DNA (SEQ ID NO: 74) was added to the third PCR reaction solution A. At this time, the total volume was adjusted to 25 μL. A 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: 75, 0.1 μM reverse primer: SEQ ID NO: 76) for synthesizing full-length double-stranded DNA was prepared. At this time, the total volume was adjusted to 25 μL.

[0179] Using a thermal cycler manufactured by TaKaRa, three short double-stranded DNA fragments were synthesized by primer extension PCR reaction. The temperature conditions for the PCR reaction at this time were 98°C for 2 minutes, followed by 10 cycles of the following temperature cycle.

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

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

[0182] Using a thermal cycler manufactured by TaKaRa, a full-length double-stranded DNA was synthesized by multi-stage ligation of three short double-stranded DNA fragments by overlap extension PCR reaction. The temperature conditions for the PCR reaction at this time were 98°C for 2 minutes, followed by 20 cycles of the following temperature cycle.

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

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

[0185] A part of the DNA amplification products of the double-stranded DNA fragments obtained by the conventional synthesis method (i) and the synthesis method (ii) 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 9.

[0186] As shown in Fig. 9, in the conventional synthesis method, a DNA amplification product of a double-stranded DNA fragment having the length of the target DNA sequence could not be obtained. 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 it into the second stage of the primer extension PCR reaction and the overlap extension PCR reaction.

[0187] Since the DNA amplification product of the double-stranded DNA fragment obtained at this time can suppress the appearance of non-specific DNA amplification products (DNA amplification products shorter than the length of the target DNA sequence), without going through steps such as gel excision purification for extracting only the target double-stranded DNA fragment, DNA cloning methods such as the TA cloning method and the blunt-end cloning method can be applied, and a cloned DNA containing the target double-stranded DNA fragment can be obtained promptly.

[0188] Since the DNA sequence synthesized in this example is a DNA sequence generally referred to as a complex sequence containing a long repetitive sequence, a continuous identical base sequence, and an AT-rich region, it was found that the two-step double-stranded DNA synthesis method of the present invention can accurately synthesize the double-stranded DNA of the target DNA sequence even for the above complex DNA sequences.

[0189] In the conventional synthesis method, even when the target DNA sequence is difficult or impossible to synthesize, by applying the synthesis method of the present invention, a double-stranded DNA fragment of the target DNA sequence can be obtained. Therefore, the synthesis method of the present invention is superior to the conventional synthesis method 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 having a multi-stage annealing temperature setting in the PCR cycle, it becomes applicable to the ligation of a plurality of DNAs having various Tm values, so that the assembly of single-stranded oligo DNAs containing a large number of homologous regions can be carried out easily, accurately, efficiently, and rapidly. Further, since the double-stranded DNA synthesis method of the present invention is applicable to the ligation of DNAs having various Tm values, there is no need for the labor of optimizing the single-stranded oligo DNA sequence to be used as a material, and it is not necessary to re-set the temperature finely for each target double-stranded DNA fragment. Furthermore, according to the present invention, it is possible to synthesize easily, accurately, efficiently, and rapidly regardless of the sequence of the target double-stranded DNA fragment, so that double-stranded DNA fragments that were difficult or impossible to synthesize due to a conventional complex sequence (for example, a long repetitive sequence, a continuous identical base sequence, an AT-rich, GC-rich sequence, etc.) can also be synthesized.

Claims

1. A method for synthesizing double-stranded DNA, comprising: (a) subjecting a PCR reaction solution containing DNA to a heat denaturation step, wherein the DNA used is a pair of single-stranded DNAs each containing a region that binds complementarily to the end portions of the other; (b) subjecting the PCR reaction solution to an annealing step consisting of a plurality of stages of annealing temperature settings; and (c) subjecting the PCR reaction solution to an extension step, wherein the target double-stranded DNA is obtained by performing cycles of steps (a), (b) and (c). A method for synthesizing double-stranded DNA, which comprises performing primer extension PCR as the method for synthesizing double-stranded DNA.

2. The method for synthesizing double-stranded DNA according to claim 1, wherein the plurality of stages of annealing temperature settings are set on a temperature gradient from the heat denaturation step to the last stage of the plurality of stages of annealing temperature settings.

3. The method for synthesizing double-stranded DNA according to claim 1 or 2, wherein each of the plurality of stages of annealing temperature settings has a predetermined temperature holding time at a predetermined temperature.

4. The method for synthesizing double-stranded DNA according to any one of claims 1 to 3, wherein the plurality of stages of annealing temperature settings are from 2 to 20 stages.

5. The method for synthesizing double-stranded DNA according to any one of claims 1 to 4, wherein the plurality of stages of annealing temperature settings include a stage of temperature setting in the range of 60°C to 85°C.

6. The method for synthesizing double-stranded DNA according to claim 5, wherein each of the plurality of stages of annealing temperature settings is in the range of 60°C to 85°C.

7. The method for synthesizing double-stranded DNA according to any one of claims 1 to 6, wherein the plurality of stages of annealing temperature settings include a stage of temperature setting having a temperature holding time of 10 seconds or more and 2 minutes or less.

8. The method for synthesizing double-stranded DNA according to claim 7, wherein the temperature holding time of each of the plurality of stages of annealing temperature settings is 10 seconds or more and 2 minutes or less.

9. A method for synthesizing double-stranded DNA, comprising the process of a first PCR reaction and the process of a second PCR reaction, wherein the process of the first PCR reaction comprises: (a) subjecting a first PCR reaction solution containing a pair of single-stranded oligo DNAs having regions that bind complementarily to the end portions of the other to a heat denaturation step; and (b) subjecting the first PCR reaction solution to an annealing step consisting of a plurality of stages of annealing temperature settings. ​ (c) subjecting the first PCR reaction solution to an extension step; including; By performing the cycles of steps (a), (b) and (c), a double-stranded DNA of the first target is obtained. The process of the second PCR reaction is as follows: (a') preparing a second PCR reaction solution containing a plurality of the double-stranded DNAs of the first target having overlapping regions with each other; (b') subjecting the second PCR reaction solution to a heat denaturation step; (c') subjecting the second PCR reaction solution to an annealing step consisting of a plurality of stages of annealing temperature settings; (d') subjecting the second PCR reaction solution to an extension step including; By performing the cycles of steps (b'), (c') and (d'), a double-stranded DNA of the second target is obtained. A method for synthesizing double-stranded DNA.

10. A program for executing the double-stranded DNA synthesis method according to any one of Claims 1 to 9.

11. An apparatus for implementing the double-stranded DNA synthesis method according to any one of Claims 1 to 9, comprising means for adjusting the temperature of a PCR reaction solution according to the cycle, and optionally incorporating the program according to Claim 10.

12. A system for implementing the double-stranded DNA synthesis method according to any one of Claims 1 to 9, incorporating the program according to Claim 10 and / or comprising the apparatus according to Claim 11.

13. The method for synthesizing double-stranded DNA according to any one of Claims 1 to 8, wherein the plurality of stages of annealing temperature settings include a stage of temperature setting having a temperature holding time of 50 seconds or more and 2 minutes or less.

14. The method for synthesizing double-stranded DNA according to any one of Claims 1 to 8, wherein the plurality of stages of annealing temperature settings are 8 to 20 stages, and the plurality of stages of annealing temperature settings have a temperature holding time of 30 seconds or more and 2 minutes or less.

15. The method for synthesizing double-stranded DNA according to Claim 14, wherein the plurality of stages of annealing temperature settings have a temperature holding time of 50 seconds or more and 2 minutes or less.

16. The method for synthesizing double-stranded DNA according to any one of Claims 1 to 8 and 13 to 15, excluding the method in which the PCR reaction solution contains polyethylene glycol.

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