Method for producing synthetic DNA molecule

Abstract

To accurately obtain a target DNA molecule in a short time without requiring a cloning process or a sequencing process.SOLUTION: Provided is a method for producing a synthetic DNA molecule consisting of a desired sequence. The method comprises the steps of: providing a plurality of template oligonucleotides each having a portion of the desired sequence; preparing a plurality of splint oligonucleotides having sequences complementary to the sequences at the ends of adjacent oligonucleotides when the plurality of template oligonucleotides are arranged in a desired sequence; annealing the plurality of splint oligonucleotides to the plurality of template oligonucleotides; after the step of annealing, ligating the plurality of template oligonucleotides each other to synthesize a circular single-stranded DNA containing the plurality of splint oligonucleotides as complementary strands; and synthesizing double-stranded DNA using φ29 DNA polymerase using the circular single-stranded DNA as a template.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for producing a synthetic DNA molecule, and more particularly to a method for producing a long-chain synthetic DNA molecule.

Background Art

[0002] Generally, when synthesizing DNA, the polymerase chain reaction (PCR) method is widely used. In recent years, various methods for producing synthetic DNA molecules have been devised for gene downstream applications where the template DNA is not available and thus cannot be obtained by the PCR method, or for sequence utilization of genes with only sequence information. Among these, the Polymerase Cycling Assembly (PCA) method is the most widely used (Non-Patent Document 1, etc.). In the PCA method, a plurality of sequences are synthesized so that oligonucleotides of accurate 80 bases to 200 bases overlap each other by about 15 bases to 30 bases, and are extended from the 3'-end of each overlapping region by DNA polymerase. This makes it possible to synthesize DNA with a length of several kb.

[0003] In addition to the PCA method, the Ligation Chain Reaction (LCR) method is known as a method for producing a synthetic DNA molecule (Non-Patent Document 2, etc.). The LCR method prepares a plurality of oligonucleotides of 60 bases to 100 bases, and repeats a thermal cycle more than 50 times using a heat-resistant ligase to increase the ligation efficiency and connect the plurality of oligonucleotides to each other, finally obtaining a desired DNA sequence. The obtained DNA sequence is amplified from the end by the PCR method using it as a template, or ligated to a vector and amplified in Escherichia coli.

[0004] In addition to these, the Patch Oligodeoxynucleotide Synthesis (POS) method is also known (Non-Patent Document 3, etc.). Unlike the above LCR method, in the POS method, only one strand is ligated instead of both the sense strand and the antisense strand, and then the target DNA can be obtained by amplifying from the ends by the PCR method.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0006] In the PCA method shown in the above Non-Patent Document 1, depending on the sequence to be synthesized, the length of DNA that can usually be accurately synthesized by the PCA method is about 2 kb to 3 kb. It is difficult to synthesize DNA with a length longer than this only by the PCA method, and the probability of error increases as the length to be synthesized becomes longer, which is not preferable. Therefore, due to mutations derived from poor oligonucleotide synthesis, cloning into host cells such as Escherichia coli and sequence confirmation are required to confirm the absence of such errors, and as a result, it takes time to produce the desired DNA.

[0007] In addition, in the LCR method shown in Non-Patent Document 2, it is necessary to prepare synthetic oligonucleotides for the entire regions of both the sense strand and the antisense strand of the target DNA. Furthermore, at the end of the process, it is necessary to separate only the extended strands having the target length by the PCR method. For this reason, cloning steps and sequencing steps into host cells such as Escherichia coli for confirming the presence or absence of errors are also required, resulting in time and cost consumption. Even when PCR is not included in the process, a cloning step into host cells such as Escherichia coli is essential to eliminate errors derived from synthetic oligonucleotides, and as a result, it does not lead to time and cost reduction. The cloning step into host cells such as Escherichia coli for eliminating this mutation is a very laborious task despite being difficult to fully automate by mechanization, and it has been pointed out that it hinders high-throughput processing.

[0008] In addition, in Non-Patent Document 3, the POS method was successfully used to synthesize the YFP gene (678 bp) and rat catenin β1 (2352 bp), but the error rate has not been calculated. Similar to the LCR method, the POS method also requires separating only the extended strands that are double-stranded and have the target length by the PCR method at the end of the process. For this reason, cloning steps and sequencing steps into host cells such as Escherichia coli for confirming the presence or absence of errors are also required, resulting in time and cost consumption.

[0009] As described above, in the conventional method for manufacturing synthetic DNA molecules, since it is necessary to use the PCR method, as a result, confirmation steps for whether the target products such as cloning steps and sequencing steps into host cells such as Escherichia coli are required, and as a result, a lot of time and cost are required. Also, in any method, not only mutations derived from synthetic defects that are contained to a certain extent in the synthesized oligonucleotides, but also to eliminate mutations during PCR, cloning into a vector, introduction into host cells such as Escherichia coli, and then isolation of single clones and determination of sequences to eliminate mutations are essential. As a result, a fully automated platform for synthesizing a wide variety of sequences cannot be formed.

[0010] The present invention has been made in view of the above problems, and its object is to obtain a target DNA molecule accurately and in a short time with a minimum number of sequencing steps without the need for a cloning step into host cells such as Escherichia coli to confirm whether a desired product is obtained, even if the target DNA molecules are diverse. [Means for Solving the Problems]

[0011] In order to achieve the above object, as a result of intensive studies, the present inventors synthesized single-stranded DNA only by ligating oligonucleotides having a desired sequence via a sprint oligonucleotide having a sequence complementary to the ends of the oligonucleotides, and using φ29 DNA polymerase, a polymerase reaction was initiated from the region where the sprint oligonucleotide was bound using the synthesized single-stranded DNA as a template, and found that a long-chain DNA having a desired sequence can be accurately and simply synthesized, thus completing the present invention.

[0012] Specifically, the method for producing a synthetic DNA molecule according to the present invention is a method for producing a synthetic DNA molecule consisting of a desired sequence, comprising the steps of preparing a plurality of template oligonucleotides each having a part of the desired sequence, preparing a plurality of sprint oligonucleotides having a sequence complementary to the sequences at the ends of adjacent template oligonucleotides when the plurality of template oligonucleotides are arranged in the desired sequence, annealing the plurality of sprint oligonucleotides to the plurality of template oligonucleotides, after the annealing step, ligating the plurality of template oligonucleotides to synthesize a circular single-stranded DNA containing the plurality of sprint oligonucleotides as complementary strands, and synthesizing double-stranded DNA using φ29 DNA polymerase with the circular single-stranded DNA as a template.

[0013] In the method for producing a synthetic DNA molecule according to the present invention, instead of using the PCR method as in the prior art, oligonucleotides having a desired sequence are ligated to each other by a ligation reaction to extend and circularize single-stranded DNA, and using the obtained circular single-stranded DNA as a template, double-stranded DNA is synthesized using φ29 DNA polymerase. The ligation reaction has almost no risk of introducing base mutations, and φ29 DNA polymerase is guaranteed to have a fidelity more than 1000 times that of Taq DNA polymerase, and is more reliable than any currently commercially available PCR enzyme, and can avoid the risk of introducing base mutations. In particular, when using circular single-stranded DNA as a template, the multi-prime rolling circle amplification (MPRCA) method that repeatedly uses circular DNA as a template can be utilized, so that the amplification efficiency of double-stranded DNA can be improved. Therefore, according to the method for producing a synthetic DNA molecule according to the present invention, a long-chain DNA of a desired sequence can be accurately synthesized, so that the cloning step into host cells such as Escherichia coli and the isolation of a single clone, which are required as confirmation operations for the sequence of the synthetic DNA obtained by the conventional PCR method, can be dispensed with. Furthermore, since the double-stranded DNA prepared by this method can be directly introduced into the sequencing step even without purification, the time involved in synthesis can be shortened and the synthesis cost can also be reduced as a result. In addition, since the cloning step via host cells is not required, it becomes possible to create a fully automated platform for synthesizing a wide variety of sequences.

[0014] In the method for producing a synthetic DNA molecule according to the present invention, it is preferable that the template oligonucleotide includes a plurality of core oligonucleotides and runner oligonucleotides.

[0015] The method for producing a synthetic DNA molecule according to the present invention preferably further includes a step of diluting the circular single-stranded DNA obtained by the ligation.

[0016] By doing so, by diluting and using the single-stranded DNA product with successful cyclization, synthetically defective oligonucleotides contained only slightly in the synthetic oligonucleotides can be probabilistically removed, so that the desired double-stranded DNA can be synthesized with high efficiency.

[0017] In the method for producing a synthetic DNA molecule according to the present invention, it is preferable to amplify the double-stranded DNA by a multiple displacement amplification (MDA) method or a multiple primer rolling circle amplification (MPRCA) method.

[0018] By using these methods, the desired double-stranded DNA can be efficiently obtained.

[0019] In the method for producing a synthetic DNA molecule according to the present invention, the synthesized double-stranded DNA is subjected to control of cleavage and dephosphorylation and exonuclease treatment to prepare a single-stranded DNA sequence required, and the single-stranded DNA sequence is used as a material for the circular single-stranded DNA to synthesize the synthetic double-stranded DNA.

Effects of the Invention

[0020] According to the method for producing a synthetic DNA molecule according to the present invention, the desired DNA can be synthesized more accurately, in a shorter synthesis time, and at a lower cost than the conventional method.

Brief Description of the Drawings

[0021]

Figure 1

Figure 2

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Mode for Carrying Out the Invention

[0022] Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. The following description of the preferred embodiments is merely illustrative in nature and is not intended to limit the present invention, its application method, or its use.

[0023] The outline of a method for manufacturing a synthetic DNA molecule according to an embodiment of the present invention is shown in FIG. 1. As shown in FIG. 1, the manufacturing method according to this embodiment is a method for manufacturing a synthetic DNA molecule consisting of a desired sequence, which includes a step of preparing a template oligonucleotide consisting of a plurality of core oligonucleotides, each having a part of the desired sequence, and a runner oligonucleotide for circularizing them; a step of preparing a plurality of sprint oligonucleotides having sequences complementary to the sequences at the ends of adjacent template oligonucleotides when the plurality of template oligonucleotides are arranged in the desired sequence; a step of annealing the plurality of sprint oligonucleotides to the plurality of template oligonucleotides; after the annealing step, a step of ligating the plurality of template oligonucleotides to synthesize a circularized single-stranded DNA containing the plurality of sprint oligonucleotides; and a step of synthesizing double-stranded DNA using φ29 DNA polymerase with the circularized single-stranded DNA as a template.

[0024] In this embodiment, the template oligonucleotide is a synthetic oligo DNA of about 10 to 100 bases accurately synthesized one base at a time by a conventional method in the desired sequence. The plurality of template oligonucleotides each consist of a part of the sequence of the DNA molecule to be finally synthesized, and the sequences of these plurality of template oligonucleotides can cover the entire sequence of the desired DNA molecule. In other words, by connecting the plurality of template oligonucleotides, the entire sequence of the desired DNA molecule can be obtained.

[0025] In the present embodiment, the sprint oligonucleotide is a synthetic oligo DNA of about 20 to 30 bases accurately synthesized one base at a time by a conventional method according to a desired sequence, and is an oligo DNA used to connect template oligonucleotides by the circular assembling into ordered sequence (CAIOS) method as shown in FIG. 1. The sprint oligonucleotide has a sequence complementary to the sequence at the end of the template oligonucleotide. More specifically, when the plurality of template oligonucleotides are aligned according to the sequence of the desired DNA molecule, they are complementary to each other at the ends of adjacent template oligonucleotides. In other words, the sprint oligonucleotide has a sequence that can anneal so as to span the ends of the core oligonucleotide or the runner oligonucleotide, which is a constituent of the adjacent template oligonucleotide.

[0026] In the present embodiment, the template oligonucleotide and the sprint oligonucleotide are annealed as described above, but the solvent and temperature conditions at that time are not particularly limited, and optimal conditions can be appropriately selected by a method well known to those skilled in the art. By the annealing step, the sprint oligonucleotide having a sequence complementary to the ends of each template oligonucleotide is annealed.

[0027] In this embodiment, template oligonucleotides annealed to the sprint oligonucleotides obtained by the annealing step are ligated to each other. The solvent, ligase, and temperature conditions for the ligation are not particularly limited, and optimal conditions can be appropriately selected by methods well known to those skilled in the art. It is preferable to phosphorylate the 5'-end of the template oligonucleotide before the actual ligation step, if necessary. In this embodiment, the template oligonucleotides annealed to the sprint oligonucleotides are connected to each other by the ligation step. Specifically, according to the sequence of the desired DNA molecule, template oligonucleotides having adjacent sequences are connected to each other. Since the sprint oligonucleotides are annealed to the ends of the template oligonucleotides, nicks called breaks are generated where the ends of the template oligonucleotides are adjacent to each other via double-strands formed by the sprint oligonucleotides, and the nicked portions are ligated by ligase, thereby connecting the template oligonucleotides to each other, and the strand containing the template oligonucleotides is circularized.

[0028] In this embodiment, after ligating a plurality of template oligonucleotides to each other, double-stranded DNA is synthesized using φ29 DNA polymerase with the circular single-stranded DNA as a template. φ29 DNA polymerase guarantees a fidelity of 1000 times or more that of Taq DNA polymerase, is more reliable than any currently commercially available PCR enzyme, and can avoid the risk of base mutation introduction, which is preferable. In addition, since the reaction by φ29 DNA polymerase is an isothermal reaction, it does not require a thermal cycle, which is preferable. For the synthesis of double-stranded DNA using φ29 DNA polymerase, a multiple displacement amplification (MDA) method, a multiple primer rolling circle amplification (MPRCA) method, or the like can be used.

[0029] The MDA method is known as a DNA amplification method using a strand displacement DNA synthase and random primers (Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, p5261-5266). The MDA method generally involves the following steps: (1) Primers randomly anneal to denatured single-stranded DNA. (2) DNA synthesis is initiated from the annealed primers, and DNA synthesis continues while peeling off the complementary strand DNA synthesized earlier by other primers present in the synthesis direction from the template DNA strand. Also, new primers newly anneal to the peeled DNA strand. (3) DNA synthesis is also carried out from the primers annealed to the peeled DNA strand, and these series of reactions occur in a chain-like manner. The initial template is not used again after the synthesis from the most 3'-end is completed. The same applies to the peeled DNA strand.

[0030] In this embodiment, as shown in FIG. 1, before synthesizing single-stranded DNA obtained by ligating a plurality of template oligonucleotides into double-stranded DNA, a step of circularizing is included. By doing so, since the MPRCA method that repeatedly uses circular DNA as a template can be utilized, the amplification efficiency of double-stranded DNA can be improved. As described above, the MPRCA method is a method of amplifying DNA by repeatedly using circular DNA as a template, and is known as an application method of the rolling circle amplification (RCA) method (Genome Research, 2001, Vol. 11, p. 1095-1099). The RCA method uses circular single-stranded DNA as a template, and DNA polymerase synthesizes DNA starting from the 3'-end of a primer hybridized to the template. When DNA polymerase returns to the 5'-end of the primer after going around the circular template, DNA polymerase continues DNA synthesis while performing strand displacement to synthesize the DNA by peeling off the forward DNA strand into double-stranded DNA, and synthesizes a long single-stranded DNA in which the complementary strand sequences of the circular DNA are arranged in tandem. On the other hand, the MPRCA method is a method of performing RCA on circular DNA serving as a template using a plurality of specific primers at a plurality of starting points. This has the advantage that synthesis proceeds more rapidly at a plurality of growth points on each circular template. In this embodiment, the sprint oligonucleotide annealed to the template oligonucleotide can serve as the starting points at the above-mentioned plurality of locations.

[0031] Also, in this embodiment, the circularized single-stranded DNA obtained as described above is preferably diluted and used before synthesizing double-stranded DNA. By doing so, oligonucleotides with slightly poor synthesis contained in the synthetic oligonucleotides can be probabilistically removed by dilution, so that the desired double-stranded DNA can be synthesized with high efficiency.

[0032] In addition, in the present embodiment, it is preferable to further use the obtained double-stranded DNA for the synthesis of double-stranded DNA again. Specifically, as shown in FIG. 1, a plurality of core oligonucleotides having a desired sequence and a runner oligonucleotide for circularizing them are ligated and circularized, and after synthesizing double-stranded DNA by the MPRCA method, the connection portion between the runner oligonucleotide and the core oligonucleotide is cut with a predetermined restriction enzyme. Then, after dephosphorylation, the connection portion between the other runner oligonucleotide and the core oligonucleotide is cut with a predetermined restriction enzyme to remove the runner oligonucleotide. Then, it is made single-stranded with a predetermined exonuclease (for example, lambda exonuclease), and then oligonucleotides having a desired sequence are further ligated and circularized, and the synthesis of double-stranded DNA by the MPRCA method is repeated. As a result, a longer double-stranded DNA can be easily and quickly synthesized with an accurate sequence.

Example

[0033] Examples for explaining in detail the method for producing a synthetic DNA molecule according to the present invention are shown below.

[0034] (Oligonucleotide) In this example, the template oligonucleotide is used as a general term for both a mixture composed only of a plurality of core oligonucleotides and a mixture of a plurality of core oligonucleotides and a runner oligonucleotide. The core oligonucleotides used in this example are shown in Table 1, the sprint oligonucleotides for the core oligonucleotides are shown in Table 2, the sprint oligonucleotides for the core oligonucleotides and the runner oligonucleotides are shown in Table 3, and the runner oligonucleotides are shown in Table 4. The combinations of these oligonucleotides used in the following experiments are shown in Table 5 for linear ligation and Table 6 for the ligation of the first circularization. Further, the sprint oligonucleotides used for the ligation of the single-stranded DNA prepared from the first circularization and the double-stranded DNA synthesis are shown in Table 7, and the combinations of the oligonucleotides used are shown in Table 8.

[0035]

Table 1

[0036]

Table 2

[0037]

Table 3

[0038]

Table 4

[0039]

Table 5

[0040]

Table 6

[0041]

Table 7

[0042]

Table 8

[0043] As can be seen from Tables 1 and 2, B89 has an array that can anneal to the 5' or 3' end of Mth for pGEX2 and T90, B90 has an array that can anneal to the 5' or 3' end of T90 and T91, B91 has an array that can anneal to the 5' or 3' end of T91 and T92, and B92 has an array that can anneal to the 5' or 3' end of T92 and T93. Also, B94 has an array that can anneal to the 5' or 3' end of T94 and T95, B95 has an array that can anneal to the 5' or 3' end of T95 and T96, B96 has an array that can anneal to the 5' or 3' end of T96 and T97, and B97 has an array that can anneal to the 5' or 3' end of T97 and T98. Also, B99 has an array that can anneal to the 5' or 3' end of T99 and T100, B100 has an array that can anneal to the 5' or 3' end of T100 and T101, B101 has an array that can anneal to the 5' or 3' end of T101 and T102, and B102 has an array that can anneal to the 5' or 3' end of T102 and T103. Also, B104 has an array that can anneal to the 5' or 3' end of T104 and T105, B105 has an array that can anneal to the 5' or 3' end of T105 and T106, B106 has an array that can anneal to the 5' or 3' end of T106 and T107, and B107 has an array that can anneal to the 5' or 3' end of T107 and T108. Note that B89i, B91i, B102i, B104i, B105i, and B107i are each 5-mer longer than B89, B91, B102, B104, B105, and B107, respectively, to improve the Tm value.

[0044] <Preliminary test in linear form> (Phosphorylation of template oligonucleotide) First, it was confirmed as follows whether the template oligonucleotides were connected to each other via the sprint oligonucleotides to obtain the desired synthetic product. Phosphorylation of the 5'-end of the template oligonucleotides was performed for subsequent ligation of the template oligonucleotides to each other. For this purpose, 5 μL of 50 μM each of the combinations of template oligonucleotides (MthL_1, MthL_2, MthL_3, MthL_4) were prepared, and for each of them, 5 μL each of 10× phosphorylation buffer (200 mM Tris acetate (pH 7.8), 100 mM magnesium acetate, 1 mg albumin derived from bovine serum), 10 mM ATP, 0.5 μL of 100 mM DTT, 2 μL of T4 polynucleotide kinase (New England Biolabs), and 32.5 μL of distilled water were added to make a total volume of 50 μL. After treating the mixture at 37°C for 30 minutes for the phosphorylation reaction of the oligonucleotides, it was treated at 65°C for 20 minutes to inactivate the enzyme, and then cooled to 12°C. As a result of this reaction, the concentration of each oligonucleotide became 1 μM (1 pmol / μL).

[0045] (Annealing) After the above phosphorylation treatment, the following treatment was performed to anneal each template oligonucleotide and the sprint oligonucleotide (linear). First, 1 μL each of 10× annealing buffer (200 mM Tris acetate (pH 7.8), 2 M potassium glutamate, 100 mM magnesium acetate, 1 mM nicotinamide adenine dinucleotide (NAD), 100 mM ammonium sulfate) was dispensed into each tube. 4.5 μL of the 1 μM mixed solution of each template oligonucleotide was transferred to the tube into which the 10× annealing buffer was dispensed, and further, 4.5 μL of the 1 μM mixed solution of the sprint oligonucleotide was added to each tube. Note that B_Mth_1 was added to MthL_1, B_Mth_2 was added to MthL_2, B_Mth_3 was added to MthL_3, and B_Mth_4 was added to MthL_4. Then, each tube was heated at 95°C for 4 minutes, the temperature was slowly lowered to 10°C and maintained for 10 minutes to complete the annealing process of the template oligonucleotide and the sprint oligonucleotide.

[0046] (Ligation) Subsequently, the following procedure was performed to ligate the template oligonucleotides annealed to the sprint oligonucleotides (linear). 1 μL of 10× annealing buffer was added to each tube after the above procedure, 1 μL of E. coli DNA ligase (Takara) was added, and 8 μL of distilled water was further added to each. Thereafter, each tube was treated at 37 °C for 30 minutes for ligation reaction, then treated at 65 °C for 20 minutes to inactivate the enzyme, and then cooled to 12 °C.

[0047] (Confirmation of synthetic product) First, whether or not the template oligonucleotides annealed to the sprint oligonucleotides in each combination (MthL_1, MthL_2, MthL_3, MthL_4) of the template oligonucleotides were ligated as desired by the above ligation step was confirmed using a conventional polyacrylamide electrophoresis method. The results are shown in Figure 2.

[0048] In Figure 2, lanes 1, 2, 3, and 4 are the ligation reaction products of MthL_1, MthL_2, MthL_3, and MthL_4, respectively, and lane M is the marker. As shown in Figure 2, nucleotides clearly longer than 60 bp, which is the length of each template oligonucleotide, were generated, and the presence of a product with a length of approximately 300 bases, in which five types of template oligonucleotides in each combination of template oligonucleotides were connected, was also confirmed (arrow part in Figure 2). However, it became clear that the amount of the product with a length of approximately 300 bases, which was the target, was smaller in the ligation reaction products of MthL_3 and MthL_4 than in those of MthL_1 and MthL_2. Therefore, when the Tm values of each sprint oligonucleotide were calculated, for those that clearly showed a low Tm value in part of the sprint oligonucleotides for MthL_1, MthL_3, and MthL_4, sprint oligonucleotides (B89i, B91i, B102i, B104i, B105i, and B107i) with an oligonucleotide length extended by 5 mer to increase the Tm were created. When the same reaction was performed again using the combinations of MthL_1R (lane 1 in Figure 3), MthL_2 (lane 2 in Figure 3), MthL_3R (lane 3 in Figure 3), and MthL_4R (lane 4 in Figure 3) shown in Table 5 containing these, it became clear that the amount of the ligation reaction products of MthL_3R and MthL_4R was significantly improved (arrow part in Figure 3).

[0049] <First circularization and amplification> (Phosphorylation of template oligonucleotide) Subsequently, using the template oligonucleotide and the sprint oligonucleotide used in the above linear test together with the oligonucleotide for cyclization, it was tested whether a desired long-chain synthetic product could be obtained by repeating the cyclization and amplification of the oligonucleotide. The method and results are shown below. First, phosphorylation of the 5'-end of the core oligonucleotide and the runner oligonucleotide (circular with TypeIIS_R) was performed for subsequent ligation of the template oligonucleotides to each other. For this purpose, 5 μL each of 50 μM of each combination of the template oligonucleotides (MthL_1, MthL_2, MthL_3, MthL_4) and the runner oligonucleotide were prepared, and for each of them, 5 μL each of 10× phosphorylation buffer (200 mM Tris acetate (pH 7.8), 100 mM magnesium acetate, and 1 mg bovine serum-derived albumin), 10 mM ATP, 0.5 μL of 100 mM DTT, 2 μL of T4 polynucleotide kinase (New England Biolabs), and 32.5 μL of distilled water were added to make a total volume of 50 μL. After treating the mixed solution at 37°C for 30 minutes for the phosphorylation reaction of the oligonucleotide, it was treated at 65°C for 20 minutes to inactivate the enzyme, and then cooled to 12°C. As a result of this reaction, the concentration of each oligonucleotide became 1 μM (1 pmol / μL).

[0050] (Annealing) After the phosphorylation treatment, the following procedure was performed to anneal each template oligonucleotide and the split oligonucleotide (circular). First, 1 μL of 10× annealing buffer (200 mM Tris acetate (pH 7.8), 2 M potassium glutamate, 100 mM magnesium acetate, 1 mM nicotinamide adenine dinucleotide (NAD), 100 mM ammonium sulfate) was dispensed into each tube. 4.5 μL of each 1 μM template oligonucleotide mixture was transferred to the tube into which the 10× annealing buffer had been dispensed, and then 4.5 μL of each 1 μM split oligonucleotide mixture was added to each tube. Note that B_Mth_1_RC was added to MthL_1, B_Mth_2_C was added to MthL_2, B_Mth_3_RC was added to MthL_3, and B_Mth_4_RC was added to MthL_4. Thereafter, each tube was heated at 95°C for 4 minutes, the temperature was slowly decreased to 10°C, and maintained for 10 minutes to complete the annealing step of the template oligonucleotide and the split oligonucleotide.

[0051] (Circularization and Amplification) Subsequently, the following procedures were performed to ligate the template oligonucleotides annealed to the sprint oligonucleotides (circularized). 1 μL of 10× annealing buffer was added to each tube after the above procedures, 1 μL of E. coli DNA ligase (Takara) was added, and 8 μL of distilled water was further added to each. Thereafter, each tube was treated at 37 °C for 30 minutes for the ligation reaction, then treated at 65 °C for 20 minutes to inactivate the enzyme, and then cooled to 12 °C. To amplify by the MPRCA method, 2 μL of 5× RA buffer (150 mM Tris-HCl (pH 7.5), 100 mM potassium chloride, 40 mM magnesium chloride) and 2 μL of 100 μM 6R5S primer were added to 6 μL of this circularization reaction solution, and each tube was heated at 95 °C for 4 minutes and then rapidly cooled to 4 °C. To each tube, 2 μL of 10× RCA buffer (100 mM Tris-HCl (pH 7.5), 100 mM potassium chloride, 100 mM magnesium chloride), 2 μL of 10 mM dNTPs, 1 μL of 100 mM DTT, 0.1 μL of pyrophosphatase (New England Biolabs), 1 μL (100 ng) of φ29 DNA polymerase, and 3.9 μL of distilled water were added, and the reaction was carried out at 30 °C for 16 hours to obtain an amplification product, a double-stranded DNA in which the circularized single-stranded DNA sequence was in a tandem repeat state. After the reaction, it was treated at 65 °C for 10 minutes to inactivate the enzyme and cooled to 12 °C. The amplification product was transferred to another tube containing 180 μL of distilled water, and the viscosity of the amplification product was decreased by stirring with a vortex mixer for 15 minutes.

[0052] (Confirmation of amplification product) To cleave at the BsaI site provided in the runner oligonucleotide, 25 μL of the dilution product was taken out, transferred to a separate tube, 5 μL of 10× CutSmart buffer (New England Biolabs), 1 μL of restriction enzyme (BsaI (New England Biolabs)), and 19 μL of distilled water were added to make the total volume 50 μL. Each tube was treated at 37 °C for 1 hour, then at 65 °C for 20 minutes to inactivate the enzyme, and then cooled to 12 °C. A part (about 10 μL) was used to confirm the cleavage by the conventional agarose gel electrophoresis method. The results are shown in Figure 4.

[0053] In Figure 4, lanes 1, 2, 3, and 4 are the ligation and cyclization products of MthL_1, MthL_2, MthL_3, and MthL_4, respectively, and lane M is the marker. As shown in Figure 4, the presence of a product with a length of approximately 300 bp to which five types of template oligonucleotides in each combination of template oligonucleotides were connected was confirmed in all lanes.

[0054] It is said that oligonucleotides prepared by any synthesis method contain oligonucleotides with synthesis defects at a ratio of about 0.2 - 0.3% with respect to the total number of molecules. Therefore, when the amount of the template of the cyclization product used for amplification is limited to about 10 - 100 copies, oligonucleotides with synthesis defects can be probabilistically excluded from amplification. Thus, the cyclization product used in Figure 4 was serially diluted to determine the amplification limit point. As a result, in any of the ligation and cyclization products of MthL_1, MthL_2, MthL_3, and MthL_4, the target amplification product was confirmed even with a template diluted 10 6 ~10 7 -fold (Figure 5). Therefore, when the sequences of the amplification products of the cyclization product before dilution and the cyclization product diluted 10 6 -fold were confirmed by a sequencing reaction, a peak signal derived from an oligonucleotide with a 1-base deletion, which is a synthesis defect, was confirmed below the main peak signal in the product derived from before dilution (Figure 6, upper panel). On the other hand, for the 10 6No weak signals thought to be derived from synthetically defective oligonucleotides were detected in the amplification products of the diluted circularized products (Figure 6, lower panel). This indicates that by diluting the circularized products for use in amplification, synthetically defective oligonucleotides can be probabilistically excluded.

[0055] (Single-stranded treatment) To 25 μL of the diluted amplification product, 4.3 μL of 10× CutSmart buffer (New England Biolabs), 2 μL of BsaI, 2 μL of Quick CIP (New England Biolabs), and 16.7 μL of distilled water were added to make a total volume of 50 μL. Subsequently, each tube was treated at 37°C for 2 hours, then at 80°C for 20 minutes to inactivate both enzymes, and then cooled to 12°C. A portion (about 10 μL) was used to confirm cleavage by conventional agarose gel electrophoresis. To the remaining cleavage solution, 1 μL of 10× CutSmart buffer (New England Biolabs), 1 μL of restriction enzyme BbsI (New England Biolabs), and 8 μL of distilled water were further added to make a total volume of 50 μL, and each tube was treated at 37°C for 1 hour, then at 65°C for 20 minutes to inactivate the enzyme, and then cooled to 12°C. A portion (about 10 μL) was used to confirm cleavage by conventional agarose gel electrophoresis. To the remaining cleavage solution, 1 μL of 10× CutSmart buffer (New England Biolabs), 1 μL of lambda exonuclease (New England Biolabs), and 8 μL of distilled water were further added to make a total volume of 50 μL, and each tube was treated at 37°C for 30 minutes, then at 75°C for 10 minutes to inactivate the enzyme, and then cooled to 12°C. Whether single-stranding occurred was confirmed using a portion (about 10 μL) by conventional polyacrylamide gel electrophoresis. The results are shown in Figure 7.

[0056] In Figure 7, lanes G1, G2, G3, and G4 are the ligation and circularization products of MthL_1, MthL_2, MthL_3, and MthL_4, respectively, where ds is double-stranded DNA, ss is single-stranded DNA, and lane M is a marker. As shown in Figure 7, in all lanes, the presence of single-stranded DNA products with different mobilities of 300-bp double-stranded DNA to which five types of template oligonucleotides in each combination of template oligonucleotides were connected was recognized.

[0057] (Purification of Single-Stranded DNA) In order to recover only single-stranded DNA, the following purification was performed using the Monarch PCR & DNA Cleanup Kit (New England Biolabs). First, 350 μL, which is 7 times the amount, of the DNA clean-up binding buffer of the kit was added to each tube after the exonuclease treatment, and the mixture was pipetted. Then, a tube with the column of the kit inserted was prepared, the above mixture was transferred onto the column, and centrifugation was performed at 16,000×g for 1 minute. Then, the liquid that passed through the column was removed, 200 μL of the DNA wash buffer of the kit was injected onto the column, and centrifugation was performed at 16,000×g for 1 minute. Then, the liquid that passed through the column was removed, 200 μL of the DNA wash buffer was injected onto the column again, and centrifugation was performed at 16,000×g for 1 minute. Then, the column was transferred into another tube, 12 μL of the DNA elution buffer of the kit was injected onto the column, allowed to stand at room temperature for 1 minute, and then centrifugation was performed at 16,000×g for 1 minute. As a result, nucleotides less than 200 bases were removed, and only single-stranded DNA of 200 bases or more was obtained. The concentration of the recovered single-stranded DNA was determined using the Qubit (registered trademark) ssDNA assay kit (Invitrogen) and converted from the weight concentration (ng) to the molar concentration by calculation.

[0058] (Second Circularization and Amplification) (Phosphorylation of Template Single-Stranded DNA) The single-stranded DNAs of recovered MthL_1, MthL_2, MthL_3, and MthL_4 were calculated for the volume to have a concentration of 5 μM (5 pmol / μL) respectively from the determined concentrations, and then a mixture (combination name, For 1.2 k) was prepared. To 10 μL of it, 10× phosphorylation buffer (200 mM Tris acetate (pH 7.8), 100 mM magnesium acetate, 1 mg albumin derived from bovine serum), 5 μL each of 10 mM ATP, 0.5 μL of 100 mM DTT, 2 μL of T4 polynucleotide kinase (New England Biolabs), and 27.5 μL of distilled water were added to make a total volume of 50 μL. After treating these mixtures at 37 °C for 30 minutes for the phosphorylation reaction of oligonucleotides, they were treated at 65 °C for 20 minutes to inactivate the enzyme, and then cooled to 12 °C. As a result of this reaction, the concentration of the single-stranded DNA became 1 μM (1 pmol / μL).

[0059] After the above phosphorylation treatment, the following treatment was performed to anneal the single-stranded DNA and the sprint oligonucleotide (B for 1.2k). 1 μL of 10× annealing buffer (200 mM Tris acetate (pH 7.8), 2 M potassium glutamate, 100 mM magnesium acetate, 1 mM NAD, 100 mM ammonium sulfate) was dispensed into a tube containing 4.5 μL of the phosphorylated single-stranded DNA (concentration 1 μM (1 pmol / μL)). Further, 4.5 μL each of 1 μM sprint oligonucleotide mixture (B_for 1.2k) was added to the tube. Then, each tube was heated at 95 °C for 4 minutes, the temperature was slowly lowered to 10 °C and maintained for 10 minutes to complete the annealing process of the template single-stranded DNA and the sprint oligonucleotide.

[0060] (Circularization, Amplification, and Confirmation) Subsequently, the following procedures were performed to ligate single-stranded template DNAs annealed to sprint oligonucleotides (circularized 1.2k). To the tube after the above procedures, 1 μL of 10× annealing buffer was added to each, 1 μL of E. coli DNA ligase (Takara) was added to each, and further 8 μL of distilled water was added to each. Then, the tubes were treated at 37°C for 30 minutes for ligation reaction, then treated at 65°C for 20 minutes to inactivate the enzyme, and then cooled to 12°C. To amplify by the MPRCA method, to 6 μL of this circularization reaction solution, 2 μL of 5× RA buffer (150 mM Tris-HCl (pH 7.5), 100 mM potassium chloride, 40 mM magnesium chloride) and 2 μL of 100 μM 6R5S primer were added, and each tube was heated at 95°C for 4 minutes and then rapidly cooled to 4°C. To the tubes, 2 μL of 10× RCA buffer (100 mM Tris-HCl (pH 7.5), 100 mM potassium chloride, 100 mM magnesium chloride), 2 μL of 10 mM dNTPs, 1 μL of 100 mM DTT, 0.1 μL of pyrophosphatase (New England Biolabs), 1 μL (100 ng) of φ29 DNA polymerase, and 3.9 μL of distilled water were added, and reacted at 30°C for 16 hours to obtain an amplification product, a double-stranded DNA in which the circularized single-stranded DNA sequence was in a tandem repeat state. After the reaction, it was treated at 65°C for 10 minutes to inactivate the enzyme and cooled to 12°C.

[0061] After amplification by the MPRCA method, the amplification product was added with 180 μL of distilled water to reduce the viscosity, then 25 μL was taken out and transferred to another tube, 5 μL of 10× CutSmart buffer (New England Biolabs), 1 μL of restriction enzyme BamHI1 (New England Biolabs), and further 19 μL of distilled water were added to make the total volume 50 μL. Each tube was treated at 37°C for 1 hour, then treated at 65°C for 20 minutes to inactivate the enzyme, and then cooled to 12°C. Using a part (about 10 μL), cleavage was confirmed by the conventional agarose gel electrophoresis method. The results are shown in Figure 8.

[0062] In FIG. 8, lane 1 is the above-mentioned product, and lane M is the marker. As shown in FIG. 8, a band was observed at the position of about 1200 bp. That is, from this result, it can be said that DNA to which four 300-bp oligonucleotides were bound was obtained by the above method.

[0063] Next, PCR was performed using the 1.2-kb DNA obtained by the test of FIG. 8 above as a template. First, about 1.1 kb from the start codon in Mth for pGEX2 to the stop codon in T108, and about 970 bp from about 170 bp inside the start codon to the stop codon were amplified by the ordinary PCR method, and agarose gel electrophoresis was performed on the resulting product. The result is shown in FIG. 9A.

[0064] In FIG. 9A, lane 1 is the PCR product amplified from the start codon to the stop codon, lane 2 is the PCR product amplified from about 170 bp inside the start codon to the stop codon, lane M is the marker, the upper arrow indicates the position of 1.1 kb, and the lower arrow indicates the position of about 970 bp. As shown in FIG. 9A, it is clear that all of the PCR products are shorter than the target lengths (1.1 kb, 970 bp). Although not shown in the figure, as a result of sequencing these PCR products, a deletion of about 560 bp was confirmed (from the 252nd base to the 853rd base from the start codon).

[0065] Since this deleted portion is the region of MthL_3 and MthL_4, next, in order to be able to amplify the region of MthL_3 in particular, primers were designed at two positions upstream and two positions downstream of this region, and a total of four types of PCR were performed. The respective predicted sizes were designed to be 397 bp, 585 bp, 482 bp, and 500 bp. The results of agarose gel electrophoresis performed on these PCR products are shown in FIG. 9B.

[0066] In B of FIG. 9, lane 1 is a PCR product with an expected size of 397 bp, lane 2 is a PCR product with an expected size of 585 bp, lane 3 is a PCR product with an expected size of 482 bp, lane 4 is a PCR product with an expected size of 500 bp, and lane M is a marker. As shown in B of FIG. 9, it was revealed that all of the PCR products were shorter than the target length.

[0067] From the results of FIGS. 2 to 7, according to the method of the present invention, desired sequences (template oligonucleotides) can be ligated to each other via splint oligonucleotides and double-stranded DNA can be amplified. Furthermore, from the results of FIG. 8, it was proven that long-chain DNA can be synthesized by further repeating ligation and double-stranded DNA amplification between the products obtained by those steps. On the other hand, as shown in FIG. 9, the 1.2 kb DNA obtained by the method of the present invention cannot be amplified by the PCR method, that is, it became clear that it is highly likely that it cannot be synthesized by the conventional PCA method.

[0068] As described above, the method according to the present invention can synthesize long-chain DNA. In particular, using single-stranded DNA obtained by a ligation reaction with no risk of base mutation introduction as a template, double-stranded DNA is synthesized using φ29 DNA polymerase. Therefore, cloning steps and sequencing steps as operations for confirming the sequence of the synthesized DNA obtained by methods using the conventional PCR method can be dispensed with. As a result, the synthesis time can be shortened, the synthesis cost can also be reduced, and it can be said that it is extremely useful.

Claims

1. A method for producing a synthetic DNA molecule consisting of a desired sequence, comprising: preparing a plurality of template oligonucleotides each having a part of the desired sequence; preparing a plurality of splint oligonucleotides having sequences complementary to the sequences at the ends of adjacent template oligonucleotides when the plurality of template oligonucleotides are arranged in the desired sequence; annealing the plurality of splint oligonucleotides to the plurality of template oligonucleotides; after the annealing step, ligating the plurality of template oligonucleotides to synthesize circular single-stranded DNA containing the plurality of splint oligonucleotides as complementary strands; and synthesizing double-stranded DNA using φ29 DNA polymerase with the circular single-stranded DNA as a template. A method for producing a synthetic DNA molecule, characterized by comprising the above steps.

2. The method for producing a synthetic DNA molecule according to claim 1, wherein the template oligonucleotide contains a plurality of core oligonucleotides and runner oligonucleotides.

3. The method for producing a synthetic DNA molecule according to claim 1 or 2, further comprising a step of diluting the circular single-stranded DNA obtained by the ligation.

4. The method for producing a synthetic DNA molecule according to any one of claims 1 to 3, wherein the double-stranded DNA is amplified by a multiple displacement amplification (MDA) method or a multiple primer rolling circle amplification (MP RCA) method.

5. By performing control of cleavage and dephosphorylation and exonuclease treatment on the synthesized double-stranded DNA, a single-stranded DNA sequence required is prepared, and the single-stranded DNA sequence is used as a material for the circular single-stranded DNA to synthesize the double-stranded DNA again. The method for producing a synthetic DNA molecule according to any one of claims 1 to 4, characterized by the above steps.

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