JPWO2018147070A1 - Method for producing a DNA fragment having a desired base sequence - Google Patents

Abstract

The present invention is a method for assembling two or more DNA materials to produce a DNA fragment having a desired base sequence, and (1) linking two or more DNA materials to obtain a desired base sequence. A step of obtaining a precursor DNA fragment in which a part and a base sequence for ligation are alternately arranged; (2) DNA having a desired base sequence by treating the precursor DNA fragment with a restriction enzyme and removing the base sequence for ligation A step of obtaining a fragment, wherein the base DNA recognized by the restriction enzyme is removed from the precursor DNA fragment when the precursor DNA fragment is cleaved by treatment with the restriction enzyme and the base DNA for ligation is removed. Provided is a method in which a part of a desired base sequence adjacent to each other via a base sequence is arranged so that it can be linked at a cleaved site.

Description

  The present invention relates to a method for producing a DNA fragment having a desired base sequence.

  As a method for producing a DNA fragment having a desired base sequence, a method of linking a plurality of DNA fragments such as Golden Gate method and Gibson method is known (Non-patent Document 1).

  The Golden Gate method is a method in which a plurality of DNA fragments containing a base sequence recognized by a type IIs restriction enzyme are prepared at one or both ends and treated with a type IIs restriction enzyme and a DNA ligase. A plurality of DNA fragments are hybridized by sticky ends (sticky ends) generated by cleavage with a type IIs restriction enzyme, and then nicked by DNA ligase, whereby a DNA fragment having a desired base sequence can be produced. By designing the type and arrangement of the base sequence recognized by the type IIs restriction enzyme, a plurality of DNA fragments cleaved by the type IIs restriction enzyme are linked so that the recognition site of the restriction enzyme is eliminated, and the desired base sequence is obtained. Can be produced. A method of introducing a DNA fragment having a desired base sequence into a cloning vector using the Golden Gate method has been reported (Patent Document 1 and Non-Patent Document 1).

  The Gibson method uses a plurality of DNA fragments designed so that the ligation regions at the ends of adjacent DNA fragments that are adjacently linked overlap each other by about 15 to 80 base pairs (bp). This is a method of preparing and treating with 5 ′ exonuclease, DNA polymerase and DNA ligase. 5 'exonuclease produces single-stranded DNA partially from the end of the DNA fragment. The resulting single-stranded DNA hybridizes with the overlapping base sequence portion. Thereafter, the gap is filled with DNA polymerase, and nicks are joined with DNA ligase, whereby a DNA fragment having a desired base sequence can be produced. In the Gibson method, it is not necessary to include a base sequence recognized by a restriction enzyme, so there is no restriction on the base sequence and it is suitable for the construction of a long DNA fragment (Non-patent Document 2, Patent Document 2 and Patent). Reference 3).

US Patent Application Publication No. 2010/0291633 US Patent No. 7723077 Special table 2011-512140 gazette

PLoS One, 2009, 4 (5), e5553 Chemistry and Biology, 2016, Vol. 54, no. 10, pp. 740-746

  In the Golden Gate method, it is said that the maximum number of DNA fragments that can be ligated at one time is about 10, and there are not so many types of type IIs restriction enzymes. Moreover, since the sticky end produced by cleavage with a type IIs restriction enzyme is as short as about 4 bases, the frequency of misligation is considered high. In particular, when the target base sequence (DNA fragment having a desired base sequence) is a repeat sequence or a base sequence with a high GC content, the frequency of misligation tends to be high, and thus a plurality of DNAs can be It is difficult to connect the fragments correctly.

  In the Gibson method, when the target base sequence includes a repeat sequence or a base sequence with a high GC content, the accuracy of single-stranded DNA hybridization is reduced, and therefore the frequency of misligation is considered to be extremely high. .

  A DNA fragment comprising a repeat sequence such as a CpG island or telomere sequence cannot be accurately produced by either the Golden Gate method or the Gibson method.

  In view of the above-mentioned problems, the present invention provides a DNA fragment having a desired base sequence that can be accurately and efficiently produced even if it is a DNA fragment containing a repeat sequence and a base sequence having a high GC content. The object is to provide a method of manufacturing.

The present invention relates to the following inventions, for example.
[1]
A method of assembling two or more DNA materials to produce a DNA fragment having a desired base sequence,
(1) linking two or more DNA materials to obtain a precursor DNA fragment in which a part of a desired base sequence and a base sequence for linking are alternately arranged;
(2) treating the precursor DNA fragment with a restriction enzyme and removing the linking base sequence to obtain a DNA fragment having a desired base sequence,
The precursor DNA fragment has a nucleotide sequence that is recognized by a restriction enzyme. When the precursor DNA fragment is cleaved by treatment with the restriction enzyme, the nucleotide sequence for ligation is removed and the desired DNA sequence is adjacent via the nucleotide sequence for ligation. A part of the base sequence is arranged so that the cleaved sites can be linked to each other.
[2]
Obtaining a precursor DNA fragment comprises:
(1-1) preparing two or more DNA materials satisfying the following (a) to (e):
(A) The DNA material has a single-stranded overhang region with a protruding 3 ′ end or 5 ′ end, respectively.
(B) the single-stranded overhang region of the DNA material includes a base sequence capable of hybridizing with the single-stranded overhang region of another DNA material linked adjacently;
(C) Each of the DNA materials includes a part of a base sequence continuous with a desired base sequence and a base sequence for linking, and at least one of the base sequences for linking included in two DNA materials that are adjacently linked , The linking base sequence overlaps with all the base sequences of the other linking base sequence,
(D) each of the DNA materials has a cleavage site cleaved by a restriction enzyme at a position adjacent to the linking base sequence, and includes a base sequence recognized by the restriction enzyme;
(E) The two DNA materials that are adjacently linked contain base sequences whose cleavage sites can hybridize to each other,
(1-2) Hybridizing a single-stranded overhang region of a DNA material with a single-stranded overhang region of another DNA material that is adjacently linked, The method according to [1] .
[3]
(1-3) The method according to [2], further comprising a step of treating in the order of DNA polymerase and DNA ligase after step (1-2).
[4]
Obtaining a DNA fragment having a desired base sequence,
The method according to any one of [1] to [3], comprising treating with a restriction enzyme and then treating with a DNA ligase.
[5]
The method according to [3], wherein the DNA polymerase is at least one DNA polymerase selected from the group consisting of DNA polymerases I, II and III.
[6]
The method according to [3] or [4], wherein the DNA ligase is at least one DNA ligase selected from the group consisting of Taq ligase and Tfi DNA ligase.
[7]
The method according to [2], wherein the single-stranded overhang region of the DNA material is formed by treating double-stranded DNA with 5 ′ exonuclease.
[8]
The method according to [7], wherein the 5 ′ exonuclease is at least one 5 ′ exonuclease selected from the group consisting of T7 exonuclease, lambda exonuclease, RecE, and T5 exonuclease.
[9]
The method according to any one of [1] to [8], wherein the restriction enzyme is a type II restriction enzyme.
[10]
The method according to [9], wherein the type II restriction enzyme is a type IIs restriction enzyme.
[11]
Type IIs restriction enzymes are AarI, AcuI, AlwI, BbsI, BbvI, BccI, BceAI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsgI, BsmPI, BsmPI, BsmPI, BsmPI, BsmPI At least one type IIs restriction enzyme selected from the group consisting of BtgZI, BstCI, EarI, EciI, Ecop15I, FauI, FokI, HgaI, HphI, HpyAV, Ksp632I, MboII, MmeI, Mn1I, SapI and SfaNI, ] Method.
[12]
The method according to [11], wherein the linking base sequence has two base sequences arranged such that base sequences recognized by the same type IIs restriction enzyme have a palindromic structure.
[13]
A method of assembling two or more DNA materials to produce a DNA fragment having a desired base sequence,
(1) preparing a double-stranded (ds) DNA material satisfying the following (a) to (c):
(A) The two dsDNA materials to be linked adjacently have the same linking base sequence,
(B) Each of the linking base sequences has a base sequence arranged so that two base sequences recognized by the same type IIs restriction enzyme have a palindromic structure.
(C) The base sequence recognized by the type IIs restriction enzyme is at a position where the base sequence for ligation is removed by cleaving with the type IIs restriction enzyme after ligation of the dsDNA material.
(2) treating the dsDNA material with a 5 ′ exonuclease to form a single-stranded overhang region with a protruding 3 ′ end;
(3) hybridizing a single-stranded overhang region of a dsDNA material with a single-stranded overhang region of another dsDNA material;
(4) a step of treating with DNA polymerase and filling a gap generated by hybridization;
(5) treating with DNA ligase and joining the nicks to obtain a precursor DNA fragment;
(6) treating the precursor DNA fragment with one or more type IIs restriction enzymes that recognize a base sequence recognized by a type IIs restriction enzyme present in the linking base sequence;
(7) a step of hybridizing complementary sticky ends generated by cleavage with a type IIs restriction enzyme, treating with a DNA ligase, and joining nicks.
[14]
[13] The base sequence recognized by the type IIs restriction enzyme contained in the base sequence for ligation adjacent through a part of the desired base sequence in the precursor DNA fragment is a different base sequence. Method.
[15]
A kit for producing a DNA fragment having a desired base sequence, comprising two or more oligo DNAs,
Each of the oligo DNAs includes a partial base sequence of a desired base sequence and a base sequence for linking used for linking with other DNA materials,
In the oligo DNA, the base sequence recognized by the restriction enzyme is treated with the restriction enzyme after ligation of the oligo DNA, so that the base sequence for ligation is removed, and the desired base adjacent through the base sequence for ligation is removed. A part of the base sequence of the sequence is arranged so that it can be linked at the sites cleaved with restriction enzymes,
The oligo DNA covers the desired base sequence when all are linked.
[16]
A DNA fragment obtained by the method according to any one of [1] to [14].

  According to the present invention, a DNA fragment having a desired base sequence can be accurately and efficiently produced even when a repeat sequence and a base sequence having a high GC content are included.

It is a figure explaining the principle which obtains a target DNA fragment by the manufacturing method concerning one embodiment of the present invention. It is a figure for demonstrating an example of design of DNA material. It is a figure for demonstrating an example of the process of obtaining a precursor DNA fragment. It is a figure for demonstrating an example of the process of obtaining the target DNA fragment.

  Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

  The production method according to this embodiment relates to a method for producing a DNA fragment (target DNA fragment) having a desired base sequence by assembling two or more DNA materials.

  The production method according to the present embodiment includes (1) linking two or more DNA materials to obtain a precursor DNA fragment in which a part of a desired base sequence and a linking base sequence are alternately arranged; (2) treating the precursor DNA fragment with a restriction enzyme and removing the linking base sequence to obtain a DNA fragment having a desired base sequence. Since the production method according to this embodiment is for producing a target DNA fragment via a precursor DNA fragment, even if the target DNA fragment is a DNA fragment containing a repeat sequence and a base sequence with a high GC content, It can be manufactured accurately and efficiently.

  The precursor DNA fragment is obtained by linking two or more DNA materials, and has a base sequence in which a continuous part of a desired base sequence and base sequences for linking are alternately arranged. The linking base sequence is a base sequence that is unnecessary for the target DNA fragment. The precursor DNA fragment has a nucleotide sequence that is recognized by a restriction enzyme. When the precursor DNA fragment is cleaved by treatment with the restriction enzyme, the nucleotide sequence for ligation is removed and the desired DNA sequence is adjacent via the nucleotide sequence for ligation. A part of the base sequence is arranged so that the cleaved sites can be linked to each other.

  The length of the linking base sequence may be, for example, 20 to 100 base pairs (bp), preferably 40 to 100 bp, and more preferably 60 to 100 bp.

  When the precursor DNA fragment includes a plurality of linking base sequences, the plurality of linking base sequences preferably have different base sequences.

  FIG. 1 is a diagram for explaining the principle of obtaining a target DNA fragment by the production method according to this embodiment. In FIG. 1, “1234” and “5678” indicate base sequences (recognition sites) recognized by restriction enzymes. In FIG. 1, “1234” indicates a site (cleavage site) that is cleaved by a restriction enzyme.

  FIG. 1 (A) is a diagram schematically showing two DNA materials that are adjacently connected. The DNA material (1) is a base sequence for ligation (CA5678TCC... Used for linking a part of the base sequence (.... Nnnn1234) of the base sequence of the target DNA fragment and the DNA material (2). 8765TC). Similarly, in the DNA material (2), a part of the base sequence (1234nnn...) Of the base sequence of the target DNA fragment and the base sequence for ligation (CA5678TCC. .. 8765TC). The base sequences for linking the DNA material (1) and the DNA material (2) are unnecessary base sequences for the target DNA fragment, and are identical base sequences that overlap each other.

  FIG. 1 (B) is a diagram schematically showing a state after the DNA material (1) and the DNA material (2) shown in FIG. 1 (A) are treated with 5 'exonuclease. By the 5 'exonuclease treatment, a single-stranded overhang region (3' overhang region) in which the 3 'end protrudes is formed in both the DNA material (1) and the DNA material (2). Since the base sequences of the 3 ′ overhang regions of the DNA material (1) and the DNA material (2) are complementary to each other, the DNA material (1) and the DNA material (2) are in the 3 ′ overhang region. It can hybridize.

  In addition, although the aspect processed with 5 'exonuclease is described in FIG. 1, by preparing DNA material (1) and DNA material (2) as what has a 3' overhang area | region previously, 5 'It is also possible to start from the state of Fig. 1 (B), omitting the exonuclease treatment.

  FIG. 1C shows a precursor DNA fragment according to one embodiment. The precursor DNA fragment can be obtained by linking the DNA material (1) and the DNA material (2) shown in FIG. Ligation is performed, for example, by hybridizing the DNA material (1) and the DNA material (2) in the 3 ′ overhang region, filling the gap with a DNA polymerase as necessary, and linking nicks with a DNA ligase. be able to.

  FIG. 1 (D) is a diagram showing a state after treating the precursor DNA fragment with a restriction enzyme. By treating with a restriction enzyme, the base sequence for ligation is removed, and a sticky end having a base sequence of “1234” (the base sequence of the target DNA fragment) is generated. The restriction enzyme may recognize, for example, the base sequence of “1234” and cleave the precursor DNA fragment at the recognition site, recognizes the base sequence of “5678”, and is different from the recognition site. The precursor DNA fragment may be cleaved at the site of the base sequence of “1234”. Further, different restriction enzymes may be used for cleavage at both ends of the linking base sequence as long as complementary sticky ends are generated. The recognition sites and cleavage sites of various restriction enzymes are well known in the art, and depending on the base sequence of the target DNA fragment, the type of restriction enzyme used, the position where the base sequence for linking is incorporated, etc. Can be set.

  In addition, although the aspect which produces | generates a sticky end was shown in FIG.1 (D), as long as the effect of this invention is not impaired, even if a restriction enzyme produces | generates a blunt end (blunt end). Good.

  FIG. 1E is a view showing a target DNA fragment according to one embodiment. The target DNA fragment can be obtained by ligating the restriction enzyme-treated fragments shown in FIG. Ligation can be performed, for example, by hybridizing both restriction enzyme-treated fragments at the sticky ends and then nicking them with DNA ligase.

  The DNA material is designed so that the precursor DNA fragment obtained by ligating the DNA material has the characteristics described above. In one embodiment, each of the DNA materials is a linking base used for linking a partial base sequence (hereinafter also referred to as “target partial sequence”) of a desired base sequence and another DNA material. And an array. The DNA material is designed so that the desired base sequence can be covered when all the DNA material is linked. In addition, in DNA material, the base sequence recognized by the restriction enzyme is treated with the restriction enzyme after ligation of the DNA material, so that the base sequence for ligation is removed and the base sequence is adjacent via the base sequence for ligation. The partial sequences are arranged so that they can be linked at sites cleaved with a restriction enzyme.

  The linking base sequence is preferably located at the end of the DNA material. The length of the linking base sequence contained in the DNA material is the same as the length of the linking base sequence in the precursor DNA fragment, and may be, for example, 20 to 100 base pairs (bp), and 40 to 100 bp. It is preferable that it is 60-100 bp. It is sufficient that at least one of the linking base sequences overlaps all the base sequences of the other linking base sequences, and the linking base sequences included in the two DNA materials that are adjacently linked are used for linking the two. It is preferable that all base sequences overlap.

  Further, in view of the role of the linking base sequence described above, the linking base sequence is preferably designed so as to have high hybridization specificity (for example, avoidance of appropriate GC content and repeat sequence). When there are a plurality of linking base sequences in the precursor DNA fragment, it is preferable that each has a different base sequence.

  The DNA material is a double stranded (ds) DNA fragment. The dsDNA fragment used as the DNA material may be dsDNA having blunt ends at both ends, or dsDNA having a single-stranded overhang region at one end or both ends. When dsDNA whose both ends are blunt ends is used as the DNA material, a single-stranded overhang region can be formed by treatment with exonuclease (3 'exonuclease or 5' exonuclease). The single-stranded overhang region may be a single-stranded overhang region with a 3 ′ end protruding (3 ′ overhang region), or a single-stranded overhang region with a 5 ′ end protruding (5 ′ overhang) Area).

  The single-strand overhang region designed in advance and the single-strand overhang region formed by exonuclease treatment may contain part or all of the linking base sequence. Since the base sequences for ligation contained in two DNA materials that are adjacently linked overlap each other, this results in a single-stranded overhang region of the DNA material and a single strand of another DNA material that is adjacently linked. It includes a base sequence capable of hybridizing with the overhang region.

  The DNA material may have a cleavage site cleaved by a restriction enzyme at a position adjacent to the linking base sequence, and may contain a base sequence (recognition site) recognized by the restriction enzyme. The recognition site contained in the DNA material may be contained in the target partial sequence, depending on the site (cleavage site) where the restriction enzyme used cleaves the DNA material, and may be contained in the linking base sequence. It may also be included across the target partial sequence and the linking base sequence. Moreover, two DNA materials connected adjacently may include a base sequence whose cleavage sites can hybridize with each other.

  A restriction enzyme is an enzyme that recognizes a specific base sequence of double-stranded DNA and cleaves DNA at the site inside the recognition site or at a site different from the recognition site. As a restriction enzyme, a type II restriction enzyme is preferably used because of the high specificity of the base sequence to be recognized and the site to be cleaved. Examples of the type II restriction enzyme include IIP type, IIA type, IIB type, and IIS type.

  Examples of restriction enzymes that cleave DNA inside the recognition site include BamHI, ClaI, EcoRI, HindIII, HinfI, HpaII, KpnI, NotI, PstI, SacI, Sau3A and TaqI.

Examples of restriction enzymes that cleave a site different from the recognition site include the type IIs restriction enzymes described in Table 1.

  The DNA material designed as described above can be obtained by a conventionally known genetic engineering technique. For example, a DNA material is obtained by synthesizing an oligo DNA having a base sequence of a designed DNA material and an oligo DNA having a complementary sequence thereof according to a conventional method and then hybridizing them to prepare a ds oligo DNA. Can do.

  The manufacturing method according to the embodiment will be described more specifically with reference to FIGS.

  FIG. 2 is a diagram for explaining an example of the design of a DNA material. In FIG. 2, the regions indicated by “1”, “2”, “3”, “4” and “5” each correspond to a part of the target DNA fragment. The target DNA fragment corresponds to a DNA fragment in which “1”, “2”, “3”, “4” and “5” are connected in this order. In FIG. 2, five DNA materials (DNA material (1) to DNA material (5) including base sequences corresponding to regions indicated by “1”, “2”, “3”, “4”, and “5”, respectively. )) Is designed. The DNA material (1) includes a base sequence for ligation (a region indicated by “BbsI” in FIG. 2) at its end. The region indicated by “BbsI” includes a recognition site for the IIs type restriction enzyme BbsI (indicated by the underline in FIG. 2). The DNA material (2) includes a linking base sequence having the same base sequence as the region indicated by “BbsI” at the end linked to the DNA material (1). The DNA material (1) and the DNA material (2) are designed so that the cleavage site cleaved by BbsI exists at a position adjacent to the region (base sequence for linking) indicated by “BbsI” (FIG. 2). In the base sequence, the portion indicated by the solid line is cut). Further, when cleaved with BbsI, a sticky end made of AGGA and a sticky end made of TCCT are generated, so that the DNA material (1) and the DNA material (2) have base sequences capable of hybridizing to each other at the cleavage sites. Is included. Hereinafter, in a manner that satisfies the same conditions, a region represented by “BsgI” (including a recognition site for IIs-type restriction enzyme BsgI) and a region represented by “EarI” (IIs-type restriction enzyme EarI The region indicated by “SapI” (including the recognition site of type IIs restriction enzyme SapI) can be designed.

  For the designed DNA material (1) to DNA material (5), for example, an oligo DNA having the base sequence of the designed DNA material and an oligo DNA having a complementary sequence thereof are synthesized according to a conventional method, and these are hybridized. It can be obtained by preparing ds oligo DNA.

  In the DNA material (1) to DNA material (5) designed and synthesized as described above, the base sequences for ligation have the same base sequences as the DNA materials to be linked adjacently. Using this base sequence for ligation, for example, the precursor DNA fragment can be obtained by ligation by utilizing the Gibson method described in Patent Document 3. Alternatively, a commercially available Gibson Assembly Master mix (manufactured by New England Biolabs) may be used to link DNA material (1) to DNA material (5) to obtain a precursor DNA fragment.

  FIG. 3 is a diagram for explaining an example of a process for obtaining a precursor DNA fragment. As shown in FIG. 3, first, the DNA material (1) and the DNA material (2) are treated with 5 'exonuclease (step 1). As a result, a 3 'overhang region including linking base sequences complementary to each other is formed in the DNA material (1) and the DNA material (2). Step 1 is intended to form a single-stranded overhang region in order to achieve specific hybridization in the next step. Therefore, in the example shown in FIG. 3, the treatment with 5 'exonuclease is performed, but the object of Step 1 can also be achieved by treating with 3' exonuclease instead of 5 'exonuclease. When treated with 3 'exonuclease, a 5' overhang region containing linking base sequences complementary to each other is formed.

  Next, the 3 'overhang regions of the DNA material (1) and the DNA material (2) are hybridized (step 2). When treated with 3 'exonuclease in step 1 instead of 5' exonuclease, the 5 'overhang region is hybridized.

  After hybridization, a gap (that is, a base sequence other than the base sequence for ligation digested with 5 ′ exonuclease is treated with DNA polymerase to replace 3 ′ exonuclease with 5 ′ exonuclease in Step 1. In the case of the treatment, a base sequence other than the base sequence for ligation digested by 3 ′ exonuclease is filled (step 3).

  Then, by treating with DNA ligase, the nicks (ie, the portion lacking the phosphodiester bond) are joined together (step 4), and a precursor DNA fragment is obtained (step 5). In FIG. 3, for the sake of simplicity, only the connecting portion of the DNA material (1) and the DNA material (2) is shown, but all of the DNA material (1) to the DNA material (5) are mixed. In the state, the process shown in FIG. 3 can also be performed.

  In addition, when synthesizing DNA material (1) to DNA material (5), a ds oligo DNA having a single-stranded overhang region at one end or both ends is designed and set so as to be in the state shown in Step 4 of FIG. By synthesizing, Step 1 to Step 3 in FIG. 3 (that is, 5 ′ exonuclease treatment and DNA polymerase treatment) can be omitted.

  The 5 ′ exonuclease treatment of Step 1 was performed using T5 exonuclease, T7 exonuclease, lambda exonuclease (Lambda phage RedA), Rac prophage RecE, 3 ′ non-thermophilic 5 ′ exonuclease lacking exonuclease activity, dNTP In the absence of 5 'exonuclease such as T5 DNA polymerase. The 3 'exonuclease treatment can be performed with a 3' exonuclease such as exonuclease III or KOD DNA polymerase. The single-stranded overhang region formed by 5 'exonuclease treatment, 3' exonuclease treatment or the like preferably has a sufficient length for hybridization. A person skilled in the art can easily set the optimum length to achieve specific hybridization. Specifically, the length of the single-stranded overhang region may be 20 to 100 bases, preferably 40 to 100 bases, and more preferably 60 to 100 bases.

  Hybridization in step 2 can be performed according to a conventional method. Further, at the time of hybridization, hybridization can be promoted by adding a crowding agent such as polyethylene glycol (PEG), Ficoll, or dextran.

  The DNA polymerase treatment in Step 3 is a treatment in which a single-stranded gap portion remaining after hybridization is made into a double strand by a DNA extension reaction with DNA polymerase. Examples of the DNA polymerase to be used include DNA polymerases such as DNA polymerase I, DNA polymerase II, and DNA polymerase III. More specific examples of the DNA polymerase include Phusion ™ polymerase, VENTRTM polymerase, Taq polymerase, Deep Vent polymerase, Pfu polymerase, 9 ° Nm polymerase, and the like. Among them, Phusion ™ polymerase is desirable because it exhibits high accuracy.

  The DNA ligase treatment in step 4 is a treatment for sealing the formed nick with DNA ligase. Examples of the DNA ligase include Taq ligase, Amprigase Thermostable DNA ligase (manufactured by Epicentre Biotechnologies), Thermostable Tfi DNA ligase (manufactured by Bioneer, Inc) and the like.

  The precursor DNA fragment obtained as described above is treated with an appropriate restriction enzyme (here, type IIs restriction enzymes BbsI, BsgI, EarI, and SapI) to remove unnecessary sequences (ligation base sequences). A target DNA fragment having a desired base sequence (here, a DNA fragment in which “1”, “2”, “3”, “4” and “5” are connected in this order) can be obtained.

  FIG. 4 is a diagram for explaining an example of a process for obtaining a target DNA fragment. First, the precursor DNA fragment is cleaved by treating with type IIs restriction enzyme BbsI. Since the BbsI cleavage sites (2 sites) are cohesive ends having complementary base sequences, the base sequences for ligation (regions indicated by “BbsI”) are removed from the precursor DNA fragments by hybridizing them. Can be obtained. As can be seen from the base sequence of the region indicated by “BbsI” shown in FIG. 2 and FIG. 4, the DNA material (1) and the DNA material (2) have a linking base sequence containing a recognition site for a type IIs restriction enzyme. It is designed to have a structure, and the recognition site is arranged in such a direction as to cut the inside of the DNA material (1) and the DNA material (2) when treated with the type IIs restriction enzyme. Therefore, by treating with a type IIs restriction enzyme that recognizes the recognition site, the linking base sequence (unnecessary sequence) containing the recognition site can be removed. In addition, the region indicated by “BsgI”, the region indicated by “EarI”, and the region indicated by “SapI”, which are linking base sequences, are similarly designed (not shown).

  By appropriately designing the type of IIs restriction enzyme and the recognition site to be used, a plurality of type IIs restriction enzymes are continuously treated in the same reaction vessel to remove a plurality of linking base sequences (unnecessary sequences). It is possible. For example, in the precursor DNA fragment, the base sequences recognized by the type IIs restriction enzyme contained in the base sequence for linking adjacent to each other through the target partial sequence are set to different base sequences (that is, different type IIs restriction). Base sequence recognized by the enzyme).

  The optimum conditions for restriction enzyme treatment can be the conditions recommended by the manufacturer of the restriction enzyme, that is, the recommended temperature and time using a buffer attached to the manufacturer. Moreover, it is preferable to add SAM as needed like restriction enzyme BsgI.

  Nicks generated by the restriction enzyme treatment can be sealed with DNA ligase, as in the production of the precursor DNA fragment.

  The present invention also relates to a DNA fragment obtained by the method described above.

  The present invention also relates to a kit for producing a DNA fragment having a desired base sequence, which contains the above-described DNA material as an oligo DNA. The kit according to one embodiment includes two or more oligo DNAs, and each oligo DNA is a base for ligation used for linking a partial base sequence of a desired base sequence and another DNA material. In the oligo DNA, the base sequence recognized by the restriction enzyme is treated with the restriction enzyme after the oligo DNA is ligated to remove the base sequence for ligation, and A part of the base sequence that is adjacent to the desired base sequence is arranged so that it can be ligated at the sites cleaved by restriction enzymes, and the oligo DNA covers the desired base sequence when all of them are linked. To do.

  The kit according to this embodiment may further include at least one selected from the group consisting of DNA ligase, DNA polymerase, and restriction enzyme. Preferred embodiments of the DNA ligase, DNA polymerase and restriction enzyme are as described above.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

[Example 1: Production of target DNA fragment (high GC content)]
A DNA fragment having the base sequence represented by SEQ ID NO: 11 designed with reference to the base sequence derived from spider silk protein was produced by the following method.

(1) Design and synthesis of oligo DNA A total of 10 types of oligo DNAs having the nucleotide sequences shown in SEQ ID NOs: 1 to 10 (hereinafter also simply referred to as oligo DNAs of SEQ ID NO: 1) were designed and synthesized (stocks) Company Fasmac commissioned synthesis).

  SEQ ID NO: 1 oligo DNA (Seg1 seal30 F), SEQ ID NO: 2 oligo DNA (Seg1 seal30 R), SEQ ID NO: 3 oligo DNA (Seg2 seal30 F), SEQ ID NO: 4 oligo DNA (Seg2 seal30 R), SEQ ID NO: 5 oligo DNA (Seg3seal30F), SEQ ID NO: 6 oligo DNA (Seg3seal30R), SEQ ID NO: 7 oligo DNA (Seg4seal30F), SEQ ID NO: 8 oligo DNA (Seg4seal30R), and SEQ ID NO: 9 Each of the oligo DNA (Seg5 seal30 F) and the oligo DNA of SEQ ID NO: 10 (Seg5 seal30 R) have complementary base sequences.

The region of 30 base pairs at the 3 ′ end of the oligo DNA of SEQ ID NO: 1 has the same base sequence as the region of 30 base pairs at the 5 ′ end of the oligo DNA of SEQ ID NO: 3 (linkage base sequence) The base sequence for ligation includes the base sequence (5′-GAAGAC-3 ′) recognized by the type IIs restriction enzyme BbsI.
The 28 base pair region at the 3 ′ end of the oligo DNA of SEQ ID NO: 3 has the same base sequence as the 28 base pair region at the 5 ′ end of the oligo DNA of SEQ ID NO: 5 (linkage base sequence). The base sequence for ligation includes a base sequence (5′-GTGCAG-3 ′) recognized by the type IIs restriction enzyme BsgI.
The region of 30 base pairs at the 3 ′ end of the oligo DNA of SEQ ID NO: 5 has the same base sequence as the region of 30 base pairs at the 5 ′ end of the oligo DNA of SEQ ID NO: 7 (linking base sequence) In the linking base sequence, a base sequence (5′-CTCTTC-3 ′) recognized by the IIs type restriction enzyme EarI is included.
The 30 base pair region at the 3 ′ end of the oligo DNA of SEQ ID NO: 7 has the same base sequence as the 30 base pair region at the 5 ′ end of the oligo DNA of SEQ ID NO: 9 (base sequence for linking) The base sequence for ligation includes a base sequence (5′-GCTCTTC-3 ′) recognized by the type IIs restriction enzyme SapI.
In addition, sticky ends (sticky ends) generated after cleaving with a type IIs restriction enzyme have base sequences that are complementary to each other.

(2) Ligation of ds oligo DNA (synthesis of precursor DNA fragment)
The oligo DNA of SEQ ID NO: 1 and the oligo DNA of SEQ ID NO: 2 were hybridized to obtain a ds oligo DNA (Seg1 seal30). The oligo DNA of SEQ ID NO: 3 and the oligo DNA of SEQ ID NO: 4 were hybridized to obtain ds oligo DNA (Seg2 seal30). The oligo DNA of SEQ ID NO: 5 and the oligo DNA of SEQ ID NO: 6 were hybridized to obtain ds oligo DNA (Seg3 seal30). The oligo DNA of SEQ ID NO: 7 and the oligo DNA of SEQ ID NO: 8 were hybridized to obtain a ds oligo DNA (Seg4 seal30). The oligo DNA of SEQ ID NO: 9 and the oligo DNA of SEQ ID NO: 10 were hybridized to obtain a ds oligo DNA (Seg5 seal30).

  Five types of ds oligo DNA of Seg1 seal30 to Seg5 seal30 were mixed so that the molar concentration {concentration (ng / μL) / fragment length (bp)} was equivalent. An equal amount of Gibson Assembly Master mix (manufactured by New England Biolabs) was added to the mixture (10 μL), and the mixture was reacted at 50 ° C. for 1 hour. That is, 5 ′ exonuclease generated single-stranded overhang regions with protruding 3 ′ ends at both ends of each ds oligo DNA. The overhang region includes part or all of the base sequence portion for linking each ds oligo DNA, and is annealed with a single-stranded overhang region of another ds oligo DNA having a complementary base sequence. Next, the gap between the annealed ds oligo DNA fragments was filled with DNA polymerase, and nicks were joined and ligated with DNA ligase to obtain a precursor DNA fragment. The precursor DNA fragment comprises a base sequence represented by SEQ ID NO: 1-a base sequence represented by SEQ ID NO: 3-a base sequence represented by SEQ ID NO: 5-a base sequence represented by SEQ ID NO: 7-a base sequence represented by SEQ ID NO: 9 The base sequences are connected in this order, and each base sequence has a base sequence from which one base sequence for connection is excluded.

  Instead of the oligo DNAs of SEQ ID NOs: 1 to 10, a short oligo DNA in which the base sequence for ligation of about 30 bp at the 3 ′ end or 5 ′ end is deleted from the oligo DNA of SEQ ID NOs: 1 to 10 is used. Thus, ds oligo DNA having a single-stranded overhang region can be prepared at the time of hybridization. In this case, the treatment with the 5 ′ exonuclease and DNA polymerase can be omitted, and the precursor DNA fragment can be ligated by ligation by ligation by treatment with DNA ligase following the annealing of the single-stranded overhang region. Can be obtained.

  The obtained precursor DNA fragment was cloned into a cloning vector obtained by improving pUC19 (having a sequence in which the region of lacZ was deleted from lac promoter and the EarI recognition site CTCTTC contained in the sequence was replaced with CTCCTC). NEB (R) Turbo Competent E.I. 4 μL of the cloning vector was added to 100 μL of E. coli competent cells (manufactured by New England Biolabs (product number: C2984I)) and mixed gently. After standing for 15 minutes on ice, a heat shock was performed at 42 ° C. for 30 seconds. Return to ice again and let stand for 2 minutes, then add 900 μL of SOC medium (sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, glucose 40 mM, magnesium sulfate 20 mM, magnesium chloride 20 mM) at 37 ° C. Incubated for about 30 minutes. After incubation, the mixture was centrifuged at 5000 rpm for 3 minutes. 900 μL of the supernatant was removed, and the precipitated fraction was resuspended with the remaining liquid and applied to an LB + amp plate. After culturing at 37 ° C. for 12 hours, colony PCR was performed on the formed colonies by the following method.

The formed colonies were picked up with a toothpick, 9.2 μL of sterile water, Primer F (SEQ ID NO: 12) 0.4 μL, Primer R (SEQ ID NO: 13) 0.4 μL, PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio) Aseptically added to a 1.5 ml tube containing 10 μL. A tube was set in a thermal cycler (Applied Biosystems Veriti), and PCR was performed under the following conditions.
(I) 1 minute at 98 ° C (ii) 10 times at 98 ° C, 5 seconds at 50 ° C, and 20 seconds at 72 ° C 30 times (iii) 2 minutes at 72 ° C

After PCR, the amplified DNA fragment was used and stored at 4 ° C. until a sequencing reaction was performed by the following method.
To 3 μL of the amplified DNA fragment (200 to 600 ng / μL), sterilized water 11 μL, 5 × Sequence Buffer 4 μL, Primer R (10 pmol) 1 μL (SEQ ID NO: 13) and BigDye (registered trademark) Terminator v3.1 Ready Reaction Mix ( 1 μL of Fisher Scientific Inc.) (1) 96 ° C. for 1 minute, (2) 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, 60 ° C. for 4 minutes 25 times, and store at 15 ° C. did.

  To 20 μL of the sequencing reaction completed solution stored at 15 ° C., 5 μL of 125 mM EDTA and 60 μL of 100% ethanol were added, mixed, and allowed to stand in the dark for 15 minutes. After standing, it was centrifuged at 10,000 × g for 20 minutes, and the supernatant was removed with a pipette. 60 μL of 70% ethanol was added to the precipitate fraction, centrifuged at 10,000 × g for 10 minutes, and the supernatant was removed with a pipette. It was left to stand in a 65 ° C. incubator for 10 minutes to dry. After drying, 20 μL of Hidi formamide was added and stirred thoroughly. The entire amount of the stirring solution was added to a 96-well plate for sequencing, and set in a sequencer (Applied Biosystems, 3730 DNA Analyzer) to confirm the base sequence. The size of the amplified DNA fragment was confirmed by agarose gel electrophoresis. As a result, a band was confirmed in the vicinity of the theoretical size (1948 bp), and it was confirmed that the base sequence also coincided with the theoretical sequence.

(3) Removal of unnecessary sequence (base sequence for ligation) by restriction enzyme treatment The unnecessary sequence (base sequence for ligation) was removed from the plasmid DNA containing the precursor DNA fragment obtained in (2) above by the following method.

[BbsI processing]
50 μg of the plasmid DNA containing the precursor DNA fragment obtained in the above (2), sterilized water 890 μL, Cutsmart Buffer (New England Biolabs Japan Inc., product number: B7204S) 100 μL, type IIs restriction enzyme BbsI (5 U / μL) The mixture was added and reacted at 37 ° C for 60 minutes, and then treated at 65 ° C for 20 minutes. After the reaction solution is cooled to 15 ° C., 10 μL of 100 mM ATP and 1 μL of T4 DNA ligase (2000 U / μL) are added to 1000 μL of the reaction solution, reacted at 16 ° C. for 60 minutes, and then at 65 ° C. Treated for 10 minutes.

[BsgI processing]
After cooling the treatment solution after the treatment with BbsI to 15 ° C., 10 μL of BsgI (5 U / μL) and 2.5 μL of SAM (80 mM) are added to the treatment solution, reacted at 37 ° C. for 60 minutes, and then at 65 ° C. for 20 minutes. Processed. After the reaction solution is cooled to 15 ° C., 10 μL of 100 mM ATP and 1 μL of T4 DNA ligase (2000 U / μL) are added to 1000 μL of the reaction solution, reacted at 16 ° C. for 60 minutes, and then at 65 ° C. Treated for 10 minutes.

[EarI processing]
After the treatment solution after the treatment with BbsI and BsgI was cooled to 15 ° C., 2.5 μL of EarI (20 U / μL) was added to the treatment solution, reacted at 37 ° C. for 60 minutes, and then treated at 65 ° C. for 20 minutes. After the reaction solution is cooled to 15 ° C., 10 μL of 100 mM ATP and 1 μL of T4 DNA ligase (2000 U / μL) are added to 1000 μL of the reaction solution, reacted at 16 ° C. for 60 minutes, and then at 65 ° C. Treated for 10 minutes.

[SapI processing]
The treatment solution after the treatment with BbsI, BsgI and EarI was cooled to 15 ° C., 5 μL of SapI (10 U / μL) was added to the treatment solution, reacted at 37 ° C. for 60 minutes, and then treated at 65 ° C. for 20 minutes. After the reaction solution is cooled to 15 ° C., 10 μL of 100 mM ATP and 1 μL of T4 DNA ligase (2000 U / μL) are added to 1000 μL of the reaction solution, reacted at 16 ° C. for 60 minutes, and then at 65 ° C. Treated for 10 minutes.

[Purification]
Plasmid DNA is purified from the treated solution after treatment with BbsI, BsgI, EarI and SapI using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega, product number A9280) according to the attached catalog. did.

[Transformation]
NEB (R) Turbo Competent E.I. 4 μL of the recovered plasmid DNA was added to 100 μL of E. coli competent cells (manufactured by New England Biolabs (product number: C2984I)) and mixed gently. After standing for 15 minutes on ice, a heat shock was performed at 42 ° C. for 30 seconds. After returning to ice again and allowing to stand for 2 minutes, 900 μL of SOC medium was added and incubated at 37 ° C. for about 30 minutes. After incubation, the mixture was centrifuged at 5000 rpm for 3 minutes. 900 μL of the supernatant was removed, and the precipitated fraction was resuspended with the remaining liquid and applied to an LB + amp plate.

[Plasmid DNA extraction]
After culturing at 37 ° C. for 12 hours, the formed colony was picked up with a toothpick and aseptically added to a culture tube containing 1.5 ml of LB + amp solution. The lid was capped and cultured overnight at 37 ° C. and 200 rpm. The culture was transferred to a 1.5 ml tube and centrifuged at 14,000 rpm for 5 minutes. The supernatant was discarded, and the plasmid was extracted using AccuPrep (registered trademark) Plasmid Extraction Kit (manufactured by Bioneer, (product number: K3030)) according to the manual attached to the kit.

[Sequence analysis]
To 1 μL of the extracted plasmid DNA (200 to 600 ng / μL), sterilized water 11 μL, 5 × Sequence Buffer 4 μL, Primer R (10 pmol) 1 μL (SEQ ID NO: 13) and BigDye® Terminator v3.1 Ready Reaction Mix (Ready Reaction Mixer) 1 μL of Fisher Scientific Inc.) (1) 96 ° C. for 1 minute, (2) 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, 60 ° C. for 4 minutes 25 times, and store at 15 ° C. did.

  To 20 μL of the sequencing reaction completed solution stored at 15 ° C., 5 μL of 125 mM EDTA and 60 μL of 100% ethanol were added, mixed, and allowed to stand in the dark for 15 minutes. After standing, it was centrifuged at 10,000 × g for 20 minutes, and the supernatant was removed with a pipette. 60 μL of 70% ethanol was added to the precipitate fraction, and the supernatant was removed with a pipette. It was left to stand in a 65 ° C. incubator for 10 minutes to dry. After drying, 20 μL of Hi-di formamide was added and sufficiently stirred. The whole amount of the stirring solution was added to a 96-well plate for sequencing, and set in a sequencer (Applied Biosystems, 3730 DNA Analyzer) to confirm the sequence.

  As a result, a DNA fragment having a base sequence (desired base sequence) that matches the theoretical sequence (SEQ ID NO: 11) from which the linking base sequence (unnecessary sequence) including the base sequence recognized by the restriction enzyme has been removed is obtained. I was able to confirm.

Claims (16)

A method of assembling two or more DNA materials to produce a DNA fragment having a desired base sequence,
(1) linking two or more DNA materials to obtain a precursor DNA fragment in which a part of a desired base sequence and a base sequence for linking are alternately arranged;
(2) treating the precursor DNA fragment with a restriction enzyme and removing the linking base sequence to obtain a DNA fragment having a desired base sequence,
The precursor DNA fragment has a nucleotide sequence that is recognized by a restriction enzyme. When the precursor DNA fragment is cleaved by treatment with the restriction enzyme, the nucleotide sequence for ligation is removed and the desired DNA sequence is adjacent via the nucleotide sequence for ligation. A part of the base sequence is arranged so that the cleaved sites can be linked to each other. Obtaining a precursor DNA fragment comprises:
(1-1) preparing two or more DNA materials satisfying the following (a) to (e):
(A) The DNA material has a single-stranded overhang region with a protruding 3 ′ end or 5 ′ end, respectively.
(B) the single-stranded overhang region of the DNA material includes a base sequence capable of hybridizing with the single-stranded overhang region of another DNA material linked adjacently;
(C) Each of the DNA materials includes a part of a base sequence continuous with a desired base sequence and a base sequence for linking, and at least one of the base sequences for linking included in two DNA materials that are adjacently linked , The linking base sequence overlaps with all the base sequences of the other linking base sequence,
(D) each of the DNA materials has a cleavage site cleaved by a restriction enzyme at a position adjacent to the linking base sequence, and includes a base sequence recognized by the restriction enzyme;
(E) The two DNA materials that are adjacently linked contain base sequences whose cleavage sites can hybridize to each other,
(1-2) Hybridizing a single-stranded overhang region of a DNA material with a single-stranded overhang region of another DNA material that is adjacently linked to the DNA material. .   (1-3) The method according to claim 2, further comprising a step of treating in the order of DNA polymerase and DNA ligase after step (1-2). Obtaining a DNA fragment having a desired base sequence,
The method according to any one of claims 1 to 3, comprising treating with a restriction enzyme and then treating with a DNA ligase.   The method according to claim 3, wherein the DNA polymerase is at least one DNA polymerase selected from the group consisting of DNA polymerases I, II and III.   The method according to claim 3 or 4, wherein the DNA ligase is at least one DNA ligase selected from the group consisting of Taq ligase and Tfi DNA ligase.   The method according to claim 2, wherein the single-stranded overhang region of the DNA material is formed by treating double-stranded DNA with 5 'exonuclease.   The method according to claim 7, wherein the 5 'exonuclease is at least one 5' exonuclease selected from the group consisting of T7 exonuclease, lambda exonuclease, RecE and T5 exonuclease.   The method according to any one of claims 1 to 8, wherein the restriction enzyme is a type II restriction enzyme.   The method according to claim 9, wherein the type II restriction enzyme is a type IIs restriction enzyme.   Type IIs restriction enzymes are AarI, AcuI, AlwI, BbsI, BbvI, BccI, BceAI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsgI, BsmPI, BsmPI, BsmPI, BsmPI, BsmPI BtgZI, BstCI, EarI, EciI, Ecop15I, FauI, FokI, HgaI, HphI, HpyAV, Ksp632I, MboII, MmeI, Mn1I, SapI and SfaNI. 10. The method according to 10.   The method according to claim 11, wherein the linking base sequence has two base sequences arranged such that base sequences recognized by the same type IIs restriction enzyme have a palindromic structure. A method of assembling two or more DNA materials to produce a DNA fragment having a desired base sequence,
(1) preparing a double-stranded (ds) DNA material satisfying the following (a) to (c):
(A) The two dsDNA materials to be linked adjacently have the same linking base sequence,
(B) Each of the linking base sequences has a base sequence arranged so that two base sequences recognized by the same type IIs restriction enzyme have a palindromic structure.
(C) The base sequence recognized by the type IIs restriction enzyme is at a position where the base sequence for ligation is removed by cleaving with the type IIs restriction enzyme after ligation of the dsDNA material.
(2) treating the dsDNA material with a 5 ′ exonuclease to form a single-stranded overhang region with a protruding 3 ′ end;
(3) hybridizing a single-stranded overhang region of a dsDNA material with a single-stranded overhang region of another dsDNA material;
(4) a step of treating with DNA polymerase and filling a gap generated by hybridization;
(5) treating with DNA ligase and joining the nicks to obtain a precursor DNA fragment;
(6) treating the precursor DNA fragment with one or more type IIs restriction enzymes that recognize a base sequence recognized by a type IIs restriction enzyme present in the linking base sequence;
(7) a step of hybridizing complementary sticky ends generated by cleavage with a type IIs restriction enzyme, treating with a DNA ligase, and joining nicks.   The base sequence recognized by the type IIs restriction enzyme contained in the base sequence for ligation adjacent through a part of the desired base sequence in the precursor DNA fragment is a different base sequence, respectively. Method. A kit for producing a DNA fragment having a desired base sequence, comprising two or more oligo DNAs,
Each of the oligo DNAs includes a partial base sequence of a desired base sequence and a base sequence for linking used for linking with other DNA materials,
In the oligo DNA, the base sequence recognized by the restriction enzyme is treated with the restriction enzyme after ligation of the oligo DNA, so that the base sequence for ligation is removed, and the desired base adjacent through the base sequence for ligation is removed. A part of the base sequence of the sequence is arranged so that it can be linked at the sites cleaved with restriction enzymes,
The oligo DNA covers the desired base sequence when all are linked.   A DNA fragment obtained by the method according to any one of claims 1 to 14.

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