WO2018147071A1 - Method for obtaining target dna fragments - Google Patents

Method for obtaining target dna fragments Download PDF

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WO2018147071A1
WO2018147071A1 PCT/JP2018/001958 JP2018001958W WO2018147071A1 WO 2018147071 A1 WO2018147071 A1 WO 2018147071A1 JP 2018001958 W JP2018001958 W JP 2018001958W WO 2018147071 A1 WO2018147071 A1 WO 2018147071A1
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dsdna
fragment
type iis
iis restriction
restriction enzyme
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PCT/JP2018/001958
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French (fr)
Japanese (ja)
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望 谷内江
足立　大輔
知香子 佐藤
潤一 菅原
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Ｓｐｉｂｅｒ株式会社
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Priority to JP2018567351A priority Critical patent/JPWO2018147071A1/en
Publication of WO2018147071A1 publication Critical patent/WO2018147071A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

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  • the present invention relates to a method for obtaining a target DNA fragment.
  • the type IIs restriction enzyme is a restriction enzyme in which the base sequence (recognition site) recognized by the restriction enzyme is different from the site to be cleaved (cleavage site).
  • Non-Patent Document 1 a method of introducing a recognition site of a type IIs restriction enzyme into a cloning vector, subcloning a DNA fragment in a site-specific manner using a type IIs restriction enzyme (Patent Document 1), and a recognition site and a cleavage site
  • Patent Document 1 a method of introducing a recognition site of a type IIs restriction enzyme into a cloning vector, subcloning a DNA fragment in a site-specific manner using a type IIs restriction enzyme (Patent Document 1), and a recognition site and a cleavage site
  • a method Non-Patent Document 1 is known in which nine double-stranded (ds) DNA fragments are ligated in a specific order in a desired order.
  • Non-Patent Document 1 a target DNA fragment can be obtained in one reaction container using one type of type IIs restriction enzyme.
  • the number of dsDNA fragments that can be ligated in one reaction vessel is limited, and the length of dsDNA fragments that can be prepared without requiring complicated operations is about 100 to 1000 base pairs (bp).
  • the method described in Non-Patent Document 1 is not suitable for obtaining a target DNA fragment of a polymer.
  • the present invention provides a method for obtaining a target DNA fragment by orderly linking a plurality of DNA fragments using a type IIs restriction enzyme even if the target DNA fragment is a polymer.
  • the purpose is to provide.
  • the present invention relates to the following inventions, for example.
  • the first to m 1 m 1 DNA fragments are the first to n 1 n 1 types (where n 1 and m 1 represent a natural number of 3 or more, and n 1 ⁇ m 1 .
  • Dividing the reaction system into one or more reaction systems comprising: (Iv) In one or a plurality of reaction systems, treatment with n1 type IIs type restriction enzymes is performed, and DNA ligation fragments in the reaction system are ligated in a specific order in a sticky end formed by cleavage with a type IIs restriction enzyme. Process, (V) repeating steps (iii) and (iv) until the target DNA fragment is obtained; A method comprising: [2] The first to m 2 m 2 DNA fragments are the first to n 2 n 2 types (where n 2 and m 2 represent a natural number of 3 or more and n 2 ⁇ m 2 .
  • step (1) to (5) (1) below (a) providing - a m 2 pieces of double-stranded (ds) DNA fragment that satisfies (e), (A) the first dsDNA fragment has a base sequence recognized by the first type IIs restriction enzyme at the end opposite to the end linked to the second dsDNA fragment; At the end portion on the side to be linked to the dsDNA fragment, the base sequence recognized by the second type IIs restriction enzyme and the sticky end formed by cleavage with the second type IIs restriction enzyme are the second dsDNA.
  • the k 2 dsDNA fragment (where k 2 represents a natural number of 2 or more and (n 2 ⁇ 1) or less) is connected to the terminal portion on the side linked to the (k 2 ⁇ 1) dsDNA fragment.
  • nucleotide sequence recognized by a type IIs restriction enzyme of the k 2 is a sticky end dsDNA fragment of the (k 2 -1) has a nucleotide sequence comprising a complementary, and the end portion of the side connecting the dsDNA fragment of the (k 2 +1), nucleotide sequences recognized by the type IIs restriction enzyme of the (k 2 +1), and the sticky end formed by being cut with a type IIs restriction enzyme (k 2 +1) has a nucleotide sequence comprising the complement of the sticky ends of dsDNA fragments of the (k 2 +1), (C) dsDNA fragments of the n 2 is first in the end on the side connected to the dsDNA fragment (n 2 -1), the base sequence recognized by a type IIs restriction enzyme of the n 2, and the n 2 A sticky end formed by cleaving with a type IIs restriction enzyme has a base sequence
  • the sticky end has a base sequence that is complementary to the sticky end of the (l 2 -1) dsDNA fragment, and the end portion on the side linked to the (l 2 +1) dsDNA fragment has ( l 2 + 1 ⁇ a ⁇ n 2 ) type IIs restriction enzyme and a sticky end formed by cleavage with the (l 2 + 1 ⁇ a ⁇ n 2 ) type IIs restriction enzyme having a sticky end and a base sequence consisting complementary of dsDNA fragments (l 2 +1) (provided that a is a number in the integer portion of a l 2 / n 2.), (E) The m 2 dsDNA fragment has a base that is recognized by the (m 2 -b ⁇ n 2 ) type IIs restriction enzyme at the end
  • the target DNA fragment can be obtained by linking a plurality of DNA fragments in a specific order using a type IIs restriction enzyme.
  • the method of the present invention according to the above [2] has an advantage that automation is easy because the dsDNA fragment or the dsDNA ligation fragment is regularly and mechanically subjected to a ligation reaction.
  • a method for obtaining a target DNA fragment comprises the steps of: 1st to m 1 m 1 DNA fragments, 1 to n 1 n 1 types (where n 1 and m 1 are 3 or more)
  • the target DNA fragment is ligated in ascending order from the first DNA fragment to the m1 DNA fragment using a type IIs restriction enzyme that represents a natural number and n 1 ⁇ m 1 ).
  • m 1 DNA fragments include k 1 (where k 1 represents a natural number of 2 or more and k 1 ⁇ n 1 ⁇ 1).
  • each of the m 1 DNA fragments has a base sequence recognized by different type IIs restriction enzymes at both ends, and the adjacent DNA fragments to be ligated are the same at the ligation ends. And a base sequence that forms a complementary sticky end when cleaved by the type IIs restriction enzyme.
  • the method for obtaining the DNA fragment of interest according to another embodiment, the first to m 2 m 2 of pieces of DNA fragments, the first to n 2 of n 2 kinds (although, n 2 and m 2 is 3 or more It represents a natural number, and a n 2 ⁇ m 2.) using type IIs restriction enzymes, the target DNA fragments by connecting to line up in ascending order from the first DNA fragment to a DNA fragment of the m 2
  • the following steps (1) to (5) are provided.
  • the first dsDNA fragment has a base sequence recognized by the first type IIs restriction enzyme at the end opposite to the end linked to the second dsDNA fragment; At the end portion on the side to be linked to the dsDNA fragment, the base sequence recognized by the second type IIs restriction enzyme and the sticky end formed by cleavage with the second type IIs restriction enzyme are the second dsDNA. It has a base sequence that is complementary to the sticky end of the fragment.
  • the k 2 dsDNA fragment (where k 2 represents a natural number of 2 or more and (n 2 ⁇ 1) or less) is connected to the terminal portion on the side linked to the (k 2 ⁇ 1) dsDNA fragment.
  • nucleotide sequence recognized by a type IIs restriction enzyme of the k 2 is a sticky end dsDNA fragment of the (k 2 -1) has a nucleotide sequence comprising a complementary, and the end portion of the side connecting the dsDNA fragment of the (k 2 +1), nucleotide sequences recognized by the type IIs restriction enzyme of the (k 2 +1), and the sticky end formed by being cut with a type IIs restriction enzyme (k 2 +1) has a nucleotide sequence comprising the complement of the sticky ends of dsDNA fragments of the (k 2 +1).
  • (C) dsDNA fragments of the n 2 is first in the end on the side connected to the dsDNA fragment (n 2 -1), the base sequence recognized by a type IIs restriction enzyme of the n 2, and the n 2
  • a sticky end formed by cleaving with a type IIs restriction enzyme has a base sequence complementary to the sticky end of the (n 2 -1) dsDNA fragment, and the (n 2 +1) dsDNA fragment;
  • the base sequence recognized by the first type IIs restriction enzyme and the sticky end formed by cleavage with the first type IIs restriction enzyme are the (n 2 +1) th dsDNA. It has a base sequence that is complementary to the sticky end of the fragment.
  • the l 2nd dsDNA fragment (where l 2 represents a natural number not less than (n 2 +1) and not more than (m 2 ⁇ 1)) is connected to the (l 2 ⁇ 1) dsDNA fragment.
  • the end portion is formed by cutting with type IIs restriction enzymes of the (l 2 -a ⁇ n 2) nucleotide sequences recognized by the type IIs restriction enzymes, and the (l 2 -a ⁇ n 2)
  • the sticky end has a base sequence that is complementary to the sticky end of the (l 2 -1) dsDNA fragment, and the end portion on the side linked to the (l 2 +1) dsDNA fragment has ( l 2 + 1 ⁇ a ⁇ n 2 ) type IIs restriction enzyme and a sticky end formed by cleavage with the (l 2 + 1 ⁇ a ⁇ n 2 ) type IIs restriction enzyme having a sticky end and a base sequence consisting complementary of dsDNA fragments (l 2 +1) (provided that a is
  • the m 2 dsDNA fragment has a base that is recognized by the (m 2 -b ⁇ n 2 ) type IIs restriction enzyme at the end of the side linked to the (m 2 -1) dsDNA fragment. And a base sequence in which the sticky end formed by cleavage with the (m 2 -b ⁇ n 2 ) type IIs restriction enzyme is complementary to the sticky end of the (m 2 -1) dsDNA fragment And is recognized by the (m 2 + 1 ⁇ b ⁇ n 2 ) type IIs restriction enzyme at the end opposite to the end connected to the (m 2 ⁇ 1) dsDNA fragment. (Where b is the numerical value of the integer part of m 2 / n 2 ).
  • dsDNA ligated fragments are placed in different reaction vessels, each containing (n 2 ⁇ 1) so as to contain continuous dsDNA fragments, and if necessary, the remaining dsDNA ligated fragments are A step of placing in a reaction vessel and further ligating the dsDNA ligation fragment by the same operation as in step (3). (5) Repeating step (4) until all dsDNA fragments are linked.
  • FIG. 1 is a diagram for explaining the principle of obtaining a target DNA fragment by a method according to an embodiment of the present invention.
  • ten dsDNA fragments (DNA fg1 to fg10) are used as a first dsDNA fragment (DNA fg1) using three types of type IIs restriction enzymes (first to third RE).
  • first to third RE three types of type IIs restriction enzymes
  • regions indicated by “first RE” to “third RE” are regions including base sequences recognized by the first to third type IIs restriction enzymes, respectively.
  • each of the regions indicated by the first to third REs is a region comprising a part of the base sequence of the target DNA fragment ( It is designed so that a sticky end having a base sequence complementary to the sticky end of the dsDNA fragment that is cut off from the white region in FIG.
  • FIG. 1 (A) shows 10 dsDNA fragments (DNA fg1 to fg10).
  • each of the 10 dsDNA fragments has a base sequence recognized by a different type IIs restriction enzyme at both ends, and the adjacent DNA fragments to be ligated are linked to each other.
  • the first dsDNA fragment (DNA fg1) has a first type IIs restriction enzyme at the end opposite to the end linked to the second dsDNA fragment (DNA fg2).
  • a region containing the base sequence recognized by (1) RE (first RE), and the end portion on the side linked to the second dsDNA fragment contains the base sequence recognized by the second type IIs restriction enzyme
  • the end has a base sequence that is complementary to the sticky end of the second dsDNA fragment.
  • the second dsDNA fragment has a region (second RE) containing a base sequence recognized by the second type IIs restriction enzyme at the terminal end linked to the first dsDNA fragment; Furthermore, the sticky end formed by cleaving with the second type IIs restriction enzyme is complementary to the sticky end of the first dsDNA fragment in a region consisting of a part of the base sequence of the target DNA fragment (open region). It has a base sequence that becomes The second dsDNA fragment also has a region (third RE) containing a base sequence recognized by the third type IIs restriction enzyme at the terminal portion on the side linked to the third dsDNA fragment (DNA fg3). And a sticky end formed by cleaving with a third type IIs restriction enzyme in a region consisting of a part of the base sequence of the target DNA fragment (outlined region) is the third dsDNA fragment The base sequence is complementary to the sticky end of.
  • the third dsDNA fragment has a region (third RE) containing a base sequence recognized by the third type IIs restriction enzyme at the terminal portion on the side linked to the second dsDNA fragment. Furthermore, the sticky end formed by cleaving with the third type IIs restriction enzyme in the region consisting of a part of the base sequence of the target DNA fragment (open region) is the sticky end of the second dsDNA fragment. And has a base sequence that is complementary.
  • the third dsDNA fragment also has a region (first RE) containing a base sequence recognized by the first type IIs restriction enzyme at the end portion on the side linked to the fourth dsDNA fragment (DNA fg4). And a sticky end formed by cleaving with the first type IIs restriction enzyme in a region consisting of part of the base sequence of the target DNA fragment (outlined region) is sticky to the fourth dsDNA fragment It has a base sequence that is complementary to the terminal.
  • the l-th dsDNA fragment (DNA fgl: where l represents a natural number of 4 or more and 9 or less) is attached to the end (l ⁇ ) of the side linked to the (l-1) -th dsDNA fragment.
  • the l-th dsDNA fragment is also a region containing the base sequence recognized by the (l + 1-a ⁇ 3) type IIs restriction enzyme (the (( (l + 1-a ⁇ 3) RE), and the (l + 1-a ⁇ 3) type IIs restriction enzyme in the region consisting of a part of the base sequence of the target DNA fragment (outlined region)
  • the sticky end formed by cleaving in step 1 has a base sequence that is complementary to the sticky end of the (l + 1) th dsDNA fragment.
  • the tenth dsDNA fragment (DNA fg10) is a base that is recognized by the (10-b ⁇ 3) type IIs restriction enzyme at the end linked to the ninth dsDNA fragment (DNA fg9). It has a region containing the sequence (first RE) and is further cleaved with the first type IIs restriction enzyme in a region consisting of part of the base sequence of the target DNA fragment (outlined region)
  • the sticky end to be prepared has a base sequence that is complementary to the sticky end of the ninth dsDNA fragment.
  • the tenth dsDNA fragment also contains a base sequence recognized by the (10 + 1-b ⁇ 3) type IIs restriction enzyme at the end opposite to the end linked to the ninth dsDNA fragment. It has a region (second RE).
  • b is a numerical value of the integer part of m / n, and is 3 in the example of FIG.
  • the dsDNA fragment described above may be dsDNA having blunt ends at both ends, or dsDNA having a single-stranded overhang region at one end or both ends.
  • the dsDNA fragment can be obtained by a conventionally known genetic engineering technique.
  • a dsDNA fragment is obtained by synthesizing an oligo DNA having a designed base sequence of a dsDNA fragment and an oligoDNA having a complementary sequence thereof according to a conventional method and then hybridizing them to prepare a ds oligo DNA. Can do.
  • reaction system 1 includes DNA fg1 and DNA fg2
  • reaction system 2 includes DNA fg3 and DNA fg4
  • reaction system 3 includes DNA fg5 and DNA fg6
  • reaction system 4 includes DNA fg7 and DNA fg6.
  • the reaction system 5 contains fg8 and DNA fg9 and DNA fg10. If there is a remaining dsDNA fragment (for example, if DNA fg11 is present), the remaining dsDNA fragment may be used as the reaction system 6.
  • All the first to third types of IIs type restriction enzymes are added to each reaction system.
  • one type IIs type restriction enzyme solution (a solution containing all type IIs type restriction enzymes) may be prepared and dispensed to each reaction system, which simplifies the process and facilitates automation. become.
  • type IIs restriction enzymes examples include type IIs restriction enzymes described in Table 1.
  • each dsDNA fragment is cleaved with a type IIs restriction enzyme.
  • the adjoining dsDNA fragments each form a complementary sticky end at the end of the connecting side.
  • the adjoining dsDNA fragment specifically hybridizes via the sticky end.
  • Hybridization can be promoted by adding a crowding agent such as polyethylene glycol (PEG), Ficoll, or dextran to the reaction system.
  • PEG polyethylene glycol
  • Ficoll Ficoll
  • dextran dextran
  • nicks that is, a portion lacking a phosphodiester bond
  • DNA ligase examples include Taq ligase, Amprigase Thermostable DNA ligase (Epicentre Biotechnologies), Thermostable Tfi DNA ligase (Bioneer, Inc).
  • reaction system 1 a dsDNA-linked fragment in which DNA fg1 and DNA fg2 are linked in the region indicated by the second RE is obtained.
  • the region indicated by the second RE has been removed, and the region indicated by the first RE and the region indicated by the third RE are included at both ends.
  • reaction systems 2-5 The same applies to reaction systems 2-5.
  • the obtained dsDNA ligation fragments are put in two, each in a different reaction vessel so as to contain continuous dsDNA fragments.
  • the dsDNA ligation fragment obtained in the reaction system 1 and the dsDNA ligation fragment obtained in the reaction system 2 were placed in the first reaction vessel and obtained in the reaction system 3.
  • the dsDNA ligation fragments and the dsDNA ligation fragments (two in total) obtained in the reaction system 4 are put in the second reaction vessel.
  • the dsDNA ligation fragment obtained in the reaction system 1 includes a first dsDNA fragment and a second dsDNA fragment
  • the dsDNA ligation fragment obtained in the reaction system 2 includes the third dsDNA fragment and the fourth dsDNA fragment. Therefore, the first reaction vessel (reaction system) containing these (two pieces) contains the first to fourth continuous dsDNA fragments.
  • the dsDNA ligation fragment obtained in the reaction system 5 is the remaining one.
  • the dsDNA ligation fragment (one piece) obtained in the reaction system 5 is put into the third reaction vessel.
  • each dsDNA ligation fragment is cleaved by the type IIs restriction enzyme, and each adjoining dsDNA ligation fragment forms a complementary sticky end at the end of the ligation side. Then, the adjoining dsDNA ligation fragments specifically hybridize via the sticky ends, and nicks are joined by DNA ligase to obtain a larger dsDNA ligation fragment.
  • the details of the reaction are as described above.
  • the region indicated by the first RE and the region indicated by the second RE are included at both ends of the obtained target DNA fragment. These regions can be used for operations such as cloning of a target DNA fragment into a cloning vector, for example.

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Abstract

The present invention provides a method whereby n types of IIs restriction enzymes are used, first to m-th DNA fragments are linked so as to be arranged in ascending order, and a target DNA fragment is obtained. The method comprises: a step (i) in which an m number of DNA fragments are separated into a plurality of reaction systems; a step (ii) in which DNA fragments in each reaction system are linked order-specifically, using IIs restriction enzymes; a step (iii) in which the obtained linking DNA fragments are separated into at least one reaction system; a step (iv) in which the linking DNA fragments in each reaction system are linked order-specifically, using IIs restriction enzymes; and a step (v) in which steps (iii) and (iv) are repeated until the target DNA fragment is obtained.

Description

目的ＤＮＡ断片を得る方法Method for obtaining a target DNA fragment
　本発明は、目的ＤＮＡ断片を得る方法に関する。 The present invention relates to a method for obtaining a target DNA fragment.
　ＩＩｓ型制限酵素は、当該制限酵素により認識される塩基配列（認識部位）と、切断される部位（切断部位）が異なる制限酵素である。 The type IIs restriction enzyme is a restriction enzyme in which the base sequence (recognition site) recognized by the restriction enzyme is different from the site to be cleaved (cleavage site).
　これまでに、ＩＩｓ型制限酵素の認識部位をクローニングベクターに導入し、ＩＩｓ型制限酵素を使用して部位特異的にＤＮＡ断片をサブクローニングする方法（特許文献１）、及び認識部位と切断部位との位置関係を適切に設計することにより、ＩＩｓ型制限酵素による切断で形成される粘着末端（スティッキー・エンド）の塩基配列を制御し、１反応容器中で１種類のＩＩｓ型制限酵素を利用して、９個の二本鎖（ｄｓ）ＤＮＡ断片を目的とする順に順特異的に連結する方法（非特許文献１）が知られている。 Up to now, a method of introducing a recognition site of a type IIs restriction enzyme into a cloning vector, subcloning a DNA fragment in a site-specific manner using a type IIs restriction enzyme (Patent Document 1), and a recognition site and a cleavage site By appropriately designing the positional relationship, the base sequence of the sticky end formed by cleavage with a type IIs restriction enzyme is controlled, and one type of type IIs restriction enzyme is used in one reaction vessel. In addition, a method (Non-Patent Document 1) is known in which nine double-stranded (ds) DNA fragments are ligated in a specific order in a desired order.
米国特許出願公開第２０１０／０２９１６３３号明細書US Patent Application Publication No. 2010/0291633
　非特許文献１に記載の方法では、１種類のＩＩｓ型制限酵素を利用して１反応容器中で目的ＤＮＡ断片を得ることができる。しかしながら、１反応容器中で連結できるｄｓＤＮＡ断片の数には限りがあり、また、煩雑な操作を要することなく準備できるｄｓＤＮＡ断片の長さが１００～１０００塩基対（ｂｐ）程度であることから、非特許文献１に記載の方法は、高分子の目的ＤＮＡ断片を得ることには向いていない。 In the method described in Non-Patent Document 1, a target DNA fragment can be obtained in one reaction container using one type of type IIs restriction enzyme. However, the number of dsDNA fragments that can be ligated in one reaction vessel is limited, and the length of dsDNA fragments that can be prepared without requiring complicated operations is about 100 to 1000 base pairs (bp). The method described in Non-Patent Document 1 is not suitable for obtaining a target DNA fragment of a polymer.
　本発明は、上述した問題に鑑み、目的ＤＮＡ断片が高分子の場合であっても、ＩＩｓ型制限酵素を利用して、複数のＤＮＡ断片を順特異的に連結して目的ＤＮＡ断片を得る方法を提供することを目的とする。 In view of the above-described problems, the present invention provides a method for obtaining a target DNA fragment by orderly linking a plurality of DNA fragments using a type IIs restriction enzyme even if the target DNA fragment is a polymer. The purpose is to provide.
　本発明は、例えば、以下の各発明に関する。
［１］
　第１～第ｍ１のｍ１個のＤＮＡ断片を、第１～第ｎ１のｎ１種類（ただし、ｎ１及びｍ１は３以上の自然数を表し、かつｎ１＜ｍ１である。）のＩＩｓ型制限酵素を使用して、第１のＤＮＡ断片から第ｍ１のＤＮＡ断片まで昇順で並ぶように連結することにより目的ＤＮＡ断片を得る方法であって、
　ｍ１個のＤＮＡ断片はそれぞれ、両末端部に異なるＩＩｓ型制限酵素により認識される塩基配列を有し、かつ隣接して連結するＤＮＡ断片はそれぞれ、連結する側の末端部に同一のＩＩｓ型制限酵素により認識される塩基配列、及び当該ＩＩｓ型制限酵素で切断されたときに相補的な粘着末端を形成する塩基配列を有しており、
（ｉ）ｍ１個のＤＮＡ断片を、ｋ１個（ただし、ｋ１は２以上の自然数を表し、かつｋ１≦ｎ１－１である。）の連続したＤＮＡ断片を含む複数の反応系に分ける工程と、
（ｉｉ）各反応系において、ｎ１種類のＩＩｓ型制限酵素で処理し、ＩＩｓ型制限酵素による切断で形成された粘着末端で反応系中のＤＮＡ断片を順特異的に連結する工程と、
（ｉｉｉ）得られたＤＮＡ連結断片を、連続したＤＮＡ断片を含むようにｌ１個（ただし、ｌ１は２以上の自然数を表し、かつｌ１≦ｎ１－１である。）のＤＮＡ連結断片を含む１又は複数の反応系に分ける工程と、
（ｉｖ）１又は複数の各反応系において、ｎ１種類のＩＩｓ型制限酵素で処理し、ＩＩｓ型制限酵素による切断で形成された粘着末端で反応系中のＤＮＡ連結断片を順特異的に連結する工程と、
（ｖ）目的ＤＮＡ断片が得られるまで（ｉｉｉ）及び（ｉｖ）の工程を繰り返す工程と、
を備える、方法。
［２］
　第１～第ｍ２のｍ２個のＤＮＡ断片を、第１～第ｎ２のｎ２種類（ただし、ｎ２及びｍ２は３以上の自然数を表し、かつｎ２＜ｍ２である。）のＩＩｓ型制限酵素を使用して、第１のＤＮＡ断片から第ｍ２のＤＮＡ断片まで昇順で並ぶように連結することにより目的ＤＮＡ断片を得る方法であって、
　以下の工程（１）～工程（５）：
（１）以下の（ａ）～（ｅ）を満たすｍ２個の二本鎖（ｄｓ）ＤＮＡ断片を準備する工程、
　（ａ）第１のｄｓＤＮＡ断片は、第２のｄｓＤＮＡ断片と連結する側の末端部とは反対の末端部に、第１のＩＩｓ型制限酵素により認識される塩基配列を有し、かつ第２のｄｓＤＮＡ断片と連結する側の末端部には、第２のＩＩｓ型制限酵素により認識される塩基配列、及び第２のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第２のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する、
　（ｂ）第ｋ２のｄｓＤＮＡ断片（ただし、ｋ２は２以上（ｎ２－１）以下の自然数を表す。）は、第（ｋ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第ｋ２のＩＩｓ型制限酵素により認識される塩基配列、及び第ｋ２のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｋ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｋ２＋１）のｄｓＤＮＡ断片と連結する側の末端部には、第（ｋ２＋１）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｋ２＋１）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｋ２＋１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する、
　（ｃ）第ｎ２のｄｓＤＮＡ断片は、第（ｎ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第ｎ２のＩＩｓ型制限酵素により認識される塩基配列、及び第ｎ２のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｎ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｎ２＋１）のｄｓＤＮＡ断片と連結する側の末端部には、第１のＩＩｓ型制限酵素により認識される塩基配列、及び第１のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｎ２＋１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する、
　（ｄ）第ｌ２のｄｓＤＮＡ断片（ただし、ｌ２は（ｎ２＋１）以上（ｍ２－１）以下の自然数を表す。）は、第（ｌ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第（ｌ２－ａ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｌ２－ａ×ｎ２）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｌ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｌ２＋１）のｄｓＤＮＡ断片と連結する側の末端部には、第（ｌ２＋１－ａ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｌ２＋１－ａ×ｎ２）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｌ２＋１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する（ただし、ａはｌ２／ｎ２の整数部分の数値である。）、
　（ｅ）第ｍ２のｄｓＤＮＡ断片は、第（ｍ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第（ｍ２－ｂ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｍ２－ｂ×ｎ２）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｍ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｍ２－１）のｄｓＤＮＡ断片と連結する側の末端部とは反対側の末端部には、第（ｍ２＋１－ｂ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列を有する（ただし、ｂはｍ２／ｎ２の整数部分の数値である。）、
（２）ｍ２個のｄｓＤＮＡ断片を、第１のｄｓＤＮＡ断片から昇順で、（ｎ２－１）個づつ、それぞれ異なるｃ個の反応容器（ただし、ｃはｍ／（ｎ－１）の整数部分の数値である。）に入れ、更に必要に応じて残余のｄｓＤＮＡ断片をｃ個の反応容器とは異なる１個の反応容器に入れる工程、
（３）ｄｓＤＮＡ断片を含む反応容器それぞれに第１～第ｎ２のｎ２種類のＩＩｓ型制限酵素を全て入れ、全ＩＩｓ型制限酵素で処理した後、切断により形成された粘着末端で反応容器中のｄｓＤＮＡ断片を順特異的に連結する工程、
（４）得られたｄｓＤＮＡ連結断片を、連続したｄｓＤＮＡ断片を含むように（ｎ２－１）個づつ、それぞれ異なる反応容器に入れ、更に必要に応じて残余のｄｓＤＮＡ連結断片を異なる１個の反応容器に入れ、工程（３）と同様の操作でｄｓＤＮＡ連結断片を更に連結する工程、
（５）全てのｄｓＤＮＡ断片が連結されるまで工程（４）を繰り返す工程、
を備える、方法。
［３］
　ＩＩｓ型制限酵素がＡａｒＩ、ＡｃｕＩ、ＡｌｗＩ、ＢｂｓＩ、ＢｂｖＩ、ＢｃｃＩ、ＢｃｅＡＩ、ＢｃｉＶＩ、ＢｃｏＤＩ、ＢｆｕＡＩ、ＢｍｒＩ、ＢｐｍＩ、ＢｐｕＥＩ、ＢｓａＩ、ＢｓｇＩ、ＢｓｍＡＩ、ＢｓｍＢＩ、ＢｓｍＦＩ、ＢｓｐＭＩ、ＢｓｐＰＩ、ＢｓｐＱＩ、ＢｓｒＤＩ、ＢｔｇＺＩ、ＢｓｔＣＩ、ＥａｒＩ、ＥｃｉＩ、Ｅｃｏｐ１５Ｉ、ＦａｕＩ、ＦｏｋＩ、ＨｇａＩ、ＨｐｈＩ、ＨｐｙＡＶ、Ｋｓｐ６３２Ｉ、ＭｂｏＩＩ、ＭｍｅＩ、Ｍｎ１Ｉ、ＳａｐＩ及びＳｆａＮＩからなる群より選ばれる少なくとも１種のＩＩｓ型制限酵素である、［１］又は［２］に記載の方法。 The present invention relates to the following inventions, for example.
[1]
The first to m 1 m 1 DNA fragments are the first to n 1 n 1 types (where n 1 and m 1 represent a natural number of 3 or more, and n 1 <m 1 . ) using type IIs restriction enzymes, a method for obtaining the desired DNA fragment by ligating to line up in ascending order from the first DNA fragment to a DNA fragment of the m 1,
m 1 pieces each DNA fragment of a nucleotide sequence recognized by a different type IIs restriction enzyme at both ends, and each DNA fragment to be connected adjacent to, the same type IIs the distal end of the side connecting A base sequence recognized by a restriction enzyme, and a base sequence that forms a complementary sticky end when cleaved by the type IIs restriction enzyme;
The (i) m 1 pieces of DNA fragments, k 1 pieces (where, k 1 represents 2 or more natural number, and a k 1 ≦ n 1 -1.) A plurality of reaction system containing a continuous DNA fragment Dividing the process into
(Ii) in each reaction system, a step of treatment with n 1 kind of type IIs restriction enzymes, ligating the DNA fragments in the reaction system in sticky ends formed by cleavage with type IIs restriction enzymes forward specifically,
(Iii) 11 ligated DNA ligated fragments (where l 1 represents a natural number of 2 or more and l 1 ≦ n 1 −1) so that the obtained DNA ligated fragments include continuous DNA fragments. Dividing the reaction system into one or more reaction systems comprising:
(Iv) In one or a plurality of reaction systems, treatment with n1 type IIs type restriction enzymes is performed, and DNA ligation fragments in the reaction system are ligated in a specific order in a sticky end formed by cleavage with a type IIs restriction enzyme. Process,
(V) repeating steps (iii) and (iv) until the target DNA fragment is obtained;
A method comprising:
[2]
The first to m 2 m 2 DNA fragments are the first to n 2 n 2 types (where n 2 and m 2 represent a natural number of 3 or more and n 2 <m 2 . ) using type IIs restriction enzymes, a method for obtaining the desired DNA fragment by ligating to line up in ascending order from the first DNA fragment to a DNA fragment of the m 2,
The following steps (1) to (5):
(1) below (a) providing - a m 2 pieces of double-stranded (ds) DNA fragment that satisfies (e),
(A) the first dsDNA fragment has a base sequence recognized by the first type IIs restriction enzyme at the end opposite to the end linked to the second dsDNA fragment; At the end portion on the side to be linked to the dsDNA fragment, the base sequence recognized by the second type IIs restriction enzyme and the sticky end formed by cleavage with the second type IIs restriction enzyme are the second dsDNA. It has a base sequence that is complementary to the sticky end of the fragment,
(B) The k 2 dsDNA fragment (where k 2 represents a natural number of 2 or more and (n 2 −1) or less) is connected to the terminal portion on the side linked to the (k 2 −1) dsDNA fragment. , nucleotide sequence recognized by a type IIs restriction enzyme of the k 2, and sticky ends are formed by being cut with a type IIs restriction enzyme of the k 2 is a sticky end dsDNA fragment of the (k 2 -1) has a nucleotide sequence comprising a complementary, and the end portion of the side connecting the dsDNA fragment of the (k 2 +1), nucleotide sequences recognized by the type IIs restriction enzyme of the (k 2 +1), and the sticky end formed by being cut with a type IIs restriction enzyme (k 2 +1) has a nucleotide sequence comprising the complement of the sticky ends of dsDNA fragments of the (k 2 +1),
(C) dsDNA fragments of the n 2 is first in the end on the side connected to the dsDNA fragment (n 2 -1), the base sequence recognized by a type IIs restriction enzyme of the n 2, and the n 2 A sticky end formed by cleaving with a type IIs restriction enzyme has a base sequence complementary to the sticky end of the (n 2 -1) dsDNA fragment, and the (n 2 +1) dsDNA fragment; At the terminal end on the ligation side, the base sequence recognized by the first type IIs restriction enzyme and the sticky end formed by cleavage with the first type IIs restriction enzyme are the (n 2 +1) th dsDNA. It has a base sequence that is complementary to the sticky end of the fragment,
(D) the l 2nd dsDNA fragment (where l 2 represents a natural number not less than (n 2 +1) and not more than (m 2 −1)) is connected to the (l 2 −1) dsDNA fragment. the end portion, is formed by cutting with type IIs restriction enzymes of the (l 2 -a × n 2) nucleotide sequences recognized by the type IIs restriction enzymes, and the (l 2 -a × n 2) The sticky end has a base sequence that is complementary to the sticky end of the (l 2 -1) dsDNA fragment, and the end portion on the side linked to the (l 2 +1) dsDNA fragment has ( l 2 + 1−a × n 2 ) type IIs restriction enzyme and a sticky end formed by cleavage with the (l 2 + 1−a × n 2 ) type IIs restriction enzyme having a sticky end and a base sequence consisting complementary of dsDNA fragments (l 2 +1) (provided that a is a number in the integer portion of a l 2 / n 2.),
(E) The m 2 dsDNA fragment has a base that is recognized by the (m 2 -b × n 2 ) type IIs restriction enzyme at the end of the side linked to the (m 2 -1) dsDNA fragment. And a base sequence in which the sticky end formed by cleavage with the (m 2 -b × n 2 ) type IIs restriction enzyme is complementary to the sticky end of the (m 2 -1) dsDNA fragment And is recognized by the (m 2 + 1−b × n 2 ) type IIs restriction enzyme at the end opposite to the end connected to the (m 2 −1) dsDNA fragment. (Where b is the numerical value of the integer part of m 2 / n 2 ),
(2) m 2 dsDNA fragments in ascending order from the first dsDNA fragment, each having (n 2 -1) c different reaction vessels (where c is an integer of m / (n-1)) A step of placing the remaining dsDNA fragments in one reaction vessel different from c reaction vessels, if necessary.
(3) Put all n 2 kinds of first to n 2 type IIs restriction enzyme to each reaction vessel containing the dsDNA fragment, after treatment with all type IIs restriction enzyme, the reaction vessel at a sticky end formed by the cut Linking the dsDNA fragments therein in a forward specific manner,
(4) The obtained dsDNA ligated fragments are placed in different reaction vessels, each containing (n 2 −1) so as to contain continuous dsDNA fragments, and if necessary, the remaining dsDNA ligated fragments are Placing in a reaction vessel and further ligating the dsDNA ligation fragment in the same manner as in step (3),
(5) repeating step (4) until all dsDNA fragments are ligated,
A method comprising:
[3]
Type IIs restriction enzymes are AarI, AcuI, AlwI, BbsI, BbvI, BccI, BceAI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsgI, BsmBI, BsmBs, BsmBs, BsmB , BstCI, EarI, EciI, Ecop15I, FauI, FokI, HgaI, HphI, HpyAV, Ksp632I, MboII, MmeI, Mn1I, SapI, and SfaNI, [1] Or the method as described in [2].
　本発明によれば、目的ＤＮＡ断片が高分子の場合であっても、ＩＩｓ型制限酵素を利用して、複数のＤＮＡ断片を順特異的に連結して目的ＤＮＡ断片を得ることができる。また、上記［２］に係る本発明の方法は、ｄｓＤＮＡ断片又はｄｓＤＮＡ連結断片を規則的かつ機械的に連結反応に供するものであることから、自動化が容易であるという利点もある。 According to the present invention, even if the target DNA fragment is a polymer, the target DNA fragment can be obtained by linking a plurality of DNA fragments in a specific order using a type IIs restriction enzyme. Further, the method of the present invention according to the above [2] has an advantage that automation is easy because the dsDNA fragment or the dsDNA ligation fragment is regularly and mechanically subjected to a ligation reaction.
本発明の一実施形態に係る方法により、目的ＤＮＡ断片を得る原理を説明する図である。It is a figure explaining the principle which obtains a target DNA fragment by the method concerning one embodiment of the present invention.
　以下、本発明を実施するための形態について詳細に説明する。ただし、本発明は以下の実施形態に限定されるものではない。 Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
　一実施形態に係る目的ＤＮＡ断片を得る方法は、第１～第ｍ１のｍ１個のＤＮＡ断片を、第１～第ｎ１のｎ１種類（ただし、ｎ１及びｍ１は３以上の自然数を表し、かつｎ１＜ｍ１である。）のＩＩｓ型制限酵素を使用して、第１のＤＮＡ断片から第ｍ１のＤＮＡ断片まで昇順で並ぶように連結することにより目的ＤＮＡ断片を得る方法に関し、（ｉ）ｍ１個のＤＮＡ断片を、ｋ１個（ただし、ｋ１は２以上の自然数を表し、かつｋ１≦ｎ１－１である。）の連続したＤＮＡ断片を含む複数の反応系に分ける工程と、（ｉｉ）各反応系において、ｎ１種類のＩＩｓ型制限酵素で処理し、ＩＩｓ型制限酵素による切断で形成された粘着末端で反応系中のＤＮＡ断片を順特異的に連結する工程と、（ｉｉｉ）得られたＤＮＡ連結断片を、連続したＤＮＡ断片を含むようにｌ１個（ただし、ｌ１は２以上の自然数を表し、かつｌ１≦ｎ１－１である。）のＤＮＡ連結断片を含む１又は複数の反応系に分ける工程と、（ｉｖ）１又は複数の各反応系において、ｎ１種類のＩＩｓ型制限酵素で処理し、ＩＩｓ型制限酵素による切断で形成された粘着末端で反応系中のＤＮＡ連結断片を順特異的に連結する工程と、（ｖ）目的ＤＮＡ断片が得られるまで（ｉｉｉ）及び（ｉｖ）の工程を繰り返す工程と、を備える。ここで、ｍ１個のＤＮＡ断片はそれぞれ、両末端部に異なるＩＩｓ型制限酵素により認識される塩基配列を有し、かつ隣接して連結するＤＮＡ断片はそれぞれ、連結する側の末端部に同一のＩＩｓ型制限酵素により認識される塩基配列、及び当該ＩＩｓ型制限酵素で切断されたときに相補的な粘着末端を形成する塩基配列を有している。 According to one embodiment, a method for obtaining a target DNA fragment comprises the steps of: 1st to m 1 m 1 DNA fragments, 1 to n 1 n 1 types (where n 1 and m 1 are 3 or more) The target DNA fragment is ligated in ascending order from the first DNA fragment to the m1 DNA fragment using a type IIs restriction enzyme that represents a natural number and n 1 <m 1 ). Regarding the obtaining method, (i) m 1 DNA fragments include k 1 (where k 1 represents a natural number of 2 or more and k 1 ≦ n 1 −1). a step of dividing the plurality of reaction system in each reaction system (ii), was treated with n 1 kind of type IIs restriction enzyme, the DNA fragments in the reaction system in sticky ends formed by cleavage with type IIs restriction enzyme sequence A step of specific ligation, and (iii) the obtained DNA ligation fragment , L 1 or to include a contiguous DNA fragment (however, l 1 represents 2 or more natural number, and a l 1 ≦ n 1 -1.) DNA contigs one or more of the reaction system containing the a And (iv) in one or a plurality of reaction systems, each of the reaction systems is treated with one type IIs type restriction enzyme, and the DNA ligation fragments in the reaction system are sequentially processed at the sticky ends formed by cleavage with the type IIs restriction enzyme. Specific ligation, and (v) repeating steps (iii) and (iv) until the target DNA fragment is obtained. Here, each of the m 1 DNA fragments has a base sequence recognized by different type IIs restriction enzymes at both ends, and the adjacent DNA fragments to be ligated are the same at the ligation ends. And a base sequence that forms a complementary sticky end when cleaved by the type IIs restriction enzyme.
　他の実施形態に係る目的ＤＮＡ断片を得る方法は、第１～第ｍ２のｍ２個のＤＮＡ断片を、第１～第ｎ２のｎ２種類（ただし、ｎ２及びｍ２は３以上の自然数を表し、かつｎ２＜ｍ２である。）のＩＩｓ型制限酵素を使用して、第１のＤＮＡ断片から第ｍ２のＤＮＡ断片まで昇順で並ぶように連結することにより目的ＤＮＡ断片を得る方法に関し、以下の工程（１）～工程（５）を備える。
（１）以下の（ａ）～（ｅ）を満たすｍ２個の二本鎖（ｄｓ）ＤＮＡ断片を準備する工程。
　（ａ）第１のｄｓＤＮＡ断片は、第２のｄｓＤＮＡ断片と連結する側の末端部とは反対の末端部に、第１のＩＩｓ型制限酵素により認識される塩基配列を有し、かつ第２のｄｓＤＮＡ断片と連結する側の末端部には、第２のＩＩｓ型制限酵素により認識される塩基配列、及び第２のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第２のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する。
　（ｂ）第ｋ２のｄｓＤＮＡ断片（ただし、ｋ２は２以上（ｎ２－１）以下の自然数を表す。）は、第（ｋ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第ｋ２のＩＩｓ型制限酵素により認識される塩基配列、及び第ｋ２のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｋ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｋ２＋１）のｄｓＤＮＡ断片と連結する側の末端部には、第（ｋ２＋１）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｋ２＋１）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｋ２＋１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する。
　（ｃ）第ｎ２のｄｓＤＮＡ断片は、第（ｎ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第ｎ２のＩＩｓ型制限酵素により認識される塩基配列、及び第ｎ２のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｎ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｎ２＋１）のｄｓＤＮＡ断片と連結する側の末端部には、第１のＩＩｓ型制限酵素により認識される塩基配列、及び第１のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｎ２＋１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する。
　（ｄ）第ｌ２のｄｓＤＮＡ断片（ただし、ｌ２は（ｎ２＋１）以上（ｍ２－１）以下の自然数を表す。）は、第（ｌ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第（ｌ２－ａ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｌ２－ａ×ｎ２）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｌ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｌ２＋１）のｄｓＤＮＡ断片と連結する側の末端部には、第（ｌ２＋１－ａ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｌ２＋１－ａ×ｎ２）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｌ２＋１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する（ただし、ａはｌ２／ｎ２の整数部分の数値である。）。
　（ｅ）第ｍ２のｄｓＤＮＡ断片は、第（ｍ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第（ｍ２－ｂ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｍ２－ｂ×ｎ２）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｍ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｍ２－１）のｄｓＤＮＡ断片と連結する側の末端部とは反対側の末端部には、第（ｍ２＋１－ｂ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列を有する（ただし、ｂはｍ２／ｎ２の整数部分の数値である。）。
（２）ｍ２個のｄｓＤＮＡ断片を、第１のｄｓＤＮＡ断片から昇順で、（ｎ２－１）個づつ、それぞれ異なるｃ個の反応容器（ただし、ｃはｍ／（ｎ－１）の整数部分の数値である。）に入れ、更に必要に応じて残余のｄｓＤＮＡ断片をｃ個の反応容器とは異なる１個の反応容器に入れる工程。
（３）ｄｓＤＮＡ断片を含む反応容器それぞれに第１～第ｎ２のｎ２種類のＩＩｓ型制限酵素を全て入れ、全ＩＩｓ型制限酵素で処理した後、切断により形成された粘着末端で反応容器中のｄｓＤＮＡ断片を順特異的に連結する工程。
（４）得られたｄｓＤＮＡ連結断片を、連続したｄｓＤＮＡ断片を含むように（ｎ２－１）個づつ、それぞれ異なる反応容器に入れ、更に必要に応じて残余のｄｓＤＮＡ連結断片を異なる１個の反応容器に入れ、工程（３）と同様の操作でｄｓＤＮＡ連結断片を更に連結する工程。
（５）全てのｄｓＤＮＡ断片が連結されるまで工程（４）を繰り返す工程。 The method for obtaining the DNA fragment of interest according to another embodiment, the first to m 2 m 2 of pieces of DNA fragments, the first to n 2 of n 2 kinds (although, n 2 and m 2 is 3 or more It represents a natural number, and a n 2 <m 2.) using type IIs restriction enzymes, the target DNA fragments by connecting to line up in ascending order from the first DNA fragment to a DNA fragment of the m 2 The following steps (1) to (5) are provided.
(1) A step of preparing m 2 double-stranded (ds) DNA fragments satisfying the following (a) to (e).
(A) the first dsDNA fragment has a base sequence recognized by the first type IIs restriction enzyme at the end opposite to the end linked to the second dsDNA fragment; At the end portion on the side to be linked to the dsDNA fragment, the base sequence recognized by the second type IIs restriction enzyme and the sticky end formed by cleavage with the second type IIs restriction enzyme are the second dsDNA. It has a base sequence that is complementary to the sticky end of the fragment.
(B) The k 2 dsDNA fragment (where k 2 represents a natural number of 2 or more and (n 2 −1) or less) is connected to the terminal portion on the side linked to the (k 2 −1) dsDNA fragment. , nucleotide sequence recognized by a type IIs restriction enzyme of the k 2, and sticky ends are formed by being cut with a type IIs restriction enzyme of the k 2 is a sticky end dsDNA fragment of the (k 2 -1) has a nucleotide sequence comprising a complementary, and the end portion of the side connecting the dsDNA fragment of the (k 2 +1), nucleotide sequences recognized by the type IIs restriction enzyme of the (k 2 +1), and the sticky end formed by being cut with a type IIs restriction enzyme (k 2 +1) has a nucleotide sequence comprising the complement of the sticky ends of dsDNA fragments of the (k 2 +1).
(C) dsDNA fragments of the n 2 is first in the end on the side connected to the dsDNA fragment (n 2 -1), the base sequence recognized by a type IIs restriction enzyme of the n 2, and the n 2 A sticky end formed by cleaving with a type IIs restriction enzyme has a base sequence complementary to the sticky end of the (n 2 -1) dsDNA fragment, and the (n 2 +1) dsDNA fragment; At the terminal end on the ligation side, the base sequence recognized by the first type IIs restriction enzyme and the sticky end formed by cleavage with the first type IIs restriction enzyme are the (n 2 +1) th dsDNA. It has a base sequence that is complementary to the sticky end of the fragment.
(D) the l 2nd dsDNA fragment (where l 2 represents a natural number not less than (n 2 +1) and not more than (m 2 −1)) is connected to the (l 2 −1) dsDNA fragment. the end portion, is formed by cutting with type IIs restriction enzymes of the (l 2 -a × n 2) nucleotide sequences recognized by the type IIs restriction enzymes, and the (l 2 -a × n 2) The sticky end has a base sequence that is complementary to the sticky end of the (l 2 -1) dsDNA fragment, and the end portion on the side linked to the (l 2 +1) dsDNA fragment has ( l 2 + 1−a × n 2 ) type IIs restriction enzyme and a sticky end formed by cleavage with the (l 2 + 1−a × n 2 ) type IIs restriction enzyme having a sticky end and a base sequence consisting complementary of dsDNA fragments (l 2 +1) (provided that a is a number in the integer portion of a l 2 / n 2.).
(E) The m 2 dsDNA fragment has a base that is recognized by the (m 2 -b × n 2 ) type IIs restriction enzyme at the end of the side linked to the (m 2 -1) dsDNA fragment. And a base sequence in which the sticky end formed by cleavage with the (m 2 -b × n 2 ) type IIs restriction enzyme is complementary to the sticky end of the (m 2 -1) dsDNA fragment And is recognized by the (m 2 + 1−b × n 2 ) type IIs restriction enzyme at the end opposite to the end connected to the (m 2 −1) dsDNA fragment. (Where b is the numerical value of the integer part of m 2 / n 2 ).
(2) m 2 dsDNA fragments in ascending order from the first dsDNA fragment, each having (n 2 -1) c different reaction vessels (where c is an integer of m / (n-1)) And the remaining dsDNA fragment is placed in one reaction vessel different from c reaction vessels as necessary.
(3) Put all n 2 kinds of first to n 2 type IIs restriction enzyme to each reaction vessel containing the dsDNA fragment, after treatment with all type IIs restriction enzyme, the reaction vessel at a sticky end formed by the cut A step of ligating the dsDNA fragments therein in a specific order.
(4) The obtained dsDNA ligated fragments are placed in different reaction vessels, each containing (n 2 −1) so as to contain continuous dsDNA fragments, and if necessary, the remaining dsDNA ligated fragments are A step of placing in a reaction vessel and further ligating the dsDNA ligation fragment by the same operation as in step (3).
(5) Repeating step (4) until all dsDNA fragments are linked.
　図１は、本発明の一実施形態に係る方法により、目的ＤＮＡ断片を得る原理を説明する図である。図１に示す方法は、１０個のｄｓＤＮＡ断片（ＤＮＡ　ｆｇ１～ｆｇ１０）を、３種類のＩＩｓ型制限酵素（第１～第３のＲＥ）を使用して、第１のｄｓＤＮＡ断片（ＤＮＡ　ｆｇ１）から第１０のｄｓＤＮＡ断片（ＤＮＡ　ｆｇ１０）まで昇順で並ぶように連結することにより目的ＤＮＡ断片を得る方法である（ｍ＝１０，ｎ＝３）。 FIG. 1 is a diagram for explaining the principle of obtaining a target DNA fragment by a method according to an embodiment of the present invention. In the method shown in FIG. 1, ten dsDNA fragments (DNA fg1 to fg10) are used as a first dsDNA fragment (DNA fg1) using three types of type IIs restriction enzymes (first to third RE). To the 10th dsDNA fragment (DNA fg10) so as to be arranged in ascending order to obtain the target DNA fragment (m = 10, n = 3).
　図１中、「第１のＲＥ」～「第３のＲＥ」で示される領域は、それぞれ第１～第３のＩＩｓ型制限酵素により認識される塩基配列を含む領域である。また、各ｄｓＤＮＡ断片は、第１～第３のＩＩｓ型制限酵素により切断されたときに、それぞれ第１～３のＲＥで示される領域が、目的ＤＮＡ断片の塩基配列の一部からなる領域（図１中、白抜きの領域）から切り離され、かつ隣接して連結するｄｓＤＮＡ断片の粘着末端と相補的な塩基配列を有する粘着末端が形成されるように設計されている。 In FIG. 1, regions indicated by “first RE” to “third RE” are regions including base sequences recognized by the first to third type IIs restriction enzymes, respectively. In addition, when each dsDNA fragment is cleaved by the first to third type IIs restriction enzymes, each of the regions indicated by the first to third REs is a region comprising a part of the base sequence of the target DNA fragment ( It is designed so that a sticky end having a base sequence complementary to the sticky end of the dsDNA fragment that is cut off from the white region in FIG.
　図１（Ａ）は、１０個のｄｓＤＮＡ断片（ＤＮＡ　ｆｇ１～ｆｇ１０）が示されている。図１（Ａ）に示すとおり、１０個のｄｓＤＮＡ断片はそれぞれ、両末端部に異なるＩＩｓ型制限酵素により認識される塩基配列を有し、かつ隣接して連結するＤＮＡ断片はそれぞれ、連結する側の末端部に同一のＩＩｓ型制限酵素により認識される塩基配列、及び当該ＩＩｓ型制限酵素で切断されたときに相補的な粘着末端を形成する塩基配列を有している。 FIG. 1 (A) shows 10 dsDNA fragments (DNA fg1 to fg10). As shown in FIG. 1 (A), each of the 10 dsDNA fragments has a base sequence recognized by a different type IIs restriction enzyme at both ends, and the adjacent DNA fragments to be ligated are linked to each other. Have a base sequence that is recognized by the same type IIs restriction enzyme and a base sequence that forms a complementary sticky end when cleaved by the type IIs restriction enzyme.
　具体的には、（ａ）第１のｄｓＤＮＡ断片（ＤＮＡ　ｆｇ１）は、第２のｄｓＤＮＡ断片（ＤＮＡ　ｆｇ２）と連結する側の末端部とは反対の末端部に、第１のＩＩｓ型制限酵素により認識される塩基配列を含む領域（第１のＲＥ）を有し、かつ第２のｄｓＤＮＡ断片と連結する側の末端部には、第２のＩＩｓ型制限酵素により認識される塩基配列を含む領域（第２のＲＥ）を有しており、更に目的ＤＮＡ断片の塩基配列の一部からなる領域（白抜きの領域）中に、第２のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第２のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する。 Specifically, (a) the first dsDNA fragment (DNA fg1) has a first type IIs restriction enzyme at the end opposite to the end linked to the second dsDNA fragment (DNA fg2). A region containing the base sequence recognized by (1) RE (first RE), and the end portion on the side linked to the second dsDNA fragment contains the base sequence recognized by the second type IIs restriction enzyme An adhesion formed by cleaving with a second type IIs restriction enzyme in a region (outlined region) having a region (second RE) and further comprising a part of the base sequence of the target DNA fragment The end has a base sequence that is complementary to the sticky end of the second dsDNA fragment.
　（ｂ）第２のｄｓＤＮＡ断片は、第１のｄｓＤＮＡ断片と連結する側の末端部に、第２のＩＩｓ型制限酵素により認識される塩基配列を含む領域（第２のＲＥ）を有し、更に目的ＤＮＡ断片の塩基配列の一部からなる領域（白抜きの領域）中に、第２のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第１のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する。第２のｄｓＤＮＡ断片はまた、第３のｄｓＤＮＡ断片（ＤＮＡ　ｆｇ３）と連結する側の末端部には、第３のＩＩｓ型制限酵素により認識される塩基配列を含む領域（第３のＲＥ）を有しており、更に目的ＤＮＡ断片の塩基配列の一部からなる領域（白抜きの領域）中に、第３のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第３のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する。 (B) the second dsDNA fragment has a region (second RE) containing a base sequence recognized by the second type IIs restriction enzyme at the terminal end linked to the first dsDNA fragment; Furthermore, the sticky end formed by cleaving with the second type IIs restriction enzyme is complementary to the sticky end of the first dsDNA fragment in a region consisting of a part of the base sequence of the target DNA fragment (open region). It has a base sequence that becomes The second dsDNA fragment also has a region (third RE) containing a base sequence recognized by the third type IIs restriction enzyme at the terminal portion on the side linked to the third dsDNA fragment (DNA fg3). And a sticky end formed by cleaving with a third type IIs restriction enzyme in a region consisting of a part of the base sequence of the target DNA fragment (outlined region) is the third dsDNA fragment The base sequence is complementary to the sticky end of.
　（ｃ）第３のｄｓＤＮＡ断片は、第２のｄｓＤＮＡ断片と連結する側の末端部に、第３のＩＩｓ型制限酵素により認識される塩基配列を含む領域（第３のＲＥ）を有しており、更に目的ＤＮＡ断片の塩基配列の一部からなる領域（白抜きの領域）中に、第３のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第２のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する。第３のｄｓＤＮＡ断片はまた、第４のｄｓＤＮＡ断片（ＤＮＡ　ｆｇ４）と連結する側の末端部には、第１のＩＩｓ型制限酵素により認識される塩基配列を含む領域（第１のＲＥ）を有し、更に目的ＤＮＡ断片の塩基配列の一部からなる領域（白抜きの領域）中に、第１のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第４のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する。 (C) The third dsDNA fragment has a region (third RE) containing a base sequence recognized by the third type IIs restriction enzyme at the terminal portion on the side linked to the second dsDNA fragment. Furthermore, the sticky end formed by cleaving with the third type IIs restriction enzyme in the region consisting of a part of the base sequence of the target DNA fragment (open region) is the sticky end of the second dsDNA fragment. And has a base sequence that is complementary. The third dsDNA fragment also has a region (first RE) containing a base sequence recognized by the first type IIs restriction enzyme at the end portion on the side linked to the fourth dsDNA fragment (DNA fg4). And a sticky end formed by cleaving with the first type IIs restriction enzyme in a region consisting of part of the base sequence of the target DNA fragment (outlined region) is sticky to the fourth dsDNA fragment It has a base sequence that is complementary to the terminal.
　（ｄ）第ｌのｄｓＤＮＡ断片（ＤＮＡ　ｆｇｌ：ただし、ｌは４以上９以下の自然数を表す。）は、第（ｌ－１）のｄｓＤＮＡ断片と連結する側の末端部に、第（ｌ－ａ×３）のＩＩｓ型制限酵素により認識される塩基配列を含む領域（第（ｌ－ａ×３）のＲＥ）を有しており、更に目的ＤＮＡ断片の塩基配列の一部からなる領域（白抜きの領域）中に、第（ｌ－ａ×３）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｌ－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する。第ｌのｄｓＤＮＡ断片はまた、第（ｌ＋１）のｄｓＤＮＡ断片と連結する側の末端部には、第（ｌ＋１－ａ×３）のＩＩｓ型制限酵素により認識される塩基配列を含む領域（第（ｌ＋１－ａ×３）のＲＥ）を有しており、更に目的ＤＮＡ断片の塩基配列の一部からなる領域（白抜きの領域）中に、第（ｌ＋１－ａ×３）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｌ＋１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する。ただし、ａはｌ／３の整数部分の数値であり、例えば、ｌ＝５の場合は１であり、ｌ＝７の場合は２である。 (D) The l-th dsDNA fragment (DNA fgl: where l represents a natural number of 4 or more and 9 or less) is attached to the end (l−) of the side linked to the (l-1) -th dsDNA fragment. a region containing a base sequence recognized by the type IIs restriction enzyme (a × 3) (RE of (1-a × 3)), and a region comprising a part of the base sequence of the target DNA fragment ( In the white area), the base in which the sticky end formed by cleavage with the (l-a × 3) type IIs restriction enzyme is complementary to the sticky end of the (l-1) dsDNA fragment Has an array. The l-th dsDNA fragment is also a region containing the base sequence recognized by the (l + 1-a × 3) type IIs restriction enzyme (the (( (l + 1-a × 3) RE), and the (l + 1-a × 3) type IIs restriction enzyme in the region consisting of a part of the base sequence of the target DNA fragment (outlined region) The sticky end formed by cleaving in step 1 has a base sequence that is complementary to the sticky end of the (l + 1) th dsDNA fragment. However, a is a numerical value of an integer part of 1/3, for example, 1 when l = 5 and 2 when l = 7.
　（ｅ）第１０のｄｓＤＮＡ断片（ＤＮＡ　ｆｇ１０）は、第９のｄｓＤＮＡ断片（ＤＮＡ　ｆｇ９）と連結する側の末端部に、第（１０－ｂ×３）のＩＩｓ型制限酵素により認識される塩基配列を含む領域（第１のＲＥ）を有しており、更に目的ＤＮＡ断片の塩基配列の一部からなる領域（白抜きの領域）中に、第１のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第９のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する。第１０のｄｓＤＮＡ断片はまた、第９のｄｓＤＮＡ断片と連結する側の末端部とは反対側の末端部に、第（１０＋１－ｂ×３）のＩＩｓ型制限酵素により認識される塩基配列を含む領域（第２のＲＥ）を有する。ただし、ｂはｍ／ｎの整数部分の数値であり、図１の例では３である。 (E) The tenth dsDNA fragment (DNA fg10) is a base that is recognized by the (10-b × 3) type IIs restriction enzyme at the end linked to the ninth dsDNA fragment (DNA fg9). It has a region containing the sequence (first RE) and is further cleaved with the first type IIs restriction enzyme in a region consisting of part of the base sequence of the target DNA fragment (outlined region) The sticky end to be prepared has a base sequence that is complementary to the sticky end of the ninth dsDNA fragment. The tenth dsDNA fragment also contains a base sequence recognized by the (10 + 1-b × 3) type IIs restriction enzyme at the end opposite to the end linked to the ninth dsDNA fragment. It has a region (second RE). However, b is a numerical value of the integer part of m / n, and is 3 in the example of FIG.
　上述したｄｓＤＮＡ断片は、両端が平滑末端であるｄｓＤＮＡであってもよく、一端又は両端に一本鎖オーバーハング領域を有するｄｓＤＮＡであってもよい。ｄｓＤＮＡ断片は、従来公知の遺伝子工学的手法により得ることができる。例えば、設計されたｄｓＤＮＡ断片の塩基配列を有するオリゴＤＮＡ、及びその相補配列を有するオリゴＤＮＡを常法に従って合成し、これらをハイブリダイズさせてｄｓオリゴＤＮＡを調製することにより、ｄｓＤＮＡ断片を得ることができる。 The dsDNA fragment described above may be dsDNA having blunt ends at both ends, or dsDNA having a single-stranded overhang region at one end or both ends. The dsDNA fragment can be obtained by a conventionally known genetic engineering technique. For example, a dsDNA fragment is obtained by synthesizing an oligo DNA having a designed base sequence of a dsDNA fragment and an oligoDNA having a complementary sequence thereof according to a conventional method and then hybridizing them to prepare a ds oligo DNA. Can do.
　図１に示す例では、まず（Ａ）において、１０個のｄｓＤＮＡ断片を、第１のｄｓＤＮＡ断片から昇順で２個づつそれぞれ異なる５つの反応系（例えば、異なる反応容器）に分ける。すなわち、反応系１は、ＤＮＡ　ｆｇ１及びＤＮＡ　ｆｇ２を含み、反応系２は、ＤＮＡ　ｆｇ３及びＤＮＡ　ｆｇ４を含み、反応系３は、ＤＮＡ　ｆｇ５及びＤＮＡ　ｆｇ６を含み、反応系４は、ＤＮＡ　ｆｇ７及びＤＮＡ　ｆｇ８を含み、反応系５は、ＤＮＡ　ｆｇ９及びＤＮＡ　ｆｇ１０を含む。もし残余のｄｓＤＮＡ断片があれば（例えば、ＤＮＡ　ｆｇ１１があれば）、残余のｄｓＤＮＡ断片を反応系６とすればよい。 In the example shown in FIG. 1, first, in (A), 10 dsDNA fragments are divided into five different reaction systems (for example, different reaction vessels), each two in ascending order from the first dsDNA fragment. That is, reaction system 1 includes DNA fg1 and DNA fg2, reaction system 2 includes DNA fg3 and DNA fg4, reaction system 3 includes DNA fg5 and DNA fg6, and reaction system 4 includes DNA fg7 and DNA fg6. The reaction system 5 contains fg8 and DNA fg9 and DNA fg10. If there is a remaining dsDNA fragment (for example, if DNA fg11 is present), the remaining dsDNA fragment may be used as the reaction system 6.
　各反応系には、それぞれ第１～第３の３種類のＩＩｓ型制限酵素を全て添加する。このようにすることで、１種類のＩＩｓ型制限酵素溶液（全てのＩＩｓ型制限酵素を含む溶液）を用意して各反応系に分注すればよいため、工程が簡便化され、自動化が容易になる。 All the first to third types of IIs type restriction enzymes are added to each reaction system. In this way, one type IIs type restriction enzyme solution (a solution containing all type IIs type restriction enzymes) may be prepared and dispensed to each reaction system, which simplifies the process and facilitates automation. become.
　ＩＩｓ型制限酵素としては、例えば、表１に記載したＩＩｓ型制限酵素を挙げることができる。
Figure JPOXMLDOC01-appb-T000001
 Examples of type IIs restriction enzymes include type IIs restriction enzymes described in Table 1.
Figure JPOXMLDOC01-appb-T000001
　次いで、ＩＩｓ型制限酵素により各ｄｓＤＮＡ断片が切断される。これにより、隣接して連結するｄｓＤＮＡ断片はそれぞれ、連結する側の末端部に相補的な粘着末端が形成される。すると、隣接して連結するｄｓＤＮＡ断片は、粘着末端を介して特異的にハイブリダイズすることになる。反応系に、ポリエチレングリコール（ＰＥＧ）、フィコール（Ｆｉｃｏｌｌ）、デキストラン等の密集剤を添加することにより、ハイブリダイズを促進することができる。 Next, each dsDNA fragment is cleaved with a type IIs restriction enzyme. As a result, the adjoining dsDNA fragments each form a complementary sticky end at the end of the connecting side. Then, the adjoining dsDNA fragment specifically hybridizes via the sticky end. Hybridization can be promoted by adding a crowding agent such as polyethylene glycol (PEG), Ficoll, or dextran to the reaction system.
　ハイブリダイズした後、反応系にＤＮＡリガーゼを添加しておくことで、ニック（すなわち、ホスホジエステル結合が欠如している部分）が繋ぎ合わされ、ｄｓＤＮＡ連結断片が得られることになる。 After hybridization, by adding DNA ligase to the reaction system, nicks (that is, a portion lacking a phosphodiester bond) are joined together to obtain a dsDNA-linked fragment.
　ＤＮＡリガーゼとしては、例えば、Ｔａｑリガーゼ、Ａｍｐｌｉｇａｓｅ　Ｔｈｅｒｍｏｓｔａｂｌｅ　ＤＮＡリガーゼ（Ｅｐｉｃｅｎｔｒｅ　Ｂｉｏｔｅｃｈｎｏｌｏｇｉｅｓ社製）、Ｔｈｅｒｍｏｓｔａｂｌｅ　Ｔｆｉ　ＤＮＡリガーゼ（Ｂｉｏｎｅｅｒ，　Ｉｎｃ社製）等を挙げることができる。 Examples of the DNA ligase include Taq ligase, Amprigase Thermostable DNA ligase (Epicentre Biotechnologies), Thermostable Tfi DNA ligase (Bioneer, Inc).
　上述した反応を経ることにより、例えば、図１（Ｂ）に示すとおり、反応系１では、第２のＲＥで示される領域部分でＤＮＡ　ｆｇ１とＤＮＡ　ｆｇ２が連結したｄｓＤＮＡ連結断片が得られる。当該ｄｓＤＮＡ連結断片は、第２のＲＥで示される領域は、除去されており、また両末端部に第１のＲＥで示される領域、及び第３のＲＥで示される領域を含んでいる。反応系２～５についても同様である。 Through the above-described reaction, for example, as shown in FIG. 1B, in the reaction system 1, a dsDNA-linked fragment in which DNA fg1 and DNA fg2 are linked in the region indicated by the second RE is obtained. In the dsDNA ligation fragment, the region indicated by the second RE has been removed, and the region indicated by the first RE and the region indicated by the third RE are included at both ends. The same applies to reaction systems 2-5.
　次いで、得られたｄｓＤＮＡ連結断片を、連続したｄｓＤＮＡ断片を含むように２個づつ、それぞれ異なる反応容器に入れる。図１に示す例では、反応系１で得られたｄｓＤＮＡ連結断片と、反応系２で得られたｄｓＤＮＡ連結断片（計２個）を第１の反応容器に入れ、反応系３で得られたｄｓＤＮＡ連結断片と、反応系４で得られたｄｓＤＮＡ連結断片（計２個）を第２の反応容器に入れている。例えば、反応系１で得られたｄｓＤＮＡ連結断片は、第１のｄｓＤＮＡ断片及び第２のｄｓＤＮＡ断片を含んでおり、反応系２で得られたｄｓＤＮＡ連結断片は、第３のｄｓＤＮＡ断片及び第４のｄｓＤＮＡ断片を含んでいるため、これら（２個）を入れた第１の反応容器（反応系）には、第１～第４の連続したｄｓＤＮＡ断片が含まれている。 Next, the obtained dsDNA ligation fragments are put in two, each in a different reaction vessel so as to contain continuous dsDNA fragments. In the example shown in FIG. 1, the dsDNA ligation fragment obtained in the reaction system 1 and the dsDNA ligation fragment obtained in the reaction system 2 (two in total) were placed in the first reaction vessel and obtained in the reaction system 3. The dsDNA ligation fragments and the dsDNA ligation fragments (two in total) obtained in the reaction system 4 are put in the second reaction vessel. For example, the dsDNA ligation fragment obtained in the reaction system 1 includes a first dsDNA fragment and a second dsDNA fragment, and the dsDNA ligation fragment obtained in the reaction system 2 includes the third dsDNA fragment and the fourth dsDNA fragment. Therefore, the first reaction vessel (reaction system) containing these (two pieces) contains the first to fourth continuous dsDNA fragments.
　図１に示す例では、反応系５で得られたｄｓＤＮＡ連結断片が残余のものとなっている。この場合、反応系５で得られたｄｓＤＮＡ連結断片（１個）を第３の反応容器に入れることになる。 In the example shown in FIG. 1, the dsDNA ligation fragment obtained in the reaction system 5 is the remaining one. In this case, the dsDNA ligation fragment (one piece) obtained in the reaction system 5 is put into the third reaction vessel.
　第１～第３の反応容器には、それぞれ第１～第３の３種類のＩＩｓ型制限酵素を全て添加する。これにより、ＩＩｓ型制限酵素により各ｄｓＤＮＡ連結断片が切断され、隣接して連結するｄｓＤＮＡ連結断片はそれぞれ、連結する側の末端部に相補的な粘着末端が形成される。すると、隣接して連結するｄｓＤＮＡ連結断片は、粘着末端を介して特異的にハイブリダイズし、ＤＮＡリガーゼにより、ニックが繋ぎ合わされ、より大きなｄｓＤＮＡ連結断片が得られることになる。当該反応の詳細は上述したとおりである。 In the first to third reaction vessels, the first to third three types of type IIs restriction enzymes are all added. As a result, each dsDNA ligation fragment is cleaved by the type IIs restriction enzyme, and each adjoining dsDNA ligation fragment forms a complementary sticky end at the end of the ligation side. Then, the adjoining dsDNA ligation fragments specifically hybridize via the sticky ends, and nicks are joined by DNA ligase to obtain a larger dsDNA ligation fragment. The details of the reaction are as described above.
　そして、図１（Ｃ）～（Ｅ）に示すとおり、上述した反応を繰り返すことで、全てのｄｓＤＮＡ断片が連結されて目的ＤＮＡ断片が得られることになる。 Then, as shown in FIGS. 1 (C) to (E), by repeating the above-described reaction, all dsDNA fragments are ligated to obtain the target DNA fragment.
　また、図１に示した例では、得られた目的ＤＮＡ断片の両末端部に第１のＲＥで示される領域、及び第２のＲＥで示される領域が含まれている。これらの領域は、例えば、クローニングベクターに目的ＤＮＡ断片をクローニングする等の操作に利用することができる。 Further, in the example shown in FIG. 1, the region indicated by the first RE and the region indicated by the second RE are included at both ends of the obtained target DNA fragment. These regions can be used for operations such as cloning of a target DNA fragment into a cloning vector, for example.

Claims (3)

  1. 　第１～第ｍ１のｍ１個のＤＮＡ断片を、第１～第ｎ１のｎ１種類（ただし、ｎ１及びｍ１は３以上の自然数を表し、かつｎ１＜ｍ１である。）のＩＩｓ型制限酵素を使用して、第１のＤＮＡ断片から第ｍ１のＤＮＡ断片まで昇順で並ぶように連結することにより目的ＤＮＡ断片を得る方法であって、
    　ｍ１個のＤＮＡ断片はそれぞれ、両末端部に異なるＩＩｓ型制限酵素により認識される塩基配列を有し、かつ隣接して連結するＤＮＡ断片はそれぞれ、連結する側の末端部に同一のＩＩｓ型制限酵素により認識される塩基配列、及び当該ＩＩｓ型制限酵素で切断されたときに相補的な粘着末端を形成する塩基配列を有しており、
    （ｉ）ｍ１個のＤＮＡ断片を、ｋ１個（ただし、ｋ１は２以上の自然数を表し、かつｋ１≦ｎ１－１である。）の連続したＤＮＡ断片を含む複数の反応系に分ける工程と、
    （ｉｉ）各反応系において、ｎ１種類のＩＩｓ型制限酵素で処理し、ＩＩｓ型制限酵素による切断で形成された粘着末端で反応系中のＤＮＡ断片を順特異的に連結する工程と、
    （ｉｉｉ）得られたＤＮＡ連結断片を、連続したＤＮＡ断片を含むようにｌ１個（ただし、ｌ１は２以上の自然数を表し、かつｌ１≦ｎ１－１である。）のＤＮＡ連結断片を含む１又は複数の反応系に分ける工程と、
    （ｉｖ）１又は複数の各反応系において、ｎ１種類のＩＩｓ型制限酵素で処理し、ＩＩｓ型制限酵素による切断で形成された粘着末端で反応系中のＤＮＡ連結断片を順特異的に連結する工程と、
    （ｖ）目的ＤＮＡ断片が得られるまで（ｉｉｉ）及び（ｉｖ）の工程を繰り返す工程と、
    を備える、方法。     The first to m 1 m 1 DNA fragments are the first to n 1 n 1 types (where n 1 and m 1 represent a natural number of 3 or more, and n 1 <m 1 . ) using type IIs restriction enzymes, a method for obtaining the desired DNA fragment by ligating to line up in ascending order from the first DNA fragment to a DNA fragment of the m 1,
    m 1 pieces each DNA fragment of a nucleotide sequence recognized by a different type IIs restriction enzyme at both ends, and each DNA fragment to be connected adjacent to, the same type IIs the distal end of the side connecting A base sequence recognized by a restriction enzyme, and a base sequence that forms a complementary sticky end when cleaved by the type IIs restriction enzyme;
    The (i) m 1 pieces of DNA fragments, k 1 pieces (where, k 1 represents 2 or more natural number, and a k 1 ≦ n 1 -1.) A plurality of reaction system containing a continuous DNA fragment Dividing the process into
    (Ii) in each reaction system, a step of treatment with n 1 kind of type IIs restriction enzymes, ligating the DNA fragments in the reaction system in sticky ends formed by cleavage with type IIs restriction enzymes forward specifically,
    The (iii) the resulting DNA ligated fragment, l 1 or to include a contiguous DNA fragment (however, l 1 represents 2 or more natural number, and a l 1 ≦ n 1 -1.) DNA ligation Dividing into one or more reaction systems containing fragments;
    (Iv) In one or a plurality of reaction systems, n treatments with one type IIs type restriction enzyme, and the DNA ligation fragments in the reaction system are ligated in a specific order with sticky ends formed by cleavage with a type IIs restriction enzyme. And a process of
    (V) repeating steps (iii) and (iv) until the target DNA fragment is obtained;
    A method comprising:
  2. 　第１～第ｍ２のｍ２個のＤＮＡ断片を、第１～第ｎ２のｎ２種類（ただし、ｎ２及びｍ２は３以上の自然数を表し、かつｎ２＜ｍ２である。）のＩＩｓ型制限酵素を使用して、第１のＤＮＡ断片から第ｍ２のＤＮＡ断片まで昇順で並ぶように連結することにより目的ＤＮＡ断片を得る方法であって、
    　以下の工程（１）～工程（５）：
    （１）以下の（ａ）～（ｅ）を満たすｍ２個の二本鎖（ｄｓ）ＤＮＡ断片を準備する工程、
    　（ａ）第１のｄｓＤＮＡ断片は、第２のｄｓＤＮＡ断片と連結する側の末端部とは反対の末端部に、第１のＩＩｓ型制限酵素により認識される塩基配列を有し、かつ第２のｄｓＤＮＡ断片と連結する側の末端部には、第２のＩＩｓ型制限酵素により認識される塩基配列、及び第２のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第２のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する、
    　（ｂ）第ｋ２のｄｓＤＮＡ断片（ただし、ｋ２は２以上（ｎ２－１）以下の自然数を表す。）は、第（ｋ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第ｋ２のＩＩｓ型制限酵素により認識される塩基配列、及び第ｋ２のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｋ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｋ２＋１）のｄｓＤＮＡ断片と連結する側の末端部には、第（ｋ２＋１）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｋ２＋１）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｋ２＋１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する、
    　（ｃ）第ｎ２のｄｓＤＮＡ断片は、第（ｎ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第ｎ２のＩＩｓ型制限酵素により認識される塩基配列、及び第ｎ２のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｎ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｎ２＋１）のｄｓＤＮＡ断片と連結する側の末端部には、第１のＩＩｓ型制限酵素により認識される塩基配列、及び第１のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｎ２＋１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する、
    　（ｄ）第ｌ２のｄｓＤＮＡ断片（ただし、ｌ２は（ｎ２＋１）以上（ｍ２－１）以下の自然数を表す。）は、第（ｌ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第（ｌ２－ａ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｌ２－ａ×ｎ２）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｌ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｌ２＋１）のｄｓＤＮＡ断片と連結する側の末端部には、第（ｌ２＋１－ａ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｌ２＋１－ａ×ｎ２）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｌ２＋１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有する（ただし、ａはｌ２／ｎ２の整数部分の数値である。）、
    　（ｅ）第ｍ２のｄｓＤＮＡ断片は、第（ｍ２－１）のｄｓＤＮＡ断片と連結する側の末端部に、第（ｍ２－ｂ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列、及び第（ｍ２－ｂ×ｎ２）のＩＩｓ型制限酵素で切断されて形成される粘着末端が、第（ｍ２－１）のｄｓＤＮＡ断片の粘着末端と相補的になる塩基配列を有し、かつ第（ｍ２－１）のｄｓＤＮＡ断片と連結する側の末端部とは反対側の末端部には、第（ｍ２＋１－ｂ×ｎ２）のＩＩｓ型制限酵素により認識される塩基配列を有する（ただし、ｂはｍ２／ｎ２の整数部分の数値である。）、
    （２）ｍ２個のｄｓＤＮＡ断片を、第１のｄｓＤＮＡ断片から昇順で、（ｎ２－１）個づつ、それぞれ異なるｃ個の反応容器（ただし、ｃはｍ／（ｎ－１）の整数部分の数値である。）に入れ、更に必要に応じて残余のｄｓＤＮＡ断片をｃ個の反応容器とは異なる１個の反応容器に入れる工程、
    （３）ｄｓＤＮＡ断片を含む反応容器それぞれに第１～第ｎ２のｎ２種類のＩＩｓ型制限酵素を全て入れ、全ＩＩｓ型制限酵素で処理した後、切断により形成された粘着末端で反応容器中のｄｓＤＮＡ断片を順特異的に連結する工程、
    （４）得られたｄｓＤＮＡ連結断片を、連続したｄｓＤＮＡ断片を含むように（ｎ２－１）個づつ、それぞれ異なる反応容器に入れ、更に必要に応じて残余のｄｓＤＮＡ連結断片を異なる１個の反応容器に入れ、工程（３）と同様の操作でｄｓＤＮＡ連結断片を更に連結する工程、
    （５）全てのｄｓＤＮＡ断片が連結されるまで工程（４）を繰り返す工程、
    を備える、方法。     The first to m 2 m 2 DNA fragments are the first to n 2 n 2 types (where n 2 and m 2 represent a natural number of 3 or more and n 2 <m 2 . ) using type IIs restriction enzymes, a method for obtaining the desired DNA fragment by ligating to line up in ascending order from the first DNA fragment to a DNA fragment of the m 2,
    The following steps (1) to (5):
    (1) below (a) providing - a m 2 pieces of double-stranded (ds) DNA fragment that satisfies (e),
    (A) the first dsDNA fragment has a base sequence recognized by the first type IIs restriction enzyme at the end opposite to the end linked to the second dsDNA fragment; At the end portion on the side to be linked to the dsDNA fragment, the base sequence recognized by the second type IIs restriction enzyme and the sticky end formed by cleavage with the second type IIs restriction enzyme are the second dsDNA. It has a base sequence that is complementary to the sticky end of the fragment,
    (B) The k 2 dsDNA fragment (where k 2 represents a natural number of 2 or more and (n 2 −1) or less) is connected to the terminal portion on the side linked to the (k 2 −1) dsDNA fragment. , nucleotide sequence recognized by a type IIs restriction enzyme of the k 2, and sticky ends are formed by being cut with a type IIs restriction enzyme of the k 2 is a sticky end dsDNA fragment of the (k 2 -1) has a nucleotide sequence comprising a complementary, and the end portion of the side connecting the dsDNA fragment of the (k 2 +1), nucleotide sequences recognized by the type IIs restriction enzyme of the (k 2 +1), and the sticky end formed by being cut with a type IIs restriction enzyme (k 2 +1) has a nucleotide sequence comprising the complement of the sticky ends of dsDNA fragments of the (k 2 +1),
    (C) dsDNA fragments of the n 2 is first in the end on the side connected to the dsDNA fragment (n 2 -1), the base sequence recognized by a type IIs restriction enzyme of the n 2, and the n 2 A sticky end formed by cleaving with a type IIs restriction enzyme has a base sequence complementary to the sticky end of the (n 2 -1) dsDNA fragment, and the (n 2 +1) dsDNA fragment; At the terminal end on the ligation side, the base sequence recognized by the first type IIs restriction enzyme and the sticky end formed by cleavage with the first type IIs restriction enzyme are the (n 2 +1) th dsDNA. It has a base sequence that is complementary to the sticky end of the fragment,
    (D) the l 2nd dsDNA fragment (where l 2 represents a natural number not less than (n 2 +1) and not more than (m 2 −1)) is connected to the (l 2 −1) dsDNA fragment. the end portion, is formed by cutting with type IIs restriction enzymes of the (l 2 -a × n 2) nucleotide sequences recognized by the type IIs restriction enzymes, and the (l 2 -a × n 2) The sticky end has a base sequence that is complementary to the sticky end of the (l 2 -1) dsDNA fragment, and the end portion on the side linked to the (l 2 +1) dsDNA fragment has ( l 2 + 1−a × n 2 ) type IIs restriction enzyme and a sticky end formed by cleavage with the (l 2 + 1−a × n 2 ) type IIs restriction enzyme having a sticky end and a base sequence consisting complementary of dsDNA fragments (l 2 +1) (provided that a is a number in the integer portion of a l 2 / n 2.),
    (E) The m 2 dsDNA fragment has a base that is recognized by the (m 2 -b × n 2 ) type IIs restriction enzyme at the end of the side linked to the (m 2 -1) dsDNA fragment. And a base sequence in which the sticky end formed by cleavage with the (m 2 -b × n 2 ) type IIs restriction enzyme is complementary to the sticky end of the (m 2 -1) dsDNA fragment And is recognized by the (m 2 + 1−b × n 2 ) type IIs restriction enzyme at the end opposite to the end connected to the (m 2 −1) dsDNA fragment. (Where b is the numerical value of the integer part of m 2 / n 2 ),
    (2) m 2 dsDNA fragments in ascending order from the first dsDNA fragment, each having (n 2 -1) c different reaction vessels (where c is an integer of m / (n-1)) A step of placing the remaining dsDNA fragments in one reaction vessel different from c reaction vessels, if necessary.
    (3) Put all n 2 kinds of first to n 2 type IIs restriction enzyme to each reaction vessel containing the dsDNA fragment, after treatment with all type IIs restriction enzyme, the reaction vessel at a sticky end formed by the cut Linking the dsDNA fragments therein in a forward specific manner,
    (4) The obtained dsDNA ligated fragments are placed in different reaction vessels, each containing (n 2 −1) so as to contain continuous dsDNA fragments, and if necessary, the remaining dsDNA ligated fragments are Placing in a reaction vessel and further ligating the dsDNA ligation fragment in the same manner as in step (3),
    (5) repeating step (4) until all dsDNA fragments are ligated,
    A method comprising:
  3. 　ＩＩｓ型制限酵素がＡａｒＩ、ＡｃｕＩ、ＡｌｗＩ、ＢｂｓＩ、ＢｂｖＩ、ＢｃｃＩ、ＢｃｅＡＩ、ＢｃｉＶＩ、ＢｃｏＤＩ、ＢｆｕＡＩ、ＢｍｒＩ、ＢｐｍＩ、ＢｐｕＥＩ、ＢｓａＩ、ＢｓｇＩ、ＢｓｍＡＩ、ＢｓｍＢＩ、ＢｓｍＦＩ、ＢｓｐＭＩ、ＢｓｐＰＩ、ＢｓｐＱＩ、ＢｓｒＤＩ、ＢｔｇＺＩ、ＢｓｔＣＩ、ＥａｒＩ、ＥｃｉＩ、Ｅｃｏｐ１５Ｉ、ＦａｕＩ、ＦｏｋＩ、ＨｇａＩ、ＨｐｈＩ、ＨｐｙＡＶ、Ｋｓｐ６３２Ｉ、ＭｂｏＩＩ、ＭｍｅＩ、Ｍｎ１Ｉ、ＳａｐＩ及びＳｆａＮＩからなる群より選ばれる少なくとも１種のＩＩｓ型制限酵素である、請求項１又は２に記載の方法。 Type IIs restriction enzymes are AarI, AcuI, AlwI, BbsI, BbvI, BccI, BceAI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsgI, BsmBI, BsmBs, BsmBs, BsmB 2. At least one type IIs restriction enzyme selected from the group consisting of BstCI, EarI, EciI, Ecop15I, FauI, FokI, HgaI, HphI, HpyAV, Ksp632I, MboII, MmeI, Mn1I, SapI and SfaNI. Or the method of 2.
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