JP4581838B2 - DNA fragment ligation method and kit - Google Patents

Description

  The present invention makes it possible to link a plurality of DNA fragments without restriction while maintaining the direction of the gene, rather than linking the target DNA fragment by a restriction enzyme recognition site, thereby accurately constructing the gene. On how to make it possible to do.

  The present invention also relates to a DNA fragment ligation kit for use in the above method.

  With the maturation of gene recombination technology, a large number of genes related to the modification of organisms by gene recombination have been isolated. In some cases, breeding can be carried out by introducing these single genes. However, studies have been conducted to increase breeding efficiency by simultaneously introducing a plurality of useful genes related to a plurality of traits. Moreover, in order to control a branched metabolic system in a complicated manner, it is essential to control gene expression by introducing multiple genes (Non-patent Document 1).

  When a plurality of target DNA fragments are ligated, it is generally constructed by cutting a part of the vector with a restriction enzyme and inserting the target DNA fragment here. At that time, a method for ligation by providing an appropriate restriction enzyme recognition site so that the target DNA fragment has directivity has been performed. In this method, it is necessary to find an appropriate restriction enzyme recognition site every time one DNA fragment of interest is ligated.

  Furthermore, when a plurality of target DNA fragments are ligated using a restriction enzyme recognition site, the property that one end of the target DNA fragment can be made into a sequence that cannot be recognized by a restriction enzyme may be used. For example, the ends cleaved with restriction enzymes SalI and XhoI are ligated together, and the resulting sequence is different from the recognition sequence of either restriction enzyme and cannot be cleaved. Therefore, the restriction sites for SalI and XhoI restriction enzymes are recognized at both ends. It is possible to continuously link the target DNA fragment to the SalI or XhoI restriction enzyme recognition site (Non-patent Document 2). However, this method has a drawback that it cannot be used when a SalI or XhoI restriction enzyme recognition site is present in the target DNA fragment.

  Furthermore, the restriction enzyme recognition sequence is added to the end of the amplified DNA fragment by amplifying the target gene with a complementary strand synthesis reaction initiation primer containing a restriction enzyme recognition sequence ahead of the sequence complementary to the target DNA fragment. A method for producing a target DNA fragment that can retain the target has been disclosed (Non-patent Document 3). However, this method is limited by the restriction enzyme recognition site used for ligation existing on the vector, as in the above method.

  On the other hand, in order to reduce the frequency of occurrence of restriction enzyme recognition sites in the target DNA fragment and to regulate the ligation direction of the DNA fragment, SfiI is a restriction enzyme that generates a non-pain sequence in the vector. A method for producing a vector that can be cloned in a certain direction by inserting a restriction enzyme recognition site to be cleaved with an adapter or the like is disclosed (Patent Document 1). However, when linking a plurality of target DNA fragments, there are disadvantages in that there are many linking steps in addition to the necessity of preparing many adapters in order to regulate the linking direction.

  Also disclosed is a method of sequentially ligating DNA fragments of interest while specifying the ligation direction without cloning the DNA fragments of interest by ligating the DNA fragments of interest using non-pain sequences in a solid phase system. (Patent Document 2). However, as the ligation step progresses and the ligated DNA fragments become longer, the ligation efficiency decreases.

On the other hand, a kit (MultiSite Gateway Three-Fragment Vector Construction Kit) capable of transferring up to three target DNA fragments to one vector using site-specific recombination DNA sequence and site-specific recombination enzyme. ) Is commercially available from Invitrogen (http://www.invitrogen.co.jp/products/molecular_biology/12533701.shtml). However, since the number of target DNA fragments that can be introduced with this kit is limited to the number of site-specific recombinant DNA sequences, it was impossible to link four or more target DNA fragments.
US Patent 5,595,895 JP2002-262870 Plant Metabolic Engineering Handbook: Supervision by Shinna Akihiko and Yoshida Kazuya, page 854, NTS Corporation, June 2002 N. Kitamoto et al., Appl. Microbiol. Biotechnol. , 50: 558, 1998 PCR Technology, 61-63, Stockton Press, New York, 1989

  An object of the present invention is to provide a method for ligating a large number of target DNA fragments without restriction by the length of the target DNA fragment while defining the ligation direction, and a kit therefor.

  By designing donor vectors, acceptor vectors and conversion vectors containing multiple sets of site-specific recombinant DNA sequences placed at specific positions, and using these vectors and site-specific recombinant enzymes, The inventors succeeded in ligating a plurality of target DNA fragments by a method different from the conventional method. According to this novel method, it is possible to link a large number of target DNA fragments without restriction by the length of the target DNA fragment while defining the connection direction.

SUMMARY OF THE INVENTION The present invention is summarized below.
In the first aspect, the present invention is the donor vector comprising the target DNA fragment between two sets of each of two sets of site-specific recombinant DNA sequences, and outside the two sets of two sets of the two types of the donor vectors. A site-specific recombination reaction between two types of site-specific recombination DNA sequences and a recipient vector containing two types of site-specific recombination DNA sequences capable of causing site-specific recombination. The step of performing, as well as the resulting recombinant vector, and the outside of the vector capable of causing site-specific recombination with the remaining one type and two sets of site-specific recombinant DNA sequences from the donor vector in the vector Site-specific between a pair of site-specific recombinant DNA sequences arranged in one type and a conversion vector comprising the two types of site-specific recombinant DNA sequences from the recipient vector placed inside A recombination reaction Comprising the step of preparing a recombinant vector containing the reproduced said one two sets of site-specific recombination DNA sequences from the restrictor, which provides a method of connecting one or more target DNA fragment.

  In the second aspect, the present invention also includes, in the second embodiment, two sets of each two types of site-specific recombinant DNA sequences L1, R4, R3, L2 in this order, and inserting a target DNA fragment between the DNA sequences. A donor vector containing two sets of site-specific recombinant DNA sequences R1 and R2 and two sets of two site-specific recombinant DNA sequences L4, R1, R2 and L3 in this order. A method for ligating one or more DNA fragments using a conversion vector comprising the above and a site-specific recombinase, the step of inserting the target DNA fragment into the donor vector, the DNA fragment insertion donor vector And the acceptor vector in the presence of a site-specific recombination enzyme to cause site-specific recombination reaction to cause site-specific recombination between L1 and R1 and between L2 and R2, respectively. First recombinant vector And a site-specific recombination reaction between the first recombination vector and the conversion vector in the presence of a site-specific recombinase, and between R3 and L3 and between R4 and L4 And a cycle comprising the steps of producing site-specific recombination with each other to produce a second recombination vector containing regenerated R1 and R2.

  In the first embodiment, the method further comprises a site-specific recombination reaction between a vector obtained by inserting another DNA fragment of interest into the donor vector and the recombinant vector from the final step of the cycle. All steps of the cycle including the step of performing a recombinant vector by repeating a plurality of cycles while changing the type of the target DNA fragment, and sequentially linking a plurality of different target DNA fragments.

In a second embodiment, the recombinant vector includes a selectable marker sequence.
In the third embodiment, the method further comprises transforming the recombinant vector into a microbial cell, culturing in a selective medium, selecting a cell to which the target DNA fragment is linked, and Recovering a nucleic acid containing the target DNA fragment from a cell.
In a fourth embodiment, the donor vector contains a restriction enzyme recognition site that is uniquely present on the vector between the site-specific recombinant DNA sequences R3 and R4 inside.
In a fifth embodiment, the conversion vector includes a restriction enzyme recognition site that is uniquely present on the vector between the site-specific recombinant DNA sequences R1 and R2 inside.

In the sixth embodiment, the acceptor vector is linearized in the site-specific recombination reaction.
In the seventh embodiment, the recombinant vector is linearized during the site-specific recombination reaction.
In the eighth embodiment, four or more kinds of target DNA fragments can be ligated.

  In the third aspect, the present invention further provides two types of site-specific recombinant DNA sequences (L1, L2) arranged on the outside and two types of site-specific recombinant DNA sequences arranged on the inside (L1, L2). R3, R4), and a restriction enzyme recognition site or a site-specific recombinant DNA sequence that makes it possible to insert a target DNA fragment between the outer DNA sequence and the inner DNA sequence. A featured donor vector is provided.

  The present invention further provides, in the fourth aspect, one type and two sets of site-specific recombinant DNA sequences (L3, L4) arranged on the outside and one type and two sets of site-specific recombinant DNA sequences arranged on the inside (L R1, R2), wherein the DNA sequences L3 and L4 are arranged in the innermost part of the recombinant vector obtained by site-specific recombination of the donor vector and the acceptor vector. 13. A set of site-specific recombinant DNA sequences R3 and R4, respectively, capable of site-specific recombination, wherein the acceptor vector comprises the DNA sequences R1 and R2 and the DNA sequence is claim 13. A conversion vector is provided which makes it possible to cause site-specific recombination with two sets of site-specific recombination DNA sequences (L1, L2) arranged outside the donor vector.

  The present invention further includes, in the fifth aspect, one type 2 comprising two types of site-specific recombinant DNA sequences (R1, R2) and the DNA sequence arranged outside the donor vector according to claim 12. A site-specific recombination of the donor vector containing the target DNA fragment into a recipient vector capable of causing site-specific recombination with a pair of site-specific recombinant DNA sequences (L1, L2), respectively, A restriction enzyme reaction is carried out using a restriction enzyme recognition site located inside two types of site-specific recombinant DNA sequences (R3, R4) arranged at the innermost side of the donor vector to obtain a linear DNA. The present invention provides a method for ligating DNA fragments, comprising site-specific recombination of the recombinant vector with the above-described conversion vector, thereby ligating the target DNA fragment into a receiving vector.

  The present invention further provides a method for linking a plurality of DNA fragments characterized in that, in the embodiment, the method for linking DNA fragments is repeated to link a plurality of target DNA fragments to a receiving vector.

The present invention further relates to the sixth aspect, wherein the donor vector comprises the donor vector, two types of site-specific recombinant DNA sequences (R1, R2), and the DNA sequence is arranged outside the donor vector. Provided is a DNA fragment ligation kit comprising a receiving vector that enables site-specific recombination with a pair of site-specific recombination DNA sequences (L1, L2), and the conversion vector in separate containers. To do.
According to that embodiment, the kit further comprises at least one site-specific recombinant enzyme.

Explanation of Terms Terms used in this specification will be described below.
“Target DNA fragment” means an individual DNA fragment to be ligated. Depending on the purpose of use, the type and size can be appropriately selected. For example, the kind of DNA fragment can be derived from animals, plants, yeasts, fungi, bacteria and other organisms, or viruses. The size of the DNA fragment is usually less than 1 Mb, preferably less than several hundred kb, more preferably less than 200 kb.

  “Site-specific recombination” means that various site-specific recombination enzymes identify desired specific nucleotide sequences and recombination occurs. This recombination can be divided into conserved site-specific recombination and transfer-type site-specific recombination by transposon. Conservative site-specific recombination is a recombination involved in the integration and excision of a phage genome into a chromosome, and occurs at a specific short homologous base sequence between the phage and the host chromosome. For example, lambda phage uses the attP site to recombine the phage genome to the attB site of Escherichia coli, Cre-lox site-specific recombination of bacteriophage P1, and the like. By utilizing these site-specific recombination, gene regions having no homology between DNAs can be moved within or between genomes.

  “Two sets of two each” refers to four site-specific recombinant DNA sequences on a donor or conversion vector. In the case of the donor vector, two site-specific recombination DNA sequences on the acceptor vector and two site-specific (ie, one set, two sets) site-specific recombinations that are capable of undergoing site-specific recombination. A recombinant DNA sequence and two other site-specific recombinant DNA sequences arranged on the conversion vector and two other sites arranged inside that are capable of site-specific recombination (ie, another one It means four DNA sequences consisting of two sets of species-specific site-specific recombinant DNA sequences. In the case of a conversion vector, two site-specific recombination DNA sequences derived from the inside of the donor vector and two arranged on the outside capable of causing site-specific recombination (that is, two sets of one type) Site-specific recombinant DNA sequences and two (ie, another set of two types) site-specific sets that are the same as the two site-specific recombinant DNA sequences on the acceptor vector It means four DNA sequences consisting of a replacement DNA sequence.

  “Donor vector” refers to two sets of site-specific recombinant DNA sequences (L1, L2 in FIGS. 1 and 2) arranged on the outside and two sets of site-specific recombinant DNA arranged on the inside. A restriction enzyme recognition site or site-specific containing a sequence (R3, R4 in FIG. 1 and FIG. 2) and enabling insertion of a target DNA fragment between the outer DNA sequence and the inner DNA sequence It means a vector containing a recombinant DNA sequence.

  “Receptor vector” refers to two sets of one type containing two sets of site-specific recombinant DNA sequences (R1, R2 in FIGS. 1 and 2), and the DNA sequences are arranged outside the donor vector. And a site-specific recombination DNA sequence (L1 and L2 in FIGS. 1 and 2), respectively, and vectors capable of causing site-specific recombination.

  “Conversion vector” means two types of site-specific recombinant DNA sequences (L3 and L4 in FIGS. 1 and 2) arranged on the outside and one type and two sets of site-specific recombinant DNA arranged on the inside. And the DNA sequences L3 and L4 are arranged on the innermost side of the recombinant vector obtained by site-specific recombination of the donor vector and the acceptor vector. It means a vector that enables site-specific recombination with each of two types of site-specific recombinant DNA sequences R3 and R4. This vector is for regenerating or restoring the site-specific recombinant DNA sequences R1 and R2 on the acceptor vector.

  “Regenerated” or “regenerated” means that the conversion vector restores the same two sets of site-specific recombinant DNA sequences on the recombinant vector as in the recipient vector.

  “Site-specific recombination enzyme” means an enzyme or a mixture of enzymes that catalyzes a site-specific recombination reaction between a donor vector and an acceptor vector or between a recombinant vector and a conversion vector.

  “Selectable marker” is a drug resistance marker incorporated into an introduction vector to select a transformed host microorganism containing a single target DNA fragment or a plurality of linked target DNA fragments, and complements auxotrophy It means a marker such as a marker.

  “Transformation” means a method or process by which heterologous DNA is incorporated into a biological cell and a new trait is expressed.

  The “selective medium” is a medium for selecting microbial cells in which the target DNA fragment is incorporated, and is a medium in which a drug such as an antibiotic is added to the medium or a specific nutrient is depleted from the medium. Means.

The “rare cutter site” means a restriction enzyme recognition site that recognizes 8 or more bases.
“Multiple cloning site” means a site for restriction cleavage composed of a plurality of restriction enzyme recognition sites.
“Linearization” means that a vector is linearized by cutting with a specific restriction enzyme.

  By “palindrome sequence” is meant a sequence whose nucleotide sequence is identical to the nucleotide sequence of the complementary strand. On the other hand, when the nucleotide sequence is different from the nucleotide sequence of the complementary strand, it is referred to as “non-palindromic sequence”.

  The present invention is a method that differs from conventional methods by utilizing donor vectors, acceptor vectors and conversion vectors, and site-specific recombinant enzymes comprising a plurality of sets of site-specific recombinant DNA sequences placed at specific positions. Provides a method of linking a plurality of target DNA fragments, thereby defining the linking direction of a plurality of target DNA fragments without being limited by the length of the target DNA fragment (or longer DNA fragments) It gives the advantage of allowing unlimited connections.

The present invention will be described more specifically.
Briefly, the present invention is to provide a method of ligating a plurality of target DNA fragments using four sets of two types of site-specific recombinant DNA sequences and a site-specific recombinant enzyme. At that time, the target DNA fragment is placed on a donor vector having two sets of two types of site-specific recombination DNA sequences, and a site-specific recombination reaction is used to store the target gene and one type and two sets of sites on the accepting vector. A specific recombinant DNA sequence is transferred to prepare a recombinant vector in which the first target DNA fragment is linked. Next, site-specific recombination is performed with two sets of site-specific recombinant DNA sequences into a recombinant vector in which the first target DNA fragment is ligated using a conversion vector. Since the vector thus obtained has the same two types of site-specific recombinant DNA sequences as the acceptor vector, the second target DNA fragment inserted into another donor vector is used as the first target It can be ligated to a DNA fragment. By repeating the above reaction n times, n target DNA fragments can be ligated to a receiving vector.

  The concept of a recycle vector that enables unlimited connection of a plurality of target DNA fragments of the present invention is shown in FIG. The procedure for introducing one DNA fragment into a vector includes a step of inserting a target DNA fragment into a donor vector, a step of producing a first recombinant vector from the resulting donor vector and acceptor vector, and the first recombination vector. It consists of three steps, the step of creating a second recombinant vector from a vector and a conversion vector. By repeating this three-step one cycle, a plurality of target DNA fragments can be added or linked one by one without limitation. .

  The vector used in each step is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, shuttle vectors, helper plasmids, etc., and plasmids derived from E. coli (for example, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5 etc.), yeast-derived plasmids (eg, YEp13, YCp50 etc.) and the like. Furthermore, animal virus vectors such as retrovirus or vaccinia virus, insect virus vectors such as baculovirus, and the like can also be used.

  In the donor vector, two sets of site-specific recombinant DNA sequences L1, R4, R3, and L2 are arranged in the order shown in FIG. 1 using appropriate restriction enzyme recognition sites existing on the vector. . That is, two sets of DNA sequences L1 and L2 of one kind are arranged on the outside, and this DNA sequence is used for recombination with a site-specific recombinant DNA sequence in a receiving vector. Another set of two DNA sequences R4 and R3 is placed inside, and this DNA sequence is retained in the first recombinant vector and then recombined with the site-specific recombinant DNA sequence in the conversion vector. Used for.

  A restriction enzyme site or a multiple cloning site suitable for inserting a target DNA fragment inside one type and two sets of site-specific recombinant DNA sequences outside one type and two sets of site-specific recombinant DNA sequences of the donor vector Alternatively, a site-specific recombinant DNA sequence such as Cre-lox (or FLP-frt) (Cosmo Bio) can be placed. In addition, a multiple cloning site having a plurality of restriction enzyme recognition sites (rare cutter sites) that recognize 8 bases or more between two types of site-specific recombinant DNA sequences (R4 and R3) inside the donor vector. It is preferable to arrange in.

  The target DNA fragment may be of any origin such as animal, plant, microorganism and virus. The size (ie, size) is usually less than 1 Mb, preferably less than several hundred kb, such as less than 800 kb, 700 kb, 600 kb, 500 kb, 400 kb or 300 kb, more preferably less than 200 kb, such as 100 kb, 90 kb, 80 kb, 70 kb. , 60 kb, 50 kb, 40 kb, 30 kb, 20 kb, 10 kb, 5 kb, or less. After inserting the target DNA fragment into a cloning vector, this vector is introduced into a suitable cell, bacterial cell such as Escherichia coli or Bacillus subtilis, yeast cell, insect cell, plant cell, or animal cell by a well-known technique. Can be cloned. In cloning, the target DNA fragment can be amplified by performing PCR using the primer containing a sequence complementary to each end of the sense strand and antisense strand of the DNA fragment and using the vector DNA as a template.

  Cloning vectors include plasmids, phages, viruses and the like, such as pUC118, pUC119, pUC18, pUC19, pBluescript, yeast chromosome vector YAC, Ti plasmid, cosmid vector pJBB, and the like. The target DNA fragment can be inserted into a suitable unique restriction site in the vector DNA using ligase. Vector DNA includes a promoter, a replication origin, a ribosome binding site (eg, SD sequence or Kozak sequence), a selection marker such as a drug resistance gene or a gene that complements auxotrophy, a transcription initiation site, a terminator, an RNA splice site, polyadenyl An element such as an activation signal can be included.

Promoters, such as adenovirus or SV40 promoter, the E. coli lac or trp promoter, phage lambda _{P L} promoter, ADH as yeast, PHO5, GPD, PGK, AOX1 promoter, the nuclear polyhedrosis virus-derived promoter as a silkworm cells And CaMV promoter for plant cells.

  Examples of drug resistance genes include ampicillin resistance gene, chloramphenicol resistance gene, gentamicin resistance gene, hygromycin resistance gene, kanamycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene and the like.

  Examples of genes that complement auxotrophy include thymidine kinase gene, dihydrofolate reductase gene, Leu2, Trp1, and Ura3 genes.

  PCR can be performed, for example, under conditions of denaturation 94 ° C., about 30 seconds to 2 minutes; annealing 57 ° C., about 1 to 3 minutes; extension 70 ° C., about 1 to 10 minutes as one cycle and 20 to 40 cycles. . The size of the primer is at least 10 bases, preferably at least 15 bases, more preferably from about 20 to about 30 bases.

  In the receiving vector, two types of site-specific recombinant DNA sequences R1 and R2 are arranged in the order shown in FIG. 1 using an appropriate restriction enzyme recognition site or the like existing on the vector. The two types of site-specific recombinant DNA sequences of the recipient vector are the two types of site-specific recombinant DNA sequences L1, outside the two types of the two types of site-specific recombinant DNA sequences of the donor vector. Arranged so that site-specific recombination reaction with L2 is possible. Furthermore, since the recipient vector is used as a vector in which the target DNA fragment is linked, for example, when a plant cell is used as a host, for example, pBI101 or the like can be suitably used as a binary vector. When an animal cell is used as a host, for example, a retrovirus vector pDON-AI (Takara Bio Inc.) can be preferably used.

  In the conversion vector, two sets of site-specific recombinant DNA sequences L4, R1, R2, and L3 are arranged in the order shown in FIG. 1 using appropriate restriction enzyme recognition sites existing on the vector. . Two sets of site-specific recombinant DNA sequences L4 and L3 outside the two sets of two types of site-specific recombinant DNA sequences of the conversion vector are two sets of site-specific recombination of two types of donor vectors. It arrange | positions so that site-specific recombination reaction may be carried out with 1 type 2 sets of site-specific recombination DNA sequence R4, R3 inside a DNA sequence. In addition, a unique restriction enzyme recognition site such as a multiple cloning site having a rare cutter site is arranged between two types of site-specific recombinant DNA sequences R1 and R2 inside the conversion vector.

  The “site-specific recombination DNA sequence” used in the present invention is not limited to a specific site-specific recombination DNA sequence, but is based on, for example, a site-specific recombination reaction of lambda phage in E. coli. The four attL and attR sites developed in the present invention can be used, and “site-specific recombination enzymes” that can be used at this time are Int (Integrate), IHF (Integration Host Factor), and Xis (Exclusionase). LR clonase enzyme (Invitrogen) can be used. Alternatively, four sets of attP sites and attB sites can be used as another “site-specific recombinant DNA sequence”, and BP clonase enzyme (Invitrogen) containing Int and IHF can be used as “site-specific recombinant enzyme”. .

  In the method of the present invention, in order to insert a target DNA fragment into a donor vector, first, the purified DNA is cleaved with a suitable restriction enzyme and ligated to a restriction enzyme site of a suitable vector DNA by a ligase reaction, or A method of ligating to a vector using a site-specific recombination reaction such as Cre-lox (or FLP-frt) is adopted (step (a) in FIG. 1). The enzyme used for the ligase reaction is not particularly limited as long as it can link DNA molecules to each other. For example, T4 DNA ligase can be preferably used. It is also possible to use Escherichia coli ligase or ligase of Tag or Pfu which is a thermostable enzyme. The ligase reaction using T4 DNA ligase is usually performed using Tris-HCl (pH 7.6) as a buffer in the presence of Mg, DTT, and ATP. Furthermore, reaction efficiency can also be improved by adding PEG. The reaction is usually carried out at about 16 ° C. for 2 hours or longer. As a reagent for the ligase reaction, a commercially available product (for example, DNA Ligation Kit manufactured by Takara Bio Inc.) can be used. In ligation between double-stranded DNA molecules, for example, recombinase (lifetech) or TOPO isomerase I (invitrogen) can be used in addition to ligase.

  Next, the donor vector having the target DNA fragment obtained in step (a) and the acceptor vector linearized by cleavage with an appropriate restriction enzyme at a rare cutter site or the like are mixed, and a site-specific recombination reaction such as clonase is performed. (Invitrogen) is used to insert a target DNA fragment into a donor vector by LR reaction, and then a recipient vector obtained by recombination is selected (step (b) in FIG. 1). As a host for constructing the above acceptor vector into which the target DNA fragment has been inserted, E. coli or the like can be used, and a selective medium supplemented with antibiotics or the like that can selectively grow Escherichia coli having the acceptor vector after transformation, etc. To select the recombinant vector 1 having the target DNA fragment. Depending on the type of drug resistance marker introduced into the vector, an antibiotic such as ampicillin, chloramphenicol, gentamicin, hygromycin, kanamycin, spectinomycin, or tetracycline is added to the selective medium.

  In the method of the present invention, in order to insert the next target DNA fragment, the recombinant vector 1 having the target DNA fragment obtained in the step (b) has two types of site-specific recombinant DNAs in the receiving vector. The arrays R1 and R2 are regenerated or restored. For this purpose, the conversion vector is mixed with the linearized recombinant vector 1 obtained by cleaving the recombinant vector 1 obtained in step (b) with a restriction enzyme at a unique restriction enzyme recognition site such as a rare cutter site. Specific recombination reaction, for example, a site-specific recombination enzyme such as clonase (Invitrogen) is used to insert a target DNA fragment into a donor vector by LR reaction, and recombination vector 2 obtained by recombination is selected ( Step (c) in FIG. E. coli etc. can be used as a host for constructing the above-mentioned recombinant vector 2 in which one set and two sets of site-specific recombinant DNA sequences resurrected in the acceptor vector are selected. Recombinant vector 2 is selected using a selective medium supplemented with antibiotics that can grow naturally.

  In the present invention, the above steps (a), (b) and (c) can be repeated according to the number of target DNA fragments to be ligated (step (d) in FIG. 1). Thereby, a large number of target DNA fragments can be sequentially linked.

  The number of target DNA fragments that can be ligated by the method of the present invention is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more.

  Furthermore, if necessary, the target DNA fragment that is multiply linked using a specific restriction enzyme can be excised and recovered from the finally obtained recombinant vector.

  Multiple ligated target DNA fragments obtained by the method of the present invention are produced by producing a fusion polypeptide in which polypeptides encoded by a plurality of target DNA fragments from which it is derived, production of transgenic organisms, genetic diseases It can be used for production of therapeutic vectors, gene function analysis, and the like.

  In the production of a fusion polypeptide, a nucleic acid linked with at least two different DNA fragments produced by the method of the present invention is incorporated into an expression vector containing a regulatory sequence such as a promoter, and then the vector is transformed into eukaryotic or The nucleic acid is introduced into a prokaryotic cell, the nucleic acid is expressed in the cell, and the obtained fusion polypeptide is recovered. At this time, the fusion polypeptide can be secreted outside the cell by ligating the DNA sequence encoding the signal peptide to the nucleic acid. For this purpose, any commercially available or literature vector can be used. For example, bacterial vectors include, for example, pQE70, pQE60, pQE-9 (Qiagen), pBluescript II KS, ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), pET-11a (Novagen). Examples of eukaryotic vectors include pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVL SV40 (Pharmacia), and the like. Furthermore, in order to facilitate recovery of the produced fusion polypeptide, a pET vector (Novagen) into which a His-tag is inserted can also be used. Alternatively, in order to fuse green fluorescent protein (GFP) or luciferase (Luc) to the target DNA fragment, for example, by using a vector such as pQBI25 (TAKARA), pQBI63 (TAKARA), or pGL vector (promega). Polypeptide recovery can also be facilitated.

  In the production of transgenic organisms, for example, in the case of transgenic plants, properties such as resistance to plant diseases caused by microorganisms and viruses, resistance to insect pests or insecticides, resistance to agricultural chemicals to control weeds, etc. The method of the present invention can be used to produce a plant to which is given. Further, in the case of a transgenic animal, for example, in order to investigate the cause of a disease including a hereditary disease or a treatment method thereof, a multi-ligated DNA fragment produced by the method of the present invention may be introduced into the chromosome of the animal. it can. Such a DNA fragment can have a DNA sequence for disrupting the gene or a DNA sequence for returning the defective gene to normal.

  Known general techniques can be used for the production of transgenic animals or plants. In the case of transgenic plants, for example, the Ti plasmid / Agrobacterium method can be used. For example, E. coli transformed with a vector containing multiple ligated DNA fragments is contacted with Agrobacterium, and the vector is transferred to Agrobacterium through conjugation to produce a recombinant Agrobacterium. Plant cells can be regenerated by inoculating and transforming plant cells. In the case of transgenic animals, a method of microinjecting a target DNA fragment into the nucleus of a fertilized egg, or incorporating a target DNA fragment into an artificial chromosome such as YAC or HAC, A transgenic animal can be produced using a method for introducing a foreign gene. For example, supervised by Tsutsuo Matsuhashi et al., Watson Recombinant DNA Molecular Biology 2nd Edition, Maruzen, 1995, JP-A No. 2002-017186, JP-A No. 2001-352951, JP-A No. 2001-309436, JP-A No. 11-239430 Gazette, JP-A-11-220975, JP-A-11-192036, JP-A-11-155420, JP-A-10-304790, JP-A-10-150983, Special table 2004-520048, Special table 2003-530888 and the like describe a method for producing a transgenic animal or plant, and the disclosure can be used in the present invention.

  In the production of a genetic disease treatment vector, vectors for gene transfer such as adeno-associated virus, adenovirus, retrovirus (RSV, HMS, MMS, etc.), HSV virus, herpes virus, cytomegalovirus, and pox are used. A therapeutic vector can be produced by incorporating a multiligated DNA fragment produced by the method of the present invention into a viral vector such as a virus. Alternatively, the DNA fragment can be encapsulated in a liposome vector. Regarding such vectors and their production, for example, JP 2004-180689, JP 2001-292770, JP 10-127288, JP 08-238092, JP 07-069933, Special Table 2004. -533827, Special Table 2002-529097, No. 98/027217, etc., the disclosure of which can be used in the present invention.

  In gene function analysis, a transgenic plant or animal is prepared as described above, and the function of the introduced DNA fragment is compared with that of a control plant or animal into which the DNA fragment is not introduced, for example, biological, physiological, biochemical. And morphological comparisons can be made to determine gene function. The introduced DNA fragment may be introduced so as to allow high expression, or may be introduced into the chromosome in the form of knockout or knockin.

  The present invention further provides a DNA fragment ligation kit comprising the above donor vector, the above acceptor vector, and the above conversion vector in separate containers.

  The donor vector contains two sets of two each site-specific recombinant DNA sequences L1, R4, R3, L2. Two sets of DNA sequences L1, L2 of one kind are arranged on the outside, and this DNA sequence is used for recombination with a site-specific recombinant DNA sequence in a receiving vector. Another set of two DNA sequences R4 and R3 is placed inside, and this DNA sequence is retained in the first recombinant vector and then recombined with the site-specific recombinant DNA sequence in the conversion vector. Used for. A restriction enzyme site or multiple cloning suitable for inserting the target DNA fragment between the two types of site-specific recombinant DNA sequences on the inner side and the two types of site-specific recombinant DNA sequences on the outer side. Site-specific recombinant DNA sequences such as sites or Cre-lox (or FLP-frt) (Cosmo Bio) can be placed. In addition, a unique restriction enzyme recognition site such as a multiple cloning site having a rare cutter site can be arranged between two types of site-specific recombinant DNA sequences R4 and R3 inside the donor vector.

  The acceptor vector contains two sets of site-specific recombinant DNA sequences R1 and R2. These site-specific recombinant DNA sequences are divided into two types and two sets of site-specific recombinant DNA sequences L1 and L2 outside the donor vector and two sets of site-specific recombinant DNA sequences. It arrange | positions so that replacement | exchange reaction can be performed.

  The conversion vector contains two sets of two types of site-specific recombinant DNA sequences L4, R1, R2, and L3. The two types of site-specific recombinant DNA sequences L4 and L3 outside these site-specific recombinant DNA sequences are located inside the two sets of two types of site-specific recombinant DNA sequences of the donor vector. The two species of site-specific recombination DNA sequences R4 and R3 are arranged so as to allow site-specific recombination reaction. In addition, a unique restriction enzyme recognition site, for example, a multiple cloning site having a rare cutter site, can be placed between two types of site-specific recombinant DNA sequences R1 and R2 inside the conversion vector.

  The kit of the present invention can further contain at least one site-specific recombinase. An example of this enzyme is clonase. Examples of other enzymes are integrase, Cre-lox enzyme and the like.

  If the site-specific recombinant DNA sequence uses, for example, four sets of attL and attR sites for site-specific recombination reaction of lambda phage in E. coli, site-specific recombination of attL × attR → attB × attP LR clonase (Invitrogen) including Int (Integrate), IHF (Integration Host Factor) and Xis (Exclusionase), which perform the reaction, can be used. Alternatively, when four pairs of attP sites and attB sites are used as site-specific recombinant DNA sequences, a site-specific recombination reaction of attB × attP → attL × attP is performed. BP clonase containing Int and IHF (Invitrogen) ) Can be used.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.

Construction of Recombinant Vector Containing Four Different Kinds of Ligated (1) Ligation of First DNA Fragment BamHI Amplified by Escherichia coli β-Glucuronidase Gene (GUS) and Introduced into N, C-Terminal Primer Part The SacI restriction enzyme recognition site was cleaved with a restriction enzyme, and then ligated to the BamHI and SacI restriction enzyme recognition sites of the pUC19 vector having the CaMV35S promoter and nos terminator (nosT) to construct a CaMV35 promoter: GUS: nos terminator cassette. The CaMV35S-GUS-nosT cassette DNA fragment was excised from the pUC19 vector at the HindIII and EcoRI restriction enzyme recognition sites and subcloned into the HindIII and EcoRI restriction enzyme recognition sites of the donor vector pRED419 (step of the first DNA fragment (a)). The obtained plasmid pRED419-GUS and the acceptor vector pGWB1 were mixed with a plasmid cleaved at the XhoI restriction enzyme recognition site, subjected to LR reaction using Invitrogen's clonase, transformed into Escherichia coli DH5α, and kanamycin (50 mg / mg). L) and hygromycin (50 mg / L) in LB medium (first DNA fragment step (b)) (FIG. 2). The obtained plasmid pGWB1-GUS was cleaved with restriction enzyme SwaI and AscI restriction enzyme recognition sites, and then LR-reacted with the conversion vector pCON and clonase, and transformed into E. coli DH5α in the same manner as above, and a medium containing kanamycin and hygromycin Selected with. Through a series of operations, the CaMV35S-GUS-nosT cassette DNA fragment was introduced into the acceptor vector pGWB1, and the vector pGWB1-GUS-CON was regenerated from the site-specific recombinant DNA sequence R1-R2 region (first DNA fragment). Step (c)) (FIG. 3).

(2) Ligation of the second DNA fragment A gene encoding the Aequorea fluorescens protein GFP was amplified by PCR, and subcloned between the CaMV35S promoter and nosT on the pUC19 vector at the BamHI and SacI restriction enzyme recognition sites introduced at both ends. (Second DNA fragment step (a)). The resulting CaMV35S: GFP: nosT cassette DNA fragment was excised at the HindIII and EcoRI restriction enzyme recognition sites and ligated to the HindIII and EcoRI restriction enzyme recognition sites of the donor vector pRED419 to obtain pRED419-GFP. The pGWB1-GUS-CON vector was cleaved at SwaI and AscI restriction enzyme recognition sites, reacted with pRED419-GFP, transformed into E. coli DH5α, and then selected with hygromycin and kanamycin (second DNA fragment step) (B)). The obtained vector pGWB1-GUS-GFP was cleaved with SwaI and AscI restriction enzyme recognition sites, subjected to LR reaction with pCON vector and clonase, transformed into Escherichia coli DH5α, selected with hygromycin and kanamycin, and plasmid pGWB1- GUS-GFP-CON was prepared (second DNA fragment step (c)).

(3) Ligation of the third DNA fragment The firefly luciferase gene synthesized by the PCR method was cleaved at the BamHI and SacI restriction enzyme recognition sites introduced at both ends and subcloned between the CaMV35S promoter and nosT on the pUC19 vector. A CaMV35S: luc: nosT cassette DNA fragment was excised from HindIII and EcoRI restriction enzyme recognition sites from the resulting plasmid and ligated to the HindIII and EcoRI restriction enzyme recognition sites of donor vector pRED419 to obtain vector pRED419-luc ( Step (a)) of the third DNA fragment. PGWB1-GUS-GFP-CON and pRED419-luc cleaved at SwaI and AscI restriction enzyme recognition sites were subjected to LR reaction with clonase, transformed into Escherichia coli DH5α, and then selected in a medium containing hygromasin and kanamycin. 3 DNA fragment step (b)). The resulting plasmid pGWB1-GUS-GFP-luc was cleaved at SwaI and AscI restriction enzyme recognition sites, subjected to LR reaction with pCON vector and clonase, transformed into DH5α, selected with hygromycin and kanamycin, pGWB1-GUS-GFP-luc-CON was obtained (step (c) of the third DNA fragment).

(4) Ligation of the fourth DNA fragment Amplification of the active site 627 bp (from amino acid 29 asparagine to 237th serine) of the Clostridium thermocellum xylanase A gene (GenBank AB010958) by PCR, and cleaves at the BamHI and SacI restriction enzyme recognition sites. And subcloned between the CaMV35S promoter and nosT on the pUC19 vector. A CaMV35S: XYL: nosT cassette DNA fragment was excised at the HindIII and EcoRI restriction enzyme recognition sites from the resulting plasmid and ligated to the HindIII and EcoRI restriction enzyme recognition sites of the donor vector pRED419 to obtain pRED419-XYL (the fourth DNA fragment). Step (a)). pGWB1-GUS-GFP-luc-CON was cleaved at SwaI and AscI restriction enzyme recognition sites, LR reaction was performed with pRED419-XYL and clonase, E. coli DH5α was transformed, and selection with kanamycin and hygromycin was performed. (Step (b) of the fourth DNA fragment). The obtained plasmid pGWB1-GUS-GFP-luc-XYL was introduced into Agrobacterium tumefaciens EHA 101 strain by electroporation and selected with hygromycin to obtain a transformant.

(5) Analysis of the ligated DNA product The plasmid pGWB1-GUS-GFP-luc-XYL obtained in (4) above was cleaved at the SwaI and SacI restriction enzyme recognition sites and electrophoresed to link the genes. This was confirmed (FIG. 4).

  According to the present invention, it is possible to define the direction of the target DNA fragment and to connect a plurality of target DNA fragments without limitation, thereby reliably connecting a large number of target DNA fragments without using a special apparatus. It became possible to connect. The method of the present invention is effective in ligation of a large number of target DNA fragments frequently used in molecular biology techniques. In addition, by using the method of the present invention, it can be used for the production of transgenic organisms, for example, for functional analysis of genes.

It is a figure which shows the concept of a recycle vector. In the figure, L1, L2, L3, and L4 and R1, R2, R3, and R4 are DNA sequences that cause site-specific recombination, respectively. B1, B2, B3, and B4 are DNA sequences generated by site-specific recombination of L1 and R1, L2 and R2, L3 and R3, and L4 and R4. It is a figure which shows the process (a) of a 1st DNA fragment using a recycle vector, and a process (b). It is a figure which shows the process (c) of the 1st DNA fragment using a recycle vector. It is an electrophoretogram showing the result of ligation of four DNA fragments using a recycle vector. M: NEB 2-LOG DNA ladder marker, band of sample (1-5) is vector and first DNA fragment (GUS) (over 10 kb) from top, next is third DNA fragment (LUC) (about 4 kb), 3 The second is the second DNA fragment (GFP) and the fourth DNA fragment (XYL) (both overlap at about 3 kbp), the fourth is the rest of the vector fragment (1287 bp), and samples 1, 2, 3, and 4 can be constructed correctly. It is a thing.

Claims (16)

A donor vector containing the target DNA fragment between L1 and R4 or between R3 and L2 of two sets of each two types of site-specific recombinant DNA sequences (L1, R4, R3, L2 in order) ; Two types of site-specific recombination that can cause site-specific recombination with two types of site-specific recombination DNA sequences (L1, L2) on the outside of the two types of each of the two types of the donor vector Performing a site-specific recombination reaction with a recipient vector containing a recombinant DNA sequence (R1, R2) , and the resulting recombinant vector and the rest of the vector from the donor vector one two sets of site-specific recombination DNA sequence (R4, R3) and one disposed on the outside which can cause site-specific recombination two sets of site-specific recombination DNA sequence (L4, L3) And the 1 type 2 derived from the acceptor vector arranged inside A site-specific recombination reaction is carried out with a conversion vector comprising a set of site-specific recombinant DNA sequences (R1, R2), and the regenerated one-species two sets of site-specific sets derived from the acceptor vector method comprising the step of preparing a recombinant vector, connecting the purpose DNA fragment containing the recombinant DNA sequence (R1, R2). Two sets of two types of each site-specific recombinant DNA sequence L1, R4, R3, L2 are included in this order , and a target DNA fragment is inserted between L1 and R4 of the DNA sequence or between R3 and L2. A donor vector that can be inserted, a recipient vector containing two sets of site-specific recombinant DNA sequences R1, R2, and two sets of site-specific recombinant DNA sequences L4, R1, R2, L3 each of two types A method of linking one or a plurality of DNA fragments using a conversion vector comprising the above-mentioned in this order and a site-specific recombinase, the step of inserting the target DNA fragment into the donor vector, A site-specific recombination reaction is performed between the insertion donor vector and the acceptor vector in the presence of a site-specific recombination enzyme, and site-specific recombination is performed between L1 and R1 and between L2 and R2, respectively. Raised the first recombination A step of producing a vector, and a site-specific recombination reaction between the first recombination vector and the conversion vector in the presence of a site-specific recombination enzyme, and between R3 and L3 and R4 and L4 The method comprising a cycle comprising the steps of producing site-specific recombination with each other to produce a second recombination vector containing regenerated R1 and R2.   Including a step of producing a recombinant vector by performing a site-specific recombination reaction between a vector obtained by inserting another DNA fragment of interest into the donor vector and the recombinant vector from the final step of the cycle. The method according to claim 2, further comprising sequentially linking a plurality of different target DNA fragments sequentially by repeating a plurality of cycles while changing the type of the target DNA fragment.   4. A method according to claim 2 or 3, wherein the recombinant vector comprises a selectable marker sequence.   The recombinant vector is transformed into a microbial cell, cultured in a selective medium, a cell to which the target DNA fragment is linked is selected, and, if necessary, a nucleic acid containing the target DNA fragment is recovered from the cell. The method according to any one of claims 2 to 4, further comprising:   6. The donor vector according to any one of claims 1 to 5, wherein the donor vector comprises a restriction enzyme recognition site that is uniquely present on the vector between the site-specific recombinant DNA sequences R3 and R4 inside. Method.   7. The conversion vector according to any one of claims 1 to 6, wherein the conversion vector contains a restriction enzyme recognition site that exists uniquely on the vector between the site-specific recombinant DNA sequences R1 and R2 located inside the conversion vector. Method.   The method according to any one of claims 1 to 7, wherein the acceptor vector is linearized during the site-specific recombination reaction.   The method according to any one of claims 1 to 8, wherein the recombinant vector is linearized in the site-specific recombination reaction.   The method according to any one of claims 1 to 9, wherein four or more kinds of target DNA fragments can be ligated. 2 types of site-specific recombinant DNA sequences (L1, L2) arranged on the outside and 2 types of site-specific recombinant DNA sequences (R3, R4) arranged on the inside, characterized in that it comprises a restriction enzyme recognition site or site-specific recombination DNA sequence makes it possible to insert the DNA fragment of interest between the DNA sequence of the DNA sequence and the inner, more of claims 1 to 10 A donor vector for use in the method of claim 1 . A conversion vector comprising two sets of site-specific recombinant DNA sequences (L3, L4) arranged on the outside and two sets of site-specific recombinant DNA sequences (R1, R2) arranged on the inside. Thus, the DNA sequences L3 and L4 are one type and two sets of site-specific recombination arranged at the innermost side of the recombinant vector obtained by site-specific recombination of the donor vector and the acceptor vector according to claim 11. A site-specific recombination with DNA sequences R3 and R4, respectively, wherein the acceptor vector comprises the DNA sequences R1 and R2 and the DNA sequence is located outside the donor vector according to claim 11. The method according to any one of claims 1 to 10 , which makes it possible to cause site-specific recombination with each of two sets of site-specific recombination DNA sequences (L1, L2). change for Vector. 12. Two sets of site-specific recombinant DNA sequences (L1) comprising two sets of site-specific recombinant DNA sequences (R1, R2) of one type and arranged on the outside of the donor vector according to claim 11. , L2) and the acceptor vector capable of causing site-specific recombination, respectively, to site-specifically recombine the donor vector containing the target DNA fragment, and then place it inside the donor vector. A recombinant vector obtained by subjecting a restriction enzyme reaction to a linear DNA by using a restriction enzyme recognition site present inside two types of site-specific recombinant DNA sequences (R3, R4) A method for ligating DNA fragments, comprising site-specific recombination with the conversion vector according to claim 12 and thereby ligating the target DNA fragment into a receiving vector.   A method for linking a plurality of DNA fragments, wherein the method for linking DNA fragments according to claim 13 is repeated to link a plurality of target DNA fragments to a receiving vector. 12. A donor vector according to claim 11, comprising two sets of site-specific recombinant DNA sequences (R1, R2) of one type, wherein the DNA sequence is located outside the donor vector of claim 11. A kit for ligating DNA fragments, comprising a receiving vector capable of causing site-specific recombination with the specific recombinant DNA sequences (L1, L2) and the conversion vector according to claim 12 , respectively, in separate containers.   The kit according to claim 15, further comprising at least one site-specific recombinase.

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