JP2004180564A - Method for dna chain binding, cloning vector and method for dna cloning - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To synthesize an industrially useful DNA chain in which a DNA chain prepared by a DNA fragment, chemical synthesis, or the like, is efficiently bound, a gene is encoded, or the like. <P>SOLUTION: The DNA chain is cloned on a vector in which which restriction enzyme recognition sites having a cleavage site different from a recognition base sequence are arranged at both sides of a cloning site. The border between the cloned inserted DNA chain and the vector or the inside of the inserted DNA chain is cleft with a restriction enzyme having a cleavage site different from its recognition base sequence. Consequently, the cleft DNA fragment is provided with a preset specific adhesion terminal, and a plurality of DNA fragments having the specific adhesion terminal obtained by the operation are bonded to bind the DNA chain. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】本発明による、ＤＮＡ鎖を認識塩基配列とは異なる切断部位を持つ制限酵素部位をクローニング部位の両脇に配置したベクターにクローニングし（図１−あ）、認識塩基配列とは異なる切断部位を持つ制限酵素にてベクターと挿入ＤＮＡ鎖の境界または挿入ＤＮＡ鎖内部を切断し特異的な粘着末端を生じせしめ（図１−い）、得られた特異的な粘着末端を持つＤＮＡ鎖を連結し（図１−う）、連結したＤＮＡ鎖の両端の特異的な粘着末端に相補する粘着末端を持つベクターで再クローニングする方法を示す概念図。図１において、Ｘ、Ｙ、Ｗ、Ｚ、Ｕ、Ｖ、Ｅ、Ｆ、Ｐ、Ｑ、Ｎ、Ｒ、ＫはＤＮＡ鎖の塩基を示す。ＸとＹ、ＷとＺ、ＵとＶ、ＥとＦ、ＰとＱ、ＲとＫは相補的な塩基である。ＲＲＲＲＲＲは認識塩基配列とは異なる切断部位を持つ制限酵素の認識配列を示す。５’ ＲＲＲＲＲＲＮ ＮＫＫＫＫＫＫ ３’は、ベクターのクローニング部位である、また、ここでのＮとＮの間のような空欄はそこでＤＮＡ鎖が切られていることを示す。図のＸ、Ｙ、Ｗ、Ｚ、Ｕ、Ｖ、Ｅ、Ｆ、Ｐ、Ｑ、Ｎ、Ｒ、Ｋの個数は一例であり、図１での数に限定されるものではない。図１−いの塩基配列上にかかれた実線は、制限酵素による切断部位を示す。
【図２】あらかじめ設定した特異的な粘着末端を生じせしめるためのベクターのクローニング部位の配列を示す概念図。図２において、Ｎ、Ｒ、ＫはＤＮＡ鎖の塩基を示す。
ＲとＫは相補的な塩基である。Ａで示される領域は平滑末端を生じる制限酵素認識部位、Ｂで示される領域は認識塩基配列とは異なる切断部位を持つ制限酵素認識部位を示す。５’ ＲＲＲＲＲＲＮ ＮＫＫＫＫＫＫ ３’は、ベクターのクローニング部位である、また、ここでのＮとＮの間のような空欄はそこでＤＮＡ鎖が切られていることを示す。図２のＮ、Ｒ、Ｋの個数は一例であり、図２での数に限定されるものではない。
【図３】連結したＤＮＡ鎖を再クローニングするベクターの作製法を示す概念図。連結したＤＮＡ鎖末端部分の配列を両端部分に持つＤＮＡ鎖をクローニングし、認識塩基配列とは異なる切断部位を持つ制限酵素で切断時に、連結したＤＮＡ鎖の末端部分の配列に相補的となる粘着末端を生じるようにする。このベクターにより、連結したＤＮＡ鎖を再クローニングできる。図において、Ｘ、Ｙ、Ｐ、Ｑ、Ｅ、Ｆ、Ｎ、Ｒ、ＫはＤＮＡ鎖の塩基を示す。ＸとＹ、ＰとＱ、ＥとＦ、ＲとＫは相補的な塩基である。ＲＲＲＲＲＲは認識塩基配列とは異なる切断部位を持つ制限酵素の認識配列を示す。５’ ＲＲＲＲＲＲＮ ＮＫＫＫＫＫＫ ３’は、ベクターのクローニング部位である、また、ここでのＮとＮの間のような空欄はそこでＤＮＡ鎖が切られていることを示す。図３のＸ、Ｙ、Ｐ、Ｑ、Ｅ、Ｆ、Ｎ、Ｒ、Ｋの個数は一例であり、図３での数に限定されるものではない。
【図４】第３実施例で調製されたＤＮＡ断片を用いて、ＤＮＡ鎖を連結した産物をアガロースゲル電気泳動により分析したもの。各ＤＮＡ断片が効率よく結合され、Ｈ１０−１、Ｈ１０−２、Ｈ１０−３、Ｈ１０−４断片の４断片の連結でも副産物が少なく、効率よく連結されていることがわかる。[0001]
[Industrial applications]
The present invention relates to a method for ligating a DNA chain, a cloning vector, and a method for cloning a DNA, and aims to obtain a novel method for synthesizing a DNA chain by efficiently ligating DNA fragments or DNA chains. is there.
[0002]
[Prior art]
A DNA chain becomes a gene that encodes a certain protein, and since it can be applied to protein production and the like by genetic engineering, artificial synthesis of a DNA chain as a gene from chemically synthesized DNA has been conventionally performed. Was. Techniques for collecting genes from living organisms and using them have also been developed, but methods for artificially producing genes cannot obtain genes from the living organism and can only obtain amino acid sequence information. In addition, when inserted into a host such as Escherichia coli, it is possible to increase the expression efficiency by preparing the base sequence based on the translation codon that is optimal for the host, to impart new properties such as heat resistance to the protein, etc. It is useful as a method for synthesizing a gene not found in the above. Conventionally, an artificial gene is prepared by creating a restriction map of the nucleotide sequence of the gene, annealing and ligating the chemically synthesized DNA strand, connecting several strands, and presenting the nucleotide sequence inside the DNA strand. Using a restriction enzyme site, a linked DNA strand consisting of several chemically synthesized DNA strands is cloned into a plasmid vector or the like. At this time, if there is an error in the base sequence, correction is performed, and the cloned fragment is further linked. Then, the target gene was prepared.
[0003]
[Problems to be solved by the invention]
However, this method has the following problems in addition to the problem that it is complicated and requires a period of several months to six months or more. First, one of the problems is that there is often an error in the chemically synthesized DNA used first. In fact, when synthesizing an oligonucleotide using a DNA synthesizer, it is not rare that an error occurs in the base sequence, but the error increases as the length of the oligonucleotide increases. The second problem is that it is not easy to further link DNA strands obtained by linking chemically synthesized DNAs unless the purity is sufficiently high. This is because by-products are produced with the ligation.
[0004]
The above two problems are that the DNA strand being prepared is cloned into a plasmid vector or the like by using a restriction enzyme site existing on the base sequence inside the DNA strand, to increase the purity, and to correct the base sequence error at this stage. You can do it. However, it is not possible to use a restriction enzyme cleavage site that can be used within the gene of interest.
[0005]
Further, a wheat lectin gene consisting of about 500 base pairs was prepared by using a restriction enzyme site existing inside the base sequence (European Journal Biochemistry (Eur. J. Biochem, 210: 989-997, 1992)). . In this example, a DNA strand of about 150 base pairs is prepared from the oligonucleotide, these are cloned into separate plasmid vectors using restriction enzyme sites existing inside the nucleotide sequence, and the cloned DNA strands are ligated. , Has obtained the gene for wheat lectin. This method was a complicated method using various restriction enzymes.
[0006]
In addition, since DNAse I of bovine pancreas contains a large amount of RNAase in the pancreas, it is difficult to obtain messenger RNA therefrom. In some cases, DNAase I gene is synthesized from oligonucleotide based on amino acid sequence. (The Journal of Biological Chemistry, Vol. 265, pp. 21889-21895, 1990 (The Journal of Biological Chemistry, 265: 21889-21895, 1990)). In this case, the restriction enzyme site existing inside the base sequence is not used, and the clone is not cloned into a plasmid vector until about 800 base pairs are prepared. Therefore, it is not possible to know a base sequence error during the preparation. In this method, a DNA strand of about 50 base pairs is prepared from an oligonucleotide, bound with T4 ligase, separated and purified by polyacrylamide gel electrophoresis, and a highly pure DNA strand is used. After the I gene has been prepared, it is cloned into a plasmid vector, a base sequence error is discovered, an internal restriction enzyme site is used to cut out a base sequence of about 50 base pairs, and this is replaced with a correct DNA strand. The method has forced modification of the base sequence.
[0007]
Furthermore, in recent years, there has been an example in which a gene is produced by a method of linking DNA chains by a polymerase chain reaction, which is more complicated than the conventional method. However, the same problem as the above-mentioned conventional method remains as to the error generated on the base sequence. In addition, a method of ligating DNA chains by a method called ligase chain reaction has also been carried out (Biochemistry Biophysics Research Communication, 248, 200 to 203, 1998 (Biochem Biophys Res Commun 248: 200-203, 1998). )). This method increases the accuracy of linking DNA strands and reduces the purification of by-products. However, there remains a problem that an error in a chemically synthesized DNA chain used first is detected and corrected during the production process.
[0008]
In addition, in order to avoid certain errors in chemically synthesized DNA used for gene production and to increase the purity of a DNA chain to which the chemically synthesized DNA is linked, it is necessary to clone the DNA into a plasmid vector or the like. In this method, a usable restriction enzyme cleavage site is located inside the target gene, and a vector that can be cloned at the same restriction enzyme cleavage site will be prepared. However, it is not possible to use a restriction enzyme cleavage site that can be used within the gene of interest.
[0009]
The present invention is intended to solve the problems as described above, and in an industrially useful method for linking DNA strands, such as encoding a gene, the method uses DNA errors according to errors in chemically synthesized DNA strands and ligation reaction. It is an object of the present invention to provide a method for ligating a DNA chain, a cloning vector, and a method for cloning a DNA, which can avoid a decrease in the purity of a strand and can easily perform ligation of DNA strands.
[0010]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the first invention is to clone a DNA chain into a vector in which restriction enzyme recognition sites having a cleavage site different from the recognition base sequence are arranged on both sides of the cloning site, and By cutting the boundary between the inserted DNA strand and the vector or the inside of the inserted DNA strand with a restriction enzyme having a cleavage site different from its recognition base sequence, a specific sticky end preset on the cut DNA fragment is set. And joining a plurality of DNA fragments having specific sticky ends obtained by this operation.
[0011]
Further, the second invention is a cloning vector for cloning DNA, which comprises a restriction enzyme recognition site (this enzyme is referred to as an enzyme A) which generates one blunt end serving as a cloning site of the vector. Locates a restriction enzyme recognition site having a different cleavage site (this enzyme is referred to as enzyme B) symmetrically without overlapping or partly with the restriction enzyme recognition site of enzyme A, and a cleavage site different from the recognition base sequence. Upon digestion with a restriction enzyme having a site, a DNA fragment having a sticky end capable of binding is produced by cutting the boundary between the inserted DNA strand and the vector or both ends inside the inserted DNA strand.
[0012]
Further, the third invention is directed to a double-stranded DNA having the sequence of the terminal portion of the target DNA fragment at both ends and a restriction enzyme recognition site generating one blunt end to be a cloning site (this enzyme is referred to as enzyme A). A restriction enzyme recognition site having a cleavage site different from the recognition base sequence (this enzyme is referred to as enzyme B), and a symmetrical arrangement of the restriction enzyme recognition site of enzyme A with partial or no overlap By cloning with a restriction enzyme having a cleavage site different from the recognition base sequence to prepare a vector having a sticky end that is complementary to the sequence of the terminal portion of the DNA fragment. This is a DNA cloning method for cloning the DNA fragment.
[0013]
Further, the fourth invention is directed to a restriction enzyme recognition site having a cleavage site different from a recognition base sequence (this restriction enzyme recognition site which generates one blunt end serving as a cloning site (this enzyme is referred to as enzyme A)). The enzyme is referred to as enzyme B), and the restriction enzyme recognition site having a cleavage site different from the recognition base sequence is located at the cloning site. By cloning the DNA strand into a vector arranged on both sides, and cutting the boundary between the cloned inserted DNA strand and the vector or the inside of the inserted DNA strand with a restriction enzyme having a cleavage site different from its recognition base sequence, A predetermined specific sticky end is added to the cut DNA fragment, and a plurality of DNA fragments having a specific sticky end obtained by this operation are ligated. That is a method of connecting the DNA strand.
[0014]
Further, the fifth invention is directed to a restriction enzyme recognition site having a cleavage site different from the recognition base sequence (this restriction enzyme recognition site which generates one blunt end serving as a cloning site (this enzyme is referred to as enzyme A)). The enzyme is referred to as enzyme B), and the restriction enzyme recognition site having a cleavage site different from the recognition base sequence is located at the cloning site. By cloning the DNA strand into a vector arranged on both sides, and cutting the boundary between the cloned inserted DNA strand and the vector or the inside of the inserted DNA strand with a restriction enzyme having a cleavage site different from its recognition base sequence, A predetermined specific sticky end is added to the cut DNA fragment, and a plurality of DNA fragments having a specific sticky end obtained by this operation are ligated. A double-stranded DNA having the sequence of the terminal portion of the DNA fragment to be cloned obtained at the both ends at the both ends is obtained by a restriction enzyme recognition site which generates one blunt end to serve as a cloning site. A restriction enzyme recognition site having a cleavage site different from the recognition base sequence (this enzyme is referred to as enzyme B) is partially or overlapped with the restriction enzyme recognition site of enzyme A. A vector having a sticky end that is complementary to the sequence of the terminal portion of the DNA fragment by inserting into a cloning site of a symmetrically arranged vector and cutting with a restriction enzyme having a cleavage site different from the recognition base sequence And cloning the DNA fragment with this vector.
[0015]
In addition, the vector has a restriction enzyme recognition site having a cleavage site different from the recognition base sequence (this enzyme is referred to as enzyme A) centering on a restriction enzyme recognition site that generates one blunt end serving as a cloning site (this enzyme is referred to as enzyme A). Enzyme B) is symmetrically arranged without any overlap or overlap with the restriction enzyme recognition site of enzyme A, ggtctcgcgagacc (SEQ ID NO: 1 in the sequence listing) or gaagaccccggttctc (SEQ ID NO: 2 in the sequence listing). It may be.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
According to the first and fourth inventions, the method for ligating a DNA chain involves cloning the DNA chain into a vector in which restriction enzyme sites having a cleavage site different from the recognition base sequence are arranged on both sides of the cloning site (FIG. 1-A). A step of cleaving the boundary between the vector and the inserted DNA strand or the inside of the inserted DNA strand with a restriction enzyme having a cleavage site different from the recognition base sequence to generate a preset specific sticky end (FIG. 1-i) The target gene is prepared by the step of ligating the obtained DNA chain having a specific sticky end (FIG. 1-D).
[0017]
In the DNA cloning methods according to the third and fifth aspects of the present invention, the DNA strand is cloned into a vector in which restriction enzyme sites having a cleavage site different from the recognition base sequence are arranged on both sides of the cloning site (FIG. 1-A). ), A step of cleaving the boundary between the vector and the inserted DNA strand or the inside of the inserted DNA strand with a restriction enzyme having a cleavage site different from the recognition base sequence to generate a predetermined specific sticky end (FIG. 1-I). ), Ligating the obtained DNA strand having a specific sticky end (FIG. 1-U), and recloning with a vector having a sticky end complementary to the specific sticky end at both ends of the ligated DNA strand. The target gene is prepared according to (FIG. 1-E).
[0018]
In the second invention, a cloning vector for generating a specific sticky end preset in the second invention has a restriction enzyme recognition site that generates one blunt end serving as a cloning site (this enzyme is referred to as enzyme A). A restriction enzyme recognition site having a cleavage site different from the recognition base sequence (this enzyme is referred to as enzyme B) is placed as a target, and the DNA strand inserted at the time of cleavage with a restriction enzyme having a cleavage site different from the recognition base sequence is targeted. The DNA fragment having a sticky end capable of binding is produced by cutting the boundary between the DNA fragment and the both ends of the inserted DNA strand (referred to as a vector having a sticky end in the third to fifth aspects of the present invention). The nucleotide sequences recognized by the enzymes A and B may or may not partially overlap as shown in FIG. By using such a cloning vector, ligation of DNA chains and cloning of DNA can be performed efficiently.
[0019]
The enzyme B cuts the boundary between the vector and the inserted DNA strand or the inside of the inserted DNA strand when the inserted sequence is located at the restriction enzyme cleavage site serving as the cloning site. Examples of such restriction enzymes include Bbv I, BciV I, Bmr I, Bpm I, Bsa I, BseRI, Bsg I, BsmA I, BsnB I, BsnF I, BspM I, BsrD I, Bts I, Bbs I I, Ear I, Eci I, Fok I, Hga I, Hph I I, Mbo II, Ple I, Sap I, SfaN I, BstF5 I, Fau I, etc., but the recognition base sequence and the single-stranded portion of cohesive end Bbs I, Bsa I and BspMI are preferred. When the cleavage site of enzyme B arranged on both sides is that of Bsa I, the restriction enzyme cleavage site serving as a cloning site is preferably Nru I, and in the case of Bbs I, Sma I is preferred.
[0020]
For example, when Bsa I and Nru I are selected, the nucleotide sequence around the cloning site is: ggtctc g c gagacc (SEQ ID NO: 1 in the sequence listing) is preferred. In this case, the recognition sites of Bsa I are ggtctc and gagacc indicated by underlines. The recognition site of Nru I is tcg cga, and its cleavage site is indicated by a blank.
[0021]
When Bbs I and Sma I were selected, the nucleotide sequence around the cloning site was: gaaccc c g ggtctttc (SEQ ID NO: 2 in the sequence listing) is preferred. In this case, the recognition sites of Bbs I are gaagac and gtctttc indicated by underlines. The recognition site of Sma I is ccc ggg, and its cleavage site is indicated by a blank.
[0022]
Next, the steps of ligation of DNA strands in the method of ligation of DNA strands of the first and fourth inventions and the method of cloning DNA of the fifth invention shown in FIG. The DNA chains X, Z, and V constituting the gene G to be prepared are cloned into the cloning vector having sticky ends as described above. At this time, bases removed when the cloned DNA strand is cut out are overlapped. Specifically, when a vector having a combination of Bsa I and Nru I is used (in this case, RRRRRRN in FIG. 1 is ggtctcg), when the X, Z, and V DNA chains bind in this order (X The upstream side is the strand side and the downstream side is the V chain side), and the four base pairs on the downstream side of the X chain and the upstream side of the Z chain are selected so as to overlap each other. When G is produced by three consecutive DNA strands of X, Z, and V, each is cloned into the cohesive end-donating vector of the present invention which has been cut with enzyme A beforehand, and cut with enzyme B to produce X, Z, V Ends of the DNA strand are cut out with sticky ends for binding in this order, and are joined by specific sticky ends.
[0023]
In the third and fifth inventions for recloning the linked DNA strands, the vector having a sticky end (cloning vector) according to the second invention is used. A restriction enzyme recognition site (this enzyme is referred to as enzyme A) having a cleavage site different from the recognition base sequence around the restriction enzyme recognition site that generates one blunt end to be a cloning site of this vector (this enzyme is referred to as an enzyme) B) is cloned into the cloning site of the vector arranged for the target, and the DNA strand having the sequence of the terminal portion of the ligated DNA strand at both ends is cloned, and digested with a restriction enzyme having a cleavage site different from the recognition base sequence. In order to generate a sticky end that is complementary to the sequence of the terminal portion of the ligated DNA strand (FIG. 3). The DNA linked by this vector can be efficiently recloned to produce a gene having a large molecular weight (FIG. 1-E).
[0024]
In addition, by repeating the operation of the fifth invention (FIGS. 1A to 1E), a plurality of DNA chains having a plurality of DNA fragments linked thereto can be further linked, thereby efficiently synthesizing long chain DNA. It can be carried out.
[0025]
【Example】
Hereinafter, the present invention will be described in detail with reference to examples. The first and second embodiments are cloning vectors and show the procedure for their production. The third embodiment relates to a method for ligating DNA strands and a method for cloning DNA using the cloning vector of the first embodiment. The fourth embodiment relates to a method for ligating DNA strands and a method for cloning DNA using the cloning vector of the second embodiment.
[0026]
First Example (Cloning Vector: Preparation of BBS Plasmid)
After digestion of plasmid pUC19 (5.0 μg) with two restriction enzymes, HindIII and EcoRI, the digest was subjected to 0.7% agarose gel electrophoresis. After the electrophoresis, the agarose gel was immersed in a 10 μg / ml aqueous solution of ethidium bromide for 2 hours, and then the agarose gel was placed on an ultraviolet irradiator for DNA, irradiated with ultraviolet light, and illuminated in an orange color of about 2.7 kb. The DNA band was cut out with a razor, purified using a Qiaquick Gel Extraction Kit (manufactured by Qiagen Co., Ltd.), dissolved in 50 μl of 10 mM Tris-HCl (pH 8.0), and stored as HindIII and EcoRI-cut pUC19.
[0027]
On the other hand, two types of DNA strands having the following sequences were prepared by a DNA synthesizer, and 100 pmol of each DNA strand was taken, and 2 μl of 0.1 M MgCl 2 was prepared. ₂ In a total of 20 μl of a reaction solution containing 0.66 M Tris-Cl (pH 7.6), 10 units of T4 polynucleotide kinase and 20 nmol of ATP at 37 ° C. for 1 hour, and the 5 ′ side of the DNA strand Was phosphorylated. Thereafter, the reaction solution was heated to 65 ° C. for 5 minutes and left at room temperature for 30 minutes.
5 'agctttgaagaccccgggttctctcg 3' (SEQ ID NO: 3 in Sequence Listing)
5 'aattcgagaagaccccgggtcttca 3' (SEQ ID NO: 4 in Sequence Listing)
[0028]
9 μl of the reaction solution mixed with the above-mentioned DNA chain was mixed with 1 μl of HindIII and EcoRI-cut pUC19, mixed with 10 μl of Ligation High (manufactured by Toyobo Co., Ltd.), and reacted at 16 ° C. for 15 hours. Prepare a competent cell and 100 μl of XL1-Blue E. coli in a 1.5-ml tube, add 3.5 μl of 2-mercaptoethanol diluted 20-fold to this, shake occasionally on ice for 10 minutes, and react at 16 ° C. described above. 2 μl of the DNA solution thus added was left on ice for 30 minutes, then immersed in a thermostat kept at 42 ° C. for 45 seconds, immediately returned to ice, and 900 μl of LB (1% bacto-tryptone, 0.5% bacto-easerase). extract, 1% sodium chloride), and cultured at 37 ° C. for 1 hour, inoculated on an LB agar plate containing 50 μg / ml ampicillin, and kept at 37 ° C. for 18 hours. Some of the resulting E. coli colonies were inoculated into 3 ml of LB medium containing 50 μg / ml of ampicillin, cultured at 37 ° C. for 18 hours, and the culture was centrifuged at 9,000 rpm for 5 minutes to precipitate the cells. The body was recovered. A plasmid was prepared from the cells using Qiagen Plasmid Purification Kit (manufactured by Qiagen).
[0029]
The nucleotide sequence of the plasmid obtained above was examined by ABI PRISM Genetic Analyzer (manufactured by Applied Biosystems Japan), and the sequence in which the DNA strand was correctly inserted into the HindIII and EcoRI cleavage sites of the pUC19 vector, that is, the pUC19 vector A plasmid having the following base sequences of HindIII site and EcoRI site was selected. This selected plasmid is designated as BBS plasmid. The underlined portions indicate the recognition site for HindIII restriction enzyme (agacttt) and the recognition site for EcoRI restriction enzyme (gaattc).
5 ' aagctt gaagaccccggggtcttctc gaattc 3 '(SEQ ID NO: 5 in Sequence Listing)
[0030]
Next, the BBS plasmid (5.0 μg) selected above was digested with a restriction enzyme, SmaI, and then subjected to agarose gel electrophoresis as described above, purified, and finally added to 50 μl of TE buffer. Dissolved and stored.
[0031]
Second Example (Cloning Vector: Preparation of BSA Plasmid)
1. Modification of the Bsa I site of plasmid pUC19
After digesting plasmid pUC19 (5.0 μg) with two restriction enzymes, Bpm I and Eam 1105 I, the digest was donated to 0.7% agarose gel electrophoresis and about 2.7 kb of DNA was removed from the agarose gel. The obtained product was purified by Qiaquick Gel Extraction Kit, dissolved in 50 μl of 10 mM Tris-HCl (pH 8.0), and stored as pUC19 cut with BpmI and Eam1105I.
[0032]
On the other hand, a DNA chain having the following sequence was prepared by a DNA synthesizer. This DNA strand is a Bsa I site ggtc contained in the plasmid pUC19. t c is ggtc g This is changed to c (t is changed to g as indicated by an underline). 300 pmol of each chain and 4 μl of a 0.33 M Tris-acetic acid (pH 7.9) buffer containing 4 μl of 0.1 M magnesium acetate, 0.66 M potassium acetate, 5 mM DDT and 0.1% BSA (bovine serum albumin), 4 units Was reacted at 37 ° C. for 20 minutes in a total of 40 μl of a reaction solution containing 5 nmol of dATP, dGTP, dTTP, and dCTP, followed by treatment at 65 ° C. for 30 minutes. In the following sequences, the underlined portions are complementary sequences.
5'taaattctgggagccgggtgagcgtgggtcgc gcggtatcattgcagcactgggggccagatgggtaagccctcccgtatcg 3 '(28th t from 5' side changed to g) (SEQ ID NO: 6 in Sequence Listing)
5 'tgcctgactccccgtcgtgttagataactactgata cggggggggtaccatctgggccccaggtgctgcaatgataccgc 3 '(SEQ ID NO: 7 in Sequence Listing)
[0033]
After adding 4 μl of 3M sodium acetate (pH 7.0) to the above 40 μl of the DNA solution, ethyl alcohol was added to a final concentration of 70%, and the mixture was frozen in a −80 ° C. freezer. Twenty minutes later, it was taken out of the freezer, centrifuged at 15,000 rpm for 10 minutes, and the precipitate was dried using a rotary evaporator. The dried precipitate was dissolved in 30 μl of 10 mM Tris-HCl (pH 8.0), digested with Bpm I and Eam1105 I, extracted with phenol / chloroform, and added with 3 M sodium acetate (pH 7.0). After ethanol precipitation, the precipitate was dissolved in 30 μl of 10 mM Tris-HCl (pH 8.0) (this is referred to as a DNA strand BPEA).
[0034]
Next, 1 μl of Bpm I and Eam1105 I-cut pUC19 and 4 μl of the above-prepared DNA strand BPEA were mixed, and 5 μl of the reagent Ligation High was mixed with the mixture, and the mixture was reacted at 16 ° C. for 15 hours. XL1-Blue E. coli was transformed and inoculated on LB agar plates containing 50 μg / ml ampicillin. Some of the resulting Escherichia coli colonies were cultured in an LB medium containing ampicillin, and a plasmid was prepared from the cells using a Qiagen Plasmid Purification Kit. The nucleotide sequence of the obtained plasmid was examined by ABI PRISM Genetic Analyzer, and the Bsa I site was found to be ggtc. g A plasmid modified to c was selected and used as a BPEA plasmid.
[0035]
2. Add cloning site
1. After digesting the BPEA plasmid prepared in Step 2 with two restriction enzymes, HindIII and EcoRI, the digest is supplied to 0.7% agarose gel electrophoresis, and about 2.7 kb of DNA is obtained from the agarose gel, and the Qiaquick Gel is obtained. It was purified by Extraction Kit, dissolved in 50 μl of 10 mM Tris-HCl (pH 8.0), and stored as HindIII and EcoRI digested BPEA plasmid.
[0036]
On the other hand, two kinds of DNA strands having the following sequences were prepared by a DNA synthesizer, and 100 pmol of each strand was taken, and 2 μl of 0.1 M MgCl 2 was prepared. ₂ In a total of 20 μl of a reaction solution containing 0.66 M Tris-Cl (pH 7.6), 10 units of T4 polynucleotide kinase and 20 nmol of ATP at 37 ° C. for 1 hour, and the 5 ′ side of the DNA strand Was phosphorylated. Thereafter, the reaction solution was heated to 65 ° C. for 5 minutes and left at room temperature for 30 minutes.
5 'agctttggggtctcgcgagacccctcg 3' (SEQ ID NO: 8 in Sequence Listing)
5 'aattcgagggtctcgcgagacccca 3' (SEQ ID NO: 9 in Sequence Listing)
[0037]
4 μl of the reaction mixture obtained by mixing the above two types of DNA chains and 1 μl of the precipitate obtained by drying the HindIII and EcoRI digested BPEA plasmid dissolved in 10 mM Tris-HCl were mixed with 1 μl. For 15 hours, and used to transform XL1-Blue E. coli and inoculated on LB agar plates containing 50 μg / ml ampicillin. Some of the resulting Escherichia coli colonies were cultured in an LB medium containing ampicillin, and a plasmid was prepared from the cells using a Qiagen Plasmid Purification Kit.
The nucleotide sequence of the obtained plasmid was examined by ABI PRISM Genetic Analyzer, and the sequence in which the DNA strand was correctly inserted into the HindIII and EcoRI cleavage sites of the BPEA plasmid, that is, the nucleotide sequences of the HindIII site and the EcoRI site of the BPEA plasmid were as follows: (This is referred to as BSA plasmid).
5 ' aagctt gggtctcgcgagacccctc gaattc 3 '(SEQ ID NO: 10 in Sequence Listing)
In the above description, the underlined portions are the HindIII restriction enzyme recognition site (aagctt) and the EcoRI restriction enzyme recognition site (gaattc).
[0038]
The plasmid (5.0 μg) selected above is digested with a restriction enzyme, Nru I, then subjected to agarose gel electrophoresis as described above, purified, and finally dissolved in 50 μl of TE buffer and stored. did.
[0039]
Third embodiment
1. DNA strand design
First, the nucleotide sequence of human Hsp10 is shown in SEQ ID NO: 11 in the sequence listing. This human Hsp10 protein translation region is encoded from a at position 1 to c at position 306. Further, agt from the first position is a start codon, and tga from the 307th position is a stop codon. Based on the nucleotide sequence of human Hsp10 shown in SEQ ID NO: 11, a sequence that is a part of the sequence of the sense strand starting from the protein translation initiation codon (atg from the first position in SEQ ID NO: 11) of the gene encoding human Hsp10 Four single-stranded DNAs of SEQ ID NOS: 12 to 15 (referred to as S1, S2, S3, and S4) in the column list, and genes of human Hsp10 encoding SEQ ID NOs: 16 to 19 (referred to as A1, A2, A3, and A4) A total of eight single-stranded DNAs, which are part of the antisense strand, were produced by a DNA synthesizer. The 20 nucleotides on the 3 'side of the S1 chain of SEQ ID NO: 12 and the 20 nucleotides on the 3' side of the A1 chain of SEQ ID NO: 16 are complementary sequences. Similarly, the S2 chain of Sequence Listing 13 and the A2 chain of SEQ ID NO: 17, the S3 chain of SEQ ID NO: 14 and the A3 chain of SEQ ID NO: 18, the S4 chain of SEQ ID NO: 15 and the A4 chain of SEQ ID NO: 19 are similarly paired. The 20 nucleotides on the 3 ′ side of the sense strand and the 3 ′ side of the antisense strand are complementary sequences.
[0040]
2. Cloning of double-stranded DNA into BBS plasmid
The S1 chain and its paired A1 chain were each 300 pmol, and 0.33 M Tris-acetic acid (pH 7) containing 4 μl of 0.1 M magnesium acetate, 0.66 M potassium acetate, 5 mM DDT and 0.1% BSA (bovine serum albumin). .9) Reaction in a total of 40 μl of a reaction solution containing a buffer solution, 4 units of T4 polymerase and 5 nmol of dATP, dGTP, dTTP, and dCTP, respectively, at 37 ° C. for 20 minutes, followed by treatment at 65 ° C. for 30 minutes did. After adding 4 μl of 3 M sodium acetate (pH 7.0) to the 40 μl of the DNA solution, ethyl alcohol was added to a final concentration of 70%, and the mixture was frozen in a minus 80 ° C. freezer. Twenty minutes later, it was taken out of the freezer, centrifuged at 15,000 rpm for 10 minutes, and the precipitate was dried using a rotary evaporator.
[0041]
Next, the dried precipitate was dissolved in 30 μl of TE buffer. Take 10 μl of this and add 2 μl of 0.1 M MgCl ₂ And 0.66 M Tris-Cl (pH 7.6), 10 units of T4 polynucleotide kinase and 20 nmol of ATP were added, and the total volume was adjusted to 20 μl with pure water, reacted at 37 ° C. for 1 hour, and the 5 ′ side of the DNA strand was Phosphorylated. 9 μl of the phosphorylated DNA solution and 1 μl of the BBS plasmid digested with Sma I prepared in Example 1 were mixed, and 10 μl of a ligation reagent DNA Ligation kit I solution was added thereto, followed by reaction at 16 ° C. for 1 hour. Was.
[0042]
Competent cells and 100 μl of XL1-Blue E. coli were prepared in a 1.5 ml tube, and 3.5 μl of 20-fold diluted 2-mercaptoethanol was added thereto. The mixture was shaken occasionally on ice for 10 minutes and reacted at 16 ° C. 5 μl of the reaction mixture was added, left on ice for 30 minutes, immersed in a thermostat kept at 42 ° C. for 45 seconds, immediately returned to ice, added with 900 μl of LB, and cultured at 37 ° C. for 1 hour. 50 μl of 100 mM IPTG and 20 mg / ml X-Gal (5-bromo-4-chloro-3-I indolyl-bD) were placed on an LB agar plate (8.5 cm in diameter, 25 ml of agar medium) containing 50 μg / ml ampicillin. -Dissolved in galactoside and dimethylformamide), and inoculated with the bacterial solution described above, followed by incubation at 37 ° C. for 18 hours. Among the resulting E. coli colonies, several white colonies were inoculated into 3 ml of LB medium containing 50 μg / ml ampicillin, cultured at 37 ° C. for 18 hours, and centrifuged at 9,000 rpm for 5 minutes. , And the cells were collected. Plasmids were prepared from the cells using Qiagen Plasmid Purification Kit.
[0043]
The nucleotide sequence of the plasmid obtained above was examined by ABI PRISM Genetic Analyzer (manufactured by Applied Biosystems Japan), and the sequence in which the DNA strand was correctly inserted into the SmaI cleavage site of the BBS plasmid, that is, is indicated by an underline. A sequence surrounded by Bbs I restriction enzyme recognition sites (gaagac on the 5 'side and gtctttc on the 3' side) having the following sequence was selected and designated as plasmid H10-1. In the following, the boundaries between the vector and the inserted DNA are shown in blank columns.
Sequence prepared using S1 and A1 chains (SEQ ID NO: 20 in Sequence Listing)
5 ' gaacac cc atggcaggacaaggtttagaaaagtttctttccactctttgaccgagtattggttgaaaaggaggtgctgctgaaactgtaaccaccaagggggcattatggttgggg gtctttc 3 '
[0044]
Similarly, a double-stranded DNA consisting of the S2 and A2 chains, the S3 and A3 chains, and the S4 and A4 chains was also cloned into a BBS plasmid, and a plasmid having a sequence in which the DNA chain was correctly inserted into the SmaI cleavage site, And plasmids H10-2, H10-3, and H10-4. At this time, the base sequence surrounded by the Bbs I restriction enzyme cleavage sites (gaagac on the 5 'side and gtctttc on the 3' side indicated by underlines) is as follows. In the following, the boundary between the vector and the inserted DNA is shown in blank.
[0045]
Sequence prepared using S2 and A2 chains (SEQ ID NO: 21 in Sequence Listing)
5 ' gaacac cc gctttccagaaaaatctcaaggaaaaagtattgcaagcaagagtgtcgctgttggatcgggttctaaaagggaaagg gggggagatttcaccaggtt ggg gtctttc 3 '
Sequence prepared using S3 and A3 chains (SEQ ID NO: 22 in Sequence Listing)
5 ' gaacac cc agtagcgtgaaaagttggagataaaagttcttctcccagaatatggaggcaccacaagtagtttctaggatgacaagg attatttttcctatttaggaga gg gtctttc 3 '
Sequence prepared using S4 and A4 chains (SEQ ID NO: 23 in Sequence Listing)
5 ' gaacac cc gagatggtgcattctttggaaagtacgttagactagaatactattgaaatggcatcaacatgatgctgcccattccactgaagtctctggggg gtctttc 3 '
[0046]
3. Cutting out DNA fragments
Plasmid H10-1, 40 µg, containing 20 units / µl restriction enzyme Bbs I, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl ₂ , 50 mM NaCl, 1 mM DTT (dithiothreitol) in a buffer at 37 ° C. overnight for digestion. Digested plasmid DNA was subjected to 0.7% agarose gel electrophoresis. The separated DNA band of about 100 bp was cut out with a razor, purified with Qiaquick Gel Extraction Kit, dissolved in 30 μl of TE buffer, and stored as an H10-1 fragment. Similarly, H10-2, H10-3, and H10-4 fragments were prepared from plasmids H10-2, H10-3, and H10-4.
[0047]
4. Ligation of DNA fragments
6 μl of each of the prepared H10-1 fragment and H10-2 fragment and 6 μl of a ligation reagent DNA Ligation kit I solution (manufactured by Takara Shuzo Co., Ltd.) were added, and the mixture was reacted at room temperature for 2 hours. The prepared H10-3 fragment and H10-4 fragment were ligated in the same manner to prepare a mixed solution 2. Next, 6 μl of each of the mixed solutions 1 and 2 and 6 μl of a ligation reagent DNA Ligation kit I solution were added, and the mixture was reacted at room temperature for 2 hours and 30 minutes. This was designated as mixture 3.
[0048]
5. Construction of plasmid vector for recloning
In order to reclond a DNA chain of about 400 bp in the mixture 3, the following two types of chemically synthesized DNA chains were used to prepare a BBT plasmid described below from the BBS plasmid.
5 ′ atggcaggatcctgaagtttctga 3 ′ (SEQ ID NO: 24 in Sequence Listing)
5 'ttagactacttaggatcctgccat 5' (SEQ ID NO: 25 in Sequence Listing)
[0049]
The two types of DNA strands consisting of the above 23 bases were prepared by a DNA synthesizer, and each DNA strand was made 100 pmol and 2 μl of 0.1 M MgCl 2. ₂ 0.66 M Tris-Cl (pH 7.6) containing 10 units of T4 polynucleotide kinase and 20 nmol of ATP, make up to a total of 20 μl with pure water, react at 37 ° C. for 1 hour, and remove the 5 ′ side of the DNA strand. Phosphorylated. Thereafter, the reaction solution was heated at 65 ° C. for 5 minutes, and then left at room temperature for 30 minutes. 4 μl of the phosphorylated DNA solution and 1 μl of the BBS plasmid (digested with Sma I) prepared in Example 1 were mixed, and 5 μl of a ligation reagent DNA Ligation kit I solution was added thereto, followed by reaction at 16 ° C. for 2 hours. I let it.
[0050]
Competent cells and 100 μl of XL1-Blue E. coli were prepared in a 1.5 ml tube, and 3.5 μl of 20-fold diluted 2-mercaptoethanol was added thereto. The mixture was shaken occasionally on ice for 10 minutes and reacted at 16 ° C. 5 μl of the reaction mixture was added, left on ice for 30 minutes, immersed in a thermostat kept at 42 ° C. for 45 seconds, immediately returned to ice, added with 900 μl of LB, and cultured at 37 ° C. for 1 hour. 50 μl of 100 mM IPTG and 20 mg / ml X-Gal were applied to an LB agar plate (8.5 cm in diameter, 25 ml of agar medium) containing 50 μg / ml of ampicillin, and the above-mentioned bacterial solution was inoculated at 37 ° C. It was kept warm for 18 hours. Among the resulting E. coli colonies, several white colonies were inoculated into 3 ml of LB medium containing 50 μg / ml ampicillin, cultured at 37 ° C. for 18 hours, and centrifuged at 9,000 rpm for 5 minutes. , And the cells were collected. Plasmids were prepared from the cells using Qiagen Plasmid Purification Kit.
[0051]
The plasmid obtained above was examined for its nucleotide sequence by ABI PRISM Genetic Analyzer, and the sequence in which the DNA strand was correctly inserted into the SmaI cleavage site of the BBS plasmid, that is, the BbsI restriction enzyme cleavage site (5 ′ The sequence surrounded by gaagac on the side and gtctttc on the 3 ′ side was selected as follows, and this plasmid was designated as BBT plasmid. The boundaries between the vector and the inserted DNA are shown in blank columns.
5 ' gaacac cc atggcaggatcctgaagtttctga gg gtctttc 3 '(SEQ ID NO: 26 in Sequence Listing)
[0052]
Next, 5.0 μg of the above-mentioned BBT plasmid was digested with the restriction enzyme BbsI, separated and purified by agarose gel electrophoresis, and dissolved in 50 μl of TE buffer. In the BBT plasmid, the sequence surrounded by the Bbs I restriction enzyme cleavage sites (gaagac on the 5 'side and gtctc on the 3' side) indicated by underlines is cut as follows to generate protruding ends. For details of the following reaction, see FIG.
5 ' gaacac cc (cut) ctgagg gtctttc 3 '
3 ' cttctg ggtacc (cut) cc cagaag 5 '
[0053]
6. Recloning of ligated DNA
1 μl of the prepared BBT plasmid (digested with Bbs I), 4 μl of a mixed solution 3 in which four DNA fragments were ligated, 5 μl of a ligation reagent DNA Ligation kit I solution were added thereto, and the mixture was reacted at 16 ° C. for 2 hours. . 100 μl of XL1-Blue Escherichia coli was transformed with the reaction solution, and colonies were formed on an LB agar plate containing ampicillin. Ten of these colonies were arbitrarily selected, and 3 ml of LB medium containing 50 μg / ml ampicillin was selected. , And cultured at 37 ° C. for 18 hours, and a plasmid was prepared from the culture solution. When the nucleotide sequence of the obtained plasmid was examined by ABI PRISM Genetic Analyzer, all the plasmids showed BbsI restriction enzyme sites (gaagac and 3 'on the 5 ′ side of SEQ ID NO: 27) as shown in SEQ ID NO: 27 in the sequence listing. The sequence surrounded by 'gtcttc) had the gene sequence of Hsp10.
[0054]
In addition, FIG. 4 shows a product obtained by analyzing a product obtained by linking DNA chains in this example by agarose gel electrophoresis. Lane 1 is a molecular weight marker obtained by digesting lambda phage DNA with a restriction enzyme EcoT14. Lane 2 is also a molecular weight marker and shows DNA of 700, 525, 500, 400, 300, 200, 100, and 50 base pairs from the top of the band shown in FIG. Lane 3 is a DNA fragment H10-1 consisting of the S1 chain and the A1 chain. Lane 4 is a mixture 1 in which the H10-1 fragment and the H10-2 fragment composed of the S2 and A2 chains were ligated. Lane 5 is the H10-3 fragment composed of the S3 and A3 chains and the S4 chain. And a mixture 2 obtained by ligating pieces of H10-4 comprising A4 chains. Lane 6 is a mixture obtained by ligating the mixture 2 and the H10-1 fragment, and lane 7 is a mixture 3 obtained by ligating the mixture 1 and the mixture 2. According to the analysis results, each DNA fragment was efficiently bound, and by ligation of the four fragments H10-1, H10-2, H10-3, and H10-4, there were few by-products and the fragments were efficiently ligated. Understand.
[0055]
Fourth embodiment
1. DNA strand design
In order to prepare a DNA probe for the human Hsp70 gene, based on the human Hsp70 gene base sequence shown in SEQ ID NO: 28 in the sequence listing, sense strands shown in SEQ ID NOS: 29 to 31, S21, S22, S23 and SEQ ID NO: Antisense strands A21, A22, and A23 shown in 32-34 were produced by a DNA synthesizer. In addition, the protein translation region of human Hsp70 starts from the start codon of atg from the first position. The base sequence shown in SEQ ID NO: 28 is a sequence encoding 100 amino acid residues from the N-terminal of human Hsp70 protein.
[0056]
2. Cloning of double-stranded DNA into BSA plasmid
0.33 M Tris-acetic acid (pH 7.0) containing 300 pmol of the S21 chain and its paired A21 chain and 4 μl of 0.1 M magnesium acetate, 0.66 M potassium acetate, 5 mM DDT, and 0.1% BSA (bovine serum albumin). 9) Reaction was performed at 37 ° C. for 20 minutes in a total of 40 μl of a reaction solution containing a buffer solution, 4 units of T4 polymerase, and 5 nmol of dATP, dGTP, dTTP, and dCTP, followed by treatment at 65 ° C. for 30 minutes. . This 40 μl DNA solution was precipitated with ethanol, and the precipitate was dissolved in 30 μl of TE buffer.
[0057]
Take 10 μl of this and add 2 μl of 0.1 M MgCl ₂ 0.66M Tris-Cl (pH 7.6) containing 10 units of T4 polynucleotide kinase and 20 nmol of ATP, make up to a total of 20 μl with pure water, react at 37 ° C. for 1 hour, and make the 5 ′ side of the DNA strand Phosphorylated. 9 μl of the phosphorylated DNA solution and 1 μl of the BSA plasmid digested with Nru I prepared in Example 2 were mixed, ligated, and used to transform XL1-Blue Escherichia coli. , IPTG and X-Gal were inoculated on an LB agar plate, and several white colonies of the resulting E. coli colonies were cultured, and a plasmid was prepared from the cells.
[0058]
The nucleotide sequence of the plasmid obtained above was examined by ABI PRISM Genetic Analyzer, and the sequence in which the DNA strand was correctly inserted into the NruI cleavage site of the BSA plasmid, that is, the BsaI restriction enzyme recognition site (5 ' The base sequence enclosed by ggtctc on the side and gagacc on the 3 ′ side) was selected as follows and designated as plasmid H70-P1. The border between the vector and the inserted DNA is shown in blank.
Plasmid H70-P1
Sequence prepared using S21 and A21 chains (SEQ ID NO: 35 in Sequence Listing)
5 ' ggtctc g atggccaaagccgcggcgatcggcatcgacctggggcaccacctactcctgcgtggggggtgttccaacacggcaaggtggagcatcatcgccacacgc gagacc ctcg 3 '
[0059]
Similarly, a double-stranded DNA consisting of the S22 and A22 chains and the S23 and A23 chains was also cloned into the BSA plasmid, and plasmids having the sequence in which the DNA chain was correctly inserted at the NruI cleavage site, plasmids H70-P2 and H70- I chose P3 each. At this time, the base sequence surrounded by the Bsa I restriction enzyme cleavage sites (ggtctc on the 5 ′ side and gagacc on the 3 ′ side indicated by underlines) is as follows.
Plasmid H70-P2
Sequence prepared using S22 chain and A22 chain (SEQ ID NO: 36 in Sequence Listing)
5 ' ggtctc g aacgaccagggcaaccgcaccacccccgagtacgttgccttcacggacaccgagcggctcatcgggggatgcggcccaagaacaccggtggcgctgaacccgcagacac gagacc 3 '
Plasmid H70-P3
Sequence prepared using S23 chain and A23 chain (SEQ ID NO: 37 in Sequence Listing)
5 ' ggtctc g agaccaccggtttttgacgcgaagcggctgatcgggccgcaagttcggcgacccggtggtgcagtcggacatgaagcactgggcctttccaggtgatcaacgacggagagagag gagacc 3 '
[0060]
3. Cutting out DNA fragments
2 above. 40 μg of the plasmid H70-P1 prepared in the above, containing 20 units / μl of restriction enzyme BsaI, 50 mM Tris-HCl (pH 7.9), 10 mM MgCl 2 ₂ , 100 mM NaCl, 1 mM DTT (dithiothreitol) in a buffer at 37 ° C. overnight for digestion. Digested plasmid DNA was subjected to 0.7% agarose gel electrophoresis. The separated DNA band of about 100 bp was cut out, purified by Qiaquick Gel Extraction Kit, dissolved in 30 μl of TE buffer, and stored as an H70-P1 fragment. In a similar manner, H70-P2 and H70-P3 fragments were prepared from plasmids H70-P2 and H70-P3.
[0061]
4. Ligation of DNA fragments
3 above. 6 μl each of the H70-P1 fragment and the H70-P2 fragment prepared in the above, and 6 μl of a ligation reagent DNA Ligation kit I solution (manufactured by Takara Shuzo Co., Ltd.) were added, and reacted at room temperature for 2 hours to obtain a mixed solution P1. . Next, 6 μl of the H70-P3 fragment and the ligation reagent DNA Ligation kit I solution were added to 18 μl of the mixed solution P1, and reacted at room temperature for 2 hours and 30 minutes. This was designated as mixture P2.
[0062]
5. Construction of plasmid vector for recloning
Two types of DNA strands consisting of 22 bases described below were prepared by a DNA synthesizer.
5 ′ atggccaaaaaggacggagacaag 3 ′ (SEQ ID NO: 38 in Sequence Listing)
5'ctttgtctccgttccttttggccat 3 '(SEQ ID NO: 39 in Sequence Listing)
[0063]
100 pmol of each of the above DNA strands and 2 μl of 0.1 M MgCl ₂ 0.66M Tris-Cl (pH 7.6) containing 10 units of T4 polynucleotide kinase and 20 nmol of ATP, make up to a total of 20 μl with pure water, react at 37 ° C. for 1 hour, and make the 5 ′ side of the DNA strand Phosphorylated. Thereafter, the reaction solution was heated at 65 ° C. for 5 minutes, and then left at room temperature for 30 minutes. 4 μl of this phosphorylated DNA solution and 1 μl of Nru I digested BSA plasmid were mixed, and 5 μl of a ligation reagent DNA Ligation kit I solution was added thereto, followed by reaction at 16 ° C. for 2 hours. Using this, XL1-Blue Escherichia coli was transformed, inoculated on an LB agar plate containing ampicillin, IPTG, and X-Gal, and kept at 37 ° C. for 18 hours. Several white colonies were cultured from the resulting E. coli colonies to prepare plasmids.
[0064]
The plasmid obtained above was examined for its nucleotide sequence by ABI PRISM Genetic Analyzer, and the sequence in which the DNA strand was correctly inserted into the NruI cleavage site of the BSA plasmid, that is, the BsaI restriction enzyme cleavage site (5 ′ The base sequence surrounded by ggtctc and 3 ′ side gagacc) was selected as follows, and this plasmid was designated as BST plasmid. The boundaries between the vector and the inserted DNA are shown in blank columns. 5 ' ggtctc g atggccaaaggacgggagaacag c gagacc 3 '(SEQ ID NO: 40 in Sequence Listing)
[0065]
Next, 5.0 μg of the above BST plasmid was digested with a restriction enzyme BsaI, separated and purified by agarose gel electrophoresis, and dissolved in 50 μl of TE buffer. In the BST plasmid, the base sequence surrounded by the Bsa I restriction enzyme cleavage sites (ggtctc on the 5 ′ side and gagacc on the 3 ′ side) indicated by underlines is cut as follows to generate protruding ends. 5 ' ggtctc g (cut) caagc gagacc 3 '
3 ' cccagg ctacc (cut) g ctctgg 5 '
[0066]
6. Recloning of ligated DNA
5 above. 1 μl of the BST plasmid (digested with Bsa I) prepared in 1), 4 μl of a mixed solution P2 in which three DNA fragments were ligated, and 5 μl of a ligation reagent DNA Ligation kit I solution were added thereto, and the mixture was reacted at 16 ° C. for 2 hours. . XL1-Blue Escherichia coli was transformed with this reaction solution, and colonies were formed on an LB agar plate containing ampicillin. Several colonies were arbitrarily selected and cultured, and a plasmid was prepared from the culture solution. When the base sequence of the obtained plasmid was examined by ABI PRISM Genetic Analyzer, all the plasmids were found to have a Bsa I restriction enzyme site (ggtctcg and 3 on the 5 ′ side of SEQ ID NO: 41) as shown in SEQ ID NO: 41 in the sequence listing. The sequence surrounded by 'caagcgacacc' on the 'side had the human Hsp70 gene shown in SEQ ID NO: 28 in the sequence listing and had the first to 300th sequences.
[0067]
【The invention's effect】
The present invention is configured as described above, and enables the synthesis of industrially useful DNA strands, such as efficiently linking DNA fragments or DNA strands produced by chemical synthesis and encoding genes. First, even if there is no source of a gene library such as a genome or mRNA, a target gene can be prepared using only information on the nucleotide sequence. In addition, even for a protein or the like that is difficult to obtain, it can be prepared using only the information on the base sequence, or the base sequence can be made a codon suitable for expression. In addition, by arbitrarily selecting a base sequence, a gene that does not exist in nature can be prepared, and an artificial mutation such as a single base or several bases can be arbitrarily created.
[0068]
Conventionally, only two DNA fragments could be ligated, but the method of ligation of DNA strands of the present invention can ligate three or more DNA fragments by a single cloning operation. Step can be greatly reduced, and the number of days for producing a DNA chain can be shortened. Further, by using the cloning vector of the present invention, a DNA fragment can be cloned into a vector without using a restriction enzyme cleavage site inherent in the DNA fragment. In addition, as compared with the method using PCR, the error of the base sequence, that is, the probability of occurrence of mutation is extremely small. In addition, the DNA cloning method of the present invention can correct errors in the base sequence of a chemically synthesized DNA chain, and can efficiently obtain highly accurate DNA with fewer base sequence errors.
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows a DNA strand according to the present invention, which is cloned into a vector having restriction enzyme sites having cleavage sites different from the recognition base sequence and arranged on both sides of a cloning site (FIG. 1-A), and different from the recognition base sequence. A restriction enzyme having a cleavage site cuts the boundary between the vector and the inserted DNA strand or the inside of the inserted DNA strand to generate a specific sticky end (FIG. 1-B), and the resulting DNA strand having a specific sticky end Fig. 1 is a conceptual diagram showing a method of ligation (Fig. 1-U) and recloning with a vector having sticky ends complementary to specific sticky ends at both ends of a ligated DNA strand. In FIG. 1, X, Y, W, Z, U, V, E, F, P, Q, N, R, and K represent bases of a DNA chain. X and Y, W and Z, U and V, E and F, P and Q, R and K are complementary bases. RRRRRR indicates a recognition sequence of a restriction enzyme having a cleavage site different from the recognition base sequence. 5′RRRRRRRN NKKKKKK 3 ′ is a cloning site of the vector, and a blank such as between N and N indicates that the DNA strand has been cut there. The number of X, Y, W, Z, U, V, E, F, P, Q, N, R, and K in the figure is an example, and is not limited to the number in FIG. The solid line on the base sequence shown in FIG. 1 indicates a cleavage site by the restriction enzyme.
FIG. 2 is a conceptual diagram showing a sequence of a cloning site of a vector for generating a predetermined specific sticky end. In FIG. 2, N, R, and K indicate bases of a DNA chain.
R and K are complementary bases. The region indicated by A indicates a restriction enzyme recognition site generating a blunt end, and the region indicated by B indicates a restriction enzyme recognition site having a cleavage site different from the recognition base sequence. 5'RRRRRRRN NKKKKKK 3 'is a cloning site for the vector, and blanks such as between N and N indicate that the DNA strand has been cut there. The numbers of N, R, and K in FIG. 2 are examples, and are not limited to the numbers in FIG.
FIG. 3 is a conceptual diagram showing a method for preparing a vector for recloning a ligated DNA strand. Cloning of a DNA strand having the sequence of the end portion of the linked DNA chain at both ends, when the DNA sequence is cleaved with a restriction enzyme having a cleavage site different from the recognition base sequence, an adhesive sequence which is complementary to the sequence of the end portion of the linked DNA chain. Make the ends come up. With this vector, the ligated DNA strand can be recloned. In the figure, X, Y, P, Q, E, F, N, R, and K indicate bases of a DNA chain. X and Y, P and Q, E and F, and R and K are complementary bases. RRRRRR indicates a recognition sequence of a restriction enzyme having a cleavage site different from the recognition base sequence. 5′RRRRRRRN NKKKKKK 3 ′ is a cloning site of the vector, and a blank such as between N and N indicates that the DNA strand has been cut there. The number of X, Y, P, Q, E, F, N, R, and K in FIG. 3 is an example, and is not limited to the number in FIG.
FIG. 4 shows a product obtained by linking DNA strands using the DNA fragment prepared in Example 3 and analyzed by agarose gel electrophoresis. It can be seen that the DNA fragments are efficiently linked, and the four fragments of H10-1, H10-2, H10-3, and H10-4 are ligated efficiently with few by-products.

Claims (8)

The DNA strand is cloned into a vector in which a restriction enzyme recognition site having a cleavage site different from the recognition base sequence is arranged on both sides of the cloning site, and the boundary between the cloned inserted DNA strand and the vector or the inside of the inserted DNA strand is By cutting with a restriction enzyme having a cleavage site different from the recognition base sequence, a predetermined specific sticky end is added to the cut DNA fragment, and the DNA having a specific sticky end obtained by this operation is obtained. A method for joining DNA chains, comprising joining a plurality of fragments. A cloning vector for cloning DNA. A restriction site having a cleavage site different from the recognition base sequence around a restriction enzyme recognition site (this enzyme is referred to as enzyme A) which generates one blunt end serving as a cloning site of this vector. The enzyme recognition site (this enzyme is referred to as enzyme B) is symmetrically arranged without overlapping or partly with the restriction enzyme recognition site of enzyme A, and digested with a restriction enzyme having a cleavage site different from the recognition base sequence. A cloning vector characterized by occasionally cutting a boundary between the inserted DNA strand and the vector or both ends inside the inserted DNA strand to generate a DNA fragment having a sticky end capable of binding. The double-stranded DNA having the sequence of the terminal portion of the target DNA fragment at both ends is converted into a recognition base sequence centered on a restriction enzyme recognition site (this enzyme is referred to as enzyme A) which generates one blunt end serving as a cloning site. A restriction enzyme recognition site having a cleavage site different from the above (this enzyme is referred to as enzyme B) is inserted into a cloning site of a vector symmetrically arranged without or partially overlapping with the restriction enzyme recognition site of enzyme A, By cutting with a restriction enzyme having a cleavage site different from the recognition base sequence, a vector having a sticky end complementary to the sequence of the terminal portion of the DNA fragment is prepared, and the DNA fragment is cloned with this vector. A method for cloning DNA, characterized by the following. Restriction enzyme recognition site (this enzyme is called enzyme B), which has a cleavage site different from the recognition base sequence around a restriction enzyme recognition site that generates one blunt end to be a cloning site (this enzyme is called enzyme A) Is arranged symmetrically without any overlap or overlap with the restriction enzyme recognition site of enzyme A, so that a restriction enzyme recognition site having a cleavage site different from the recognition base sequence is placed on both sides of the cloning site. The DNA strand is cloned, and the boundary between the cloned inserted DNA strand and the vector or the inside of the inserted DNA strand is cleaved with a restriction enzyme having a cleavage site different from the recognition base sequence, thereby obtaining a cut DNA fragment in advance. The method is characterized in that a specific sticky end is set and a plurality of DNA fragments having a specific sticky end obtained by this operation are bound. Method of connecting the NA chain. Restriction enzyme recognition site (this enzyme is called enzyme B), which has a cleavage site different from the recognition base sequence around a restriction enzyme recognition site that generates one blunt end to be a cloning site (this enzyme is called enzyme A) Is arranged symmetrically without any overlap or overlap with the restriction enzyme recognition site of enzyme A, so that a restriction enzyme recognition site having a cleavage site different from the recognition base sequence is placed on both sides of the cloning site. The DNA strand is cloned, and the boundary between the cloned inserted DNA strand and the vector or the inside of the inserted DNA strand is cleaved with a restriction enzyme having a cleavage site different from the recognition base sequence, thereby obtaining a cut DNA fragment in advance. The specified specific sticky ends are given, and a DNA chain is created by joining a plurality of DNA fragments having the specific sticky ends obtained by this operation. The double-stranded DNA having the sequence of the terminal portion of the DNA fragment to be cloned obtained at the both ends at the both ends is converted to a restriction enzyme recognition site that generates one blunt end to be a cloning site (this enzyme is referred to as enzyme A). ), A restriction enzyme recognition site having a cleavage site different from the recognition base sequence (this enzyme is referred to as enzyme B) is arranged symmetrically without overlapping or partially overlapping with the restriction enzyme recognition site of enzyme A. The vector was inserted into the cloning site of the vector and cut with a restriction enzyme having a cleavage site different from the recognition base sequence to prepare a vector having a sticky end that was complementary to the sequence of the terminal portion of the DNA fragment. A method for cloning DNA, characterized in that said DNA fragment is cloned by the following method. A restriction enzyme recognition site having a cleavage site different from the recognition base sequence (this enzyme is referred to as enzyme B), with a restriction enzyme recognition site that generates one blunt end serving as a vector cloning site (this enzyme is referred to as enzyme A). The sequence symmetrically arranged without overlapping or partially overlapping with the restriction enzyme recognition site of enzyme A is ggtctcgcgagacc (SEQ ID NO: 1 in the Sequence Listing) or gaagaccccggtctttc (SEQ ID NO: 2 in the Sequence Listing). The method for ligating DNA strands according to claim 4, characterized in that: A restriction enzyme recognition site having a cleavage site different from the recognition base sequence (this enzyme is referred to as enzyme B), with a restriction enzyme recognition site that generates one blunt end serving as a vector cloning site (this enzyme is referred to as enzyme A). The sequence symmetrically arranged without overlapping or partially overlapping with the restriction enzyme recognition site of enzyme A is ggtctcgcgagacc (SEQ ID NO: 1 in the Sequence Listing) or gaagaccccggtctttc (SEQ ID NO: 2 in the Sequence Listing). The cloning vector according to claim 2, characterized in that: A restriction enzyme recognition site having a cleavage site different from the recognition base sequence (this enzyme is referred to as enzyme B), with a restriction enzyme recognition site that generates one blunt end serving as a vector cloning site (this enzyme is referred to as enzyme A). The sequence symmetrically arranged without overlapping or partially overlapping with the restriction enzyme recognition site of enzyme A is ggtctcgcgagacc (SEQ ID NO: 1 in the Sequence Listing) or gaagaccccggtctttc (SEQ ID NO: 2 in the Sequence Listing). The method for cloning DNA according to claim 3 or 5, wherein:

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