JP2022048163A - Method for preparing long single-stranded dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for preparing a long single-stranded DNA that has an accurate sequence neither containing internal mutations nor terminal deletions, is uniform, and is mixed with no double-stranded DNA.

SOLUTION: A long single-stranded DNA of interest is prepared by cloning a DNA of interest using a vector with a nicking endonuclease recognition site, or a nicking endonuclease recognition site and sequence-specific double-stranded cleavage endonuclease recognition site, cleaving the cloned DNA using an appropriate enzyme, subjecting the cleaved DNA to electrophoresis, then cutting out a gel containing a single-stranded DNA of interest.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2022,JPO&INPIT

Description

Patent Law Article 30, Paragraph 2 Application Applicable January 20, 2016, ssODN-mediated knock-in with CRISPR-Cas forrange genomic regions in zygotes. Nat Commun. Published at 20; 7: 10431

Application for application of Article 30, Paragraph 2 of the Patent Act On February 09, 2016, a sales representative of Biodynamics Research Institute Co., Ltd. prepares a long-chain single-stranded DNA invented by Hitoshi Nagato in-house. (DS620 Long ssDNA Preparation Kit for 3.0 kb (LsODN Preparation Kit)) is on sale.

Application for application of Article 30, Paragraph 2 of the Patent Act April 15, 2016, Funakoshi News April 15, 2016 issue (No. 609), pp. Published on 3, 32

Application for application of Article 30, Paragraph 2 of the Patent Act June 01, 2016, Funakoshi News June 01, 2016 issue (No. 612), p. Published at 9

Patent Law Article 30, Paragraph 2 Application Applicable May 26, 2016, Genome Editing 2016 (Potential for Genome Editing 2016) Published in Room 281 on the 2nd floor of Chuo University Surugadai Memorial Hall

Patent Law Article 30, Paragraph 2 Application Applicable February 26, 2016, Website https: // www. funakoshi. co. Published on jp / contents / 64803

Patent Law Article 30, Paragraph 2 Application Applicable February 16, 2016, Website https: // www. funakoshi. co. Published at jp / contents / 80479

Application for application of Article 30, Paragraph 2 of the Patent Act April 2016, website https://fnkprdddata. blob. core. windows. net / domestic / download / pdf / BDL. _SlODN_ex_flyer. Published on pdf

Application for application of Article 30, Paragraph 2 of the Patent Act April 2016, website http: // www. diagnocine. com / download / lsODNkit. Published on pdf

Application for application of Article 30, Paragraph 2 of the Patent Act April 2016, website http: // www. bio-rev. com / wp-content / uploads / 2010/11 / BDL_160401_BR_lsODNkit. Published on pdf

Patent Law Article 30, Paragraph 2 Application Applicable March 15, 2016, Website http: // m. blog. naver. Published at com / osteorich74 / 2206555319395

Patent Law Article 30, Paragraph 2 Application Applicable March 15, 2016, Website http: // blog. naver. Published at com / osteorich74 / 2206555319395

Patent Law Article 30, Paragraph 2 Application Applicable May 17, 2016, Website http: // m. blog. daum. net / coresciences / 108? Published at categoryId = 2

Application for application of Article 30, Paragraph 2 of the Patent Act March 2016, website http: // www. ibric. org / myboard / read. php? Published on Board = new_protech & id = 135251

Patent Law Article 30, Paragraph 2 Application Applicable March 11, 2016, Website http: // www. Biodynamics. co. jp / prd_ds610. Published on http

Patent Law Article 30, Paragraph 2 Application Applicable March 11, 2016, Website http: // www. Biodynamics. co. jp / e / prd_ds610. Published on http

The present invention relates to a method for preparing long single-stranded DNA. Specifically, the present invention relates to a method for preparing a long-chain single-stranded DNA by cleaving the recognition site using a vector having a nickel endonuclease recognition site or a DNA strand of interest.

Single-stranded DNA is used in many molecular biological experiments such as DNA sequencing, SNP analysis, DNA chips, and SCSP analysis SELEX. Several methods are used for the preparation, and four types of methods are roughly known. The first is chemical synthesis. In recent years, the chemical synthesis method of DNA has been improved, and single-stranded DNA can be produced at low cost (Non-Patent Document 1). The second is a method using a λ exonuclease, in which phosphoric acid is introduced into only the 5'end of one strand by a PCR reaction using a phosphorylated DNA oligomer, and only one of the strands phosphorylated by the λ exonuclease. The other single-stranded DNA that remains undegraded can be obtained by degrading the DNA (Non-Patent Document 2). The third is a method using biotin, in which biotin is introduced into only one strand by a PCR reaction using a biotinylated DNA oligomer, and after alkali denaturation, only the target strand can be recovered with avidin-coated magnetic beads (non-). Patent Document 3). The fourth method is to use RNA, which can be obtained by using RNA as a starting material, obtaining single-stranded DNA by reverse transcriptase, and then degrading RNA with RNase (non-patented). Document 4).

However, each of the above-mentioned methods has some problems when used in actual molecular biological experiments. For example, when the first chemical synthesis is used, the length of bases that can be synthesized is limited to about 200 bases (Non-Patent Document 1), and this length is the entire gene of general size. It is too short to encode (around 1,000 bases) (Non-Patent Document 5). In addition, it is known that chemical synthesis has a very high error rate, and although the error rate of the replication mechanism of prokaryotes and eukaryotes is ^10-7 to ^10-8 , it is typical of chemical synthesis. The error rate of the artificial gene is ^10-2 to ^10-3 (Non-Patent Document 6). Calculating from this value, when an organism makes 200-base double-stranded DNA, 99.998% to 99.9998% is the correct sequence, but the same 200-base double-stranded DNA is also obtained from chemically synthesized oligomers. When created, only 13.4% to 81.9% have the correct sequence. In addition, the error rate of this artificially synthesized gene is the result of making full use of various error elimination techniques currently available, and when simply chemically synthesizing a long-chain single-stranded DNA of 200 bases, The error rate is higher than this value (Non-Patent Document 6).

In addition, in the second method using λ exonuclease, in addition to having to use a synthetic DNA oligomer that is not highly accurate as described above, the target single strand is accompanied by PCR. A mutation is introduced into the DNA, the target single-stranded DNA is also degraded by a side reaction of the λ exonuclease, and the double-stranded DNA remains without completing the reaction of the λ exonuclease. (Non-Patent Document 2).

In the third method using biotin, as in the second method, in addition to having to use a synthetic DNA oligomer that is not highly accurate, one of the purposes associated with performing PCR is one. There is a possibility that a mutation may be introduced into the double-stranded DNA, and when only the target strand is recovered with the avidin-coated magnetic beads after the alkali denaturation, the denaturation is not sufficient and the double-stranded DNA remains. There are problems such as (Non-Patent Document 3).

In the method using the fourth RNA, as in the above method, in addition to having to use a synthetic DNA oligomer that is not highly accurate as a primer for the reverse transcription reaction, reverse transcription by reverse transcriptase The accuracy of the reaction is comparable to that with Taq DNA cDNA, it is by no means high, mutations may be introduced, and the entire process is transcriptional RNA. The acquisition of cDNA, the production of cDNA by reverse transcriptase, and the degradation of RNA by RNase require complicated and complicated procedures. There was a problem that a stopped product, that is, a product having a deletion at the 5'end was completed, and a mixture of a normal product and a product having a deletion was produced (Non-Patent Document 4).

Kosuri S, Church GM (2014) Large-scale de novo DNA synthesis: technologies and applications. Nat Methods. 11 (5): 499-507. Wakimoto Y, Jiang J, Wakimoto H. (2014) Isolation of single-stranded DNA. Curr Protoc Mol Biol. 1; 107: 2.15.1-9. Avci-Adali M, Paul A, Wilhelm N, Ziemer G, Wendel HP. (2009) Upgrading SELEX technology by using lambda exonuclease digestion for single-stranded DNA generation. Molecules. 24; 15 (1): 1-11. Miura H, Gurumurthy CB, Sato T, Sato M, Ohtsuka M. (2015) CRISPR / Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA. Sci Rep. 10.1038 / srep12799. Mashiko D1, Young SA, Muto M, Kato H, Nozawa K, Ogawa M, Noda T, Kim YJ, Satouh Y, Fujihara Y, Ikawa M. (2014) Feasibility for a large scale mouse mutagenesis by injecting CRISPR / Cas plasmid into zygotes. Dev Growth Differ. 56 (1): 122-9 Ma S, Saaem I, Tian J. (2012) Error correction in gene synthesis technology. Trends Biotechnol. 30 (3): 147-54.

By the way, in recent years, with the development of genome editing technology, the demand for long-chain single-stranded DNA that cannot be obtained by the above-mentioned method is increasing (Non-Patent Document 4). For example, in genome editing using fertilized eggs of animals including rats and mice, CRISPR-CAS or TALEN can be used to knock out specific positions or genes on the genome, or even ssODN. By using (single-strand oligodeoxynucleotide; short single-stranded DNA) as a donor, accurate modification and introduction (knock in) in a small region such as base substitution or SNP mutation can be performed. On the other hand, there is also a desire to modify a larger region, that is, to introduce an entire specific gene, or to introduce an entire gene such as GFP or Cre recombinase for functional analysis.

In the case of genome editing using fertilized eggs, it is not possible to make selections by drugs as in the case of genome editing using cells, so in order to perform genome editing efficiently, it is necessary to increase the efficiency of knock in itself. There is. However, although the conventional method using ssODN as a donor is certainly very efficient and useful as compared with the method using double-stranded DNA as a donor, it is one with a length of 200 bases or more as described above. Since it is technically difficult to accurately produce double-stranded DNA (Non-Patent Document 4). Therefore, even if a single-stranded DNA with a length of 200 bases or more can be created, the double-stranded DNA may be contaminated, mutations may be introduced at the ends or inside, or the DNA may be non-uniform. , It was necessary to overcome qualitative problems in order to introduce it into fertilized eggs.

In addition, when knocking in the target DNA by genome editing, it has been pointed out that if the double-stranded DNA is mixed, the double-stranded DNA will be integrated into a place other than the target place on the genome. (Non-Patent Document 5).

Furthermore, when knocking in the target DNA by genome editing, the introduction efficiency is about 10% to 20% at the highest, and when using fertilized eggs, it takes 1 to 2 months to obtain an individual. .. In addition, since it deals with individuals rather than cells, it takes extra time and effort. Therefore, if mutations or deletions occur at a high rate in the single-stranded DNA that should be the basis of genome editing using fertilized eggs, it will take a great deal of effort to obtain accurate knock-in individuals. Will end up.

Therefore, a method for preparing a long-chain single-stranded DNA having an accurate sequence and being uniform and free from contamination with double-stranded DNA has been desired.

The present invention has been made in view of such a situation, and is frequently used in molecular biological experiments by using a nickel endonuclease recognition site when preparing long-chain single-stranded DNA. It is an object of the present invention to provide a method for preparing an accurate long-chain single-stranded DNA, which is also desired in the field of genome editing.

In order to solve such a problem, the present inventors have investigated whether or not a target long-stranded single-stranded DNA can be prepared based on the double-stranded DNA. As a result of repeated diligent research, the double-stranded DNA was denatured or separated by introducing a nick into a vector obtained by cloning the double-stranded DNA having the desired single-stranded DNA using a nickel endonuclease. It has been found that long-chain single-stranded DNA can be prepared.

Specifically, according to the first main aspect of the present invention, it is a method for preparing long-chain single-stranded DNA, in which (1) and (a) at least one nickeling end nuclease is used at both ends of the cloning site. A vector having a recognition site in which the same chain is cleaved by a nickel end nuclease that recognizes the nickel end nuclease recognition site, or (b) at least one nickel end nuclease recognition site at one end of the cloning site, and the above. A step of cloning a DNA strand of interest into a vector having at least one sequence-specific double-strand break end nuclease recognition site at the other end of the cloning site, and (2) the DNA strand of interest by the Nicking End nuclease. The DNA strand of interest is cleaved by a sequence-specific double-strand break end nuclease that cleaves the cloned vector (a) or recognizes the nickel-specific double-strand break end nuclease recognition site. The step of cleaving the cloned vector (b) to generate at least three types of single-stranded DNA having different molecular weights, and (3) adding an appropriate amount of a modifier to the at least three types of single-stranded DNA. Then, the step of denaturing the single-stranded DNA and
(4) Provided is a method comprising a step of separating at least three kinds of denatured single-stranded DNAs by an arbitrary separation means to prepare a desired single-stranded DNA.

Further, according to the second main aspect of the present invention, it is a method for preparing a long-chain single-stranded DNA, and the purpose is to have at least one nickel-end nuclease recognition site at both ends of (1) and (a). A DNA strand of interest whose same strand is cleaved by a nickel-end nuclease that recognizes the nickel-end nuclease recognition site, or (b) at least one nickel-end nuclease recognition site at one end and at least one at the other end. The target DNA strand having one sequence-specific double-strand break end nuclease recognition site was cloned into a vector by cloning the target DNA strand into a vector, and (2) the nickel end nuclease cloned the target DNA strand of (a). A vector in which the DNA strand of interest (b) is cloned by a sequence-specific double-strand break end nuclease that cleaves a vector or recognizes the nickel-specific double-strand break end nuclease recognition site. To generate at least three types of single-stranded DNA having different molecular weights, and (3) adding an appropriate amount of a denaturing agent to the at least three types of single-stranded DNA to obtain the single-stranded DNA. Provided is a method comprising a step of denaturing and (4) a step of separating at least three kinds of the denatured single-stranded DNA by an arbitrary separation means to prepare a desired single-stranded DNA.

According to such a configuration, there is no mutation or deletion of the terminal, the single-stranded DNA has an accurate sequence, and the single-stranded DNA in the form of a uniform length up to several thousand bases is not contaminated with the double-stranded DNA. Can be provided.

Further, according to one embodiment of the present invention, it is preferable that the target DNA strand is 200 bases or more in the above method. Further, according to another embodiment of the present invention, the DNA strand of interest is at least 300 bases, at least 400 bases, at least 500 bases, at least 600 bases, at least 700 bases, at least 800 bases, at least 900 bases, or. At least 1000 bases.

Further, according to another embodiment of the present invention, the arbitrary separation means is gel electrophoresis. In this case, the gel electrophoresis includes non-denaturing agarose gel electrophoresis without a denaturing agent, modified agarose gel electrophoresis containing a denaturing agent, non-denaturing acrylamide gel electrophoresis without a denaturing agent, or modified acrylamide containing a denaturing agent. Gel electrophoresis is preferred.

Furthermore, according to another embodiment of the invention, the optional separation means is gel column chromatography. In this case, according to one embodiment of the present invention, gel column chromatography is preferably gel filtration column chromatography, ion exchange gel column chromatography, or affinity gel column chromatography.

Further, according to one embodiment of the present invention, the number of bases of the nickel endonuclease recognition site is at least 3 bases. Further, according to another embodiment of the present invention, the number of bases of the nickel endonuclease recognition site is preferably 3 bases, 4 bases, 5 bases, 6 bases, or 7 bases. In this case, according to one embodiment of the present invention, the nickel endonuclease is Nb. BbvCI, Nb. BsmI, Nb. BtsI, Nb. BsrDI, Nt. BspQI, Nt. BbvCI, Nt. AlwI, Nt. BsmAI, Nt. BstNBI, Nt. CviPII, Nb. Mva1269I, Nt. Bpu10I, or Nb. BssSI is preferred.

Further, according to another embodiment of the present invention, a guide RNA can be bound to the nickel endonuclease recognition site. In this case, the nickel endonuclease is preferably a D10A variant of Cas9.

Further, according to another embodiment of the present invention, the sequence-specific double-strand break endonuclease recognition site is an enzyme selected from the group consisting of restriction enzymes and meganucleases, or a TALEN recognition site. Can be done. In this case, the meganuclease is preferably I-CeuI, I-SceI, PI-PspI, or PI-SceI.

Furthermore, according to another embodiment of the present invention, a guide RNA or a guide DNA can be bound to the sequence-specific double-strand break endonuclease recognition site. in this case,
The sequence-specific double-strand break endonuclease is preferably Cas9 or Argonaute.

Further, according to another embodiment of the present invention, the denaturing agent is formamide, glycerol, urea, thiourea, ethylene glycol, or sodium hydroxide. In this case, the denaturing agent is preferably formamide or glycerol.

Further, according to the third main aspect of the present invention, the kit used in the method of the first or second main aspect of the present invention described above, (a) at least one at each end of the cloning site. A vector having a nickel endonuclease recognition site in which the same strand is cleaved by a nickel endonuclease that recognizes the nickel endonuclease recognition site, and (b) at least one nickel endonuclease recognition site at one end of the cloning site. And a kit comprising at least one vector selected from vectors having at least one sequence-specific double-strand break endonuclease recognition site at the other end of the cloning site.

Further, according to one embodiment of the present invention, there is provided a kit which is the above-mentioned kit and further includes a reagent containing a denaturing agent for denaturing DNA.

Further, according to the fourth main aspect of the present invention, there is provided a kit used in the method of the second main aspect of the present invention described above, which comprises a vector containing no nickel endonuclease recognition site. Will be done.

Further, according to another embodiment of the present invention, there is provided a kit which is the above-mentioned kit and further includes a reagent containing a denaturing agent for denaturing DNA.

The features and remarkable actions / effects of the present invention other than those described above will be clarified to those skilled in the art by referring to the sections and drawings of the following embodiments of the present invention.

FIG. 1 is a conceptual schematic diagram of a method according to an embodiment of the present invention. FIG. 2 shows a plasmid map of the vector used in the method according to the embodiment of the present invention (FIG. 2A), a multi-cloning site in the vector (FIG. 2B), and an enzyme for cloning to obtain single-stranded DNA. 2C (FIG. 2C) and the entire sequence (FIG. 2D, SEQ ID NO: 1). FIG. 3 is a sequence (1,500 bp, SEQ ID NO: 2) of a target DNA fragment derived from λ phage DNA, which is used in the method according to the embodiment of the present invention. FIG. 4 shows the results of electrophoresis obtained by using the method according to the embodiment of the present invention. Formamide was added to the pLSODN-1 (1.5 kb fragment) plasmid into which two nicks were introduced, and after heat denaturation, electrophoresis was performed on a 1.2% concentration non-denatured agarose gel using 1 × TAE buffer as a running solution. did. A 10 μg plasmid was loaded into lane 1 and a 5 μg plasmid was loaded into lane 2. FIG. 5 shows the results of electrophoresis obtained by using the method according to the embodiment of the present invention. Each lane is a pLSODN-1 (1.5 kb fragment) plasmid (200 ng: lane 1) with two nicks introduced and a purified 1.5 kb long single-stranded DNA (200 ng: lane 2). .. FIG. 6 shows a plasmid map of the cloning vector pETUK (del) used in the method according to one embodiment of the present invention (FIG. 6A), the sequence of the multicloning site (FIG. 6B), and the entire sequence (FIG. 6C, SEQ ID NO: 3) is shown. FIG. 7 is a global sequence of the GFP gene (720 bases, SEQ ID NO: 4). FIG. 8 shows the results of electrophoresis obtained by using the method according to the embodiment of the present invention. Formamide was added to the pETUK (GFP-Tyr) plasmid into which two nicks were introduced, and after heat denaturation, the mixture was electrophoresed on a 1.0% concentration 4M urea agarose gel using the same 4M urea 1 × TAE buffer as a running solution. .. Lane 1 is DynaMarker DNA High, lane 2 is pETUK (GFP-Tyr) without endonuclease treatment, and lane 3 is pETUK (GFP-Tyr) with endonuclease treatment. FIG. 9 is the result of electrophoresis of the purified long-chain single-stranded DNA (759 bases, 100 ng) of the purified GFP gene obtained by using the method according to the embodiment of the present invention. FIG. 10 shows the results of electrophoresis of a plasmid introduced with two nicks on a non-denaturing gel when various denaturing agents were used in one embodiment of the present invention.

Hereinafter, embodiments and examples according to the present invention will be described with reference to the drawings.
As described above, the method for preparing a long-chain single-stranded DNA according to an embodiment of the present invention comprises a nickel-end nuclease recognition site, or a nickel-end nuclease recognition site and a sequence-specific double-strand break end nuclease recognition site. The target DNA is cloned using the vector to be possessed, cleaved with an appropriate enzyme, electrophoresed, and then the gel containing the target single-stranded DNA is cut out to prepare the target long-chain single-stranded DNA. do.

Further, the method for preparing a long-chain single-stranded DNA according to another embodiment of the present invention has a nickel-end nuclease recognition site, or a nickel-end nuclease recognition site and a sequence-specific double-strand break end nuclease recognition site. The target long-stranded single-stranded DNA is obtained by cloning into a cloning vector using the target DNA, cleaving it with an appropriate enzyme, electrophoresis, and then cutting out a gel containing the target single-stranded DNA. adjust.

The problems of the above-mentioned four types of conventional single-stranded DNA preparation methods can be summarized as follows: (1) the problem of difficulty in obtaining long-stranded single-stranded DNA, (2) sequences such as internal mutations and terminal deletions. There are problems of accuracy and size non-uniformity, (3) problems of double-stranded DNA contamination, and (4) problems of complexity of the operation process. According to the method according to the present invention, it can be easily understood that all of them are superior to the conventional methods as described below.

First, regarding the problem of the length of the single-stranded DNA in (1), in the method according to the present invention, there are three types having different molecular weights depending on the nickel-ending endonuclease or the nickel-ending endonuclease and the sequence-specific double-stranded cleaving endonuclease. DNA molecules of the above are generated, and then denatured or separated, thereby making it possible to prepare the desired long-chain single-stranded DNA. Then, as described in the problem (3) described later, in the method according to the present invention, when gel electrophoresis is used for separation in order to prevent other DNA molecules from being mixed with the target long-chain single-stranded DNA, The size of the target long-stranded single-stranded DNA is preferably about half the size of the vector. That is, it is possible to cut out only the target band by allowing the target long-chain single-stranded DNA to flow to the tip of the gel during electrophoresis and to obtain a sufficient distance from other DNA molecules. Even if there is such a length restriction, for example, when a vector having a length of about 12,000 bases is used, a single-stranded DNA having a length of about 6,000 to 7,000 bases can be prepared. The single-stranded DNA of such a length is a length that cannot be obtained by the above-mentioned conventional method, and is sufficiently long as a sequence encoding one structural gene. Theoretically, when a cosmid vector (30 kbp to 45 kbp), a BAC vector (to 300 kbp), or a YAC vector (several Mbp) is used, a larger long-chain single-stranded DNA is used by the method according to the present invention. Can be prepared.

Next, regarding the problem of internal mutation and terminal deletion in (2), since the single-stranded DNA prepared by the method according to the present invention is derived from the DNA cloned into the vector, the error rate is the prokaryote. The error rate of the replication mechanism of living organisms is about ^10-7 to ^10-8 , which is negligibly small for normal use of single-stranded DNA (Non-Patent Document 6). Further, the single-stranded DNA prepared by the method according to the present invention is prepared by a sequence-specific double-strand break endonuclease such as a restriction enzyme with high accuracy of base recognition or a nickeling endonuclease derived from the restriction enzyme. As such, mutations and endoheterogeneity are also negligible in normal experiments.

Regarding the problem of contamination of double-stranded DNA in (3), according to the method of the present invention, the size of the target single-stranded DNA is set to about half the size of the vector during electrophoresis, and the purpose is If the molecular weight of the target single-stranded DNA is adjusted so that the single-stranded DNA flows to the tip of the gel, and electrophoresis is performed so that the target single-stranded DNA and other DNA are sufficiently separated, then two Contamination of double-stranded DNA does not occur.

Regarding the problem of complexity of the operation step (4), since the method according to the present invention uses a very simple principle, according to the present invention, as long as it is cloned into a vector, it is nicked. From cleavage with endonucleases to electrophoresis and band excision extraction can be completed in half a day, or at most one day.

FIG. 1 shows a schematic diagram showing a series of flows of the method according to the embodiment of the present invention. First, in FIG. 1A, a target DNA to be a long-chain single-stranded DNA is placed in a vector DNA having a nickel endonuclease recognition site in a direction capable of cleaving the same strand side, and the nickel endonuclease recognition site is arranged at both ends thereof. A DNA of interest to be a long-chain single-stranded DNA is added to a vector DNA having a nickel-necking endonuclease recognition site and a sequence-specific double-stranded endonuclease recognition site in such a manner that it can be cloned or cleaved on the same strand side. , The nickel end nuclease recognition site and the sequence-specific double-strand break endonuclease recognition site are cloned so as to be arranged at both ends thereof. At this time, the vector DNA may have a recognition site for a plurality of nickeling endonucleases or sequence-specific double-strand break endonucleases at both ends of the cloning site.

Further, in one embodiment of the present invention, the recognition site of the nickel end nuclease or the sequence-specific double-strand break endonuclease can be provided at both ends of the DNA to be cloned instead of the vector DNA. ..

Subsequently, in FIG. 1B, the vector in which the above-mentioned DNA of interest is cloned is cleaved using the vector DNA or the nickel endonuclease possessed by the DNA of interest, or the nickeling endonuclease and the sequence-specific double-strand break endonuclease. .. This produces three single-stranded DNAs with different molecular weights. When only the nickel endonuclease was used as the enzyme for cleavage, two linear sequences and one circular sequence were generated, and the nickel endonuclease and the sequence-specific double-stranded endonuclease were used. In some cases, three linear sequences will be generated.

Simply cleaving a vector with a nickeling endonuclease or a sequence-specific double-strand break endonuclease does not mean that the cleaved sequence is in a complete single-stranded state, but hydrogen bonds with its complementary strand. It is in a state of being connected by. Therefore, after treating the vector with a nickeling endonuclease or a sequence-specific double-strand break endonuclease, a denaturant is added to the sample obtained as described above, and appropriate treatment is performed to denature the complete single. Bring it to the state of strand DNA. Then, three single-stranded DNAs having different molecular weights are subjected to agarose gel electrophoresis and separated according to the molecular weight (FIG. 1C). At this time, examples of the "appropriate treatment" include heat treatment, incubation, and standing overnight. If three single-stranded DNAs having different molecular weights are denatured or separated by a denaturing agent, these may be used. Not limited. After separating three single-stranded DNAs having different molecular weights in this way, a gel piece containing a band of the desired single-stranded DNA can be cut out and extracted to prepare the desired single-stranded DNA. (Fig. 1D). As is clear from FIG. 1B, when three single-stranded DNAs having different molecular weights are generated by the present invention, the target single-stranded DNA always has the smallest molecular weight sequence, so that the gel is cut out after electrophoresis. Just cut out the band that has flowed to the bottom.

In the present invention, the "nicking endonuclease" is an endonuclease having a nicking activity, which recognizes a specific nucleotide sequence and cleaves only one of the strands of the double-stranded nucleic acid having the nucleotide sequence. It means something that can be done. This nickel endonuclease cleaves the phosphodiester bond in one strand of the double-stranded DNA. Many endonucleases exhibiting such activity are known together with their recognition sequences, and those skilled in the art can appropriately select from these endonucleases and use them in the present invention. In the present specification, the nickel endonuclease may be referred to as "nicking enzyme" or "nickase", both of which are agreed.

In one embodiment of the present invention, the number of bases in the recognition site of the nickel endonuclease is at least 3 bases, and may be 4 bases, 5 bases, 6 bases, or 7 bases. Examples of such endonucleases include Nb. BbvCI (number of bases in recognition site 7: 5'-GC / TGAGG-3'), Nb. BsmI (number of bases in recognition site 6: 5'-NG / CATTC-3'), Nb. BtsI (number of bases in recognition site 6: 5'-NN / CACTGC-3'), Nb. BsrDI (number of bases in recognition site 6: 5'-NN / CATTGC-3'), Nt. BspQI (number of bases in recognition site 7: 5'-GCTCTTCN / -3'), Nt. BbvCI (number of bases in recognition site 7: 5'-CC / TCAGC-3'), Nt. AlwI (number of bases in recognition site 5: 5'-GGATCNNNN / N-3'), Nt. BsmAI (number of bases in recognition site 5: 5'-GTCTCN / N-3'), Nt. BstNBI (number of bases in recognition site 5: 5'-GAGTCNNNN / N-3'), Nt. CviPII (number of bases in recognition site 3: 5'-/ CCD-3'), Nb. Mva1269I (number of bases in recognition site 6: 5'-G / CATTC-3'), Nt. Bpu10I (7: 5'-CC / TNAGC-3'bases at the recognition site), and Nb. BssSI (number of bases in recognition site 6: 5'-C / TCGTG-3') can be mentioned (the number of bases in recognition site and their sequences are shown in parentheses), but not limited to these enzymes. do not have. Here, "/" indicates a cleavage site, "N" indicates a nucleotide of A, T, G, or C, and "D" indicates a nucleotide of A, T, or G, respectively.

In recent years, CAS9 D10A nickase has been developed by introducing a D10A mutation into the CAS9 protein used for genome editing as a new artificial nickel endonuclease (Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 12; 154 (6): 1380-9.). In one embodiment of the present invention, such an artificially produced nickelase (nicking endonuclease) can also be used.

When a D10A mutant of CAS9 is used as a nickel endonuclease, one of the purposes is to utilize the characteristic of the D10A mutant of CAS9 that only one strand is cleaved and a nick is inserted without cleaving the DNA double strand. Main strand DNA can be prepared. Further, when the D10A mutant of CAS9 is used, a guide RNA having a sequence complementary to the base sequence can be bound to the "nicking endonuclease recognition site". Then, the complex of the guide RNA and the D10A mutant of CAS9 recognizes one strand of the double-stranded DNA via the guide RNA and nicks it. In one embodiment of the present invention, the length of the recognition sequence of the guide RNA is at least 5 bases, preferably about 20 bases.

Further, in the present invention, the "sequence-specific double-strand break endonuclease" may be any one capable of recognizing a specific DNA sequence and breaking the double strand, and may include various known enzymes. , Proteins with endonuclease activity can be used. Moreover, the mode of cutting may be one that produces either a sticky end or a blunt end. For example, in one embodiment of the present invention, the "sequence-specific double-strand break endonuclease" includes various restriction enzymes and meganucleases such as I-CeuI, I-SceI, PI-PspI, or PI-SceI. , Or TALEN (transcriptional activator-like effector nuclease) (Cermak T1, Doyle EL, Christian M, Wang L), which is an artificial nuclease fused with FokI, which is a restriction enzyme, as a DNA-cleaving domain and TALE protein as a DNA-binding domain. , Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF. (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. Jul; 39 (12) : e82.) Can be used, and is not limited to these.

Further, in one embodiment of the present invention, as a "sequence-specific double-strand break endonuclease", a protein having only DNA-cleaving ability and functioning with other molecules having DNA-binding ability can also be used. For example, the CAS9 protein (Jinek M1, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. . 17; 337 (6096): 816-21.) And Argonaute proteins (Gao F, Shen XZ, Jiang F, Wu Y, Han C (2016) DNA-guided genome editing using) that depend on guided DNA for DNA binding ability. The Natronobacterium gregoryi Argonaute. Nat Biotechnol. 34 (7): 768-73.) Can also be used, but is not limited to these. Further, in one embodiment of the present invention, the length of the recognition sequence of the guide RNA or the guide DNA is at least 5 bases, preferably about 20 bases.

In the present invention, a vector in which a DNA strand of interest is cloned is cleaved using a nickeling endonuclease as described above, or using a combination of a nickeling endonuclease and a sequence-specific double-strand cleavage endonuclease, and the molecular weight is reduced. At least three different types of single-stranded DNA can be produced. At this time, the recognition site of the nickel end nuclease or the sequence-specific double-strand break endonuclease may be present on either the vector side or the target DNA side.

In one embodiment of the present invention, the "target DNA strand", "target single-stranded DNA", or "long-stranded single-stranded DNA" refers to a sequence having at least 200 bases. Further, in one embodiment of the present invention, the "target DNA strand", "target single-stranded DNA", or "long-stranded single-stranded DNA" includes at least 300 bases, at least 400 bases, and at least 500 bases. At least 600 bases, at least 700 bases, at least 800 bases, at least 900 bases, at least 1,000 bases, at least 1,500 bases, at least 2,000 bases, at least 2,500 bases, at least 3,000 bases, at least 3,500 Bases, at least 4,000 bases, at least 4,500 bases, at least 5,000 bases, at least 5,500 bases, at least 6,000 bases, at least 6,500 bases, at least 7,000 bases, at least 7,500 bases, It can be at least 8,000 bases, at least 8,500 bases, at least 9,000 bases, at least 9,500 bases, and at least 10,000 bases.

In the present invention, the "arbitrary separation means" means that after denaturing at least three types of single-stranded DNA having different molecular weights, the denatured at least three types of single-stranded DNA are converted into individual single-stranded DNA. Refers to any means that can be separated. For example, optional separation means include, but are not limited to, gel electrophoresis and gel column chromatography.

Further, in one embodiment of the present invention, the gel electrophoresis may or may not contain a denaturing agent in the gel or buffer. For example, the gel electrophoresis used in one embodiment of the present invention includes non-denaturing agarose gel electrophoresis without a denaturing agent, modified agarose gel electrophoresis containing a denaturing agent, and non-denaturing acrylamide gel electrophoresis without a denaturing agent. , Or modified acrylamide gel electrophoresis containing a denaturing agent, but is not limited to this.

In one embodiment of the present invention, when gel electrophoresis is used, double-stranded DNA having a size of several thousand bases is heat-denatured by adding a physical denaturing agent that lowers Tm to obtain the state of single-stranded DNA. The DNA can be electrophoresed while maintaining a single-stranded state by simply separating it and loading it into a normal non-denatured gel. For example, in the examples described later, double-stranded DNA of 4,751 bases is used. In addition, the present inventors have confirmed that the prepared single-stranded DNA can be electrophoresed in a single-stranded state even when a double-stranded DNA of 9,547 bases is used. .. At this time, even if the concentration of the plasmid loaded on the electrophoresis gel is as high as 1 μg / μl, it is denatured into a single strand and can be separated with sufficient resolution by the difference in molecular weight.

When gel column chromatography is used as "arbitrary separation means" in one embodiment of the present invention, any gel column chromatography can be used. For example, the gel column chromatography used in one embodiment of the present invention includes, but is not limited to, gel filtration column chromatography, ion exchange gel column chromatography, or affinity gel column chromatography.

Further, in one embodiment of the present invention, the "denaturant" refers to a reagent having an action of cleaving a hydrogen bond of double-stranded DNA by a physical or chemical action to dissociate it into a single strand. In the present invention, as described above, the sample obtained by treating the vector with a nickel endonuclease or a combination of a nickeling endonuclease and a sequence-specific double-strand break endonuclease contains nicks. However, it is not completely separated into single-stranded DNA. Therefore, a denaturing agent is added to the nicked vector in order to separate it into three types of single-stranded DNA having different molecular weights. In one embodiment of the invention, the physical denaturant may be, for example, formamide, glycerol, or urea, or deprotonation of the base, which lowers the polarity of the solvent and weakens hydrophobic interactions such as stacking of base moieties of nucleic acids. Examples include, but are not limited to, sodium hydroxide, which is an alkaline reagent that induces inhibition of hydrogen bond formation due to deprotonation. Further, in one embodiment of the present invention, examples of the chemical denaturing agent include, but are not limited to, formaldehyde and glyoxal that form a Schiff base with nucleic acid. Further, in one embodiment of the present invention, thiourea, ethylene glycol and the like can also be used as the denaturing agent during electrophoresis.

Here, in the case of electrophoresis of long-chain RNA, after denaturing with a physical denaturing agent such as formamide, the denaturing state is covalently fixed with a chemical denaturing agent such as formaldehyde or glyoxal, and then. Electrophoresis is performed in an agarose gel that also contains formaldehyde. That is, RNA needs to be exposed to the chemical denaturing agent not only in the loading buffer but also in the gel to maintain the denatured state. Inferring from the case of this RNA electrophoresis, even in single-stranded DNA denaturation gel electrophoresis, agarose gel to which the same chemical denaturing agent is added after fixing the denaturation with a chemical denaturing agent before electrophoresis is used. If it is not used, it is considered that reanneaturation with the complementary strand is prevented and electrophoresis cannot be performed while maintaining the single-stranded state. However, although a covalent denaturant can reversibly break the bond, it cannot be ruled out that it may be accompanied by some side reaction. That is, the use of chemical denaturants cannot rule out the possibility of causing mutations in DNA. In addition, these chemical denaturants are extremely toxic and irritating, and care must be taken in handling when actually conducting experiments, which is dangerous and lacks convenience.

Based on the above, from the viewpoint of safety, convenience, and prevention of DNA damage, in one embodiment of the present invention, a long-chain single-stranded DNA is denatured only with a physical denaturing agent, and then a non-denaturing gel is used. It is preferable to perform gel electrophoresis using the above.

When performing electrophoresis with a physical denaturing agent, the single-stranded DNA in the wells of the electrophoresis gel leaves the source of the physical denaturing agent contained in the loading buffer when the electrophoresis is started. Then, it goes into the non-denatured gel. The inventors of the present application have discovered that single-stranded DNA after entry into a non-denatured gel does not reanneal to complementary strands as easily as previously thought. That is, the present inventors have obtained the finding that the denatured single-stranded DNA is stably electrophoresed as a single strand even in a non-denatured gel.

In addition, the present inventors also migrate single-stranded DNA electrophoresed in a non-denatured gel based on the molecular weight, and the resolution thereof is necessary for the use of the present invention for preparing single-stranded DNA. We have obtained the finding that it is sufficient. That is, since the single-stranded DNA in the gel does not have a stable structure like the double-stranded DNA or RNA whose denaturation is fixed by a chemical denaturing agent, it forms a secondary structure in the gel. Should be. Therefore, although a simple and accurate proportional relationship between the mobility of electrophoresis and the logarithm of molecular weight (log ₁₀ M) cannot be established, the present invention does not analyze subtle differences in molecular weight. It does not require such elaborate mathematical relationships.

Further, the kit for carrying out the method according to the embodiment of the present invention includes (a) a vector having at least one nickel endonuclease recognition site at both ends of the cloning site, wherein the nickel endonuclease recognition site is described. A vector in which the same strand is cleaved by a nickel endonuclease that recognizes (b) at least one nickeling endonuclease recognition site at one end of the cloning site and at least one sequence-specific double strand at the other end of the cloning site. At least one vector selected from vectors having a cleavage endonuclease recognition site is included. That is, since each of the above-mentioned vectors (a) and (b) has at least one nickel-ending endonuclease recognition site in the vector itself, it is cloned to be the source of the target single-stranded DNA. The double-stranded DNA may or may not have a nickel endonuclease recognition site.

On the other hand, in one embodiment of the present invention, as described above, recognition sites for nickel enzyme or sequence-specific double-strand break endonucleases are provided at both ends of the DNA to be cloned instead of the vector DNA. In this case, the vector included in the kit is preferably one that does not contain a nickel endonuclease recognition site.

Further, in one embodiment of the present invention, the above-mentioned kit may contain a denaturing agent for DNA denaturation.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

In Example 1 described later, the procedure for cloning the target DNA when a cloning vector in which the nickel end nuclease recognition site is arranged at the multi-cloning site is used, and in Example 2 is the procedure for digesting the vector cloned with the target DNA using the nickel end nuclease (). In FIG. 1B), in Example 3, a vector obtained by cloning a target DNA treated with a nickel-end nuclease was heat-modified with a denaturing agent, and agarose gel electrophoresis was performed under non-modification conditions to extract the target long-chain single-stranded DNA. The procedure up to purification (FIGS. 1C and 1D) is shown respectively. Example 4 shows an example in which a long-chain single-stranded DNA containing the entire GFP gene was prepared using a vector obtained by removing the nickel endonuclease recognition site from the entire region. Example 5 shows the effect of denaturation of the pLSODN-1 (1.5 Kb fragment) plasmid with two nicks introduced by various denaturants.

Cloning of target DNA into a cloning vector In Example 1, a 1.5 kb DNA fragment derived from λ phage (λ phage DNA 38,951-40,450, FIG. 3) was used as a model DNA fragment for preparing long-chain single-stranded DNA. board. As the cloning vector, pLSODN-1 in which multiple nickel endonuclease recognition sites were arranged at the multi-cloning site was used. FIG. 2A shows the plasmid map of the cloning vector pLSODN-1, FIG. 2B shows the sequence of the multi-cloning site, FIG. 2C shows the combination of enzymes for cloning to obtain single-stranded DNA, and FIG. 2D shows the whole base. Shows the sequence. In FIG. 2B, the site indicated by the box is a nickel endonuclease site, and the nick introduction position thereof is indicated by an arrow. As shown in FIG. 2B, four types of Nb-type nickel endonuclease sites and one type of Nt-type nickel endonuclease site are arranged facing each other so that DNA strands on the same side can be cleaved in the center. .. Restriction enzyme sites for 6-base or 8-base recognition that give rise to 5'overhang adhesive ends are located upstream of the Nb-type nicking enzyme site, and 3'overhanged adhesive ends are located downstream of the Nt-type nicking enzyme site. A restriction enzyme site for 6-base recognition is located. As shown in FIG. 2C, the target DNA fragment for obtaining long-stranded single-stranded DNA is between two nicking enzyme sites of Nb type and Nt type (circle number 1, central figure) or Nt type. Restriction of 6-base recognition resulting from nicking enzyme site and 5'overhang sticky end Limitation of 6-base recognition producing Nb-type nicking enzyme and 3'overhang sticky end between enzyme sites (circle 2, left figure) Cloning was performed between the enzyme sites (circle number 3, right figure). Here, we used two nickel endonuclease sites, Nb.BsrDI and Nt.BspQI.

First, a linear cloning vector pLSODN-1 was obtained by performing PCR on the cloning vector pLSODN-1 using two synthetic DNA oligomers consisting of the sequences shown below. The underlined portion in the following sequence is a homology sequence with the target DNA introduced to clone the target DNA into the vector.

Specifically, in a 400 ng cloning vector pLSODN-1, 80 pmol of each of the above two synthetic DNA oligomers, 1 × GXL buffer (Takara Bio Co., Ltd.), and 5 units of PrimeSTAR GXL DNA polymerase (Takara Bio Co., Ltd.), respectively. PCR was performed in a total of 400 μl of a reaction solution containing 80 nmol of dATP, dGTP, dTTP, and dCTP. The reaction temperature and time were first repeated 16 times at 95 ° C. for 1 minute, then at 95 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 6 minutes 16 times in this order, and finally at 72 ° C. for 10 minutes. .. The PCR reaction was donated to a 0.8% agarose gel electrophoresis containing 1.6 μg / ml Crystal Violet. After electrophoresis, a band of 3.2 kb DNA dyed in blue was cut out with a razor, purified with Qiaquick Gel Extraction Kit (Qiagen Co., Ltd.), dissolved in 50 μl of 10 mM Tris hydrochloric acid (pH 8.0), and this was dissolved. It was stored as a linear cloning vector pLSODN-1.

On the other hand, the target DNA for obtaining long-chain single-stranded DNA was obtained from λ phage DNA by performing PCR using two synthetic DNA oligomers consisting of the sequences shown below. The underlined portion in the following sequence is a homology sequence with the vector introduced to clone the target DNA into the vector. The sequence of the target DNA derived from the λ phage DNA is shown in FIG.

Specifically, the above two synthetic DNA oligomers are added to 400 ng of λ phage DNA (Promega) at 80 pmol each, 1 × GXL buffer (Takara Bio Co., Ltd.), and 5 units of PrimeSTAR GXL DNA polymerase (Takara Bio Co., Ltd.). PCR was performed in a total of 400 μl of a reaction solution containing 80 nmol of dATP, dGTP, dTTP, and dCTP, respectively. The reaction temperature and time were first repeated 16 times at 95 ° C. for 1 minute, then at 95 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 3 minutes, respectively, and finally at 72 ° C. for 10 minutes. .. The PCR reaction was donated to a 0.8% agarose gel electrophoresis containing 1.6 μg / ml Crystal Violet. After electrophoresis, a band of 1.5 kb DNA dyed in blue was cut out with a razor, purified with Qiaquick Gel Extraction Kit (Qiagen Co., Ltd.), dissolved in 50 μl of 10 mM Tris hydrochloric acid (pH 8.0), and this was dissolved. It was stored as the target DNA for obtaining long single-stranded DNA.

Next, the linear cloning vector pLSODN-1 obtained by PCR amplification and 1.5 kb of target DNA derived from λ phage DNA were introduced based on the homology sequence at the end introduced by the synthetic DNA oligomer used in the PCR reaction. Concatenated.

Specifically, to the 40 ng linear cloning vector pLSODN-1 obtained by PCR amplification, 75 ng of 1.5 kb target DNA derived from λ phage DNA also obtained by PCR amplification, 0.5 μl 1 x Cloning EZ Buffer (Genescript) and 0.5 μl of Clone EZ Enzyme (Genescript) were added, and the reaction was carried out at 22 ° C for 20 minutes in a total of 5 μl of the reaction solution, and then allowed to stand on ice for 5 minutes. Placed to complete the ligation reaction.

Subsequently, 1 μl of the ligation reaction solution was used to perform a transformation operation of a competent cell (Jet Competent Cell, Biodynamics Laboratory Co., Ltd.) as follows. First, freeze competent cells (25 μl) are placed on ice to thaw, then immediately add 1 μl of the ligation reaction solution to the competent cells, and after 5 minutes, 0.25 ml of Recovery Medium at room temperature (included with Jet Competent Cell). Moved competent cells to. Then, after allowing to stand for 5 minutes as it is, the bacterial solution suspended in Recovery Medium is inoculated into an LB agar plate (diameter 8.5 cm, agar medium volume 25 ml) containing 50 mg / ml ampicillin, and 18 at 37 ° C. I kept it warm for a while. The Escherichia coli colonies generated on the LB agar plate by this culture were taken out, inoculated into 3 ml of LB liquid medium containing 50 mg / ml ampicillin, and cultured with shaking at 37 ° C. for 18 hours. A plasmid was prepared from the cells obtained by this shaking culture using the Qiagen plasmid Purification Kit (Qiagen Co., Ltd.).

The plasmid obtained as described above was digested with BsrDI and BspQI, and a target DNA fragment derived from 1.5 kb λ phage was inserted by agarose gel electrophoresis analysis, and ABI PRISM Genetic Analyzer (Applied Biosystem Japan Co., Ltd.) By examining the base sequence by the company), a plasmid having a sequence in which the DNA strand was correctly inserted between the BsrDI site and the BspQI site of the pLSODN-1 vector was selected. This selected plasmid was designated as a pLSODN-1 (1.5 Kb fragment) plasmid.

Digestion of the vector in which the target DNA was cloned with a nickeling enzyme By digesting the plasmid pLSODN-1 (1.5 Kb fragment) prepared in Example 1 with a nickel end nuclease, on the above-mentioned plasmid having the structure of circular double-stranded DNA. Nick was introduced by cleaving only the strands on both ends and one side of the 1.5 Kb fragment, which is the target DNA derived from λ phage.

Specifically, 1 x 3.1 NEBuffer (New England Biolabs), 50 units of Nt.BspQI, and 50 units of Nb.BsrDI were added to 100 μl of plasmid pLSODN-1 (1.5 Kb fragment). The reaction was carried out in 50 μl of the reaction solution at 50 ° C. for 60 minutes, followed by 60 ° C. for 60 minutes. After the reaction, ethanol precipitation was performed as a desalting operation.

Specifically, 125 μl of ethanol was added to 50 μl of pLSODN-1 (1.5 Kb fragment) plasmid reaction solution into which two nicks were introduced and mixed. Then, this mixed solution was set in a micro high-speed centrifuge and rotated at 15,000 rpm for 10 minutes at 4 ° C. to precipitate. After removing the supernatant from the tube, add 500 μl of 70% cold ethanol to the precipitate, vortex it, and rotate it again at 15,000 rpm, 10 minutes, 4 ° C. using a micro high-speed centrifuge to precipitate again. rice field. After removing the upper stalk by suction from the tube, the tube was vacuumed and dried, 50 μl of sterile water was added, the buffer was exchanged, and two demineralized nicks were introduced into pLSODN-1 (1. 5Kb fragment) plasmid was obtained.

Heat denaturation with denaturant, separation by non-denaturing agarose gel electrophoresis, extraction and purification from gel 10 μl containing 20 μg of pLSODN-1 (1.5 Kb fragment) plasmid with two nicks introduced in Example 2. 95% formamide containing 30 μl of bromophenol blue was added to the aqueous solution to obtain a final concentration of 71% formamide solution. This was heat-treated at 70 ° C. for 5 minutes, and then immediately placed on ice and rapidly cooled. After placing on ice for 1 minute, 20 μl (10 μg) and 10 μl (5 μg) were electrophoresed on a 1.2% concentration non-denatured agarose gel using 1 × TAE buffer as a running solution. It should be noted here that it is not necessary to use a modified agarose gel containing urea or formamide. Electrophoresis was stopped when the bromophenol blue dye had moved to a suitable position in the gel. After electrophoresis, the gel was removed from the device, stained with ethidium bromide, and photographed under ultraviolet light (Fig. 4). After imaging, a gel piece containing the target 1.5 kb single-stranded DNA band having the smallest molecular weight at the tip was cut out from the three bands that appeared. Since it is at the tip, there is no possibility that other bands are mixed in. After weighing the gel pieces, the target 1.5 kb single-stranded DNA was extracted using the QIAquick Gel Extraction Kit (Qiagen). The yield of the target 1.5 kb single-stranded DNA was about 30%. The purified 1.5 kb single-stranded DNA was subjected to non-denatured agarose gel electrophoresis on an analytical scale by the same method for a small amount thereof, and it had a high degree of purification without contamination with other bands. Was confirmed (Fig. 5).

Cloning of GFP gene into cloning vector pETUK (del) In Example 4, a vector pETUK (del) having no nicking enzyme recognition site at the multicloning site in addition to the vector skeleton was used. Here, the nickel endonuclease recognition sites were introduced on both sides of the target DNA fragment by incorporating the nickel endonuclease recognition sites into the synthetic DNA oligomer used when amplifying the vector and the target DNA fragment by PCR. Moreover, in this example, a single-stranded DNA encoding the entire GFP gene was prepared. This GFP gene is bound to a homology arm consisting of 19 bases upstream and 20 bases downstream of the target site of knock-in to the rat tyrosinase (Tyr) gene by genome editing. In this example, a denatured gel, urea agarose gel, was used for the separation and preparation of single-stranded DNA.

FIG. 6A shows a plasmid map of the cloning vector pETUK (del), FIG. 6B shows the sequence of the multicloning site, and FIG. 6C shows the entire base sequence. Here, two nicking enzyme sites, Nb.BsrDI and Nt.BspQI, were used to prepare long-chain single DNA, but as mentioned above, the multicloning site of the vector itself does not have this nicking endonuclease site. Was used to introduce nickeling enzyme recognition sites on both sides of the target DNA by incorporating it into the synthetic DNA oligomer used when performing PCR.

First, a linear cloning vector pETUK (del) was obtained by performing PCR using the cloning vector pETUK (del) as a template and two synthetic DNA oligomers consisting of the sequences shown below. The underlined portion of the following sequence is a homology sequence with the target DNA introduced for cloning the target DNA into the vector. The sequence enclosed in the box is the recognition sequence of the nickel endonucleases Nb.BsrDI and Nt.BspQI introduced on both sides of the target DNA.

Specifically, 80 pmol of each of the above two synthetic DNA oligomers in a 400 ng cloning vector pETUK (del), 1 × GXL buffer (Takara Bio Co., Ltd.), and 5 units of PrimeSTAR GXL DNA polymerase (Takara Bio Co., Ltd.). 80 nmol of dATP, dGTP, dTTP, and dCTP were added, respectively, and PCR was performed in a total of 400 μl of the reaction solution. The reaction temperature and time were first repeated 16 times at 95 ° C. for 1 minute, then at 95 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 6 minutes, respectively, and finally at 72 ° C. for 10 minutes. .. The PCR reaction was donated to a 0.8% agarose gel electrophoresis containing 1.6 μg / ml Crystal Violet. After electrophoresis, a 2.67 kb DNA band dyed in blue was cut out with a razor, purified with a Qiaquick Gel Extraction Kit (Qiagen Co., Ltd.), dissolved in 50 μl of 10 mM Tris hydrochloric acid (pH 8.0), and directly dissolved. It was stored as a chain cloning vector pETUK (del).

On the other hand, the target GFP gene for obtaining long-chain single-stranded DNA is PCRed from pCMV-GFP-LC3 Expression Vector (Cell Biolabs. Inc) using two synthetic DNA oligomers consisting of the sequences shown below. Obtained by The underlined portion in the following sequence is a homology sequence with the vector side introduced to clone the GFP gene into the vector. The GFP gene fragment is cloned between the BamHI and NotI sites of the vector's multicloning site. The sequence of the GFP gene derived from pCMV-GFP-LC3 Expression Vector is shown in FIG. The parts surrounded by the box are the start codon and the stop codon of the GFP protein, respectively.

Specifically, in 400 ng of pCMV-GFP-LC3 Expression Vector, 80 pmol of each of the above two synthetic DNA oligomers, 1 × GXL buffer (Takara Bio Co., Ltd.), and 5 units of PrimeSTAR GXL DNA polymerase (Takara Bio Co., Ltd.) PCR was performed in a total of 400 μl of a reaction solution containing 80 nmol of dATP, dGTP, dTTP, and dCTP, respectively. The reaction temperature and time were first repeated 16 times at 95 ° C for 1 minute, then at 95 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 3 minutes, and finally at 72 ° C for 10 minutes. be. The PCR reaction was donated to a 0.8% agarose gel electrophoresis containing 1.6 μg / ml Crystal Violet. After electrophoresis, a band of 0.75 kb DNA dyed in blue was cut out with a razor, purified with Qiaquick Gel Extraction Kit (Qiagen Co., Ltd.), dissolved in 50 μl of 10 mM Tris hydrochloric acid (pH 8.0), and lengthened. Purpose for obtaining single-stranded DNA The whole GFP gene was stored as a DNA fragment.

Next, the linear cloning vector pETUK (del) fragment obtained by the above PCR amplification and the GFP gene whole-range DNA fragment were ligated based on the terminal homology sequence introduced by the synthetic DNA oligomer used in the PCR reaction.

Specifically, a 40 ng linear cloning vector pETUK (del) obtained by PCR amplification, a 75 ng GFP gene also obtained by PCR amplification, and 0.5 μl of 1 x Cloning EZ Buffer (Genescript). ), 0.5 μl of Clone EZ Enzyme (Genescript) was added, and the reaction was carried out at 22 ° C. for 20 minutes in a total of 5 μl of the reaction solution, and then allowed to stand on ice for 5 minutes to complete the ligation reaction.

Subsequently, 1 μl of the ligation reaction solution was used to perform a transformation operation of a competent cell (Jet Competent Cell, Biodynamics Laboratory Co., Ltd.) as follows. First, freeze competent cells (25 μl) are placed on ice to thaw, then immediately add 1 μl of the ligation reaction solution to the competent cells, and after 5 minutes, 0.25 ml of Recovery Medium at room temperature (included with Jet Competent Cell). Moved competent cells to. Then, after allowing to stand for 5 minutes as it is, the bacterial solution suspended in Recovery Medium is inoculated into an LB agar plate (diameter 8.5 cm, agar medium volume 25 ml) containing 50 mg / ml ampicillin, and 18 at 37 ° C. I kept it warm for a while. The Escherichia coli colonies generated on the LB agar plate by this culture were taken out, inoculated into 3 ml of LB liquid medium containing 50 mg / ml ampicillin, and cultured with shaking at 37 ° C. for 18 hours. A plasmid was prepared from the cells obtained by this shaking culture using the Qiagen plasmid Purification Kit (Qiagen Co., Ltd.).

The plasmid obtained as described above was digested with BsrDI and BspQI, a target GFP gene DNA fragment of 759 bases was inserted by agarose gel electrophoresis analysis, and the ABI PRISM Genetic Analyzer (Applied Biosystem Japan Co., Ltd.) The base sequence was examined, and a pETUK (del) vector having a sequence in which the GFP gene was correctly inserted together with the BsrDI site and the BspQI site was selected. This selected plasmid was designated as the pETUK (GFP-Tyr) plasmid.

The pETUK (GFP-Tyr) plasmid was then digested with two nickel endonucleases (Nb.BsrDI and Nt.BspQI) and nicks were introduced into only one strand at both ends of the GFP gene. Specifically, a total of 50 μl reaction was added to 100 μg of plasmid pETUK (GFP-Tyr) with 1 x 3.1 NEBuffer (New England Biolabs), 50 units of Nt.BspQI, and 50 units of Nb.BsrDI. The reaction was carried out in the liquid at 50 ° C. for 60 minutes, and subsequently at 60 ° C. for 60 minutes. After the reaction, ethanol precipitation was performed as a desalting operation.

Specifically, 125 μl of ethanol was added to 50 μl of the pETUK (GFP-Tyr) plasmid reaction solution into which two nicks were introduced and mixed. Then, this mixed solution was set in a micro high-speed centrifuge and rotated at 15,000 rpm for 10 minutes at 4 ° C. to precipitate. After removing the supernatant from the tube, 500 μl of 70% cold ethanol was added to the precipitate, vortexed, and again rotated at 15,000 rpm, 10 minutes, 4 ° C. in a micro high speed centrifuge to precipitate again. PETUK (GFP-Tyr) was introduced with two nicks that had been desalted by sucking and removing the upper sperm from the tube, applying the tube to a vacuum evaporator to dry it, adding 50 μl of sterile water, exchanging the buffer, and introducing the demineralized nick. A plasmid was obtained.

95% formamide containing 30 μl bromophenol blue was added to a 10 μl aqueous solution containing 20 μg of pETUK (GFP-Tyr) plasmid into which two nicks were introduced to obtain a final concentration of 71% formamide solution. This was heat-treated at 70 ° C. for 5 minutes, and then immediately placed on ice and rapidly cooled. After placing it on ice for 1 minute, 100 ng of the part was electrophoresed on a 1.0% concentration 4M urea agarose gel using a 4M urea-containing 1 × TAE buffer as a running solution for analysis. .. Electrophoresis was stopped when the bromophenol blue dye had moved to a suitable position in the gel. After electrophoresis, the gel was removed from the device, stained with ethidium bromide, and photographed under ultraviolet light (Fig. 8). Since it was confirmed by analytical electrophoresis that the separation was successful, the entire remaining amount was subjected to 4M urea agarose gel electrophoresis. After electrophoresis, the mixture was stained with ethidium bromide, and out of the three bands appearing, a gel piece containing the target 759-base GFP single-stranded DNA band having the smallest molecular weight at the tip was cut out. Since it is at the tip, there is no possibility that other bands are mixed in. After weighing the gel pieces, a 759-base GFP single-stranded DNA of interest was extracted using the QIAquick Gel Extraction Kit (Qiagen). The yield of the target 759-base GFP single-stranded DNA was about 30%. The purified 759 single-stranded DNA was electrophoresed on a 4M urea agarose gel in the same manner, and it was confirmed that it had a high degree of purification without contamination with other bands (Fig. 9). ..

Separation of individual single-stranded DNA by electrophoresis on a modified gel and a non-denatured gel of a pLSODN-1 (1.5 kb fragment) plasmid with two nicks introduced when various denaturing agents are used. Examples of nick-introduced pLSODN-1 (1.5 kb fragment) plasmid denaturation with several physical denaturants and the appearance and separation of single-stranded DNA by electrophoresis on non-denaturing gels are shown. The case of formamide, glycerol, and urea as physical denaturants and sucrose as a control is shown. Formamide, glycerol, and urea are reagents commonly used as denaturants for electrophoresis of nucleic acids.

Specifically, pLSODN-1 (1.5 kb fragment) plasmids with various concentrations of two nicks introduced were mixed with various concentrations of homuamide, glycerol, urea, or sucrose. Next, it was heated at 70 ° C. for 5 minutes and then rapidly cooled on ice. After standing on ice for 1 minute, it was subjected to 1.2% non-denatured agarose gel electrophoresis. After electrophoresis, the gel was removed and stained with 1.6% Crystal Violet overnight with shaking. The result is shown in FIG.

Formamide completely denatures the pLSODN-1 (1.5 kb fragment) plasmid with two nicks introduced at a concentration of 25%, even at a high concentration of 1 μg / μl, for the purpose of 1.5 kb. Single-stranded DNA was given. Glycerol was found to be capable of completely denaturing the plasmid at concentrations above 50%. Urea was able to denature plasmids at 0.25 μg / μl concentrations at 6 M concentrations, but was limited for plasmids at concentrations above 0.5 μg / μl. Sucrose had no denaturing effect.

In addition, the present invention is not limited to the above-described embodiment, and can be variously modified without changing the gist of the invention.

Claims (24)

A method for preparing long-chain single-stranded DNA,
(1) (a) A vector having at least one nickel-end nuclease recognition site at both ends of the cloning site, and a vector in which the same chain is cleaved by the nickel-end nuclease that recognizes the nickel-end nuclease recognition site, or (a). b) Clone the DNA strand of interest into a vector having at least one nickeling endonuclease recognition site at one end of the cloning site and at least one sequence-specific double-strand break endonuclease recognition site at the other end of the cloning site. And the process to do
(2) A sequence-specific substance that cleaves the vector of (a) in which the DNA strand of interest has been cloned by the nickel-cooking endonuclease, or recognizes the nickel-cooking endonuclease and the sequence-specific double-strand cleaved endonuclease recognition site. A step of cleaving the vector (b) in which the target DNA strand was cloned with a target double-strand break endonuclease to generate at least three types of single-stranded DNA having different molecular weights.
(3) A step of adding an appropriate amount of a denaturing agent to the at least three types of single-stranded DNA to denature the single-stranded DNA.
(4) A method comprising a step of separating at least three types of denatured single-stranded DNA by an arbitrary separation means to prepare a desired single-stranded DNA. A method for preparing long-chain single-stranded DNA,
(1) (a) A DNA strand of interest having at least one nickel-end nuclease recognition site at both ends, and the same DNA strand is cleaved by the nickel-end nuclease that recognizes the nickel-end nuclease recognition site. Or (b) a step of cloning into a vector a DNA strand of interest having at least one nickeling endonuclease recognition site at one end and at least one sequence-specific double-strand break endonuclease recognition site at the other end.
(2) The nicking endonuclease cleaves the vector into which the DNA strand of interest (a) has been cloned, or sequence-specific recognition of the nicking endonuclease and the sequence-specific double-strand cleavage endonuclease recognition site. A step of cleaving the vector into which the target DNA strand of interest (b) is cloned with a double-strand break endonuclease to generate at least three types of single-stranded DNA having different molecular weights.
(3) A step of adding an appropriate amount of a denaturing agent to the at least three types of single-stranded DNA to denature the single-stranded DNA.
(4) A method comprising a step of separating at least three types of denatured single-stranded DNA by an arbitrary separation means to prepare a desired single-stranded DNA. The method according to claim 1 or 2, wherein the target DNA strand is 200 bases or more. The method of claim 1 or 2, wherein the optional separation means is gel electrophoresis. In the method according to claim 4, the gel electrophoresis is a non-denaturing agarose gel electrophoresis without a denaturing agent, a modified agarose gel electrophoresis containing a denaturing agent, a non-denaturing acrylamide gel electrophoresis without a denaturing agent, or a modification. A method, which is a modified acrylamide gel electrophoresis containing an agent. The method according to claim 5, wherein the gel electrophoresis is a non-denaturing agarose gel electrophoresis containing no denaturant. The method according to claim 1 or 2, wherein the optional separation means is gel column chromatography. The method according to claim 7, wherein the gel column chromatography is gel filtration column chromatography, ion exchange gel column chromatography, or affinity gel column chromatography. The method according to claim 1 or 2, wherein the number of bases of the nickel endonuclease recognition site is at least 3 bases. In the method according to claim 9, the nickel endonuclease recognition site is Nb. BbvCI, Nb. BsmI, Nb. BtsI, Nb. BsrDI, Nt. BspQI, Nt. BbvCI, Nt. AlwI, Nt. BsmAI, Nt. BstNBI, Nt. CviPII, Nb. Mva1269I, Nt. Bpu10I and Nb. A method that is a recognition site for a nickel endonuclease selected from the group consisting of BssSI. The method according to claim 10, wherein the number of bases of the nickel endonuclease recognition site is 6 bases or 7 bases. In the method according to claim 11, the nickel endonuclease recognition site is Nb. BbvCI, Nb. BsmI, Nb. BtsI, Nb. BsrDI, Nb. BssSI, Nt. BspQI, Nb. Mva1269I, Nt. Bpu10I and Nt. A method that is a recognition site for a nickel endonuclease selected from the group consisting of BbvCI. The method according to claim 1 or 2, wherein the guide RNA binds to the nickel endonuclease recognition site. 13. The method of claim 13, wherein the nickel endonuclease is a D10A variant of Cas9. The method according to claim 1 or 2, wherein the sequence-specific double-strand break endonuclease recognition site is an enzyme selected from the group consisting of restriction enzymes and meganucleases, or a TALEN recognition site. The method of claim 15, wherein the meganuclease is I-CeuI, I-SceI, PI-PspI, or PI-SceI. The method of claim 1 or 2, wherein the guide RNA or guide DNA binds to the sequence-specific double-strand break endonuclease recognition site. 17. The method of claim 17, wherein the sequence-specific double-strand break endonuclease is Cas9 or Argonaute. The method according to claim 1 or 2, wherein the denaturing agent is formamide, glycerol, urea, thiourea, ethylene glycol, or sodium hydroxide. 19. The method of claim 19, wherein the denaturing agent is formamide or glycerol. A kit used for the method according to claim 1 or 2, wherein (a) a vector having at least one nickel endonuclease recognition site at both ends of the cloning site, and a nickel end that recognizes the nickel endonuclease recognition site. A vector in which the same strand is cleaved by a nuclease, and (b) at least one nickeling endonuclease recognition site at one end of the cloning site and at least one sequence-specific double-stranded endonuclease recognition site at the other end of the cloning site. A kit comprising at least one vector selected from vectors having and. A kit used for the method according to claim 21, further comprising a reagent containing a denaturing agent for denaturing DNA. A kit used for the method according to claim 2, wherein the kit has a vector containing no nickel endonuclease recognition site. 23. The kit according to claim 23, further comprising a reagent containing a denaturing agent for denaturing DNA.

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