WO2013031700A1 - Dna分子の環状化において単分子による環状化dnaのみを選別する方法 - Google Patents
Dna分子の環状化において単分子による環状化dnaのみを選別する方法 Download PDFInfo
- Publication number
- WO2013031700A1 WO2013031700A1 PCT/JP2012/071492 JP2012071492W WO2013031700A1 WO 2013031700 A1 WO2013031700 A1 WO 2013031700A1 JP 2012071492 W JP2012071492 W JP 2012071492W WO 2013031700 A1 WO2013031700 A1 WO 2013031700A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- adapter
- circular dna
- dna
- molecule
- cleavage
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2521/00—Reaction characterised by the enzymatic activity
- C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
- C12Q2521/301—Endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/30—Oligonucleotides characterised by their secondary structure
- C12Q2525/307—Circular oligonucleotides
Definitions
- the present invention relates to a method for producing a circular DNA molecule having a structure capable of distinguishing circular DNA formed from a single DNA molecule, circular DNA formed from a plurality of DNA molecules, and circular DNA derived therefrom.
- the present invention relates to a method for selecting only circular DNA formed by a single DNA molecule, a novel adapter used in the method, and a circular DNA preparation kit containing the novel adapter.
- this invention relates to the identification and / or detection method of a gene using circular DNA obtained by said method.
- the present invention relates to a method for identifying and / or detecting a fusion gene that causes various pathological conditions.
- a conventional gene analysis method includes a vector method.
- the sequence of the full length of a gene obtained by incorporating the gene to be analyzed into a vector and growing it is determined by a sequencer.
- the vector method requires a culture operation, it is necessary to analyze the full length of the gene with a sequencer.
- Fig. 1 schematically shows an outline of gene analysis by the mate pair method.
- a base sequence for binding (restriction enzyme recognition site) is bound to both ends of an analysis target gene, and the target gene is circularized.
- the circularized gene is cut mainly at the restriction enzyme recognition site, usually using Type II restriction enzyme, and cut at 15 or more bases before and after the recognition site, preferably 25 bases or more and tens of bases or less.
- the base sequence of the amplified and excised partial gene is determined.
- a mate pair is a set of sequence data of base sequences obtained by decoding both ends of a single DNA fragment.
- a method of cutting out a gene having a certain number of bases a method of cutting out a predetermined number of bases by cutting a site away from the recognition site using Type II restriction enzyme, and a circular DNA physically by sonication (Sonication) etc.
- a method is actually used in which the fragments are recovered by biotin which has been cut and attached to the linker, and the fragments are grown by PCR to determine the sequence.
- a known gene in the mate pair method, can be identified by reading a certain base sequence before and after the binding portion in a circularized gene by binding both ends of DNA. Basically, if the base sequence of a part of the head portion and tail portion of a gene is read, the sequences can be reliably differentiated for each gene, so the mate pair method has been adopted as a reliable and simple gene analysis method ( Non-Patent Documents 1 and 2). In addition, the mate-pair method is applied to next-generation sequence analysis, and is becoming increasingly important with the advent of high-speed sequencers.
- DNA when DNA is circularized for gene analysis by the mate pair method, in addition to self-circularization with a single gene, DNA (single molecule), circularization with multiple DNA (multiple molecules) or multiple molecules (2 A linear bond of more than molecules).
- a linear molecule composed of a plurality of molecules is separated and removed from a cyclic molecule by a subsequent operation.
- a cyclic molecule composed of a plurality of molecules cannot be separated from a cyclic molecule composed of a single molecule and becomes a contaminant.
- Multi-molecule circulars inhibit individual gene analysis and greatly reduce analysis specificity for the reasons described below. Specifically, when three kinds of cDNAs are to be self-circulated as shown in FIG.
- the gene analysis by the mate pair method is to specify a gene by the base sequences of both ends of the target gene. Specifically, after binding adapters for circularization are bound to both ends of each gene, the genes are bound and circularized at both adapter sites, and then a fixed number of base sequences are formed around the adapter sites. Then, the gene is identified by cutting and analyzing the partial base sequence from each end of the initial gene as a result. Therefore, in the cyclized product of a plurality of molecules, there are a plurality of adapter sites, and both ends bound to the adapter are each one end of a different gene.
- the gene analysis is performed by cutting the circularized product so as to obtain a constant base sequence at both ends binding to the adapter by either of the two methods described above. Therefore, the gene fragment for analysis obtained from the cyclized product of a plurality of molecules contains one end of each of different genes, and one gene is not analyzed. Thus, in gene analysis by the mate pair method, the presence of a cyclized product of a plurality of molecules inhibits each gene analysis.
- the probability of circularization of multiple DNA molecules usually varies from several percent to several tens of percent, depending on the method, but in the analysis of known genes, it is recognized as an abnormal base sequence and can be almost excluded from the analysis sequence. . Therefore, although complicated, the accuracy is only slightly reduced.
- the mate pair method is used to detect the presence of an abnormal gene such as a fusion gene from a group of normal genes, if multiple normal genes are circularized, it is determined that the abnormal gene is present. End up. As a result, the presence of an abnormal gene such as a fusion gene cannot be confirmed accurately.
- a fusion gene is a gene in which multiple (two) genes are joined together to construct a gene with a new function.
- cancer cells have abnormal chromosomal structures such as deletion, duplication, recombination, and translocation.
- Gene splitting and linking occur at the DNA level, and a fusion gene is formed when a structural gene is present at each breakpoint.
- fusion genes are lethal to cells, meaningless, and in many cases do not cause clinical problems.
- the fusion protein produced from the fusion gene inhibits the regulation of cell proliferation and thereby abnormally promotes cell proliferation, it becomes clinically prominent as a tumor or the like.
- the fusion gene was said to be expressed mainly in hematopoietic tumors, but in recent years, it has been estimated that the fusion gene is also involved in epithelial solid tumors (Non-patent Document 3). Among them, responsible fusion genes were discovered from prostate cancer and lung cancer (Non-patent Documents 4 and 5).
- fusion genes that is, confirmation of their presence
- cancers cancers
- rapid diagnosis of a disease state becomes possible by detecting a known fusion gene known to correspond to the disease state.
- discovery of new fusion genes will also lead to the discovery of drug discovery targets.
- Non-patent Document 6 chromosomal analysis has been limited for solid tumors, and analysis and confirmation of fusion genes has been extremely difficult, but recently, new methods such as cDNA functional expression analysis by Mano et al. Have been developed. Yes. However, these are still insufficient techniques due to operational complexity and accuracy problems (Patent Document 1). Recently, various gene next-generation high-speed sequencers have been developed, and high-speed sequence analysis of genes has been remarkably advanced, and analysis in a short time is becoming possible. This has led to the search for fusion genes by high-speed, large-scale base sequence analysis of tumor genomes and genes (Non-patent Document 6).
- FIG. 4 A schematic diagram in the case of analyzing the fusion gene by the mate pair method is shown in FIG.
- FIG. 4 A schematic diagram in the case of analyzing the fusion gene by the mate pair method is shown in FIG.
- FIG. 4 there is a possibility that a plurality of cDNAs may form one circular DNA.
- sequence analysis using the mate pair method is performed, even if it is a normal gene, it appears as if it is a fusion gene. Results. If this is excluded because it does not exist in the conventional gene sequence, the fusion gene will be excluded as well, and it will be virtually impossible to confirm the presence of the fusion gene.
- the present inventors have so far carried out a two-step ligation using an adapter having a specific structure, thereby causing only a circularization by a single DNA molecule, and circularity between multiple molecules by a plurality of DNA molecules.
- the analysis using the mate pair method particularly the analysis aiming at the search for a new fusion gene, even a very small amount of circular DNA by a plurality of genes can be a false positive clone, which is a problem.
- the present invention refers to a circular DNA formed by a single DNA molecule (hereinafter also referred to as “single molecule circular DNA”) and a circular DNA formed by a plurality of DNA molecules (hereinafter also referred to as “multimolecular circular DNA”). ) And a method for producing a circular DNA molecule having a specific structure that enables discrimination from monomolecular circular DNA derived from multimolecular circular DNA, and monomolecular circular not derived from multimolecular circular DNA in the production of circular DNA The object is to provide a method for screening only DNA.
- the present inventors introduced a single-molecule circular DNA not derived from a multi-molecular circular DNA by introducing adapters having specific structures each containing a unique sequence into a single DNA molecule, and performing two-step cleavage and ligation.
- the inventors have found that it is possible to create a circular DNA molecule having a specific structure that makes it possible to distinguish multimolecular circular DNA and single molecular circular DNA derived from multimolecular circular DNA.
- the present inventors have found a method of selecting only single-molecule circular DNA that is not derived from multi-molecular circular DNA by sequencing adapter sites after preparing a circular DNA molecule having the structure.
- single-molecule circular DNA refers to circular DNA formed by a single DNA molecule
- multi-molecular circular DNA refers to circular DNA formed by a plurality of DNA molecules.
- circular DNA molecule means a single circular DNA molecule or a plurality of circular DNA molecules.
- plurality of circular DNA molecules may be expressed as “a group of circular DNA molecules” composed of a plurality of circular DNA molecules having the same or different sequences.
- derived from XX means that the DNA molecule XX is generated from the DNA molecule XX by some kind of treatment. The term does not mean that a new molecule is created, but typically a part of the DNA molecule is separated into an independent DNA molecule, or a part of the DNA molecule is cleaved, resulting in pre-cleavage. It means that a shorter molecule is generated.
- a unimolecular circular DNA “derived from” a multimolecular circular DNA is a circular DNA formed by a single DNA molecule, and is a single DNA resulting from cleavage of the multimolecular circular DNA by restriction enzyme treatment or the like. Is a circular DNA formed by self-circularization of the linear DNA molecule.
- a unimolecular circular DNA “not derived from” a multimolecular circular DNA is a circular DNA formed by a single DNA molecule, and a single linear DNA molecule undergoes the formation of a multimolecular circular DNA. This refers to circular DNA resulting from self-circularization without.
- cleavage end “derived” from the adapter (A) or (b) refers to an end generated by cleaving the cleavage site of the adapter (A) or (b), for example, by restriction enzyme treatment. This is the case for 5 ′ or 3 ′ overhangs.
- the “unique sequence” is a sequence unique to each adapter (b), and is a sequence that is different for each adapter (b). If the types of unique sequences (types of adapter (b)) are increased sufficiently, adapters (b) having different unique sequences for different target DNAs will bind.
- the created circular DNA molecule is a unimolecular circular DNA not derived from a multimolecular circular DNA, or a multimolecular circular DNA or a monomolecular circular derived therefrom. It is possible to determine whether it is DNA.
- “selecting” only single-molecule circular DNA not derived from multi-molecular circular DNA means physically separating single-molecular circular DNA not derived from multi-molecular circular DNA from a group of circular DNA molecules. It is possible to select only information obtained from single-molecule circular DNA not derived from multi-molecular circular DNA at the data analysis stage from information obtained from a group of circular DNA molecules (for example, base sequence data). It may be.
- the “palindromic restriction enzyme site” refers to a restriction enzyme recognition site that recognizes a palindromic sequence
- the “non-palindromic restriction enzyme site” refers to a non-Parisdromic restriction enzyme site.
- the present invention in the first aspect, is a method for producing a population of circular DNA molecules, comprising the following steps, wherein the single molecule circle is not derived from a multimolecular circular DNA from the population of circular DNA molecules produced by the method.
- step 6 the following types of DNA may remain as linear DNA: (b) the unimolecular DNA that was not recombined in step 5) and recombined (very small amount), and In step 3), a plurality of DNA molecules are circularized, and in step 5), single or multiple molecules of DNA that could not be recombined after cleavage of each adapter (b) (this occupies most of linear DNA). These linear DNAs may be removed, but are not necessarily removed. This is because the DNA does not have a structure in which two identical unique sequences are arranged, so that it can be determined by sequencing the adapter (b) portion and excluded from the analysis target.
- the present invention also provides a method for producing a unimolecular circular DNA not derived from a multimolecular circular DNA, comprising the following steps: 1) a step of preparing a circular DNA molecule by the method of the first aspect of the present invention; and 2) sequencing the adapter (b) portion of the generated circular DNA molecule, not derived from a multimolecular circular DNA A step of selecting only single-molecule circular DNA, wherein, if the two unique sequences contained in the adapter (b) portion are identical, the circular DNA molecule is a single-molecule circular DNA that is not derived from multiple-molecule circular DNA; If the two unique sequences are different, the circular DNA molecule is a multimolecular circular DNA or a single molecular circular DNA derived from a multimolecular circular DNA.
- the present invention also provides, in the third aspect, a method for selecting only single-molecule circular DNA not derived from multi-molecular circular DNA in the generation of circular DNA molecules, comprising the following steps: 1) First-stage circularization adapter (A) is bound to one end of each target DNA molecule, and adapter (b) and adapter (A) for second-stage circularization are included at the other end ( B) binding step; where the adapter (B) binds to the DNA molecule via the adapter (b) side, and the adapter (A) in the adapter (B) is the DNA molecule and the adapter (b) Located outside the bond with, here, Adapter (A) contains a cleavage site that produces a cleavage end that binds non-specifically to the cleavage end of any adapter (A); Adapter (b) comprises two unique sequences that differ for each adapter (b), the two unique sequences being the same sequence oriented in the same or opposite direction; and The adapter (b) includes a cle
- the adapter (A) is a double-stranded DNA comprising a restriction enzyme site that generates a cleavage end complementary to the cleavage end of any adapter (A).
- double-stranded DNA containing a recognition site for a restriction enzyme that recognizes a palindromic sequence (“palindromic restriction enzyme site”).
- the adapter (b) includes two cleavage sites that generate a cleavage end that non-specifically binds to the cleavage end of any adapter (b). .
- the sequence between the two cleavage sites in the adapter (b) is not removed from the circular DNA molecule in which the second cleavage step has not proceeded successfully, the molecule is identified as “incomplete clone” by sequencing the adapter (b). "And can be excluded from the analysis target.
- the cleavage site contained in the adapter (b) is preferably a restriction enzyme site, for example, a palindromic restriction enzyme site.
- the number of cleavage sites included in the adapter (b) is at least one, preferably two, but may be three or more.
- pairs of nicking enzyme recognition sites that are identical to each other and oriented in the opposite directions can be used as cleavage sites contained in adapter (A) and / or adapter (b).
- the adapter (A) is a double-stranded DNA complementary to each other containing a palindromic restriction enzyme site X
- the adapter (B) is a double-stranded DNA complementary to each other having the following structure Z 1 -YZ 2 -A or Z 1 -YZ ' 2 -A:
- A is a double-stranded DNA containing a palindromic restriction enzyme site X and corresponds to the adapter (A);
- Z 1 -YZ 2 corresponds to the adapter (b) in the first to third aspects of the present invention;
- Y is a double-stranded DNA containing palindromic restriction enzyme sites y 1 and y 2 ;
- y 1 and y 2 are the same, have a different sequence from X, and give rise to a cleavage end that is not complementary to the cleavage end caused by cleavage of X;
- Z 1 and Z 2 are double stranded
- n is 1 to 40, preferably, n is 4 to 15, more preferably, n is 5 to 10. However, even if n exceeds 40, the method of the present invention can be carried out.
- the palindromic restriction enzyme site X is preferably a BamHI site, a NotI site or a BclI site, and preferably the palindromic restriction enzyme sites y 1 and y 2 are both EcoRI sites or PacI sites.
- the present invention provides an adapter for producing circular DNA comprising double-stranded DNAs complementary to each other having the following structure Z 1 -YZ 2 -A or Z 1 -YZ ′ 2 -A.
- An adapter is provided that allows for the selection of only single-molecule circular DNA that is not derived from multi-molecular circular DNA from a population of circular DNA molecules created using the adapter: [In the structure, A is a double-stranded DNA containing palindromic restriction enzyme site X; Y is a double-stranded DNA containing palindromic restriction enzyme sites y 1 and y 2 ; y 1 and y 2 are the same, have a different sequence from X, and give rise to a cleavage end that is not complementary to the cleavage end caused by cleavage of X; Z 1 and Z 2 are double stranded DNA sequences comprising unique sequences C 1 and C 2 that vary from adapter to adapter, where C 1 and C 2 are identical sequences oriented in
- n is 1 to 40, preferably, n is 4 to 15, more preferably, n is 5 to 10. However, even if n exceeds 40, the method of the present invention can be carried out.
- the adapter according to the fourth aspect of the present invention corresponds to the adapter (B) according to the first to third aspects of the present invention, and is derived from a method for creating a population of circular DNA molecules according to the present invention, a multimolecular circular DNA.
- This method is preferably used for a method for producing a single-molecule circular DNA that is not derived from a single molecule and a method for selecting only a single-molecule circular DNA that is not derived from a plurality of circular molecules.
- the palindromic restriction enzyme site X is a BamHI site, a NotI site or a BclI site, and preferably both the palindromic restriction enzyme sites y 1 and y 2 are EcoRI sites or PacI site.
- the present invention further provides an adapter (also simply referred to as adapter (B)) described in the fourth aspect, and the same restriction enzyme site X as the restriction enzyme site X contained in the adapter (B).
- a circular DNA production kit comprising an adapter composed of double-stranded DNA (also simply referred to as adapter (A)), which is not derived from a multi-molecular circular DNA from a group of circular DNA molecules produced using the kit.
- a kit that makes it possible to select only single-molecule circular DNA.
- the present invention is also a method for preparing a cDNA library using the method of the first aspect, wherein the library is not derived from a multimolecular circular DNA from the library. And a method for preparing a cDNA library consisting only of single-molecule circular DNA not derived from multi-molecular circular DNA using the method of the second or third aspect.
- the present invention further provides, in the seventh aspect, a method for identifying a gene by subjecting a circular DNA molecule to a mate pair method, comprising the following steps: 1) A population of circular DNA molecules prepared by the method of the first aspect of the present invention, a single molecule circular DNA not derived from the multimolecular circular DNA prepared by the method of the second aspect of the present invention, or the first of the present invention A step of decoding a base sequence of 15 to 600 bases adjacent to both sides of the adapter (B) in a single-molecule circular DNA not derived from a multimolecular circular DNA selected by the method of the third aspect, wherein When using a population of circular DNA molecules produced by the method of the first aspect of the invention, the sequence of the adapter (b) portion is sequenced before, simultaneously with, or after the step.
- the base sequence to be decoded is 15 to 100 bases, more preferably 25 to 35 bases.
- the method of the present invention can be carried out even if 600 bases or more are decoded.
- the present invention further provides, in the eighth aspect, a method for detecting a fusion gene by subjecting a circular DNA molecule to a mate pair method, which comprises the following steps: 1) A population of circular DNA molecules prepared by the method of the first aspect of the present invention, a single molecule circular DNA not derived from the multimolecular circular DNA prepared by the method of the second aspect of the present invention, or the first of the present invention A step of decoding a base sequence of 15 to 600 bases adjacent to both sides of the adapter (B) in a single-molecule circular DNA not derived from a multimolecular circular DNA selected by the method of the third aspect, wherein When using a population of circular DNA molecules produced by the method of the first aspect of the invention, the sequence of the adapter (b) portion is sequenced before, simultaneously with, or after the step.
- step 1) A step of comparing the base sequence decoded in step 1) with a sequence at both ends of a known gene, where the genes at both ends correspond to known different genes are not derived from the multi-molecule circular DNA
- a gene contained in a unimolecular circular DNA is identified as a fusion gene.
- the base sequence to be decoded is 15 to 100 bases, more preferably 25 to 35 bases.
- the method of the present invention can be carried out even if 600 bases or more are decoded.
- the known fusion gene when the sequences on both sides adjacent to both sides of the adapter (B) correspond to the ends on both sides of the known fusion gene, the known fusion gene is detected.
- a method for detecting a disease characterized by expression of the fusion gene using the fusion gene detected by the method of the eighth aspect as a marker is provided Is done.
- the sequences adjacent to both sides of the adapter (B) correspond to the end sequences of different genes and do not correspond to the ends of both sides of the known fusion gene
- the circular DNA molecule or the monomolecular circular DNA are identified as novel fusion genes.
- the detected novel fusion gene is suitably used for drug discovery screening.
- the accuracy of genetic analysis is dramatically improved by using the adapter and method of the present invention.
- the adapter and method of the present invention it is possible to completely discriminate between unimolecular circular DNA not derived from multimolecular circular DNA and unimolecular circular DNA derived from multimolecular circular DNA and multimolecular circular DNA. It is possible to select only single-molecule circular DNA that is not derived from multi-molecular circular DNA with a probability of almost 100%.
- unprecedented high-precision mate pair analysis is possible, and a very useful tool is provided for genome analysis.
- the method of the present invention to the preparation of a cDNA library, there is a possibility that a novel fusion gene is discovered with a high possibility.
- the present invention there is provided a method for selecting only unimolecular circular DNA not derived from multimolecular circular DNA in circularization of DNA. Thereby, the problem of contamination in gene analysis such as mate pair analysis is solved, and high-precision analysis becomes possible. Further, by applying the method of the present invention to detection / analysis of fusion genes, it becomes possible to analyze fusion genes with high accuracy and provide an effective diagnostic tool.
- FIG. 1 schematically shows an outline of gene analysis by the mate pair method.
- FIG. 2 schematically shows problems in gene analysis by the mate pair method.
- FIG. 3 shows a schematic diagram when the fusion gene is analyzed by the mate pair method.
- FIG. 4 schematically shows problems in analyzing a fusion gene by the mate pair method.
- FIG. 5 schematically shows the DNA molecule after adapter binding.
- FIG. 6 schematically shows a first cleavage, first-stage circularization, second-stage cleavage, and second-stage circularization process of the circular DNA molecule production method according to the present invention. For convenience, different unique sequences are distinguished from each other with reference symbols a, b, c, d, e and f ⁇ .
- FIG. 7 schematically shows the structure of the adapter (b) portion that can occur after the second-stage cyclization step.
- NNNNN in the figure represents a unique sequence. By sequencing such regions, target clones, non-target clones and incomplete clones can be distinguished.
- FIG. 8 schematically shows an example of a method for creating the adapter (b). NNNNN in the figure represents a unique sequence.
- FIG. 9 shows a schematic diagram when the method of the present invention is applied to cDNA synthesized from mRNA.
- FIG. 10 shows a schematic diagram when the method of the present invention is applied to a genomic library. Unlike cDNA, genomic fragments cannot distinguish left and right DNA fragments.
- both the left end linker and the right end linker are bonded together, and only those in which both linkers are appropriately bonded (a) are used as PCR methods or modified linkers, or as restriction enzyme sites X at both ends. It can be selected by using a palindromic array or the like.
- the present invention provides, as a first aspect, a method for producing a population of circular DNA molecules comprising the following steps, wherein a plurality of molecules are produced from the population of circular DNA molecules produced by the method.
- a method is provided that makes it possible to select only unimolecular circular DNA not derived from circular DNA: 1) First-stage circularization adapter (A) is bound to one end of each target DNA molecule, and adapter (b) and adapter (A) for second-stage circularization are included at the other end ( B) binding step; where the adapter (B) binds to the DNA molecule via the adapter (b) side, and the adapter (A) in the adapter (B) is the DNA molecule and the adapter (b) Located outside the bond with, here, Adapter (A) contains a cleavage site that produces a cleavage end that binds non-specifically to the cleavage end of any adapter (A); Adapter (b) comprises two unique sequences that differ for
- the first aspect of the present invention is a specific molecule that enables discrimination between unimolecular circular DNA not derived from multimolecular circular DNA and multimolecular circular DNA and single molecular circular DNA derived from multimolecular circular DNA. This is a technique that enables creation of a circular DNA molecule having a structure. Specifically, the method of the first aspect of the present invention is characterized by two-stage cyclization.
- step 1) a first-step circularization adapter (A) is bound to one end of each target DNA molecule, and the adapter (b) and the adapter (A) are included at the other end.
- step 2) a step of binding the adapter (B).
- the adapter (B) is bound to the DNA molecule via the adapter (b) side, so that the adapter (A) in the adapter (B) is outside the bond between the DNA molecule and the adapter (b). To position.
- the adapter (A) is bound to one end of each target DNA molecule, and the adapter (B) is bound to the other end, and the adapter (A) in the adapter (B) is located on the end side of the adapter (b). By doing so, the adapter (A) is positioned at both ends of each target DNA molecule.
- the adapter (A) includes a cleavage site that generates a cleavage end that binds nonspecifically to the cleavage end of any adapter (A). That is, the adapter (A) does not exhibit binding specificity, and can be non-selectively bound between the cleavage ends derived from any adapter (A).
- the adapter (b) includes two unique sequences different for each adapter (b), and the two unique sequences are the same sequence oriented in the same direction or in opposite directions.
- the adapter (b) includes a cleavage site that generates a cleavage end that non-specifically binds to the cleavage end of either adapter (b) between the two unique sequences.
- adapter (b) When the cleavage site of A) is cleaved, it is not cleaved, and a cleavage end that is not complementary to the cleavage end of the adapter (A) is generated. That is, adapter (b) does not show binding specificity, and can bind non-selectively between cleavage ends derived from any adapter (b). This is the same even when the number of cleavage sites contained in the adapter (b) is one, two, or three or more.
- Step 2) is a first cutting step of cleaving the DNA molecule obtained in step 1) at the cutting site of the adapter (A). Since the adapter (A) is bound to both ends of the DNA molecule obtained in step 1), the first cleavage step generates a cleavage end derived from the adapter (A) at both ends of each DNA molecule.
- Step 3) is a first-stage circularization step in which both ends of the DNA molecule obtained in Step 2) are combined to be circularized.
- the first-stage circularization is circularization by the adapter (A), and the cleavage ends derived from the adapter (A) generated at both ends of each DNA molecule are bonded to each other to cause circularization.
- the adapter (B) is incorporated together with the adapter (A) into the cyclic molecule during the first stage of cyclization.
- there is no specificity in the function of the adapter (A) and there is no selectivity for rebinding at both ends of the target DNA molecule.
- Step 4) is a step of removing the DNA molecules that are not circularized but linearized in Step 3).
- Linear DNA molecules can be removed by, for example, acting exonuclease.
- Step 5) is a second cleavage step of cleaving the circular DNA molecule obtained in Step 3) and Step 4) at the cleavage site of the adapter (b). That is, cleavage is performed at the adapter (b) incorporated into each DNA molecule that has been circularized in step 3) to produce a linear molecule.
- cleavage ends derived from the adapter (b) are generated at both ends of each DNA molecule.
- the cleavage end generated by cleavage is non-specifically bound to the cleavage end of any adapter (b).
- Step 6) is a second-stage circularization step in which both ends of the DNA molecule obtained in Step 5) are combined and circularized. That is, there are cleavage ends derived from the adapter (b) at both ends of each DNA molecule obtained in step 5), and the second-stage circularization is achieved by binding of the cleavage ends derived from the adapter (b). Is done. Molecules that are not circularized but remain linear can be separated and removed by, for example, acting exonuclease.
- the second-stage circularization step in addition to circularization by a single DNA molecule, circularization by a plurality of DNA molecules can also occur.
- the probability that the original plural molecules bonded before the second cutting step are rebonded and cyclized is extremely low and substantially zero as described later.
- the second stage cyclization process can yield the following five types of molecules: (1) “Monomolecular circular DNA not derived from multi-molecular circular DNA” (for example, A4 in FIG. 6), wherein the monomolecular circular DNA is a single-molecular circular DNA formed in the first-stage circularization step (for example, A single molecule DNA (for example, A3 in FIG. 6) formed by cutting A2) in FIG. 6 by the second cutting step is formed by self-circularization; (2) “Monomolecular circular DNA derived from multimolecular circular DNA” (for example, B4-1 in FIG. 6), where the monomolecular circular DNA is a multimolecular circular DNA formed in the first-stage circularization step.
- a monomolecular DNA (for example, B3 in FIG. 6) formed by cleaving (for example, B2 in FIG. 6) by the second cleavage step is formed by self-circularization; (3) “Multimolecular circular DNA-1” (for example, B4-2 in FIG. 6), wherein the DNA-1 is a multimolecular circular DNA (for example, B2 in FIG. 6) formed in the first-stage circularization step. ) Is formed by cleaving a plurality of single-molecule DNAs (for example, B3 in FIG. 6) that are produced by cleaving in the second cleaving step; (4) “Multimolecular circular DNA-2” (for example, B4-3 in FIG.
- the DNA-2 is a single molecular circular DNA formed in the first-stage circularization step (for example, A2 in FIG. 6).
- the adapter (b) part contains Since two different unique sequences exist side by side, by sequencing the sequence of the adapter (b) part, such a multimolecular circular DNA can be excluded as not being the target clone (B4 in FIG. 6). -2, B4-3 and B4-4 and Fig. 7).
- the two unique sequences in the adapter (b) are identical only in the single-molecule circular DNA ((1) above) that is not derived from the multi-molecule circular DNA ( A4 in FIG.
- the same inherent property is obtained through the second cleavage process and the second-stage circularization process.
- Unimolecular circular DNA can be generated in which two sequences are present side by side. However, as will be described later, the probability that such a unimolecular circular DNA is generated can be made as close to 0 as possible by increasing the length of the unique sequence (that is, increasing the type of adapter (b)). If the unique sequence has a certain length, it is considered to be substantially zero, so this is not a problem.
- the circular DNA molecule obtained in step 6) can be obtained by sequencing the sequence of the adapter (b) portion, so that if the two unique sequences contained in the adapter (b) portion are the same, a multimolecular circular DNA If the two unique sequences are different from each other, it can be determined that the DNA is a multimolecular circular DNA or a single molecular circular DNA derived from a multimolecular circular DNA. It is possible.
- the structures of the adapters (A) and (b) are not particularly limited as long as they fulfill the above functions.
- examples of adapters (A) include those containing palindromic restriction enzyme sites
- examples of adapters (b) include two identically oriented in the opposite directions. In which two palindromic restriction enzyme sites are included between the two unique sequences.
- the adapters (A) and (b) are not limited to these.
- the adapter (b) contains two identical unique sequences, and a cleavage site that generates a cleavage end that non-specifically binds to the cleavage end of any adapter (b) between the two unique sequences. Only one or three or more may be included.
- the resulting cleavage ends are recombined to obtain the origin of the circular DNA (that is, whether it is a unimolecular circular DNA that does not originate from multiple molecular circular DNAs). Or a multimolecular circular DNA or a single molecular circular DNA derived from a multimolecular circular DNA) as long as it forms a structure in which two identical or different unique sequences are arranged.
- non-palindromic restriction enzyme sites can be used as long as they generate complementary cleavage ends.
- restriction enzyme sites they are identical to each other oriented in opposite directions. It is also possible to use a pair of nick-created enzyme recognition sites. For example, when using the recognition site of BbsI, which is a non-palindromic restriction enzyme, the cleavage ends occur outside the recognition site. Complementary cut ends can be generated.
- the restriction enzyme site is preferably a recognition site for a restriction enzyme (so-called “rare cutter”) that recognizes a rare gene sequence that does not cut the target DNA as much as possible.
- a palindromic restriction enzyme site that recognizes 8 bases, which is a rare cutter, is the PacI site shown below.
- restriction enzyme sites such as the following SfiI site Is also possible.
- SfiI site for example, NNNNN upstream and downstream And set.
- recognition sites for Type II restriction enzymes such as the BbsI site shown below can also be introduced.
- cleavage can be performed only at the restriction enzyme site in the adapter, for example, as in the following SgeI restriction enzyme site.
- the single adapter (A) (referred to as “A1”) and the adapter (A) (“A2” included in the adapter (B)
- the sequences are designed or selected so that adapters “A1” and “A2” bind to each other while “A1” and “A2” cannot bind to each other. In this case, it is expected that the efficiency of monomolecular circular DNA formation in the first stage is slightly higher than that of the ordinary adapter (A).
- the created circular DNA is a unimolecular circular DNA not derived from a multimolecular circular DNA, or a multimolecular circular DNA or a plurality of molecules It becomes possible to discriminate whether it is a unimolecular circular DNA derived from the circular DNA (which becomes a contaminant in the analysis).
- This invention provides the preparation method of the unimolecular circular DNA which is not derived from multimolecular circular DNA including the following processes as 2nd aspect: 1) a step of preparing a circular DNA molecule by the method of the first aspect of the present invention; and 2) sequencing the adapter (b) portion of the generated circular DNA molecule, not derived from a multimolecular circular DNA A step of selecting only single-molecule circular DNA, wherein, if the two unique sequences contained in the adapter (b) portion are identical, the circular DNA molecule is a single-molecule circular DNA that is not derived from multiple-molecule circular DNA; If the two unique sequences are different, the circular DNA molecule is a multimolecular circular DNA or a single molecular circular DNA derived from a multimolecular circular DNA.
- the circular DNA molecule created by the method of the first aspect of the present invention is any one of single-molecule circular DNA not derived from multi-molecular circular DNA, multi-molecular circular DNA, or single-molecular circular DNA derived from multi-molecular circular DNA. It is.
- the second aspect of the present invention by sequencing the adapter (b) portion of a group of circular DNA molecules composed of these circular DNAs, only a single molecule circular DNA that is not derived from a multi-molecular circular DNA is selected from the group. How to get.
- the present invention provides, as the third aspect, a method for screening only unimolecular circular DNA not derived from multimolecular circular DNA in the generation of circular DNA molecules, comprising the following steps: 1) First-stage circularization adapter (A) is bound to one end of each target DNA molecule, and adapter (b) and adapter (A) for second-stage circularization are included at the other end ( B) binding step; where the adapter (B) binds to the DNA molecule via the adapter (b) side, and the adapter (A) in the adapter (B) is the DNA molecule and the adapter (b) Located outside the bond with, here, Adapter (A) contains a cleavage site that produces a cleavage end that binds non-specifically to the cleavage end of any adapter (A); Adapter (b) comprises two unique sequences that differ for each adapter (b), the two unique sequences being the same sequence oriented in the same or opposite direction; and The adapter (B) binds to the
- Steps 1) to 6) in the third aspect of the present invention are the same as Steps 1) to 6) in the first aspect of the present invention.
- step 7) in the third embodiment of the present invention the sequence of the adapter (b) part of the circular DNA molecule obtained in step 6) is sequenced, and only single-molecule circular DNA not derived from multi-molecular circular DNA is sequenced. It is a process of sorting.
- the two unique sequences in the adapter (b) are the same as a single molecule not derived from a multimolecular circular DNA. Only circular DNA. Therefore, by determining the sequence in the adapter (b) to determine whether the two unique sequences are the same or different, the circular DNA molecule obtained in step 6) is not derived from a multimolecular circular DNA. Whether it is molecular circular DNA or multimolecular circular DNA or unimolecular circular DNA derived therefrom can be discriminated, and only single molecular circular DNA not derived from multimolecular circular DNA can be selected as an analysis target.
- multi-molecular circular DNA that is a contaminant and single-molecular circular DNA derived therefrom are excluded from the analysis target, and the multi-molecular circular DNA is converted into a multi-molecular circular DNA. Only single-molecule circular DNA that is not derived can be selected for analysis.
- the adapter (A) is a double-stranded DNA complementary to each other containing a palindromic restriction enzyme site X
- the adapter (B) is a double-stranded DNA complementary to each other having the following structure Z 1 -YZ 2 -A or Z 1 -YZ ' 2 -A:
- A is a double-stranded DNA containing a palindromic restriction enzyme site X and corresponds to the adapter (A);
- Z 1 -YZ 2 corresponds to the adapter (b) in the first to third aspects of the present invention;
- Y is a double-stranded DNA containing palindromic restriction enzyme sites y 1 and y 2 ;
- y 1 and y 2 are the same, have a different sequence from X, and give rise to a cleavage end that is not complementary to the cleavage end caused by cleavage of X;
- Z 1 and Z 2 are double stranded
- n is 1 to 40, preferably, n is 4 to 15, more preferably, n is 5 to 10. However, even if n exceeds 40, the method of the present invention can be carried out.
- Such a preferred adapter (A) is a sequence for performing ligation between cleavage ends for the purpose of cleaving with a restriction enzyme in the first cleavage step and then obtaining circular DNA. Therefore, any restriction enzyme site may be included as long as it is a palindromic restriction enzyme site. Preferably, it contains a restriction enzyme site that recognizes a rare gene sequence that does not cut the target DNA as much as possible. Examples of the restriction enzyme site X contained in the adapter (A) include the following BamHI site, NotI site, and BclI site.
- SEQ ID NO: 1 forward chain
- SEQ ID NO: 2 reverse chain
- Such a preferred adapter (B) is a sequence for ligation between the cleaved ends in the second cleaving step, in which the cleaved ends are ligated in the hope of recircularization of the cleaved DNA molecule. Therefore, any restriction enzyme site that recognizes a palindromic sequence may be included. Preferably, it contains a restriction enzyme site that recognizes a rare gene sequence that does not cut the target DNA as much as possible. Examples of the restriction enzyme site contained in the Y portion of the adapter (B) include the following EcoRI site and PacI site.
- the cleavage ends generated by cleavage of the palindromic restriction enzyme sites y 1 and y 2 in the adapter (B) are both complementary to each other, and ligation between the cleavage ends results in the same in the adapter (b) part or A structure with two different unique sequences is obtained.
- such a preferred adapter (B) has two identical restriction enzyme sites (y 1 and y 2 ) in the Y portion. Therefore, a circular DNA molecule in which the sequence between the two restriction enzyme sites has not been removed after the second cleavage step can be identified and eliminated as an “incomplete clone” in which the restriction enzyme treatment in the second cleavage step has not progressed well.
- FIG. 7 There are advantages (FIG. 7).
- each step of the first and third aspects of the present invention comprises: It becomes as follows.
- Step 1) is a step of binding the first-stage circularization adapter (A) to one end of each target DNA molecule and binding the second-stage circularization adapter (B) to the other end ( FIG. 5).
- Step 2) is a first cleavage step of cleaving the DNA molecule obtained in step 1) with a restriction enzyme (BamHI in FIG. 6) that recognizes the palindromic restriction enzyme site X contained in the adapter (A). Yes (A1 and B1 in FIG. 6).
- Step 3) is a first-stage circularization step in which both ends of the DNA molecule obtained in Step 2) are ligated and circularized (A2 and B2 in FIG. 6).
- Step 4) is a step of removing the DNA molecules that are not circularized but linearized in Step 3).
- Step 5 the adapter (b) palindrome in type restriction enzyme sites y 1 and y 2 recognizing restriction enzyme (EcoRI in FIG. 6), a circular DNA molecule obtained in step 3) and step 4) It is the 2nd cutting process to cut
- Step 6) is a second-stage circularization step in which both ends of the DNA molecule obtained in step 5) are ligated and circularized (A4, B4-1, B4-2, B4-3 in FIG. 6 and B4-4). By this step, a structure in which two identical or different unique sequences are contained in the adapter (b) is obtained.
- step 7) included in the third aspect of the present invention the adapter (b) portion is sequenced for the circular DNA molecule obtained in step 6), and the two unique sequences contained therein are the same or different.
- This is a step of selecting only single-molecule circular DNA that is not derived from multi-molecular circular DNA.
- multimolecular circular DNA B4-2, B4-3 and B4-4 in FIG. 6
- single molecular circular DNA derived from the multimolecular circular DNA (B4-1 in FIG. 6) are:
- the adapter (b) has a structure in which two different unique sequences are arranged (cb, db, ec, da, ea, af in FIG. 6).
- the unimolecular circular DNA (A4 in FIG. 6) not derived from the multimolecular circular DNA has a structure in which two identical unique sequences are arranged (a-a in FIG. 6). Therefore, by selecting molecules having the same two unique sequences contained in the adapter (b) from the circular DNA molecules obtained in step 6), only single-molecule circular DNA not derived from multi-molecular circular DNA can be obtained. It becomes possible to select as analysis object.
- the adapter (b) portion after the second-stage circularization step has a structure in which two unique sequences are arranged with one restriction enzyme site in between.
- the restriction enzyme treatment is incomplete, the sequence between the restriction enzyme sites is not removed, resulting in a structure in which two restriction enzyme sites still exist (FIG. 7). Therefore, by sequencing the adapter (b) part, it is possible to discriminate the clone having the latter structure as an incomplete clone and exclude it from the analysis target.
- the length of the unique sequence in the adapter (b) ( in the adapter having the structure Z 1 -YZ 2 -A or Z 1 -YZ ′ 2 -A described above, the “n” ”) is not particularly limited, but is preferably 1 to 40 bases, more preferably 4 to 15 bases, still more preferably 5 to 10 bases. As the number of bases increases, the types of unique sequences increase, and in the first-stage circularization process, different DNA molecules having the same adapter (b) part (that is, having the same unique sequence) are combined to form a multimolecular circular DNA. The probability of doing decreases.
- the length of the unique sequence is 8 bases
- the probability that the unimolecular circular DNA (for example, B4-1 in FIG. 6) derived from the multimolecular circular DNA after the second-stage circularization process has two identical unique sequences is determined in the first-stage circularization process.
- the multimolecular circular DNA for example, B4-2, B4-3 and B4-4 in FIG.
- the adapter (b) portion containing the unique sequence after the second-stage circularization step it is a unimolecular circular DNA not derived from the multimolecular circular DNA, or derived from the multimolecular circular DNA or this. Whether it is a unimolecular circular DNA can be completely discriminated.
- the present invention relates to a suitable adapter (B) used in the method of the first to third aspects of the present invention, that is, the following structure Z 1 -Y
- a circular DNA molecule adapter composed of double-stranded DNAs complementary to each other having -Z 2 -A or Z 1 -YZ ' 2 -A, and the group of circular DNA molecules prepared using the adapter Provides an adapter that allows to select only single-molecule circular DNA from non-multimolecular circular DNA from: [In the structure, A is a double-stranded DNA containing palindromic restriction enzyme site X; Y is a double-stranded DNA containing palindromic restriction enzyme sites y 1 and y 2 ; y 1 and y 2 are the same, have a different sequence from X, and give rise to a cleavage end that is not complementary to the cleavage end caused by cleavage of X; Z 1 and Z 2 are double stranded
- n is 1 to 40, preferably, n is 4 to 15, more preferably, n is 5 to 10. However, even if n exceeds 40, the method of the present invention can be carried out.
- examples of the restriction enzyme site X contained in the A portion include a BamHI site, NotI site, BclI site, etc.
- examples of the restriction enzyme sites y 1 and y 2 included in the above include an EcoRI site and a PacI site.
- the adapter (b) having two identical unique sequences that are preferably used is prepared by, for example, using a nucleic acid having a hairpin structure and performing a three-step reaction of polymerase extension, nick generation, and polymerase extension. (FIG. 8).
- the production method is not limited to this, and a desired adapter (b) can be produced by, for example, sequence synthesis.
- the present invention relates to a circular DNA comprising a suitable adapter (A) and adapter (B) used in the method of the first to third aspects of the present invention.
- a kit for preparation which makes it possible to select only single-molecule circular DNA not derived from multi-molecular circular DNA from a group of circular DNA molecules generated using the kit. That is, the adapter (A) comprising a double-stranded DNA containing the same restriction enzyme site as the restriction enzyme site X described in the fourth aspect, and the adapter (B) described in the fourth aspect, A kit for producing circular DNA is provided.
- the kit for producing circular DNA of the present invention comprises an adapter (A) and an adapter (B), which are bound to both ends of the target DNA molecule, and the two-step circularization according to the first or third aspect of the present invention.
- a circular structure with a specific structure that makes it possible to discriminate between single-molecule circular DNA not derived from multi-molecular circular DNA and multi-molecular circular DNA and single-molecular circular DNA derived from multi-molecular circular DNA. It is possible to obtain DNA and to select only single-molecule circular DNA that is not derived from multi-molecular circular DNA.
- the sixth aspect of the present invention is a method for preparing a cDNA library using the two-step circularization method of any one of the first to third aspects of the present invention.
- the sequence of the adapter (b) part is sequenced for the members of the library, so that the single molecule is not derived from a multimolecular circular DNA.
- a cDNA library capable of selecting only members that are molecular circular DNAs can be prepared. Therefore, for example, when gene analysis is performed using such a cDNA library, after complete sequencing of the library, only single-molecule circular DNA data not derived from multi-molecular circular DNA is selected for analysis. be able to.
- a cDNA library composed only of single-molecule circular DNA not derived from multimolecular circular DNA is prepared. Can do.
- a seventh aspect of the present invention is a method for identifying a gene by subjecting a circular DNA molecule to a mate pair method, comprising the following steps: 1) A population of circular DNA molecules prepared by the method of the first aspect of the present invention, a single molecule circular DNA not derived from the multimolecular circular DNA prepared by the method of the second aspect of the present invention, or the first of the present invention A step of decoding a base sequence of 15 to 600 bases adjacent to both sides of the adapter (B) in a single-molecule circular DNA not derived from a multimolecular circular DNA selected by the method of the third aspect, wherein When using a population of circular DNA molecules produced by the method of the first aspect of the invention, the sequence of the adapter (b) portion is sequenced before, simultaneously with, or after the step.
- Step 1) of the method of the seventh aspect of the present invention includes steps adjacent to both sides of the adapter (B) in the circular DNA molecule obtained using the method according to the first to third aspects of the present invention.
- This is a step of decoding a base sequence of 15 to 600 bases.
- the base sequence to be decoded is 15 to 100 bases, more preferably 25 to 35 bases.
- the method of the present invention can be carried out even if 600 bases or more are decoded.
- the base sequence can be decoded using a method well known to those skilled in the art, for example, a sequencer.
- step 1) when the sequence of the adapter (b) part is determined “simultaneously with the step”, for example, in the sequencing reaction for decoding the base sequence adjacent to both sides of the adapter (B),
- the reading of the sequence from the outside of the base sequence to be performed toward the adapter (B) is started, the reading is proceeded into the adapter (B) as it is, and the sequence of the adapter (b) portion is simultaneously determined.
- the same sample may be divided into a plurality of samples for sequencing on the outside of the adapter (B) and sequencing for the adapter (b) portion, and these may be simultaneously subjected to a sequencing reaction to obtain sequence data. It is done.
- it is possible to select only data of a sample in which two unique sequences in the adapter (b) portion are the same that is, a monomolecular circular DNA not derived from a multimolecular circular DNA
- Step 2) of the method of the seventh aspect of the present invention is a step of identifying a gene contained in a circular DNA molecule by comparing the decoded base sequence with sequences at both ends of a known gene. If it is confirmed that the decoded base sequence corresponding to the both ends of the target DNA molecule is identical to the both ends of the known gene, the target DNA molecule is identified as the known gene. .
- the eighth aspect of the present invention is a method for detecting a fusion gene by subjecting a circular DNA molecule to a mate pair method, comprising the following steps: 1) A population of circular DNA molecules prepared by the method of the first aspect of the present invention, a single molecule circular DNA not derived from the multimolecular circular DNA prepared by the method of the second aspect of the present invention, or the first of the present invention A step of decoding a base sequence of 15 to 600 bases adjacent to both sides of the adapter (B) in a single-molecule circular DNA not derived from a multimolecular circular DNA selected by the method of the third aspect, wherein When using a population of circular DNA molecules produced by the method of the first aspect of the invention, the sequence of the adapter (b) portion is sequenced before, simultaneously with, or after the step.
- step 1) A step of comparing the base sequence decoded in step 1) with a sequence at both ends of a known gene, where the genes at both ends correspond to known different genes are not derived from the multi-molecule circular DNA
- a gene contained in a unimolecular circular DNA is identified as a fusion gene.
- Step 1) of the method of the eighth aspect of the present invention comprises steps adjacent to both sides of the adapter (B) in the circular DNA molecule obtained by using the method according to the first to third aspects of the present invention.
- This is a step of decoding a base sequence of 15 to 600 bases.
- the base sequence to be decoded is 15 to 100 bases, more preferably 25 to 35 bases.
- the method of the present invention can be carried out even if 600 bases or more are decoded.
- the base sequence can be decoded using a method well known to those skilled in the art, for example, a sequencer.
- the sequence of the adapter (b) portion is determined “simultaneously with the step” as in the seventh embodiment.
- Step 2) of the method according to the eighth aspect of the present invention is a step of comparing the decoded base sequence with sequences at both ends of a known gene.
- the gene contained in the circular DNA molecule is identified as a fusion gene. That is, the portion corresponding to one end of the decoded base sequence corresponding to the both ends of the target DNA molecule is the same as one end of the known gene, and the portion corresponding to the other end is another known If it is the same as one end of the gene, the target DNA molecule is identified as a fusion gene composed of two known genes.
- the target DNA is detected as a known fusion gene.
- the relationship between the expression of a known fusion gene and a disease is known, it is possible to detect a disease characterized by the expression of the fusion gene by using the fusion gene detected by such a method as a marker. .
- the gene contained in the circular DNA molecule is a new fusion gene. Is identified.
- the novel fusion gene detected by such a method can be used in drug discovery screening.
- Example 1 Application of the method of the present invention to discovery of a fusion gene using cDNA synthesized from mRNA
- a cDNA library is synthesized from mRNA
- the most common method currently used (Clontech SMART cDNA method)
- an oligonucleotide (1) having a polyT sequence complementary to the polyA site at the 3 ′ end of mRNA as shown in FIG. 9 first, complementary strand DNA is synthesized with reverse transcriptase.
- a specific oligonucleotide sequence (2) is incorporated at the 5 ′ end of the mRNA as shown in FIG.
- DNA synthesis is performed from oligonucleotides complementary to this specific sequence, or a cDNA library is prepared by PCR.
- the adapter (B) is introduced into the oligonucleotide (1) sequence having a polyT sequence complementary to the polyA site at the 3 ′ end of mRNA.
- An adapter (A) is added to the nucleotide (2) sequence.
- a 5 ′ phosphate group-added primer is used as an upstream primer
- a primer containing a unique sequence is amplified as a downstream primer
- a phosphate group primer is used after PCR.
- the incorporated strand is digested with ⁇ exonuclease, followed by primer extension from the upstream primer again to complete a cDNA library that maintains the diversity due to the unique sequence.
- this process is not necessary for the adapter binding method, and the following adapter binding method is performed in order to introduce the adapter of the present invention after modification such as fragmentation of the library.
- restriction enzyme sites can be introduced at the 3 ′ end and 5 ′ end, respectively, as shown in FIG. 9 (3).
- a normal palindromic restriction enzyme site can be used as the restriction enzyme site, or a non-palindromic restriction enzyme site can be used to distinguish the ends.
- Example 2 Application of the method of the present invention to mate pair analysis of genomes
- genomic fragments cannot distinguish between left and right DNA fragments. Therefore, as shown in FIG. 10, the adapter at the left end and the adapter at the right end are both bound together, and both adapters are bound appropriately, that is, only (a) in FIG. 10 is a method using a PCR method or a modified linker, or both ends.
- the restriction enzyme site X can be selected by using a non-palindromic sequence, or by using a restriction enzyme containing N region (BstXI or the like) and installing a plurality of restriction enzyme sites at the same site.
- the left end linker is modified with biotin and the right end linker is used.
- DIG digoxigenin
- the modification used is not limited to biotin or DIG, and the left end linker and the right end linker need only be modified differently. The case of using a restriction enzyme site of a non-palindromic sequence will be described in detail below.
- the adapters (A) and (B) are added to both ends of the DNA molecule by using the following procedure, respectively.
- the adapter (A) is first bound to a plurality of DNA groups having a certain concentration determined according to the number of target DNA molecules.
- the amount of adapter (A) added is less than the target DNA molecular weight present.
- it may be the same as the number of target DNA molecules, or a single base overhang may be enzymatically created in a DNA fragment and a complementary adapter may be bound.
- the concentration of may be excessive.
- the adapter (A) is bound to the end of the DNA molecule.
- the adapter (B) is bound.
- FIG. 10 (a) it is necessary to prepare a molecule in which an adapter (A) is bound to one end and an adapter (B) is bound to the other end.
- a DNA molecule (FIG. 10 (b)) bound only to the adapter (A) and a DNA molecule (FIG. 10) bound only to the adapter (B). (C)) must be eliminated. The method will be described below.
- the adapter “A1” that generates the cut end W1 is used, and the adapter (A ), The adapter “A2” that generates the cut end W2 is used.
- the cut ends W1 and W2 are complementary, while W1 and W2 are not complementary, that is, the sequence is designed or selected so that ligation is possible only between adapters “A1” and “A2” To do.
- the first-stage cyclization is possible independently.
- it After passing through the second cutting and second-stage circularization steps, it has a structure in which two different unique sequences are arranged, and this can be discriminated and eliminated.
- the structure distinguishes it from the single-molecule circular DNA that is not derived from the multimolecular circular DNA after the second-stage circularization. Can not do it. However, since it contains a binding site between the adapters (A) that is not included in the adapter (B), it can be eliminated, for example, by forming a linear molecule using the “BstXI method” described below.
- BstXI is charged outside the restriction enzyme site of the adapter (A). That is, CCANNNNNNTGG is incorporated into the restriction enzyme site BstXI site described below, for example, BamHI site so as to be CCAGGATCCTGG.
- the restriction enzyme site of the adapter (A) contained in the adapter (A) and the adapter (B) is, for example, a BamHI site.
- the sequence outside the BamHI site is, for example, a BstXI recognition sequence. That is, the adapter (A) sequence is CCAGGATCCTGG.
- the adapter (A) included in the adapter (B) includes a BamHI site, but does not incorporate a BstXI recognition sequence and prevents cleavage by BstXI.
- the adapter (A) By being able to hold, if only the adapter (A) binds to both ends and they are associated with each other, it can be opened and eliminated by cutting with BstXI. Of course, when the adapters (A) and (B) are bound, the adapter (B) does not have a BstXI recognition sequence, so it is not cleaved by BstXI.
- the method of the present invention is used to create and / or select single-molecule circular DNA that is not derived from multi-molecular circular DNA. And the accuracy of the mate pair analysis can be significantly improved.
- Example 3 Regarding the possibility of recombination of the same DNA molecules in a multi-molecule circular DNA
- (1) DNA Possibility is roughly estimated by dividing the estimation into the volume of the molecule and the amount (volume) of the reaction solution, and (2) the estimation from the number of molecules in the reaction solution.
- the Avogadro number is 6 ⁇ 10e23
- the accuracy of genetic analysis is dramatically improved by using the adapter and method of the present invention.
- the adapter and method of the present invention it is possible to completely discriminate between unimolecular circular DNA not derived from multimolecular circular DNA and unimolecular circular DNA derived from multimolecular circular DNA and multimolecular circular DNA. It is possible to select only single-molecule circular DNA that is not derived from multi-molecular circular DNA with a probability of almost 100%.
- unprecedented high-precision mate pair analysis is possible, and a very useful tool is provided for genome analysis.
- the method of the present invention to the preparation of a cDNA library, there is a possibility that a novel fusion gene is discovered with a high possibility.
- the present invention there is provided a method for selecting only unimolecular circular DNA not derived from multimolecular circular DNA in circularization of DNA. Thereby, the problem of contamination in gene analysis such as mate pair analysis is solved, and high-precision analysis becomes possible. Further, by applying the method of the present invention to detection / analysis of fusion genes, it becomes possible to analyze fusion genes with high accuracy and provide an effective diagnostic tool.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Computational Biology (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pathology (AREA)
Abstract
Description
本明細書中、「単分子環状DNA」とは、単一のDNA分子により形成される環状DNAをいい、「複数分子環状DNA」とは、複数のDNA分子により形成される環状DNAをいう。
1)各目的DNA分子の一方の末端に第一段階環状化用アダプター(A)を結合させ、他方の末端に、アダプター(b)と前記アダプター(A)を含む第二段階環状化用アダプター(B)を結合させる工程;ここで、アダプター(B)は、DNA分子にアダプター(b)側を介して結合して、アダプター(B)中のアダプター(A)は、DNA分子とアダプター(b)との結合の外側に位置する、
ここで、
アダプター(A)は、いずれのアダプター(A)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含む;
アダプター(b)は、該アダプター(b)ごとに異なる固有配列を2つ含み、該2つの固有配列は同一方向または逆方向に配向した同一の配列であり; かつ、
アダプター(b)は、該2つの固有配列の間に、いずれのアダプター(b)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含み、該切断部位は、アダプター(A)の切断部位を切断する際には切断されることがなく、かつ、アダプター(A)の切断末端とは結合しない切断末端を生じさせるものである;
2)アダプター(A)の切断部位において、工程1)で得られたDNA分子を切断する第一切断工程;
3)工程2)で得られたDNA分子の両末端を結合させて環状化させる第一段階環状化工程;
4)工程3)において環状化せず直線状となったDNA分子を除去する工程;
5)アダプター(b)の切断部位において、工程3)および工程4)で得られた環状DNA分子を切断する第二切断工程; および、
6)工程5)で得られたDNA分子の両末端を結合させて環状化させる第二段階環状化工程、
ここで、工程6)で得られる環状DNA分子は、アダプター(b)部分の配列を配列決定することにより、アダプター(b)部分に含まれる2つの固有配列が同一であれば、複数分子環状DNAに由来しない単分子環状DNAであり、該2つの固有配列が異なっていれば、複数分子環状DNAであるかまたは複数分子環状DNAに由来する単分子環状DNAであると判定されるものである。
1)本発明の第一の態様の方法によって環状DNA分子を作成する工程;および
2)該作成された環状DNA分子についてアダプター(b)部分の配列を配列決定し、複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程、ここで、アダプター(b)部分に含まれる2つの固有配列が同一であれば、当該環状DNA分子は複数分子環状DNAに由来しない単分子環状DNAであり、該2つの固有配列が異なっていれば、当該環状DNA分子は複数分子環状DNAであるかまたは複数分子環状DNAに由来する単分子環状DNAである。
1)各目的DNA分子の一方の末端に第一段階環状化用アダプター(A)を結合させ、他方の末端に、アダプター(b)と前記アダプター(A)を含む第二段階環状化用アダプター(B)を結合させる工程;ここで、アダプター(B)は、DNA分子にアダプター(b)側を介して結合して、アダプター(B)中のアダプター(A)は、DNA分子とアダプター(b)との結合の外側に位置する、
ここで、
アダプター(A)は、いずれのアダプター(A)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含む;
アダプター(b)は、該アダプター(b)ごとに異なる固有配列を2つ含み、該2つの固有配列は同一方向または逆方向に配向した同一の配列であり; かつ、
アダプター(b)は、該2つの固有配列の間に、いずれのアダプター(b)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含み、該切断部位は、アダプター(A)の切断部位を切断する際には切断されることがなく、かつ、アダプター(A)の切断末端とは結合しない切断末端を生じさせるものである;
2)アダプター(A)の切断部位において、工程1)で得られたDNA分子を切断する第一切断工程;
3)工程2)で得られたDNA分子の両末端を結合させて環状化させる第一段階環状化工程;
4)工程3)において環状化せず直線状となったDNA分子を除去する工程;
5)アダプター(b)の切断部位において、工程3)および工程4)で得られた環状DNA分子を切断する第二切断工程;
6)工程5)で得られたDNA分子の両末端を結合させて環状化させる第二段階環状化工程;および、
7)工程6)で得られた環状DNA分子が有するアダプター(b)部分の配列を配列決定し、複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程、ここで、アダプター(b)部分に含まれる2つの固有配列が同一であれば、当該環状DNA分子は複数分子環状DNAに由来しない単分子環状DNAであり、該2つの固有配列が異なっていれば、当該環状DNA分子は複数分子環状DNAであるかまたは複数分子環状DNAに由来する単分子環状DNAである。
好ましくは、アダプター(B)は、下記構造Z1-Y-Z2-AまたはZ1-Y-Z’2-Aを有する互いに相補的な二本鎖DNAである:
[構造中、
Aは、パリンドローム型制限酵素サイトXを含む二本鎖DNAであって、アダプター(A)に相当し;
Z1-Y-Z2は、本発明の第一から第三までの態様におけるアダプター(b)に相当し;
Yは、パリンドローム型制限酵素サイトy1およびy2を含む二本鎖DNAであり;
y1およびy2は同一であり、Xとは異なる配列を有し、かつ、Xの切断により生じる切断末端と相補的でない切断末端を生じさせるものであり;
Z1およびZ2は、アダプターごとに異なる固有配列C1およびC2を含む二本鎖DNA配列であり、ここで、C1とC2は、互いに逆方向に配向した同一の配列であり;
nは1以上40以下の整数であり;
N1~Nnは、それぞれ同一であっても異なっていてもよく、dAMP、dCMP、dGMPおよびdTMPからなる群から選択されるデオキシリボヌクレオチドであり;
N’1~N’nは、前記N1~Nnに対してそれぞれ下記:
または
[構造中、
Z1-Y-Z’2は、本発明の第一から第三までの態様におけるアダプター(b)に相当し;
Z1およびZ’2は、アダプターごとに異なる固有配列C1およびC’2を含む二本鎖DNA配列であり、ここで、C1とC’2は、同一方向に配向した同一の配列であり;
A、X、Y、y1、y2、n、N1~Nn、N’1~N’n、およびkの定義は、上記の構造Z1-Y-Z2-Aにおけるものと同一である]。
上記Z1-Y-Z2-AおよびZ1-Y-Z’2-Aいずれの構造においても、nは1~40であり、好ましくは、nは4~15であり、さらに好ましくは、nは5~10である。ただし、nが40を超えても本発明の方法を実施することができる。
[構造中、
Aは、パリンドローム型制限酵素サイトXを含む二本鎖DNAであり;
Yは、パリンドローム型制限酵素サイトy1およびy2を含む二本鎖DNAであり;
y1およびy2は同一であり、Xとは異なる配列を有し、かつ、Xの切断により生じる切断末端と相補的でない切断末端を生じさせるものであり;
Z1およびZ2は、アダプターごとに異なる固有配列C1およびC2を含む二本鎖DNA配列であり、ここで、C1とC2は、互いに逆向きに配向した同一の配列であり;
nは1以上40以下の整数であり;
N1~Nnは、それぞれ同一であっても異なっていてもよく、dAMP、dCMP、dGMPおよびdTMPからなる群から選択されるデオキシリボヌクレオチドであり;
N’1~N’nは、前記N1~Nnに対してそれぞれ下記:
または
[構造中、
Z1およびZ’2は、アダプターごとに異なる固有配列C1およびC’2を含む二本鎖DNA配列であり、ここで、C1とC’2は、同一方向に配向した同一の配列であり;
A、X、Y、y1、y2、n、N1~Nn、N’1~N’n、およびkの定義は、上記の構造Z1-Y-Z2-Aにおけるものと同一である]。
上記Z1-Y-Z2-AおよびZ1-Y-Z’2-Aいずれの構造においても、nは1~40であり、好ましくは、nは4~15であり、さらに好ましくは、nは5~10である。ただし、nが40を超えても本発明の方法を実施することができる。
1)本発明の第一の態様の方法によって作成された環状DNA分子の集団、本発明の第二の態様の方法によって作成された複数分子環状DNAに由来しない単分子環状DNAまたは本発明の第三の態様の方法によって選別された複数分子環状DNAに由来しない単分子環状DNAにおける、アダプター(B)の両側に隣接するそれぞれ15塩基以上600塩基以下の塩基配列を解読する工程、ここで、本発明の第一の態様の方法によって作成された環状DNA分子の集団を用いる場合には、当該工程の前に、当該工程と同時に、または当該工程の後に、アダプター(b)部分の配列を配列決定することにより複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程をさらに含む;および、
2)工程1)で解読した塩基配列を既知の遺伝子の両末端の配列と比較することにより、該複数分子環状DNAに由来しない単分子環状DNAに含まれる遺伝子を同定する工程。
好ましくは、解読する塩基配列は、15~100塩基、さらに好ましくは25~35塩基である。ただし、600塩基以上を解読しても本発明の方法を実施することができる。
1)本発明の第一の態様の方法によって作成された環状DNA分子の集団、本発明の第二の態様の方法によって作成された複数分子環状DNAに由来しない単分子環状DNAまたは本発明の第三の態様の方法によって選別された複数分子環状DNAに由来しない単分子環状DNAにおける、アダプター(B)の両側に隣接するそれぞれ15塩基以上600塩基以下の塩基配列を解読する工程、ここで、本発明の第一の態様の方法によって作成された環状DNA分子の集団を用いる場合には、当該工程の前に、当該工程と同時に、または当該工程の後に、アダプター(b)部分の配列を配列決定することにより複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程をさらに含む;および、
2)工程1)で解読した塩基配列を既知の遺伝子の両末端の配列と比較する工程、ここで、両末端の遺伝子が既知の相異なる遺伝子に対応する場合、該複数分子環状DNAに由来しない単分子環状DNAに含まれる遺伝子は融合遺伝子であると同定される。
好ましくは、解読する塩基配列は、15~100塩基、さらに好ましくは25~35塩基である。ただし、600塩基以上を解読しても本発明の方法を実施することができる。
本発明は、第一の態様として、以下の工程を含む、環状DNA分子の集団の作成方法であって、該方法により作成された環状DNA分子の集団から複数分子環状DNAに由来しない単分子環状DNAのみを選別することを可能にする方法を提供する:
1)各目的DNA分子の一方の末端に第一段階環状化用アダプター(A)を結合させ、他方の末端に、アダプター(b)と前記アダプター(A)を含む第二段階環状化用アダプター(B)を結合させる工程;ここで、アダプター(B)は、DNA分子にアダプター(b)側を介して結合して、アダプター(B)中のアダプター(A)は、DNA分子とアダプター(b)との結合の外側に位置する、
ここで、
アダプター(A)は、いずれのアダプター(A)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含む;
アダプター(b)は、該アダプター(b)ごとに異なる固有配列を2つ含み、該2つの固有配列は同一方向または逆方向に配向した同一の配列であり; かつ、
アダプター(b)は、該2つの固有配列の間に、いずれのアダプター(b)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含み、該切断部位は、アダプター(A)の切断部位を切断する際には切断されることがなく、かつ、アダプター(A)の切断末端とは結合しない切断末端を生じさせるものである;
2)アダプター(A)の切断部位において、工程1)で得られたDNA分子を切断する第一切断工程;
3)工程2)で得られたDNA分子の両末端を結合させて環状化させる第一段階環状化工程;
4)工程3)において環状化せず直線状となったDNA分子を除去する工程;
5)アダプター(b)の切断部位において、工程3)および工程4)で得られた環状DNA分子を切断する第二切断工程; および、
6)工程5)で得られたDNA分子の両末端を結合させて環状化させる第二段階環状化工程、
ここで、工程6)で得られる環状DNA分子は、アダプター(b)部分の配列を配列決定することにより、アダプター(b)部分に含まれる2つの固有配列が同一であれば、複数分子環状DNAに由来しない単分子環状DNAであり、該2つの固有配列が異なっていれば、複数分子環状DNAであるかまたは複数分子環状DNAに由来する単分子環状DNAであると判定されるものである。
工程1)は、各目的DNA分子の一方の末端に第一段階環状化用アダプター(A)を結合させ、他方の末端に、アダプター(b)と前記アダプター(A)を含む第二段階環状化用アダプター(B)を結合させる工程である。ここで、アダプター(B)は、DNA分子にアダプター(b)側を介して結合させることにより、アダプター(B)中のアダプター(A)は、DNA分子とアダプター(b)との結合の外側に位置する。即ち、各目的DNA分子の一端には、アダプター(A)を、他端にはアダプター(B)を結合させ、アダプター(B)中のアダプター(A)をアダプター(b)よりも末端側に位置させることにより、アダプター(A)を各目的DNA分子の両末端に位置させる。
(1)「複数分子環状DNAに由来しない単分子環状DNA」(例えば図6のA4)、ここで、該単分子環状DNAは、第一段階環状化工程において形成された単分子環状DNA(例えば図6のA2)が第二切断工程によって切断されて生じる単分子DNA(例えば図6のA3)が自己環状化することにより形成されるものである;
(2)「複数分子環状DNAに由来する単分子環状DNA」(例えば図6のB4-1)、ここで、該単分子環状DNAは、第一段階環状化工程において形成された複数分子環状DNA(例えば図6のB2)が第二切断工程によって切断されて生じる単分子DNA(例えば図6のB3)が自己環状化することにより形成されるものである;
(3)「複数分子環状DNA-1」(例えば図6のB4-2)、ここで、該DNA-1は、第一段階環状化工程において形成された複数分子環状DNA(例えば図6のB2)が第二切断工程によって切断されて生じる単分子DNA(例えば図6のB3)同士が複数結合して環状化することにより形成されるものである;
(4)「複数分子環状DNA-2」(例えば図6のB4-3)、ここで、該DNA-2は、第一段階環状化工程において形成された単分子環状DNA(例えば図6のA2)が第二切断工程によって切断されて生じる単分子DNA (例えば図6のA3)と、第一段階環状化工程において形成された複数分子環状DNA(例えば図6のB2)が第二切断工程によって切断されて生じる単分子DNA(例えば図6のB3)とが結合して環状化することにより形成されるものである; および
(5)「複数分子環状DNA-3」(例えば図6のB4-4)、ここで、該DNA-3は、第一段階環状化工程において形成された単分子環状DNA(例えば図6のA2)が第二切断工程によって切断されて生じる単分子DNA(例えば図6のA3)同士が複数結合して環状化することにより形成されるものである。
ここで、例えばNNNNNを上流および下流で
と設定する。
本発明は、第二の態様として、以下の工程を含む、複数分子環状DNAに由来しない単分子環状DNAの作成方法を提供する:
1)本発明の第一の態様の方法によって環状DNA分子を作成する工程;および
2)該作成された環状DNA分子についてアダプター(b)部分の配列を配列決定し、複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程、ここで、アダプター(b)部分に含まれる2つの固有配列が同一であれば、当該環状DNA分子は複数分子環状DNAに由来しない単分子環状DNAであり、該2つの固有配列が異なっていれば、当該環状DNA分子は複数分子環状DNAであるかまたは複数分子環状DNAに由来する単分子環状DNAである。
本発明は、第三の態様として、以下の工程を含む、環状DNA分子の作成において複数分子環状DNAに由来しない単分子環状DNAのみを選別する方法を提供する:
1)各目的DNA分子の一方の末端に第一段階環状化用アダプター(A)を結合させ、他方の末端に、アダプター(b)と前記アダプター(A)を含む第二段階環状化用アダプター(B)を結合させる工程;ここで、アダプター(B)は、DNA分子にアダプター(b)側を介して結合して、アダプター(B)中のアダプター(A)は、DNA分子とアダプター(b)との結合の外側に位置する、
ここで、
アダプター(A)は、いずれのアダプター(A)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含む;
アダプター(b)は、該アダプター(b)ごとに異なる固有配列を2つ含み、該2つの固有配列は同一方向または逆方向に配向した同一の配列であり; かつ、
アダプター(b)は、該2つの固有配列の間に、いずれのアダプター(b)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含み、該切断部位は、アダプター(A)の切断部位を切断する際には切断されることがなく、かつ、アダプター(A)の切断末端とは結合しない切断末端を生じさせるものである;
2)アダプター(A)の切断部位において、工程1)で得られたDNA分子を切断する第一切断工程;
3)工程2)で得られたDNA分子の両末端を結合させて環状化させる第一段階環状化工程;
4)工程3)において環状化せず直線状となったDNA分子を除去する工程;
5)アダプター(b)の切断部位において、工程3)および工程4)で得られた環状DNA分子を切断する第二切断工程;
6)工程5)で得られたDNA分子の両末端を結合させて環状化させる第二段階環状化工程;および、
7)工程6)で得られた環状DNA分子が有するアダプター(b)部分の配列を配列決定し、複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程、ここで、アダプター(b)部分に含まれる2つの固有配列が同一であれば、当該環状DNA分子は複数分子環状DNAに由来しない単分子環状DNAであり、該2つの固有配列が異なっていれば、当該環状DNA分子は複数分子環状DNAであるかまたは複数分子環状DNAに由来する単分子環状DNAである。
好ましくは、アダプター(B)は、下記構造Z1-Y-Z2-AまたはZ1-Y-Z’2-Aを有する互いに相補的な二本鎖DNAである:
[構造中、
Aは、パリンドローム型制限酵素サイトXを含む二本鎖DNAであって、アダプター(A)に相当し;
Z1-Y-Z2は、本発明の第一から第三までの態様におけるアダプター(b)に相当し;
Yは、パリンドローム型制限酵素サイトy1およびy2を含む二本鎖DNAであり;
y1およびy2は同一であり、Xとは異なる配列を有し、かつ、Xの切断により生じる切断末端と相補的でない切断末端を生じさせるものであり;
Z1およびZ2は、アダプターごとに異なる固有配列C1およびC2を含む二本鎖DNA配列であり、ここで、C1とC2は、互いに逆方向に配向した同一の配列であり;
nは1以上40以下の整数であり;
N1~Nnは、それぞれ同一であっても異なっていてもよく、dAMP、dCMP、dGMPおよびdTMPからなる群から選択されるデオキシリボヌクレオチドであり;
N’1~N’nは、前記N1~Nnに対してそれぞれ下記:
または
[構造中、
Z1-Y-Z’2は、本発明の第一から第三までの態様におけるアダプター(b)に相当し;
Z1およびZ’2は、アダプターごとに異なる固有配列C1およびC’2を含む二本鎖DNA配列であり、ここで、C1とC’2は、同一方向に配向した同一の配列であり;
A、X、Y、y1、y2、n、N1~Nn、N’1~N’n、およびkの定義は、上記の構造Z1-Y-Z2-Aにおけるものと同一である]。
アダプター(b)における固有配列の長さ(上記構造Z1-Y-Z2-AまたはZ1-Y-Z’2-Aを有するアダプターにおいては、構造中の「n」)は、特に限定されないが、好ましくは1~40塩基、より好ましくは4~15塩基、さらに好ましくは5~10塩基である。塩基数が多いほど固有配列の種類が増え、第一段階環状化工程において同じアダプター(b)部分を有する(即ち、同じ固有配列を有する)異なるDNA分子同士が結合して複数分子環状DNAを形成する確率は低下する。
本発明は、第四の態様として、上記本発明の第一から第三までの態様の方法において用いられる好適なアダプター(B)、即ち、下記構造Z1-Y-Z2-AまたはZ1-Y-Z’2-Aを有する互いに相補的な二本鎖DNAからなる環状DNA作成用アダプターであって、該アダプターを用いて作成された環状DNA分子の集団から複数分子環状DNAに由来しない単分子環状DNAのみを選別することを可能にするためのアダプターを提供する:
[構造中、
Aは、パリンドローム型制限酵素サイトXを含む二本鎖DNAであり;
Yは、パリンドローム型制限酵素サイトy1およびy2を含む二本鎖DNAであり;
y1およびy2は同一であり、Xとは異なる配列を有し、かつ、Xの切断により生じる切断末端と相補的でない切断末端を生じさせるものであり;
Z1およびZ2は、アダプターごとに異なる固有配列C1およびC2を含む二本鎖DNA配列であり、ここで、C1とC2は、互いに逆向きに配向した同一の配列であり;
nは1以上40以下の整数であり;
N1~Nnは、それぞれ同一であっても異なっていてもよく、dAMP、dCMP、dGMPおよびdTMPからなる群から選択されるデオキシリボヌクレオチドであり;
N’1~N’nは、前記N1~Nnに対してそれぞれ下記:
または
[構造中、
Z1およびZ’2は、アダプターごとに異なる固有配列C1およびC’2を含む二本鎖DNA配列であり、ここで、C1とC’2は、同一方向に配向した同一の配列であり;
A、X、Y、y1、y2、n、N1~Nn、N’1~N’n、およびkの定義は、上記の構造Z1-Y-Z2-Aにおけるものと同一である]。
上記第四の態様のアダプターにおける構造Z1-Y-Z2またはZ1-Y-Z’2に代表され、上記本発明の第一から第三までの態様において好適に使用される、同一の固有配列を2つ有するアダプター(b)は、例えば、ヘアピン構造を有する核酸を用い、ポリメラーゼ伸長、ニック作成、ポリメラーゼ伸長という3段階の反応を行うことにより作成することができる(図8)。しかし、作成方法はこれに限定されず、例えば配列合成等によって所望のアダプター(b)を作成することも可能である。
本発明は、第五の態様として、上記本発明の第一から第三までの態様の方法において用いられる好適なアダプター(A)およびアダプター(B)を含む環状DNA作成用キットであって、該キットを用いて作成された環状DNA分子の集団から複数分子環状DNAに由来しない単分子環状DNAのみを選別することを可能にするキットを提供する。即ち、上記第四の態様に記載の制限酵素サイトXと同一の制限酵素サイトを含む二本鎖DNAからなるアダプター(A)と、上記第四の態様に記載のアダプター(B)とを含む、環状DNA作成用キットを提供する。
本発明の第六の態様は、本発明の第一から第三までのいずれかの態様の二段階環状化方法を用いる、cDNAライブラリーの作成方法である。
本発明の第一の態様による方法を直鎖状cDNAからなるライブラリーに適用することにより、ライブラリーのメンバーについてアダプター(b)部分の配列を配列決定することによって複数分子環状DNAに由来しない単分子環状DNAであるメンバーのみを選別することが可能なcDNAライブラリーを作成することができる。従って、例えばかかるcDNAライブラリーを用いて遺伝子解析を行う場合、該ライブラリーについて網羅的に配列解読を行った後、複数分子環状DNAに由来しない単分子環状DNAのデータのみを解析対象として選別することができる。
本発明の第七の態様は、以下の工程を含む、環状DNA分子をメイトペア法に供することにより遺伝子を同定する方法である:
1)本発明の第一の態様の方法によって作成された環状DNA分子の集団、本発明の第二の態様の方法によって作成された複数分子環状DNAに由来しない単分子環状DNAまたは本発明の第三の態様の方法によって選別された複数分子環状DNAに由来しない単分子環状DNAにおける、アダプター(B)の両側に隣接するそれぞれ15塩基以上600塩基以下の塩基配列を解読する工程、ここで、本発明の第一の態様の方法によって作成された環状DNA分子の集団を用いる場合には、当該工程の前に、当該工程と同時に、または当該工程の後に、アダプター(b)部分の配列を配列決定することにより複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程をさらに含む;および、
2)工程1)で解読した塩基配列を既知の遺伝子の両末端の配列と比較することにより、該複数分子環状DNAに由来しない単分子環状DNAに含まれる遺伝子を同定する工程。
あるいは、同じサンプルを、アダプター(B)の外側の配列解読用およびアダプター(b)部分の配列解読用として複数に分け、これらを同時にシークエンシング反応に供試してそれぞれ配列データを取得する場合も挙げられる。
いずれにおいても、アダプター(b)部分における2つの固有配列が同一であるサンプル(即ち、複数分子環状DNAに由来しない単分子環状DNA)のデータのみを解析対象として選別することができる。
本発明の第八の態様は、以下の工程を含む、環状DNA分子をメイトペア法に供することにより融合遺伝子を検出する方法である:
1)本発明の第一の態様の方法によって作成された環状DNA分子の集団、本発明の第二の態様の方法によって作成された複数分子環状DNAに由来しない単分子環状DNAまたは本発明の第三の態様の方法によって選別された複数分子環状DNAに由来しない単分子環状DNAにおける、アダプター(B)の両側に隣接するそれぞれ15塩基以上600塩基以下の塩基配列を解読する工程、ここで、本発明の第一の態様の方法によって作成された環状DNA分子の集団を用いる場合には、当該工程の前に、当該工程と同時に、または当該工程の後に、アダプター(b)部分の配列を配列決定することにより複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程をさらに含む;および、
2)工程1)で解読した塩基配列を既知の遺伝子の両末端の配列と比較する工程、ここで、両末端の遺伝子が既知の相異なる遺伝子に対応する場合、該複数分子環状DNAに由来しない単分子環状DNAに含まれる遺伝子は融合遺伝子であると同定される。
当該工程1)において、「当該工程と同時に」アダプター(b)部分の配列を配列決定する場合については、上記第七の態様と同様である。
mRNAからcDNAライブラリーを合成する場合、現在使用されている最も一般的な方法(Clontech SMART cDNA method) では、図9のようにmRNAの3’末端のpolyAサイトに相補的なpolyT配列を有するオリゴヌクレオチド (1)を用いて、まず相補鎖DNAを逆転写酵素で合成する。合成終了時にmRNAの5’末端において図9のように特定オリゴヌクレオチド配列 (2)が取り込まれる。次にこの特定配列へ相補的なオリゴヌクレオチドからDNA合成を行うこと、あるいはPCRにより、cDNAライブラリーが作成される。
この場合、mRNAの3’末端のpolyAサイトに相補的なpolyT配列を有するオリゴヌクレオチド(1)配列にアダプター(B)を導入しておき、オリゴヌクレオチド(2)配列にアダプター(A)を付加しておく。これにより、cDNAライブラリーが合成されたときには、自動的に本発明の基本形の構造になっており、右端左端に新たな配列を結合させる必要もなく、そのまま次のステップへ進むことができる(図9)。この方法で、さらにcDNAライブラリーをPCR増幅する際に、そのまま増幅したのではある特定の固有配列が増幅されてしまう。そこで、固有配列の多様性を維持するため、例えば、上流プライマーとして5’リン酸基付加プライマーを使用し、下流プライマーには固有配列を含むプライマーを用いて増幅し、PCR後にリン酸基プライマーを取り込んだ側のストランドをλエキソヌクレアーゼで分解し、再度上流プライマーからプライマー伸長を行い、固有配列による多様性を維持したcDNAライブラリーを完成させる、等の手続きを行う。但し、アダプター結合法であればこの過程は必要なく、ライブラリーの断片化などの修飾を行った上で本発明のアダプターを導入するためには、次のアダプター結合法を行う。
cDNAライブラリーを作成した場合、図9の(3)のように3’末端、5’末端にそれぞれ制限酵素サイトを導入しておくことができる。この場合の制限酵素サイトとしては通常のパリンドローム型制限酵素サイトを使用することも、末端を区別するためにノン・パリンドローム型制限酵素サイトを使用することも可能である。また、アダプター結合を確実にするため、平滑末端ではなくA突出塩基を有する末端へアダプターを結合することも可能である。ライブラリーの修飾を行った上に本発明のアダプターを導入するためにはこちらの方法が望ましい。
ゲノムを対象とする場合はcDNAライブラリーとは状況が異なる。ゲノム断片は、cDNAと異なりDNA断片の左右を区別することができない。従って図10のように左端のアダプター、右端のアダプターを双方結合させて、双方のアダプターが適切に結合したもの、即ち図10の( a ) のみを、PCR法や修飾リンカーを用いる方法、あるいは両端の制限酵素サイトXとしてノン・パリンドローム配列を用いることや、N領域を含む制限酵素(BstXI等)を用いて同一部位に制限酵素サイトを複数設置する方法等によって選択してゆくことができる。
基本的に、以下のような手法をとることによりアダプター(A)と(B)をDNA分子の両末端にそれぞれ付加する。第一に、目的DNA分子数に応じて、ある程度の濃度を決定した複数のDNA群に、先ず、アダプター(A)を結合させる。この場合、アダプター(A)の添加量は存在する対象DNA分子量よりも少なめとする。ただし、量論的には、対象DNA分子数と同数であっても、あるいは、DNA断片に一塩基突出を酵素的に作成し、相補的なアダプターを結合させてもよく、この場合は特にアダプターの濃度は過剰であっても構わない。これにより、アダプター(A)をDNA分子の末端に結合させる。次いで、アダプター(B)を結合させる。
例えば、アダプター(A)として、切断末端W1を生じさせるアダプター"A1"を用い、アダプター(B)に含まれるアダプター(A)として、切断末端W2を生じさせるアダプター"A2"を用いる。ここで、切断末端W1とW2が相補的である一方、W1同士およびW2同士は相補的でない、即ち、アダプター"A1"と"A2"の間でのみライゲーションが可能なように配列を設計または選択する。
図10の(a) タイプの分子の場合、第一段階環状化、第二切断、第二段階環状化が問題なく行われ、問題なく目的の環状化DNAが作成される。一方、図10の(b)タイプの分子の場合、単独で第一段階環状化が可能であるが、アダプター(b)部分を有さないため、第二切断が行われずに環状DNAのまま残ってしまい、排除することができない。これを防ぐ方法例として、下記の「BstXI法」がある。
両端にアダプター(A)が付加された場合、第一段階でライゲーションされるが、第二段階の開裂が起きないため、このままでは環状のまま留まってしまう。この問題を解決する方法として、アダプター(A)の制限酵素サイトを含む外側に、例えば BstXI を仕込むことが挙げられる。つまり、下記制限酵素サイトBstXI部位において、CCANNNNNNTGGを、例えば、 CCAGGATCCTGG となるようにBamHIサイトを組み込む。
第二切断工程により分離したDNA分子同士が再び会合する可能性は確率論的に十分無視できるが、その根拠を以下に説明する。
第二切断工程により複数分子環状DNAが切断されて生じた一のDNA分子が、切断前に結合していた相手DNA分子と再び会合する可能性を確率論的に推定するため、(1)DNA分子の体積と反応溶液量(体積)で推定する場合と、(2)反応溶液中の分子数から推定する場合に分けて可能性を概算する。
まずDNA1分子を球状として体積を推定し、反応系で一度分離された分子同士が互いに球体として溶液中で会合する可能性を概算する。
一塩基の長さ:0.34 nm (0.34 x 10e-9 m = 3.4 x 10e-8 mm)
3kbp (3000 bp) のプラスミドの長さ:1 x 10e-4 mm
球体として占めると推定した体積は:4/3 x 3.14 x (1 x 10e-4) x (1 x 10e-4) x (1 x 10e-4) mm3 = 4 x 10e-12 mm3
一分子の体積を 4 x 10e-12 mm3と仮定すると 100 μL中には球体として:100 mm3 / 4 x 10e-12 mm3 = 2.5 x 10e13 個分の体積が存在することとなる。
従って、均一であるとすると、ある球体に相補的とされる同等な球体が会合する可能性は、(1 / (2.5 x 10e13)) = 4 x 10e-14と極めて小さい。
一方、分子数を計算すると、
3kbpのプラスミドの分子量は:625 x 3000 = 1.8 x 10e6
1モルのプラスミド質量は:1 mol = 1.8 x 10e6 g = 1.8 x 10e12 μg
3 μgのプラスミドのモル数は:3 μg / (1.8 x 10e12) μg = 3/1.8 x 10e-12 mol = 1.6 x 10e-12 mol
アボガドロ数を 6 x 10e23 とすると、3μgのプラスミドの分子数は、1.6 x 10e-12 x 6 x 10e23 = 1.6 x 6 x 10e11 = 1 x 10e12 個となる。
従って、例えば 100μLの反応系中に 3μgのプラスミドが存在する場合、第二切断工程において分離した一方の分子が、他方の(即ち、同一の固有配列を有する)分子と再度会合する可能性は、1 / ((1 x 10e12) -1) = 1 x 10e-12 と極めて低い。
Claims (20)
- 以下の工程を含む、環状DNA分子の集団の作成方法であって、該方法により作成された環状DNA分子の集団から複数分子環状DNAに由来しない単分子環状DNAのみを選別することを可能にする方法:
1)各目的DNA分子の一方の末端に第一段階環状化用アダプター(A)を結合させ、他方の末端に、アダプター(b)と前記アダプター(A)を含む第二段階環状化用アダプター(B)を結合させる工程;ここで、アダプター(B)は、DNA分子にアダプター(b)側を介して結合して、アダプター(B)中のアダプター(A)は、DNA分子とアダプター(b)との結合の外側に位置する、
ここで、
アダプター(A)は、いずれのアダプター(A)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含む;
アダプター(b)は、該アダプター(b)ごとに異なる固有配列を2つ含み、該2つの固有配列は同一方向または逆方向に配向した同一の配列であり; かつ、
アダプター(b)は、該2つの固有配列の間に、いずれのアダプター(b)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含み、該切断部位は、アダプター(A)の切断部位を切断する際には切断されることがなく、かつ、アダプター(A)の切断末端とは結合しない切断末端を生じさせるものである;
2)アダプター(A)の切断部位において、工程1)で得られたDNA分子を切断する第一切断工程;
3)工程2)で得られたDNA分子の両末端を結合させて環状化させる第一段階環状化工程;
4)工程3)において環状化せず直線状となったDNA分子を除去する工程;
5)アダプター(b)の切断部位において、工程3)および工程4)で得られた環状DNA分子を切断する第二切断工程; および、
6)工程5)で得られたDNA分子の両末端を結合させて環状化させる第二段階環状化工程、
ここで、工程6)で得られる環状DNA分子は、アダプター(b)部分の配列を配列決定することにより、アダプター(b)部分に含まれる2つの固有配列が同一であれば、複数分子環状DNAに由来しない単分子環状DNAであり、該2つの固有配列が異なっていれば、複数分子環状DNAであるかまたは複数分子環状DNAに由来する単分子環状DNAであると判定されるものである。 - アダプター(b)が、2つの固有配列の間に、いずれのアダプター(b)の切断末端とも非特異的に結合する切断末端を生じる切断部位を2つ含むものである、請求項1記載の方法。
- アダプター(A)が、パリンドローム型制限酵素サイトXを含む互いに相補的な二本鎖DNAであり、
アダプター(B)が、下記構造Z1-Y-Z2-AまたはZ1-Y-Z’2-Aを有する互いに相補的な二本鎖DNAである、請求項1または2記載の方法:
[構造中、
Aは、パリンドローム型制限酵素サイトXを含む二本鎖DNAであって、アダプター(A)に相当し;
Z1-Y-Z2は、請求項1または2におけるアダプター(b)に相当し;
Yは、パリンドローム型制限酵素サイトy1およびy2を含む二本鎖DNAであり;
y1およびy2は同一であり、Xとは異なる配列を有し、かつ、Xの切断により生じる切断末端と相補的でない切断末端を生じさせるものであり;
Z1およびZ2は、アダプターごとに異なる固有配列C1およびC2を含む二本鎖DNA配列であり、ここで、C1とC2は、互いに逆方向に配向した同一の配列であり;
nは1以上40以下の整数であり;
N1~Nnは、それぞれ同一であっても異なっていてもよく、dAMP、dCMP、dGMPおよびdTMPからなる群から選択されるデオキシリボヌクレオチドであり;
N’1~N’nは、前記N1~Nnに対してそれぞれ下記:
または
[構造中、
Z1-Y-Z’2は、請求項1または2におけるアダプター(b)に相当し;
Z1およびZ’2は、アダプターごとに異なる固有配列C1およびC’2を含む二本鎖DNA配列であり、ここで、C1とC’2は、同一方向に配向した同一の配列であり;
A、X、Y、y1、y2、n、N1~Nn、N’1~N’n、およびkの定義は、上記の構造Z1-Y-Z2-Aにおけるものと同一である]。 - 請求項1~3いずれかに記載の方法によって作成された環状DNA分子の集団。
- 以下の工程を含む、複数分子環状DNAに由来しない単分子環状DNAの作成方法:
1)請求項1~3のいずれかに記載の方法によって環状DNA分子を作成する工程;および
2)該作成された環状DNA分子についてアダプター(b)部分の配列を配列決定し、複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程、ここで、アダプター(b)部分に含まれる2つの固有配列が同一であれば、当該環状DNA分子は複数分子環状DNAに由来しない単分子環状DNAであり、該2つの固有配列が異なっていれば、当該環状DNA分子は複数分子環状DNAであるかまたは複数分子環状DNAに由来する単分子環状DNAである。 - 請求項5記載の方法によって作成された、複数分子環状DNAに由来しない単分子環状DNA。
- 以下の工程を含む、環状DNA分子の作成において、複数分子環状DNAに由来しない単分子環状DNAのみを選別する方法:
1)各目的DNA分子の一方の末端に第一段階環状化用アダプター(A)を結合させ、他方の末端に、アダプター(b)と前記アダプター(A)を含む第二段階環状化用アダプター(B)を結合させる工程;ここで、アダプター(B)は、DNA分子にアダプター(b)側を介して結合して、アダプター(B)中のアダプター(A)は、DNA分子とアダプター(b)との結合の外側に位置する、
ここで、
アダプター(A)は、いずれのアダプター(A)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含む;
アダプター(b)は、該アダプター(b)ごとに異なる固有配列を2つ含み、該2つの固有配列は同一方向または逆方向に配向した同一の配列であり; かつ、
アダプター(b)は、該2つの固有配列の間に、いずれのアダプター(b)の切断末端とも非特異的に結合する切断末端を生じさせる切断部位を含み、該切断部位は、アダプター(A)の切断部位を切断する際には切断されることがなく、かつ、アダプター(A)の切断末端とは結合しない切断末端を生じさせるものである;
2)アダプター(A)の切断部位において、工程1)で得られたDNA分子を切断する第一切断工程;
3)工程2)で得られたDNA分子の両末端を結合させて環状化させる第一段階環状化工程;
4)工程3)において環状化せず直線状となったDNA分子を除去する工程;
5)アダプター(b)の切断部位において、工程3)および工程4)で得られた環状DNA分子を切断する第二切断工程;
6)工程5)で得られたDNA分子の両末端を結合させて環状化させる第二段階環状化工程;および、
7)工程6)で得られた環状DNA分子が有するアダプター(b)部分の配列を配列決定し、複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程、ここで、アダプター(b)部分に含まれる2つの固有配列が同一であれば、当該環状DNA分子は複数分子環状DNAに由来しない単分子環状DNAであり、該2つの固有配列が異なっていれば、当該環状DNA分子は複数分子環状DNAであるかまたは複数分子環状DNAに由来する単分子環状DNAである。 - アダプター(b)が、2つの固有配列の間に、いずれのアダプター(b)の切断末端とも非特異的に結合する切断末端を生じる切断部位を2つ含むものである、請求項7記載の方法。
- アダプター(A)が、パリンドローム型制限酵素サイトXを含む互いに相補的な二本鎖DNAであり、
アダプター(B)が、下記構造Z1-Y-Z2-AまたはZ1-Y-Z’2-Aを有する互いに相補的な二本鎖DNAである、請求項7または8記載の方法:
[構造中、
Aは、パリンドローム型制限酵素サイトXを含む二本鎖DNAであって、アダプター(A)に相当し;
Z1-Y-Z2は、請求項7または8におけるアダプター(b)に相当し;
Yは、パリンドローム型制限酵素サイトy1およびy2を含む二本鎖DNAであり;
y1およびy2は同一であり、Xとは異なる配列を有し、かつ、Xの切断により生じる切断末端と相補的でない切断末端を生じさせるものであり;
Z1およびZ2は、アダプターごとに異なる固有配列C1およびC2を含む二本鎖DNA配列であり、ここで、C1とC2は、互いに逆方向に配向した同一の配列であり;
nは1以上40以下の整数であり;
N1~Nnは、それぞれ同一であっても異なっていてもよく、dAMP、dCMP、dGMPおよびdTMPからなる群から選択されるデオキシリボヌクレオチドであり;
N’1~N’nは、前記N1~Nnに対してそれぞれ下記:
または
[構造中、
Z1-Y-Z’2は、請求項7または8におけるアダプター(b)に相当し;
Z1およびZ’2は、アダプターごとに異なる固有配列C1およびC’2を含む二本鎖DNA配列であり、ここで、C1とC’2は、同一方向に配向した同一の配列であり;
A、X、Y、y1、y2、n、N1~Nn、N’1~N’n、およびkの定義は、上記の構造Z1-Y-Z2-Aにおけるものと同一である]。 - 請求項7~9いずれかに記載の方法により選別された、複数分子環状DNAに由来しない単分子環状DNA。
- 下記構造Z1-Y-Z2-AまたはZ1-Y-Z’2-Aを有する互いに相補的な二本鎖DNAからなる環状DNA作成用アダプターであって、該アダプターを用いて作成された環状DNA分子の集団から複数分子環状DNAに由来しない単分子環状DNAのみを選別することを可能にするための、アダプター:
[構造中、
Aは、パリンドローム型制限酵素サイトXを含む二本鎖DNAであり;
Yは、パリンドローム型制限酵素サイトy1およびy2を含む二本鎖DNAであり;
y1およびy2は同一であり、Xとは異なる配列を有し、かつ、Xの切断により生じる切断末端と相補的でない切断末端を生じさせるものであり;
Z1およびZ2は、アダプターごとに異なる固有配列C1およびC2を含む二本鎖DNA配列であり、ここで、C1とC2は、互いに逆向きに配向した同一の配列であり;
nは1以上40以下の整数であり;
N1~Nnは、それぞれ同一であっても異なっていてもよく、dAMP、dCMP、dGMPおよびdTMPからなる群から選択されるデオキシリボヌクレオチドであり;
N’1~N’nは、前記N1~Nnに対してそれぞれ下記:
または
[構造中、
Z1およびZ’2は、アダプターごとに異なる固有配列C1およびC’2を含む二本鎖DNA配列であり、ここで、C1とC’2は、同一方向に配向した同一の配列であり;
A、X、Y、y1、y2、n、N1~Nn、N’1~N’n、およびkの定義は、上記の構造Z1-Y-Z2-Aにおけるものと同一である]。 - 請求項11に記載のアダプターと、請求項11に記載のアダプターに含まれる制限酵素サイトXと同一の制限酵素サイトを含む二本鎖DNAからなるアダプターとを含む環状DNA作成用キットであって、該キットを用いて作成された環状DNA分子の集団から複数分子環状DNAに由来しない単分子環状DNAのみを選別することを可能にする、キット。
- 請求項1~3いずれかに記載の方法を用いるcDNAライブラリーの作成方法であって、該ライブラリーが、該ライブラリーから複数分子環状DNAに由来しない単分子環状DNAのみを選別することが可能なものである、方法。
- 請求項5記載の方法または請求項7~9いずれかに記載の方法を用いる、複数分子環状DNAに由来しない単分子環状DNAのみからなるcDNAライブラリーの作成方法。
- 以下の工程を含む、環状DNA分子をメイトペア法に供することにより遺伝子を同定する方法:
1)請求項1~3いずれかに記載の方法によって作成された環状DNA分子の集団または請求項6もしくは10に記載の複数分子環状DNAに由来しない単分子環状DNAにおける、アダプター(B)の両側に隣接するそれぞれ15塩基以上600塩基以下の塩基配列を解読する工程、ここで、請求項1~3いずれかに記載の方法によって作成された環状DNA分子の集団を用いる場合には、当該工程の前に、当該工程と同時に、または当該工程の後に、アダプター(b)部分の配列を配列決定することにより複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程をさらに含む;および、
2)工程1)で解読した塩基配列を既知の遺伝子の両末端の配列と比較することにより、該複数分子環状DNAに由来しない単分子環状DNAに含まれる遺伝子を同定する工程。 - 以下の工程を含む、環状DNA分子をメイトペア法に供することにより融合遺伝子を検出する方法:
1)請求項1~3いずれかに記載の方法によって作成された環状DNA分子の集団または請求項6もしくは10に記載の複数分子環状DNAに由来しない単分子環状DNAにおける、アダプター(B)の両側に隣接するそれぞれ15塩基以上600塩基以下の塩基配列を解読する工程、ここで、請求項1~3いずれかに記載の方法によって作成された環状DNA分子の集団を用いる場合には、当該工程の前に、当該工程と同時に、または当該工程の後に、アダプター(b)部分の配列を配列決定することにより複数分子環状DNAに由来しない単分子環状DNAのみを選別する工程をさらに含む;および、
2)工程1)で解読した塩基配列を既知の遺伝子の両末端の配列と比較する工程、ここで、両末端の遺伝子が既知の相異なる遺伝子に対応する場合、該複数分子環状DNAに由来しない単分子環状DNAに含まれる遺伝子は融合遺伝子であると同定される。 - アダプター(B)の両側に隣接する両側の配列が、既知融合遺伝子の両側の末端に対応する、請求項16に記載の融合遺伝子の検出方法。
- 請求項16に記載の方法により検出された融合遺伝子をマーカーとして用いる、該融合遺伝子の発現を特徴とする疾患を検出する方法。
- アダプター(B)の両側に隣接する配列が、相異なる遺伝子の末端配列に対応し、かつ既知融合遺伝子の両側の末端には対応しないことにより、環状DNA分子に含まれる遺伝子が新規融合遺伝子であると同定される請求項16に記載の方法。
- 請求項19に記載の方法により検出された新規融合遺伝子の創薬スクリーニングにおける使用。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/240,488 US9416358B2 (en) | 2011-08-31 | 2012-08-24 | Method for exclusive selection of circularized DNA from monomolecular DNA in circularizing DNA molecules |
CN201280052157.8A CN103890175B (zh) | 2011-08-31 | 2012-08-24 | 在dna分子的环化中仅选择由单分子形成的环化dna的方法 |
JP2013531289A JP6066209B2 (ja) | 2011-08-31 | 2012-08-24 | Dna分子の環状化において単分子による環状化dnaのみを選別する方法 |
EP12827670.6A EP2752486B1 (en) | 2011-08-31 | 2012-08-24 | Method for exclusive selection of circularized dna from monomolecular dna when circularizing dna molecules |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2011189280 | 2011-08-31 | ||
JP2011-189280 | 2011-08-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2013031700A1 true WO2013031700A1 (ja) | 2013-03-07 |
Family
ID=47756190
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2012/071492 WO2013031700A1 (ja) | 2011-08-31 | 2012-08-24 | Dna分子の環状化において単分子による環状化dnaのみを選別する方法 |
Country Status (5)
Country | Link |
---|---|
US (1) | US9416358B2 (ja) |
EP (1) | EP2752486B1 (ja) |
JP (1) | JP6066209B2 (ja) |
CN (1) | CN103890175B (ja) |
WO (1) | WO2013031700A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016058134A1 (zh) * | 2014-10-14 | 2016-04-21 | 深圳华大基因科技有限公司 | 一种接头元件和使用其构建测序文库的方法 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109593783B (zh) * | 2017-09-30 | 2024-05-24 | 中国科学院动物研究所 | 一种体外产生环状核酸分子的方法 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008504805A (ja) * | 2004-07-02 | 2008-02-21 | 株式会社ダナフォーム | 塩基配列タグの調製方法 |
JP2008295444A (ja) * | 2006-10-11 | 2008-12-11 | Astellas Pharma Inc | Eml4−alk融合遺伝子 |
JP2008545448A (ja) * | 2005-06-06 | 2008-12-18 | 454 ライフ サイエンシーズ コーポレイション | 両末端配列決定(pairedendsequencing) |
WO2009032167A1 (en) * | 2007-08-29 | 2009-03-12 | Illumina Cambridge | Method for sequencing a polynucleotide template |
JP2011510669A (ja) * | 2008-02-05 | 2011-04-07 | エフ.ホフマン−ラ ロシュ アーゲー | ペアエンド配列決定の方法 |
WO2011103236A2 (en) * | 2010-02-18 | 2011-08-25 | The Johns Hopkins University | Personalized tumor biomarkers |
WO2012029577A1 (ja) * | 2010-09-02 | 2012-03-08 | 学校法人 久留米大学 | 単分子dnaから形成される環状dnaの作成方法 |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007145612A1 (en) | 2005-06-06 | 2007-12-21 | 454 Life Sciences Corporation | Paired end sequencing |
CN101351552A (zh) * | 2005-06-06 | 2009-01-21 | 454生命科学公司 | 配对末端测序 |
US7897344B2 (en) | 2007-11-06 | 2011-03-01 | Complete Genomics, Inc. | Methods and oligonucleotide designs for insertion of multiple adaptors into library constructs |
US8530197B2 (en) * | 2008-01-09 | 2013-09-10 | Applied Biosystems, Llc | Method of making a paired tag library for nucleic acid sequencing |
US8352194B1 (en) | 2008-06-17 | 2013-01-08 | University Of South Florida | Method to identify cancer fusion genes |
JPWO2011043220A1 (ja) | 2009-10-06 | 2013-03-04 | 富士レビオ株式会社 | 融合遺伝子の測定方法 |
EP2586862B9 (en) | 2010-06-22 | 2016-07-13 | LSI Medience Corporation | Detection method for novel ros1 fusion product |
EP2599878A4 (en) | 2010-07-26 | 2014-02-12 | Astellas Pharma Inc | NEW METHOD FOR DETECTING FUSED BODIES RET |
ES2578370T3 (es) | 2011-07-01 | 2016-07-26 | HTG Molecular Diagnostics, Inc | Métodos para detectar fusiones génicas |
US9216172B2 (en) | 2011-08-04 | 2015-12-22 | National Cancer Center | Method for determining effectiveness of cancer treatment by assessing the presence of a KIF5B-RET chimeric gene |
-
2012
- 2012-08-24 US US14/240,488 patent/US9416358B2/en not_active Expired - Fee Related
- 2012-08-24 CN CN201280052157.8A patent/CN103890175B/zh not_active Expired - Fee Related
- 2012-08-24 EP EP12827670.6A patent/EP2752486B1/en not_active Not-in-force
- 2012-08-24 JP JP2013531289A patent/JP6066209B2/ja active Active
- 2012-08-24 WO PCT/JP2012/071492 patent/WO2013031700A1/ja active Application Filing
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008504805A (ja) * | 2004-07-02 | 2008-02-21 | 株式会社ダナフォーム | 塩基配列タグの調製方法 |
JP2008545448A (ja) * | 2005-06-06 | 2008-12-18 | 454 ライフ サイエンシーズ コーポレイション | 両末端配列決定(pairedendsequencing) |
JP2008295444A (ja) * | 2006-10-11 | 2008-12-11 | Astellas Pharma Inc | Eml4−alk融合遺伝子 |
JP4303303B2 (ja) | 2006-10-11 | 2009-07-29 | アステラス製薬株式会社 | Eml4−alk融合遺伝子 |
WO2009032167A1 (en) * | 2007-08-29 | 2009-03-12 | Illumina Cambridge | Method for sequencing a polynucleotide template |
JP2011510669A (ja) * | 2008-02-05 | 2011-04-07 | エフ.ホフマン−ラ ロシュ アーゲー | ペアエンド配列決定の方法 |
WO2011103236A2 (en) * | 2010-02-18 | 2011-08-25 | The Johns Hopkins University | Personalized tumor biomarkers |
WO2012029577A1 (ja) * | 2010-09-02 | 2012-03-08 | 学校法人 久留米大学 | 単分子dnaから形成される環状dnaの作成方法 |
Non-Patent Citations (8)
Title |
---|
"Jikken Igaku", vol. 27, 2009, article "Shikkan Idenshi no Tansaku to Chokosoku Sequence", pages: 113 - 143 |
BASHIR ET AL., PLOS COMPUTATIONAL BIOLOGY, vol. 4, no. 4, April 2008 (2008-04-01), pages EL000051 |
CHINNAIYAN ET AL., SCIENCE, vol. 310, 2005, pages 644 - 648 |
MITELMAN ET AL., NATURE GENETICS, vol. 36, no. 4, 2004, pages 331 - 334 |
RUAN Y . ET AL.: "Multiplex parallel pair-end-ditag sequencing approaches in system biology", WILEY INTERDISCIP. REV. SYST. BIOL. MED., vol. 2, no. 2, 2010, pages 224 - 234, XP002680213 * |
See also references of EP2752486A4 * |
SODA ET AL., NATURE, vol. 448, 2007, pages 561 - 566 |
TANPAKUSHITSU KAKUSAN KOSO, vol. 1233-124, August 2009 (2009-08-01), pages 1271 - 1275 |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016058134A1 (zh) * | 2014-10-14 | 2016-04-21 | 深圳华大基因科技有限公司 | 一种接头元件和使用其构建测序文库的方法 |
CN107075512A (zh) * | 2014-10-14 | 2017-08-18 | 深圳华大基因科技有限公司 | 一种接头元件和使用其构建测序文库的方法 |
CN107075512B (zh) * | 2014-10-14 | 2021-01-15 | 深圳华大智造科技股份有限公司 | 一种接头元件和使用其构建测序文库的方法 |
Also Published As
Publication number | Publication date |
---|---|
EP2752486A4 (en) | 2015-02-25 |
EP2752486A1 (en) | 2014-07-09 |
US9416358B2 (en) | 2016-08-16 |
JPWO2013031700A1 (ja) | 2015-03-23 |
JP6066209B2 (ja) | 2017-01-25 |
CN103890175B (zh) | 2015-12-09 |
CN103890175A (zh) | 2014-06-25 |
US20140303004A1 (en) | 2014-10-09 |
EP2752486B1 (en) | 2016-12-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5780527B2 (ja) | 単分子dnaから形成される環状dnaの作成方法 | |
US10538806B2 (en) | High throughput screening of populations carrying naturally occurring mutations | |
EP3423598B1 (en) | Methods and kits for tracking nucleic acid target origin for nucleic acid sequencing | |
AU2016366231B2 (en) | Improved adapters, methods, and compositions for duplex sequencing | |
JP6982087B2 (ja) | 競合的鎖置換を利用する次世代シーケンシング(ngs)ライブラリーの構築 | |
AU2006259990B2 (en) | Improved strategies for sequencing complex genomes using high throughput sequencing technologies | |
WO2011095501A1 (en) | Complexitiy reduction method | |
US20230017673A1 (en) | Methods and Reagents for Molecular Barcoding | |
JP2023513606A (ja) | 核酸を評価するための方法および材料 | |
JP6066209B2 (ja) | Dna分子の環状化において単分子による環状化dnaのみを選別する方法 | |
EP2333104A1 (en) | RNA analytics method | |
US7220548B2 (en) | Partial homologous recombination of DNA chain | |
AU3085701A (en) | Method of analyzing a nucleic acid |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12827670 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2013531289 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14240488 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2012827670 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012827670 Country of ref document: EP |