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WO2002090538A1 - Procede de synthese d'acide nucleique - Google Patents

Procede de synthese d'acide nucleique Download PDF

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Publication number
WO2002090538A1
WO2002090538A1 PCT/JP2002/004479 JP0204479W WO02090538A1 WO 2002090538 A1 WO2002090538 A1 WO 2002090538A1 JP 0204479 W JP0204479 W JP 0204479W WO 02090538 A1 WO02090538 A1 WO 02090538A1
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Prior art keywords
primer
nucleic acid
base sequence
region
complementary
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Japanese (ja)
Inventor
Kentaro Nagamine
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Eiken Chemical Co Ltd
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Eiken Chemical Co Ltd
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Publication of WO2002090538A1 publication Critical patent/WO2002090538A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • An analysis method based on the complementation of nucleobase sequences can directly analyze genetic characteristics. Therefore, it is a very effective means for identifying genetic diseases, canceration, microorganisms, and the like. In addition, in order to detect the gene itself, there is also a method that can eliminate time-consuming and labor-intensive operations such as culture.
  • PCR Polymerase Chain Reaction
  • the PCR method requires a special temperature controller to perform the procedure; the amplification reaction progresses exponentially, so there is a problem in the quantification; the sample and the reaction solution are contaminated from outside, Problems have been pointed out, such as the susceptibility to contamination, in which erroneously contaminated nucleic acids function as, and the like. ,
  • S Ps single nucleotide polymorphism
  • the LCR method (Ligase Chain Reaction, Laffler TG; Carrino JJ; Marshall RL; Ann. Biol. Clin. (Paris), 1993, 51: 9, 821-6) is performed on the sequence to be detected.
  • the basic principle is that one probe is hybridized and the two are linked by ligase. Since the two probes cannot be ligated to ⁇ where the target nucleotide sequence does not exist, the presence of the ligation product is an indicator of the target nucleotide sequence.
  • the LCR method also has the same problems as the PCR method because temperature control is required to separate the synthesized complementary strand.
  • the SM method When it is simply expressed as 5 'side or 3' side, it means the direction in the chain with the «symbol.
  • This is called the SM method because the 5 ′ double-stranded portion is replaced by a newly synthesized complementary strand.
  • the SDA method by introducing a restriction enzyme recognition sequence in advance into a sequence annealed as a primer, the temperature change step, which is indispensable in the PCR method, can be omitted.
  • the nick generated by the restriction enzyme gives the 3'-0H group that serves as the starting point for complementary strand synthesis, and the strand synthesized by strand displacement synthesis releases the previously synthesized complementary strand as a single strand. It will be reused as the type II in the next complementary strand synthesis.
  • the SM method eliminates the need for complicated temperature control, which was essential in the PCR method.
  • the method of supplying 3′-0H3 ⁇ 4 by enzymatically removing the RNA portion using a DNA-RNA chimeric oligonucleotide is also ⁇ P.
  • a chimeric oligonucleotide having a 3'-side thigh is used as a primer.
  • the RNA portion of this oligonucleotide is digested by the action of RNAseH, and new 3, -0H groups are continuously added.
  • the oligonucleotide used for the primer must be a DNA-RNA chimeric oligonucleotide.
  • an enzyme for removing RNA is also required, it can be said that it has the same problem as the above-mentioned SDA method.
  • NASBA Nucleic Acid Sequence-based Amplification
  • TMA Transcription Mediated Amplification method
  • NASBA uses a target thigh as a face to perform DNA synthesis using a DNA polymerase with a probe to which a T7 promoter has been added, and further forms a double-stranded DNA with a second probe. This is a reaction system that amplifies a large amount of thighs by performing transcription by the DNA (Nature, 350, 91-92, 1991).
  • NASBA requires several heat denaturation steps to complete double-stranded DNA, but the subsequent transcription reaction with T7 RNA polymerase proceeds isothermally.
  • RCA rolling-circle amplification
  • Combination with polymerase that catalyzes the complementary strand synthesis reaction at the strand position ⁇ S enables ligation and cyclization in the presence of the target base sequence.
  • a continuous complementary strand synthesis reaction with the padlock probe ⁇ M triggered is triggered.
  • a single-stranded nucleic acid having a structure in which a region consisting of the same base sequence is repeated continuously is generated.
  • the single-stranded nucleic acid is further annealed with a primer to synthesize its complementary strand, thereby realizing high-level amplification.
  • the need for multiple enzymes remains an issue.
  • the trigger of complementary strand synthesis depends on the ligation reaction between two adjacent regions, and its specificity is at the same level as LCR in principle.
  • 3′-0H supply a method is known in which the 3 ′ has a sequence complementary to the base sequence on the same strand to form a hairpin loop at the end (Gene 71, 29-). 40, 1988). From such a hairpin loop, complementary strand synthesis is performed with itself as a ⁇ -type, and a single-stranded nucleic acid composed of a complementary base sequence is generated.
  • a structure is realized in which the terminal portion where a complementary base sequence is linked anneals on the same strand.
  • This step is said to proceed depending on a delicate equilibrium state between complementary nucleotide sequences involving base pairing.
  • utilizing the state maintained between base pairing with the complementary strand and base pairing on the same strand only the base sequence on the same strand that anneals with the base sequence becomes the starting point for complementary strand synthesis. . Therefore, it is considered that strict setting of reaction conditions is required to achieve high reaction efficiency.
  • the primer itself forms a loop structure. Therefore, once a primer dimer is generated, the amplification reaction is automatically started regardless of the presence or absence of the target base sequence, and a nonspecific synthetic product is formed. This is a serious problem. Furthermore, the generation of primer dimers and the accompanying consumption of primers by non-specific synthesis reactions lead to a decrease in amplification efficiency of the desired reaction.
  • an oligonucleotide having a hairpin-like structure at the end is often used to supply a double-stranded promoter region (see Japanese Patent Application Laid-Open No. H05-205686). 211873). However, they do not allow a continuous supply of 3'-0H for complementary strand synthesis.
  • a hairpin loop structure in which the 3 ′ end is annealed on the same strand is used for the purpose of obtaining DNA transcribed by RNA polymerase. In this method, amplification is performed by using transcription to RA and reverse transcription from RA to DNA.
  • a reaction system cannot be constructed unless a plurality of enzymes are combined.
  • the present applicant has developed and applied for a patent for a novel method for synthesizing nucleic acids that can be carried out under isothermal conditions and that can maintain a high level of reaction specificity as compared with PCR ( W000 / 28082).
  • This method was named Loop-mediated isothermal amp lification (hereinafter abbreviated as LAMP method) ( ⁇ ⁇ Notomi et al., Nucleic Acid Res., 2000, Vol. 28, No. 12, e63).
  • LAMP method Loop-mediated isothermal amp lification
  • the present applicant has also reported a method for detecting SPs using the LAMP method (The 3rd International Workshop on Advanced Genomics 2000.
  • the LAMP method is characterized in that the nucleic acid amplification reaction is remarkably inhibited when the nucleotide sequence is different from the designed one. Due to this feature, the mutation detection method based on the LAMP method achieved high specificity without sacrificing detection sensitivity. Disclosure of the invention
  • An object of the present invention is to provide a new method for synthesizing a nucleic acid using the reaction principle of the LAMP method.
  • the LAMP method is a method for synthesizing nucleic acids that can achieve high reaction specificity at an isothermal temperature.
  • the present inventor has conducted studies to apply this method and establish a new principle of nucleic acid synthesis. As a result, they found that by applying the LAMP method with a nucleic acid having a specific structure, it was possible to synthesize a nucleic acid on a new principle. Further, based on this reaction principle, they found that various effects, which were not known by the LAMP method, could be achieved, and completed the present invention. That is, the present invention relates to the following nucleic acid synthesizing methods, kits therefor, and uses thereof.
  • a method for synthesizing a nucleic acid containing a target base sequence comprising the following steps.
  • a step of generating at least two types of single-stranded nucleic acids wherein the two types of single-stranded nucleic acids are It has a complementary base sequence at its 3 'end, and has a target:! ⁇ ⁇ Contains a base sequence selected from the sequence.
  • region (b) region (a) a region that forms a loop when hybridized to any region on the same chain, (c) a region containing a nucleotide sequence complementary to the region (a), and
  • step (1) a step of annealing at least two kinds of single-stranded nucleic acids of step (1) at their 3′-ends and synthesizing a complementary strand by a polymerase that catalyzes a complementary strand synthesis reaction involving strand displacement;
  • step (3) Anneal the 3 'of the nucleic acid synthesized in step (2) to a region consisting of a complementary nucleotide sequence on the same strand, synthesize a complementary strand starting from the 3', and synthesize the complementary strand from the target nucleotide sequence.
  • a step of synthesizing a single-stranded nucleic acid
  • At least one kind of single-stranded nucleic acid of the step (1) is a nucleic acid having a structure that forms a 3 ′ without complementary strand by hybridizing the region ( a ) and the region (c) [ 1].
  • At least one kind of the single-stranded nucleic acid in the step (1) is a nucleic acid having a structure forming a 3 ′ end with a complementary strand by hybridization of the region (a) and the region (c).
  • the constituent base sequences are complementary base sequences
  • insert primer is at least one set of insert primers having mutually complementary nucleotide sequences at the 5 'end.
  • the insert primer has a base sequence complementary to each other at the 5 ′ end thereof composed of RNA, and the 3 ′ end composed of an RNase-resistant polynucleotide.
  • the insert primer is a first insert primer serving as a starting point of complementary strand synthesis in an arbitrary region in the target base sequence, and a target base sequence contained in an extension product starting from the insert primer.
  • the method according to [4], wherein the method comprises a combination with a second insert primer which is a starting point of complementary strand synthesis in an arbitrary region therein.
  • the base sequence constituting the single-stranded nucleic acid contains a base sequence selected from the target base sequence
  • the primer having the sequence is annealed, the complementary strand is synthesized by the DNA polymerase which catalyzes the complementary strand synthesis reaction involving strand displacement starting from the 3 ′ end, and the single-stranded nucleic acid of 'A process of substituting the terminus to enable base pairing,
  • step (b) a step of annealing the insert of the product of step (a) with an insert primer and synthesizing a complementary strand starting from its 3 ′ end; wherein the insert primer is attached to an arbitrary region of the product of step (a).
  • a base sequence complementary to the base sequence is provided at the 3 'end, and the base sequence at the 5' end is complementary to the base sequence constituting the 3 'end of any of the two single-stranded nucleic acids in [1]. Consisting of a base sequence, and
  • the complementary strand synthesized in the step (b) is made into a single strand, the 3 'of which is annealed to itself, and the complementary strand is synthesized starting from the 3'5 ⁇ , Step of generating single-stranded nucleic acid
  • step (a) The method according to [9], wherein the nucleic acid containing the target base sequence in step (a) is the product of step (2) in [1].
  • a method for amplifying a nucleic acid containing a target base sequence comprising the following steps.
  • a step of generating at least two types of single-stranded nucleic acids wherein the two types of single-stranded nucleic acids are It has a complementary base sequence at its 3 'end and contains a base sequence selected from a target base sequence.
  • region (b) region (a) a region that forms a loop when hybridized to any region on the same chain,
  • step (2) a step of annealing at least two kinds of single-stranded nucleic acids in step (1) at their 3'-ends and synthesizing a complementary strand by a polymerase that catalyzes a complementary strand synthesis reaction involving strand displacement;
  • step (3) Annealing the 3 'of the nucleic acid synthesized in step (2) into a region consisting of a complementary nucleotide sequence on the same strand, synthesizing a complementary strand starting from the 3', and A step of synthesizing a single-stranded nucleic acid,
  • step (3) Using the product of step (3) as type I, complementary strand with strand displacement starting from the 3 'end of the inner primer and Z or insert primer Complementary strand by DNA polymerase that catalyzes the synthesis reaction.
  • step (3) complementary strand with strand displacement starting from the 3 'end of the inner primer and Z or insert primer Complementary strand by DNA polymerase that catalyzes the synthesis reaction.
  • a method for amplifying a nucleic acid containing a target base sequence comprising:
  • a method for synthesizing a nucleic acid containing a target base sequence comprising a step of incubating the following elements:
  • Inner primer F wherein inner primer F anneals to a region defining the 3 ′ side of one of the strands constituting the target base sequence at the 3 ′ end, and inner primer F 5 ′ * 3 ⁇ 4 has a base sequence complementary to any region of the complementary strand synthesis reaction product starting from this primer,
  • Inner primer R wherein the inner primer R anneals to a region defining the 3 ′ side of one of the strands constituting the target base sequence at the 3 ′ end, and the inner primer R 5'-dan of one R has a base sequence complementary to an arbitrary region of a complementary strand synthesis reaction product starting from this primer,
  • insert primer F has a base sequence complementary to an arbitrary region of one of the strands constituting the target base sequence at its 3 ′ end.
  • insert primer R has a base sequence complementary to an arbitrary region of the other strand constituting the target base sequence at its 3 ′ end, and
  • the synthetic product originating from each primer of insert primer F and insert primer R has a nucleotide sequence complementary to each other in the region containing 5% of both primers.
  • outer primer F is a starting point of a complementary strand synthesis reaction starting from the 3 ′ side of the region to be identified by the inner primer F in the present invention
  • outer primer R is a starting point of a complementary strand synthesis reaction starting from the 3 ′ side of the region of the key where the inner primer R should be annealed
  • Loop primer F The region containing the 3 'end of the loop primer F is the region containing the 5' end of the inner primer F, and the region containing the 5 'end of the inner primer F is any of the complementary strand synthesis reaction products starting from the inner primer F. Anneal to any region in the loop formed by hybridizing to that region, and
  • Loop primer R the region containing 3% of the loop primer R is the region containing the 5 'end of the inner primer R is any region of the complementary strand synthesis reaction product starting from the inner primer R. Anneal any region in the loop formed by hybridizing to the region
  • the target base sequence in the type I nucleic acid is double-stranded, and each primer should be annealed by incubating under conditions that allow complementary strand synthesis starting from each of the primers.
  • the method according to [17] including a step of bringing the region into a state capable of base pairing.
  • a method for detecting a mutation in a type I nucleic acid based on the method for detecting a nucleic acid according to [24], wherein the method for synthesizing a nucleic acid according to [1] or [17] comprises: A method for detecting a mutation, characterized in that a complementary strand synthesis reaction starting from the 3 'end of a main-strand nucleic acid is prevented when the base sequence of a type III nucleic acid is not a predicted base sequence.
  • a kit comprising the following elements for synthesizing a nucleic acid containing a target base sequence:
  • Inner primer F wherein inner primer F is located at the 3 'end with respect to a region defining the 3' side of one strand constituting the target base sequence.
  • the primer has a nucleotide sequence complementary to an arbitrary region of the complementary strand synthesis reaction product starting from this primer on the 5 ′ side of the inner primer F.
  • Inner primer R wherein the inner primer R is used at its 3 ′ end to a region defining the 3 ′ side of one of the strands constituting the target base sequence, and the inner primer R 5 'side of, to have a nucleotide sequence complementary to an arbitrary region of a complementary strand synthesis reaction product that this primer first and origin,
  • insert primer F has a base sequence complementary to an arbitrary region of one of the strands constituting the target base sequence at its 3 ′ end.
  • an insert primer R wherein the insert primer R has a base sequence complementary to an arbitrary region of the other strand constituting the target base sequence at its 3 ′ end, A synthetic product starting from each of the primer F and the insert primer R has a nucleotide sequence complementary to a region containing both of them.
  • outer primer F is a starting point for a complementary strand synthesis reaction starting from the 3 ′ side of the region to be identified by the inner primer F in
  • Loop primer R the region containing the 3 'portion of the loop primer R is a region containing the 5' end of the inner primer: R is located in any region of the complementary strand synthesis reaction product starting from the inner primer R.
  • the insert primer had a nucleotide sequence different from the expected nucleotide sequence, the complement was synthesized with the 3 'end of the insert primer and the 5' end of Z or the insert primer as ⁇ .
  • the 3 ′ of the inner primer F and the insert primer F serve as a starting point for synthesizing a base sequence derived from the first type I nucleic acid, and the inner primer R and the insert primer R It is a starting point for synthesizing a nucleotide sequence derived from a nucleic acid, and consists of nucleotide sequences complementary to each other in the region including the 5 'end of the force primer F and the insert primer R.
  • the kit according to [37] which comprises a region.
  • nucleic acid in the present invention, can be DNA or RNA, or a chimeric molecule thereof. Nucleic acids can be natural or artificially synthesized. Further, even a nucleotide derivative partially or wholly composed entirely of an artificial structure is included in the nucleic acid of the present invention as long as it can form a base pair bond. Examples of such a molecule include a polynucleotide derivative whose backbone is formed by a phosphotie bond.
  • the number of constituent bases of the nucleic acid in the present invention is not limited. Nucleic acid is synonymous with the term polynucleotide.
  • the term “oligonucleotide” in the present invention is used as a term indicating a polynucleotide having a particularly small number of constituent bases among polynucleotides. Oligonucleotides generally refer to oligonucleotides having a base number of 2 to 100, more generally about 2 to 50, and are referred to as oligonucleotides, but are not limited to these numerical values.
  • the nucleic acids of the invention are generally included in a biological sample.
  • Biological samples can refer to animal, plant, or microbial tissues, cells, cultures, excreta, or extracts thereof.
  • the biological sample of the invention includes genomic DNA or RNA from intracellular parasites such as viruses and mycoplasmas.
  • the nucleic acid of the present invention may be derived from the nucleic acid contained in the biological sample. For example, cDNA synthesized based on mRNA and nucleic acid amplified based on nucleic acid derived from a biological sample are typical examples of the nucleic acid in the present invention.
  • a target nucleotide sequence is a nucleotide sequence of a nucleic acid to be synthesized.
  • the base sequence constituting the nucleic acid to be synthesized in the present invention is the target base sequence.
  • the base sequence constituting the nucleic acid to be amplified is the target base sequence.
  • the base sequence of a nucleic acid describes the base sequence of the sense strand from the 5 'side to the 3' side.
  • the target base sequence in the present invention includes, in addition to the base sequence of the sense strand, its complementary strand, that is, the base sequence of the antisense strand. That is, the term “target base sequence” is used as a term meaning at least one of a base sequence to be synthesized and its complementary strand.
  • the target base sequence is not limited to the base sequence of the nucleic acid used as type III. Therefore, the target nucleotide sequence may be composed of the same nucleotide sequence as that of the recording form, or may be a different nucleotide sequence. It is also possible to introduce a mutation into the base sequence or synthesize a target base sequence consisting of a base sequence obtained by joining a part of the base sequences.
  • terminal or 5 refers to a region located not only at one terminal of either terminal but also at one terminal and containing one base. More specifically, 500 bases, desirably 100 bases, or at least 20 bases from either end are included at the 3, terminal or 5 'end. On the other hand, in order to indicate a base at a specific position that covers one or more bases at the end, the position is specified by specifying a numerical value.
  • the 3 ′ is the starting point of the complementary strand synthesis, it means that the ⁇ 0H group at the 3 ′ end is the starting point of the complementary strand synthesis.
  • the term «And complementary strand: The term « used in the present invention means a nucleic acid on the side that is the type of complementary strand synthesis.
  • a complementary strand having a base sequence complementary to « has a meaning as a strand corresponding to, but the relationship between the two is only a relative one. That is, a chain synthesized as a complementary chain can again function as a chain. That is, the complementary strand can be ⁇ .
  • the synthesis of a nucleic acid having a base sequence different from that of a nucleic acid may be aimed at by synthesizing a base sequence contained in a nucleic acid as a target base sequence as it is.
  • a nucleic acid having a nucleotide sequence different from that of a nucleic acid includes, for example, introducing a mutation into the nucleotide sequence contained in the nucleic acid, or synthesizing a region that is separated on ⁇ S nucleic acid as a continuous nucleotide sequence.
  • the target base sequence in the present invention can be a base sequence obtained by linking base sequences derived from different nucleic acids.
  • Synthesis and amplification of nucleic acid means elongation of nucleic acid from an oligonucleotide which is a starting point of synthesis. In addition to synthesis, when the generation of another nucleic acid and the elongation reaction of the generated nucleic acid occur continuously, a series of reactions is collectively referred to as amplification.
  • Anil “Anil” and “hybridize” mean that nucleic acids form a double helix structure by base pairing based on the Petson-Crick model. Therefore, even if the nucleic acid strand constituting the base pair bond is a single strand, if the complementary base sequence in the molecule forms a base pair bond, it is anneal or hybridized. In this effort, anonymous and hybridized are synonymous in that they form a double helix structure by nucleic acid; ⁇ base pairing.
  • the «paired 3 '» is the starting point for complementary strand synthesis, it is sometimes referred to as an ayur. However, this does not deny that hybridis is the starting point for complementary strand synthesis.
  • nucleotide sequences that make up the primers used in the present invention include nucleotide sequences that are completely identical or not completely complementary. It is. That is, the same as a certain sequence can include a sequence complementary to a base sequence capable of annealing to a certain sequence.
  • complementary refers to sequences that can be annealed under stringent conditions and provide a starting point for complementary strand synthesis.
  • the term “identical” means that the homology of the nucleotide sequence is, for example, 90% or more, It means 5% or more, more preferably 98% or more.
  • the term “complementary” means the same nucleotide sequence as the complementary sequence. That is, it can be said to be complementary when the homology of the base sequence to the complementary sequence is, for example, 90% or more, usually 95% or more, and more preferably 98% or more. In addition, it is preferable that at least one base at the 3 ′ end of the complementary base sequence completely coincides with the complementary sequence when it functions as a starting point of complementary strand synthesis.
  • the homology of the nucleotide sequences can be determined by a known search algorithm such as BLAST.
  • Complementary strand synthesis reaction involving strand displacement In the synthesis of the nucleic acid of the present invention, a polymerase chain catalyzing a complementary strand synthesis reaction involving strand displacement is used.
  • the complementary chain synthesis reaction involving strand displacement refers to the following reaction.
  • the complementary strand synthesis is performed while separating the polynucleotide from The reaction that proceeds is called a complementary strand synthesis reaction involving strand displacement.
  • the separated polynucleotide usually maintains its phosphodiester bond. Therefore, a polynucleotide having a length corresponding to the length of the complementary strand synthesis force S and capable of base pairing is produced.
  • the same one as the DNA polymerase used in SM and the like is used. That is, when a complementary strand is synthesized using a primer complementary to the third and third sides of a certain base sequence as a synthesis starting point, if there is a double-stranded region on the 5 ′ side, the complementary strand is synthesized while displacing the two strands. Specialized polymerases that carry out the above are known. In the present invention, a substrate necessary for complementary strand synthesis is further added.
  • the target base is simply obtained.
  • the reaction can be started simply by incubating the nucleic acid containing the sequence with the primer.
  • the nucleic acid to be type II may remain double-stranded.
  • the present invention utilizes at least two types of single-stranded nucleic acids including the following regions (a) to (d) from the 5 ′ side to the 3 ′ side, Is synthesized.
  • the two types of single-stranded nucleic acids contain a complementary base sequence at their 3, terminal and a base sequence selected from a target base sequence.
  • region (b) region (a) a region that forms a loop when hybridized to any region on the same chain,
  • the at least two types of single-stranded nucleic acids are formed by forming a continuous base sequence that constitutes one of two strands of a nucleic acid chain consisting of a set of complementary base sequences that constitute a target base sequence. Including.
  • both have nucleotide sequences complementary to each other in the 3′-terminal region (d).
  • Each of these nucleic acids has a region (a) consisting of a base sequence complementary to an arbitrary region of the same chain at its 5 '.
  • the arbitrary region of the same chain may be an arbitrary region (c) of the nucleic acid.
  • the region (a) forms a loop by hybridizing to the region (c).
  • An area corresponding to the loop formed at this time is defined as (b).
  • the above relationship can be shown, for example, as follows. (a) (b) (c) (d)
  • each nucleic acid region 1 / the same symbols (a) to (d) are given to each nucleic acid region 1 /, but the nucleotide sequences are not required to be identical. Rather, these regions usually have different base sequences. Furthermore, regions (a) and (d) of each nucleic acid must be located at the 5 'end or 3' end, while regions (b) and (c) are regions (a) and (d) ) Can be placed anywhere. Further, for the region (c), another base sequence is allowed to intervene with the region (d).
  • Desirable positional relationship is such that the number of bases constituting region (a) and region (d) is 10 to 500 bases, usually 10 to 100 bases, and preferably 20 to 50 bases.
  • the salt fiber constituting the region (c) has the number of bases corresponding to the region (a).
  • the size of the loop region (b) formed by hybridization between the region (c) and the region (a) is designed to be 1 to 200 bases, preferably 10 to 100 bases. Since the area (b) is an area sandwiched between the area (a) and the area (c), it is uniquely determined by defining these areas.
  • the region (c) exists adjacent to the region (b) and is complementary to the region (a);
  • the present invention uses a nucleic acid having a long base sequence inserted between (c) and (d). Can be carried out.
  • the target base sequence is a region from one region (a) to the other region (a) of the single-stranded nucleic acid.
  • the length of the target base sequence is determined by the number of bases in each region and the region interposed therebetween.
  • the length of the target base sequence in the present invention is Not restricted. Therefore, the length can be, for example, 30 to 500 bases, usually 50 to 1000 bases, preferably 500 bases or less, and more preferably 400 bases.
  • the nucleotide sequence of the region intervening between the normal region (c) and the region (d) is lengthened.
  • a target base sequence consisting of a long base sequence can be obtained.
  • the number of bases between F 2 ZR 2 is, for example, 90 to 240 bases (the number of bases in the region between F 1 and R 1 is 10 to 40, (Corresponding to a base group).
  • a longer target base sequence can be accurately synthesized or amplified.
  • the entire target base sequence can be constituted by combining the base sequences constituting the single-stranded nucleic acid. More specifically, one of the single-stranded nucleic acids is apparently acted on by the other nucleic acid. As a result of complementary strand synthesis, the base sequence of one nucleic acid constituting the target base sequence can be completed. .
  • the ratio of each single strand to the target base sequence is arbitrary. In order to synthesize a longer nucleotide sequence, it is usually reasonable to use a length of about 1 Z2 with respect to the target nucleotide sequence.
  • the region (c) can be arranged independently or can be arranged so as to overlap with the region (d).
  • Region (c) is arranged so as to overlap with region (d): ⁇ means that the single-stranded nucleic acid has a 5 ′ terminal nucleotide sequence and a 3 ′ nucleotide sequence in a loop region (b )) To form a stem-loop structure.
  • Such a structure is shown as 3 or 4 in FIG.
  • Stem norape refers to a structure consisting of a loop and a double-stranded stem.
  • the 3'-side nucleotide sequence and the 5'-side nucleotide sequence contained in the same molecule are composed of complementary nucleotide sequences, and when they hybridize, a stem is formed. At this time, when the base sequence on the 5 ′ side is completely complementary to the base sequence on the 3 side, the 3 ′ end becomes a double strand with a complementary strand.
  • the single-stranded nucleic acid can be synthesized enzymatically, for example, as follows. First, using four types of primers (insert primer X2 and inner primer X2) having the following characteristics, a DNA polymerase that catalyzes complementary strand synthesis involving strand displacement based on To form a complementary strand.
  • the insert primer and the inner primer will be specifically described below.
  • the insert primer is a primer that contains a target base sequence and anneals to a nucleic acid to serve as a starting point for complementary strand synthesis.
  • the inner primer is annealed to a nucleic acid synthesized by this primer.
  • a synthetic product starting from each of the insert primer F and the insert primer R has a base sequence complementary to each other in a region including the 5 'end of both.
  • the insert primer is a primer unique to the present invention, which has been devised for enzymatically synthesizing a single-stranded nucleic acid composed of the regions (a) to (d), and has novel characteristics. .
  • the insert primer is designed such that its 5 ′ overlaps with a part of the base sequence constituting the nucleic acid to be used.
  • the inner primer FIP anneals to the product of one of the insert primers R to synthesize a complementary strand.
  • the inner primer RIP anneals to the product of the other insert primer F to synthesize a complementary strand.
  • the extension products of the insert primer F and the insert primer-R have a positional relationship such that an overlapping region is synthesized.
  • the base sequence of the region including the 5 'of the insert primer can be any base sequence.
  • the primers may be used to initiate complementary strand synthesis. Therefore, in order for the 3 'ends of both to overlap, it is desirable to set the length of the overlapping portion to a number of bases that cannot be annealed under the reaction conditions.
  • the base sequence of the insert primer By selecting the base sequence of the insert primer, it is possible to synthesize a region located at a distance in the nucleic acid as a continuous base sequence. That is, in a nucleic acid, when the region A and the region B are located across the region S, a target base sequence composed of the region A and the region B can be synthesized.
  • the base sequence on the 3 'side of the insert primer should be a base sequence complementary to the boundary region of region A with region S, and similarly to the boundary region of region B with region S.
  • the 5 'side is designed so that both base sequences are complementary.
  • the base sequences of the regions A and B can be shared on the 5 'side in order to match the amino acid translation frames. That is, for example, when the 3 ′ side is composed of a base sequence complementary to the region A, the base sequence of the region B is added to the 5 ′ side. However, if the shared base sequence is long, a primer-dimer may be formed. Therefore, in order to match the translation frames, an artificial linker sequence can be intervened.
  • a nucleic acid synthesized by such an insert primer has a base sequence in which an arbitrary intermediate portion of a continuous base sequence is deleted. That is, a deletion mutant of the nucleic acid can be obtained.
  • FIG. 9 shows the relationship between the target base sequence and the target base sequence at this time. That is, a mutant (lower in FIG. 9) consisting of a nucleotide sequence in which the portion indicated by “deletion” in (, upper in FIG. 9) has been deleted can be synthesized according to the present invention.
  • One of the functional analyzes of a protein encoded by a gene is to identify the active domain of the protein.
  • a method of expressing a mutant in which a part of a gene is deleted and estimating an active domain based on the relationship between the deleted region and the activity is generally used.
  • PCR is often applied to obtain deletion mutants.
  • the target region can be easily obtained by performing PCR using primers designed for continuous regions of the gene.
  • primers can be designed only at both ends of the nucleic acid to be synthesized. Therefore, it is difficult to synthesize a mutant lacking the intermediate portion in one step.
  • an arbitrary region can be deleted, and a region that exists apart in the and can be synthesized as one continuous base sequence.
  • the features of the present invention are also useful in genome analysis.
  • genes are present in introns.
  • RNA transcribed from the genome is spliced to remove introns, resulting in exon-linked mRNA.
  • Exons and introns in the genome are finding certain laws However, at least at present, the accuracy of the prediction is not sufficient. Therefore, although the structure of the genome has been elucidated, the analysis of the genes contained therein is still a major research topic.
  • a nucleic acid in which a plurality of regions of a genome are linked can be synthesized in one step. That is, it is possible to synthesize a nucleic acid in which the predicted exons are linked. Further, the obtained nucleic acid can be further ligated as necessary.
  • the usefulness of the present invention is apparent from the fact that only one exon can be synthesized in one step by the PCR method.
  • the method for synthesizing a nucleic acid of the present invention can also be used for analyzing a transcription regulatory region.
  • the present invention is useful for searching for a region having promoter activity in the genome.
  • a promoter is a region located upstream of a gene and recognized by a transcription factor of the gene.
  • the search for a region having promoter activity using the present invention can be performed, for example, as follows.
  • an expression cassette comprising a base sequence of a genome to be analyzed and a reporter gene connected downstream thereof is synthesized according to the present invention. That is, it is only necessary to design the inner primer and the insert primer of the present invention for each of the genome and the reporter gene, and to carry out a method for synthesizing nucleic acids having different nucleic acids as templates.
  • primers for various regions in the genome region to be analyzed various types of expression cassettes can be synthesized.
  • an arbitrary region can be freely synthesized as an expression cassette by designing a primer.
  • the resulting expression cassette is actually expressed in an appropriate host, and the promoter activity can be evaluated by observing the signal of the reporter gene.
  • the method for synthesizing nucleic acids of the present invention is useful for synthesizing nucleic acids in which distant regions contained in the same type II are linked. If this principle is further developed, according to the present invention, it is also possible to synthesize a nucleic acid in which two regions included in different regions are linked. That is, the reaction can be started using nucleic acids having different base sequences derived from nucleic acids as the two types of single-stranded nucleic acids in the nucleic acid synthesis method of the present invention.
  • the resulting nucleic acid is a nucleic acid in which base sequences derived from different key nucleic acids are linked on the 5 ′ and 3 ′ sides.
  • a single-stranded nucleic acid containing a base sequence derived from a different type III nucleic acid can be obtained, for example, by the following method. Insert primers and inner primers designed to act as primers for different nucleic acids are used as primers for use in the nucleic acid synthesis method of the present invention.
  • an inner primer F and an insert primer F are designed for the ⁇ M nucleic acid F.
  • a base sequence that acts as a primer for synthesizing a region intended for the synthesis of nucleic acid F is arranged.
  • a base sequence complementary to an arbitrary region of a complementary strand synthesized starting from the 3 ′ side is arranged.
  • an inner primer R and an insert primer R are designed.
  • a base sequence that acts as a primer for synthesizing a region intended for the synthesis of nucleic acid R is arranged.
  • a base sequence complementary to an arbitrary region of the complementary strand synthesized starting from the 3' side is arranged.
  • insert primer F and insert primer It consists of a unique base sequence.
  • the base sequence on the 5 ′ side of the insert primer is designed so that the genes can be linked in frame.
  • the method for synthesizing or amplifying the nucleic acid of the present invention can be carried out under the same conditions, except that the design of such primers and the use of different nucleic acids are different. That is, mix two types of nucleic acid type II, all primers, and DNA polymerase, substrate, buffer, etc. necessary for the reaction, and incubate under appropriate conditions.
  • a complementary primer synthesis reaction may be carried out by adding a corresponding primer to a different type I nucleic acid to generate a single-stranded nucleic acid, and then the two may be mixed, and then the complementary strand synthesis reaction may be continued. it can.
  • the target base sequence can be obtained by collecting the target base sequence or a reaction product having a size larger than the target base sequence from the reaction product.
  • the recovered nucleic acid can be digested with a restriction enzyme and purified if necessary.
  • the method for obtaining a nucleic acid containing a base sequence of a different nucleic acid based on the nucleic acid synthesis method or the amplification method according to the present invention is useful, for example, in the following application fields.
  • a gene encoding a fusion protein can be synthesized according to the present invention. It has been difficult to synthesize large-sized genes simultaneously using known methods. For example, in the method, an artificial base sequence could be added to the end of the target base sequence as long as it could be synthesized as a primer. However, the length of the base sequence that can be added by this method was limited. Therefore, small proteins such as histidine tags cannot be added. On the other hand, in the present invention, since a gene encoding a protein to be added can also be synthesized by a complementary strand synthesis reaction, a gene of any length can be fused in principle.
  • a vector containing the required nucleotide sequence To add a long nucleotide sequence that is difficult to add as a primer, prepare a vector containing the required nucleotide sequence in advance and fuse it with this vector. A method of inserting a gene has been used. However, in this method, a vector containing a fusion partner must be prepared in advance to produce a fusion protein. On the other hand, in the present invention, a target region can be freely selected and fused from different ⁇ M nucleic acids.
  • a mutation or an additional nucleotide sequence can be introduced into the insert primer used in the present invention.
  • the insert primer is required to have a specific condition of base sequence in 3 and 4.
  • various nucleic acids generated by the complementary strand synthesis reaction using the base sequence of the insert primer as a face have various base sequences complementary to each other at the 5 ′ end. Can be arranged freely.
  • the introduction of a mutation or the addition of a base sequence is allowed in the middle of the insert primer.
  • nucleotide sequence obtained by adding a mutation or a nucleotide sequence to the nucleotide sequence contained in. Since the insert primer can be set at any position in the target base sequence, according to the present invention, any mutation can be introduced at any position. Mutations can be substitutions, deletions, or additions of bases. Any of these mutations can be introduced by the present invention.
  • nucleic acid synthesis methods such as PCR
  • PCR a method that can introduce an arbitrary mutation at an arbitrary position in a target base sequence as in the present invention is useful.
  • multiple ML insert primers can be used at the same time.
  • ⁇ M Mutual substitution of nucleic acids occurs, and faster complementary strand synthesis can be expected.
  • the base sequence constituting the 3 'side of the insert primer be set so that the type II nucleic acid can be used as a double-stranded nucleic acid as a starting point for complementary strand synthesis.
  • the primer can function as a primer under the same conditions as in the complementary strand synthesis reaction using the inner primer and the outer primer. Specifically, similarly to the above-mentioned region (d) and the like, it is 5-200 bases, more preferably 10-50 bases.
  • the region constituting the 5 ′ side of the insert primer provides a base sequence necessary for the complementary chain synthesized using this region to be MM, so that its 3 end is the starting point of complementary chain synthesis. Therefore, as in the case of the base sequence constituting the 3 'side, it is 5 to 200 bases, more preferably 10 to 50 salts.
  • the 5, 3 ⁇ 4 base sequence constitutes a region (a) containing 5, «of the single-stranded nucleic acid.
  • a different inner primer is used for each of the two strands constituting the target base sequence. Usually they are called forward side and reparse side.
  • the inner primer is a primer used in the known LAMP method. However, in the known LAMP method, the reaction is basically composed of an inner primer. There is currently no report on the combination of the insert primer and the inner primer.
  • nucleic acid synthesis method of the present invention it is important to first generate a single-stranded nucleic acid consisting of the regions (a) to (d).
  • a nucleic acid can be provided with the structure by a nucleic acid synthesis reaction based on the present invention using the insert primer and an inner primer having the following structure. The details of this reaction will be described later.
  • the inner primer used for the nucleic acid synthesis reaction of the present invention is composed of at least the two regions X2 and X1c.
  • X2 is a region including 3, male
  • X1c is a region including 5, terminal.
  • the structure of the inner primer in the present invention is determined by the target base sequence. At least a part of the target nucleotide sequence is known or can be estimated. The portions whose nucleotide sequences are to be clarified are the region X2c and the region X1c located on the fifth side thereof. These two regions can be assumed to be continuous ⁇ ⁇ and separated ⁇ . The relative position of the two determines the state of the loop formed when the product nucleic acid self-anneals.
  • the distance between the two is not unnecessarily large. Therefore, it is desirable that the positional relationship between the two is usually continuous over a distance of 0 to 500 bases.
  • the later-described loop formation by self-anneal it is expected that there is a case where it is disadvantageous to form a loop in a desirable state if the user is too close.
  • the loop is required to have a structure that can smoothly initiate the annealing of a new oligonucleotide and a complementary strand synthesis reaction involving strand displacement starting from the new strand.
  • the region X 2c and its 5 ′ The distance from the region X1c located on the side is designed to be 0 to 100 bases, more preferably 10 to 70 bases. This value indicates the length not including XIc and X2.
  • the number of bases constituting the loop portion is a length obtained by further adding a region corresponding to X2.
  • the regions X 2 and XIc constituting the inner primer of the present invention with respect to the target base sequence are usually arranged continuously without overlapping. Alternatively, if both base sequences have a common part, they can be partially arranged so as to overlap. Since X2 must function as a primer, it must always be at the 3 'end.
  • X 1c is located at 5 ′ because it is necessary to provide a function as a primer to the 3, 3 terminal of the complementary strand synthesized as described above.
  • the complementary strand obtained using this oligonucleotide 'as the synthesis starting point will be the one for synthesis of the complementary strand from the opposite direction, and finally the inner primer portion according to the present invention will be transferred to the complementary strand as Can be 3.
  • the end generated by copying has the base sequence X1 and anneals to X1c on the same strand to form a loop.
  • the inner primer in the present invention satisfies the two conditions of being capable of forming a complementary base pair bond with the target base sequence, and of giving the -0H group which is the starting point of complementary strand synthesis in (3) above.
  • the pack component is not necessarily limited to a phosphodiester bond.
  • it may be composed of a phosphothioate form.
  • the base may be any one that enables complementary base pairing. In its natural state, it can also be an analog, such as bromodeoxyuridine, which is generally one of the five types of ACTG and U. It is desirable that the oligonucleotide used in the present invention not only serves as a starting point of synthesis but also functions as a source of complementary strand synthesis.
  • the inner primer of the present invention is a strand that is capable of performing base pairing with a complementary strand while maintaining necessary specificity in a given environment in various nucleic acid synthesis reactions described below.
  • an excessively long base sequence is difficult to prepare by chemical synthesis, and the above-mentioned chain length is exemplified as a desirable range.
  • the chain length exemplified here is the chain length of the portion that anneals to the complementary chain.
  • the inner primer according to the invention consists of at least two regions X2 and XIc. Therefore, the chain length exemplified here should be understood as the chain length of each region constituting the in-primer. .
  • FIG. 1 and FIG. show a method for synthesizing a nucleic acid containing a target base sequence, including a nucleic acid containing a target base sequence (between F 2 ZR 2c).
  • the primers indicated by fc in the figure are as follows.
  • a base sequence complementary to an arbitrary region in the target base sequence is arranged at the 3 ′ end of the insert primer F and the insert primer R shown in the figure, and the base sequence is complemented by annealing to a nucleic acid to be It is the starting point for chain synthesis.
  • each instance The 5 'side of the primer is designed so as to have mutually complementary nucleotide sequences.
  • the two insert primers are designed so that the 5 ′ ⁇ s of the two primers overlap each other in an arbitrary region in the two strands constituting the target base sequence.
  • the outer primer F 3 or R 3 is composed of a nucleotide sequence complementary to an arbitrary region F 3c or R 3c 5 ′ from the 5 ′ end of the target nucleotide sequence.
  • the outer primer is intended to provide a starting point for a complementary strand synthesis reaction for displacement of a synthesized strand starting from the inner primer / insert primer.
  • the extension product of the insert primer-R is annealed with the inner primer-FIP to synthesize a complementary strand.
  • the synthesized phase capture strand is displaced by the outer primer F3 and released as a single-stranded nucleic acid (1).
  • This (1) is nothing but one of the nucleic acids having the regions (a) to (d) in the present invention.
  • nucleic acid (1) and nucleic acid (2) hybridize at the 5 'end with the region containing their complementary base sequence, but the region containing the 3' end is single-stranded and Has a complementary nucleotide sequence. Therefore, the region containing the 3 'end of both can hybridize.
  • the nucleic acid (1) and the nucleic acid (2) have a configuration in which the base sequence constituting the target base sequence includes the base sequence on the 5 'side thereof but lacks the base sequence on the 3 side. .
  • the 5 'base sequence of the target base sequence constituting each chain is nothing but a base sequence complementary to the 3' base sequence lacking in the other chain. Therefore, if the complementary strands are synthesized in the form of ⁇ starting from the 3 ′ of these nucleic acids, the base sequence on the 3 ′ ⁇ side of each strand that is lacking with respect to the target base sequence is synthesized. Thus, the target base sequence is completed. Thus, in the present invention, a nucleic acid comprising the target base sequence is synthesized.
  • the nucleic acid where the insert primer anneals is depicted as a single strand.
  • a denaturation step for making the nucleic acid to become single-stranded is not necessarily required.
  • Most of the complementary strand synthesis reaction constituting the present invention is performed by a DNA polymerase that catalyzes a complementary strand synthesis reaction involving strand displacement. When this type of DNA polymerase is used, it is possible to synthesize a complementary strand using a double-stranded nucleic acid under appropriate conditions.
  • the present inventors have clarified the conditions under which a double-stranded nucleic acid can be used as a single-stranded nucleic acid without denaturation. In other words, they found that the conditions under which primer-annealing and double-stranded M-dependent complementary strand synthesis for double-stranded nucleic acid can be set without denaturation step, given the conditions under which the double strand is destabilized to some extent. Also in the present invention, this condition is used for IJ, and a double-stranded nucleic acid can be used as ⁇ as it is.
  • a single-stranded nucleic acid is frequently used as a, and the temperature is lower than the temperature at which the nucleic acid is denatured into a single-stranded nucleic acid. What is necessary is just to set to the temperature which can react.
  • the temperature required for the reaction can be adjusted with a melting temperature regulator (hereinafter, the melting temperature is abbreviated as Tm).
  • Tm melting temperature regulator
  • nucleic acid containing the target base sequence which is generated based on the combination of the nucleic acid (1) and the nucleic acid (2).
  • FIG. 3 as a nucleic acid generated by such a combination, first, a product in which nucleic acid (1) and nucleic acid (2) are extended in the 3 ′ direction will be described.
  • the nucleic acid (1) and the nucleic acid (2) have a base sequence complementary to an arbitrary region of the nucleic acid at the 5 ′ end. Therefore phase
  • the nucleic acid produced as a result of complement synthesis has a base sequence complementary to itself at the 3 'end. As a result, if the region containing the 3 'becomes a single-stranded structure, the 3' end is annealed to itself, and the synthesis of the complementary chain is started.
  • This step is shown in FIG.
  • the double-stranded nucleic acid generated from the nucleic acid (1) and the nucleic acid (2) is further annealed by the insert primer to start phase-trapping synthesis, so that the region containing the 3 ′ is a single-stranded region. It is said.
  • the 3 'end anneals to itself and initiates complementary strand synthesis with itself, generating a nucleic acid with the target base sequence linked through the loop (bottom two products in Figure 3).
  • the lower two products have complementary base sequences at their 3 'and 5', and are ready for annealing of the inner primer in the loop.
  • this nucleic acid has the structure necessary to start the LAMP method. Therefore, once such a structure is generated, a nucleic acid synthesis reaction based on the known principle of the LAMP method is started. That is, the following three reactions are repeated continuously and forcelessly in theory. The result is a high degree of nucleic acid amplification.
  • FIG. 3 also shows that nucleic acid (3) and nucleic acid (4) are produced as products. These nucleic acids are generated by replacing the nucleic acid synthesized with the insert primer as a starting point with the complementary strand synthesis proceeding by allowing the above 3 to be self-assembled. These nucleic acids also contribute to the reaction for synthesizing the nucleic acid of the present invention as a material for initiating a reaction as described later.
  • a nucleic acid having a single-stranded structure originally on the 3 ′ side was used to initiate the reaction.
  • the method for synthesizing a nucleic acid of the present invention is not limited to the case where the region containing 3 ′ as a single-stranded nucleic acid is single-stranded.
  • the 3 'end Even in the case of having a double-stranded structure with a complementary strand, it is possible to initiate the synthesis of the 3'-anneal and the accompanying target base sequence.
  • the nucleic acid (3) and the nucleic acid (4) generated by the above-described reaction have complementary 5'-terminal complementary terminals. It forms a double-stranded structure with the region consisting of the base sequence.
  • Such a structure can be annealed to other nucleic acids by some means if its 3 'is made into a single strand.
  • the 3′ ⁇ S of the inner primer FIP used in the figure is composed of F2c. Since F2c is a nucleotide sequence complementary to F2, it anneals to F2c present in the loop of nucleic acid (3) and provides a starting point for complementary strand synthesis. The complementary strand synthesis initiated from within the loop proceeds in the 5 ′ direction of the nucleic acid (3), which is at the 5 ′ end. At this time, the 3 'end, which had originally hybridized to 5, 5 of the nucleic acid (3), is replaced with the synthesis of a new complementary strand, and becomes a single-stranded state. Similarly, RIP anneals to the loop portion of nucleic acid (4) and opens its 3 'end as a single strand.
  • the nucleic acid (3) and the nucleic acid (4) become single-stranded, and the nucleic acid (3) and the nucleic acid (3) are combined, and a complementary strand synthesis reaction in which the nucleic acids (1) and (2) are separated from each other is started in the same manner as the reaction using the nucleic acid (1) and the nucleic acid (2). Then, the base sequence on the third side is obtained, and the synthesis of the target base sequence is completed.
  • the single-stranded nucleic acid of the present invention can function as a starting point for complementary strand synthesis without having to positively change the 3 ′ to a single-stranded nucleic acid.
  • a double-stranded nucleic acid can be used as a nucleic acid without undergoing a denaturation step depending on conditions.
  • the nucleic acid (3) and the nucleic acid (4) can be annealed at their 3 ′ ends.
  • the reaction between the nucleic acid (3) and the nucleic acid (4) can be initiated by using the RNA-DNA chimeric primer.
  • the nucleic acid (3) and the nucleic acid (4) are composed of RNA on the 5th side, and when a complementary strand is involved, the portion composed of RA can be decomposed. .
  • Nucleic acid (3) and nucleic acid (4) have a structure in which the 3 side is hybridized to its own 5 'end region. Therefore, if the region constituting the 5 ′ end is removed, the 3 ′ side is in a state where base pairing is possible. Since the 3 'ends of both are composed of mutually complementary base sequences, the two are annealed to initiate a complementary strand synthesis reaction with each other as.
  • the RNA can be eliminated from the RNA / DNA hybrid using an enzyme such as RNAseH.
  • a primer whose 5′-containing region is composed of RNA is used as a primer for synthesizing these nucleic acids. Just use it. It is necessary that the region including the 3 'end of the primer has a structure having RNAseH resistance, such as DNA.
  • RNAseH resistance such as DNA.
  • an RNA / DNA hybrid with nucleic acid (3) or nucleic acid (4) is constructed at the stage where nucleic acid (3) or nucleic acid (4) is synthesized. This is because the 3 'end required for nucleic acid (4) is not synthesized.
  • the step of digesting RNA is common to the above-described ICAN method.
  • the principle is different from that of the ICAN method because the thigh must be on the 5 'end side of the nucleic acid (3) or the nucleic acid (4).
  • the nucleic acid (3) and the nucleic acid (4) each have a double-stranded structure at 3 and 3, it has already been described that this region becomes single-stranded by a primer that anneals to the loop.
  • the conditions under which the primer can be annealed without the step of positively making a single strand are also shown above. Further, by setting the 5 'as RNA and digesting this RNA, the 3' can be made into a state capable of base pairing.
  • the single-stranded nucleic acid having the regions (a) to (d) provides A method for synthesizing a nucleic acid is performed.
  • each of the nucleic acids generated by the combination of the nucleic acid (3) and the nucleic acid (4) has an additional base sequence in addition to the target base sequence.
  • the additional base sequence corresponds to the base sequence that nucleic acid (3) and nucleic acid (4) have from the loop to 5. Therefore, the nucleic acid generated from the combination of the nucleic acid (3) and the nucleic acid (4) has an additional nucleotide sequence on the 5 ′ side and 3 ′ side of the target nucleotide sequence.
  • the combination of the nucleic acid (3) or the nucleic acid (4) with the nucleic acid (1) or the nucleic acid (2) has an additional base sequence on either the 5 ′ side or the 3 ′ side of the target base sequence Generates nucleic acids. Since the 3 'end or 5' end having the additional base sequence is a base sequence derived from the insert primer, it does not have a base sequence complementary to itself. Therefore, the nucleic acid itself is not in a state where the reaction of the LAMP method can be directly started. However, in this nucleic acid, the base sequence from the insert primer to the inner primer is conserved in the region containing its 5 '.
  • a new nucleic acid (1) or nucleic acid (2) can be generated based on the inner primer RIP or FIP with this region as ⁇ M.
  • the generated nucleic acid (1) or nucleic acid (2) is displaced by complementary strand synthesis from another nucleic acid that anneals to the 5 side of the inner primer, and is released as a single-stranded nucleic acid .
  • the nucleic acid (1), the nucleic acid (2), the nucleic acid (3), and the nucleic acid (1) can be obtained by using the insert primer and the inner primer, more preferably the outer primer. (4) is continuously synthesized, and a nucleic acid containing the target base sequence is synthesized.
  • the primer primer insert primer is a nucleic acid in a double-stranded state.
  • the conditions that can be used are as described above.
  • the above is the reaction principle of the nucleic acid synthesis method according to the present invention.
  • the reaction as described above can be carried out.
  • all the reactions can be carried out under common conditions. That is, under desirable conditions, it is not necessary to perform a treatment such as heating for the purpose of denaturing the nucleic acid.
  • the present invention relates to a method for synthesizing a nucleic acid containing a target base sequence, comprising a step of incubating the following elements.
  • Inner primer F wherein the inner primer F anneals at its 3 'end to a region defining the 3' side of one of the strands constituting the target base sequence, and The 5 ′ end has a base sequence complementary to any region of the complementary strand synthesis reaction product starting from this primer.
  • insert primer F has a base sequence at its 3 ′ end that is complementary to an arbitrary region of one of the strands constituting the target base sequence
  • an insert primer wherein the insert primer R has a base sequence complementary to an arbitrary region of the other strand constituting the target base sequence at its 3 ′ end,
  • the synthetic product originating from each primer ⁇ "of the insert primer F and the insert primer R has a base sequence complementary to each other in a region containing 5, « of both.
  • the following components can be further added to the reaction solution.
  • outer primer F is a starting point of a complementary strand synthesis reaction starting from the 3 ′ side of the region to be annealed by the inner primer F in, and
  • Outer primer R is a starting point of a complementary strand synthesis reaction starting from the 3 ′ side of the region to be annealed by the inner primer R in the above.
  • the following element can be further added to the reaction solution.
  • loop primer F the region containing 3 of loop primer F is the region containing the 5 ′ end of the above-mentioned inner primer F, and the region containing the 5′-end of the complementary primer synthesis reaction starting from the inner primer F. Anneal to any region in the loop formed by hybridizing to any region, and
  • Loop primer R here, the region containing 3, of the norepe primer R is the region containing the 5, end of the above-mentioned inner primer R.
  • nucleic acids having loops composed of various base sequences are continuously produced.
  • the loop annealed by the inner primer is an important loop for starting complementary strand synthesis starting from the inner primer.
  • the region containing the 5 ' ⁇ of the inner primer hybridizes to an arbitrary region of the complementary strand synthesis reaction product starting from the inner primer.
  • the inner primer is not anneal to the loop formed by the step.
  • the present inventors have found that a primer that anneals to this loop to provide a starting point for complementary strand synthesis improves the reaction rate (W002 / 24902).
  • the inner primer is an anneal.
  • a primer that gives a starting point for complementary strand synthesis in a loop that does not perform is called a loop primer.
  • the loop primer in the present invention can design at least two types of loop primers, that is, a loop primer F for the inner primer F and a loop primer R for the inner primer R.
  • at least one type of loop primer designed in this manner is used.
  • Preferred loop primers are two types of loop primers, namely, a primer Primer F and a loop primer R.
  • three or more types of loop primers may be used in combination with loop primers capable of annealing to different regions in the same loop. If the loop primer is applied to the nucleic acid synthesis method of the present invention, an improvement in the reaction rate can be expected.
  • the insert primer F is, for example, 0.1 to 100 times, preferably 0.1 to 50 times, the inner primer F. It is more preferably used at a concentration of 0.2 to 5 times.
  • the concentration ratio between the insert primer R and the inner primer R can be similarly set.
  • the insert primer F and the insert primer R, or the inner primer F and the inner primer R are usually used at approximately the same concentration.
  • the amount of primers and enzymes used should be set appropriately according to the expected conditions such as the concentration of type I, reaction time, reaction temperature, and the activity of the enzyme used in the reaction. Can be set. More specifically, for example, The concentration is usually from 100 to 400 OnM, preferably from 250 to 300 OnM, more preferably from 500 to 300 OnM, so that the concentration of 6 or more copies can be obtained. Based on the results, an ascertainable level of amplification product can be generated.
  • the first purpose of the outer primer is to displace the strand synthesized from the inner primer by a complementary strand synthesis reaction from the 3 ′ side. Therefore, it is desirable that the complementary strand synthesis reaction starting from the inner primer be initiated preferentially over that of the outer primer.
  • the primer is usually designed so that the Tm of the outer primer is lower than the Tm of the inner primer.
  • the Tm of the inner primer refers to the Tm for the nucleic acid in the region including the terminal 3, which is the starting point of complementary strand synthesis.
  • the outer primer at a lower concentration than the inner primer, the complementary strand synthesis of the inner primer can be preferentially performed.
  • the working concentration of the primer primer can be, for example, 1/2 or less, preferably 1Z10 or less, or 1Z100 or less with respect to the inner primer.
  • the concentration of the loop primer used is desirably set to, for example, lZ10 to an equivalent amount with respect to the concentration of the inner primer. More specifically, it can be 1/3 to 1/2. Usually, the amount of the loop primer F is equal to that of the loop primer R.
  • the use of the outer primer is not an essential condition. Even if the inner primer is not released from »nucleic acid by displacement, the extension reaction product can function as a new type III. Even if the step of forming a single strand is omitted, the complementary strand synthesis reaction based on the primer is started with a certain probability, as described above. Since this phenomenon can be expected even with the extension product from the inner primer, there is a possibility that the complementary strand synthesis reaction starting from the insert primer or the other inner primer can be started without using the outer primer. . However, an extension product generated from the inner primer as a starting point depending on the type III nucleic acid is a product constituting the first step of the nucleic acid synthesis method of the present invention.
  • a loop primer is not required in the present invention.
  • the use of loop primers can improve the reaction rate. Therefore, the use of the loop primer is one of the desirable conditions in the nucleic acid synthesis method of the present invention.
  • primers used in the present invention can be chemically synthesized.
  • a natural nucleic acid can be cleaved with a restriction enzyme or the like, and modified or ligated so as to be composed of the above base sequence.
  • the insert primer and the inner primer in the present invention can be labeled with a known labeling substance.
  • the labeling substance include a binding ligand such as digoxin dibiotin, an enzyme, a fluorescent substance and a luminescent substance, and a sex isotope.
  • a technique of replacing a base constituting the inner primer with a fluorescent analog (W095 / 05391, Proc. Natl. Acad. Sci. USA, 91, 664 4-6648, 1994) is also known.
  • the insert primer / binner primer according to the present invention may be itself bound to a solid phase by a method that does not hinder its function.
  • any part of these primers can be labeled with a binding ligand such as biotin, and then immobilized indirectly with a binding partner such as immobilized avidin.
  • the separation is easy at the ⁇ where the solid-phased primer is used as the synthesis starting point because the nucleic acid synthesis reaction product is captured on the solid phase.
  • a nucleic acid-specific indicator or an additional labeled probe is used. Detection can also be performed by hybridizing.
  • the target nucleic acid fragment can be recovered by digestion with an arbitrary restriction enzyme.
  • the reaction solution can be composed of the above-described components and a buffer solution that maintains appropriate reaction conditions. Further, an enzyme protectant, Tm-adjusted IJ, and the like can be added to the reaction solution.
  • a series of reactions consist of a buffer that provides a suitable pH for the enzymatic reaction, salts necessary for maintaining the catalytic activity of the enzyme and for annealing, a protective agent for the enzyme, and, if necessary, adjusting the melting temperature (Tm). !] And so on.
  • a neutral to weakly alkaline buffering agent such as Tris-HC1 is used. Adjust the pH according to the DNA polymerase used. Salts KC1, NaCl, MgCl 2, MgS0 4, or (NH 4) 2 S0 4 or the like, is suitably added to the melting temperature (Tm) of the adjustment of the active maintenance and nucleic acid enzyme.
  • Serum albumin saccharide is used as a protective agent for the enzyme.
  • Tm melting temperature
  • betaine proline, formamide, dimethyl sulfoxide (hereinafter abbreviated as DMS0), or trimethylamine N-oxide (hereinafter abbreviated as T-article 0) is generally used. Used. By utilizing the melting temperature (Tm) preparation, the annealing of the oligonucleotide can be adjusted under limited temperature conditions.
  • betaine N, N, N, -trimethylglycine
  • T-article 0 trimethylamine N-oxide
  • Betaine can be expected to promote the nucleic acid amplification reaction of the present invention by adding 0.2 to 3.0 M, preferably 0.5 to 1.5 M, of the reaction solution. Since these melting temperature modifiers act in the direction of lowering the melting temperature, conditions that give appropriate stringency and reactivity should be set in consideration of other reaction conditions such as salt concentration and reaction temperature. You.
  • Tm can be calculated based on the length of the base sequence to be annealed and its GC content, salt concentration, and Tm conversion concentration.
  • the anneal of the primer to the double-stranded nucleic acid is presumably unstable.
  • the complementary strand is synthesized from the primer as a synthesis starting point while being unstable.
  • the hybridization between the synthesized complementary strand and the nucleic acid is gradually stabilized.
  • a DNA polymerase as shown below can catalyze the synthesis of a complementary strand using a primer as a starting point for synthesis of a double-stranded ⁇ M nucleic acid.
  • Supporting the method for synthesizing nucleic acids according to the present invention is a DNA polymerase that catalyzes a complementary strand synthesis reaction involving strand displacement.
  • the following are known as this kind of DNA polymerase.
  • various mutants of these enzymes can be used in the present invention as long as they have sequence-dependent #S complementary chain synthesis activity and strand displacement.
  • the term “mutant” as used herein refers to a structure in which only the structure that provides the catalytic activity required by the enzyme is extracted, or a structure in which the catalytic activity, stability, or heat resistance is altered by amino acid mutation or the like. it can.
  • DNA polymerases are used in amounts that can achieve the required reaction under the reaction conditions.
  • a sufficient amount of a synthetic product can be produced in a short time by setting the amount to 2 to 20 ml, usually 5-1 OU, per 25 ⁇ L of the reaction solution.
  • Vent Exo- DNA polymerase (Vent DNA polymerase from which etasonuclease activity has been removed)
  • Bst DNA polymerase ⁇ Bca (exo-) DNA polymerase is a particularly desirable enzyme because it has a certain degree of heat resistance and high tactility.
  • a step of using a primer as a synthesis starting point and a complementary strand synthesis reaction are optionally performed on a double-stranded nucleic acid under the same conditions. While such reactions often require some degree of heating, it is a desirable condition that the enzyme be thermostable. By using a thermostable enzyme, it can respond to a wide range of reaction conditions.
  • nt (Exo-) DNA polymerase is an enzyme that has a high degree of thermostability along with its strand placement.
  • Vent (Exo-) DNA polymer T4 gene 32 is effective as a single-stranded binding protein against lysase.
  • DNA polymerases that do not have 3, 5 'ethanonuclease activity have a phenomenon that complementary strand synthesis does not stop at the 5' end of the DNA and proceeds to the point where one base is protruded.
  • a phenomenon is undesirable because the sequence at the 3 ′ end when the complementary strand synthesis reaches the end leads to the start of the next complementary strand synthesis.
  • the addition of a base to the 3 'by a DNA polymerase is A with a high probability. Therefore, the sequence may be selected so as to start with the synthetic power from the 3 'end so that there is no problem even if dATP is added by one base by mistake.
  • a DNA polymerase such as Taq polymerase generally used in PCR and the like has substantially no strand displacement under ordinary conditions.
  • this type of DNA polymerase can be used in the present invention if it can provide conditions that allow strand displacement.
  • the nucleic acid synthesis method of the present invention uses a nucleic acid as a key.
  • the type of nucleic acid used as ⁇ is not limited. Therefore, double-stranded, single-stranded, or triple-stranded, DNA-RNA hybrids, or DNA or RNA derivatives including artificial nucleotide derivatives, etc. can be made into type III.
  • the nucleic acid of the present invention may be purified or unpurified.
  • the method of the present invention can be applied in a state where it is present in a cell (in situ). By using a double-stranded nucleic acid in a cell, in situ analysis of the genome becomes possible. Intracellular mRNA (single-stranded nucleic acid) can also be used as a key.
  • the step of synthesizing the cDNA and the method of synthesizing a nucleic acid according to the present invention can be performed under the same conditions. Brittle By synthesizing the first strand of the cDNA, a double-stranded nucleic acid based on DM-spirodipride is completed. Using this double-stranded nucleic acid in the present invention, a method for synthesizing a nucleic acid can be carried out. If the DNA polymerase used in the nucleic acid synthesis method of the present invention has reverse transcriptase activity, nucleic acid can be synthesized under the same conditions using a single enzyme.
  • ca DNA polymerase is a DNA polymerase that has strand mimicry and also has reverse transcriptase activity. It goes without saying that the nucleic acid synthesis method according to the present invention can be applied after the second strand is synthesized to obtain a complete double-stranded cDNA.
  • a nucleic acid in a double-stranded state when used as a key, an arbitrary primer is added, and the mixture is incubated under conditions capable of achieving a complementary strand synthesis reaction starting from the primer.
  • the region to be inserted by the insert primer / binner primer can be brought into a state where base pairing is possible.
  • arbitrary primer refers to a region in which various primers are to be annealed so as to be capable of base pairing. Therefore, the arbitrary primer is a primer capable of initiating complementary strand synthesis with the insert primer and the primer primer of the double-stranded nucleic acid to be used as a complementary strand to the nucleic acid strand to be affixed. There is a need. Furthermore, the complementary strand synthesis using an arbitrary primer in the present invention as a starting point of synthesis should be in a positional relationship such that the insert primer / binner primer proceeds toward the region to be annealed.
  • any primer can be composed of a nucleotide sequence complementary to any region as long as this condition is satisfied.
  • one of a set of an inner primer and an insert primer can be used as an arbitrary primer.
  • Such an embodiment is one of the desirable embodiments in the present invention because it reduces the components required for the reaction.
  • an insert primer and an inner primer are used for different nucleic acids, only one set of inner primer or insert primer is provided for one type III. In such a case, a quick reaction can be expected by adding an arbitrary primer in addition to these primers and setting a region where these primers should be annealed to be capable of base pairing.
  • a primer could not give a synthetic origin unless at least the region to be annealed was a single strand. For this reason, conventionally, in order to convert a double-stranded nucleic acid into type II, a step of making the double-stranded nucleic acid single-stranded by denaturation is always performed prior to the use of the primer. However, even if it is not necessarily a single strand, a starting point for synthesis can be given by incubating with a primer under the condition that the double strand is unstable by some means. As a condition for destabilizing the duplex, for example, a method of heating to near the melting temperature (Tm) can be shown. Alternatively, it is also effective to have Tm conversion.
  • Tm melting temperature
  • nucleic acid in the nucleic acid synthesis method or amplification method of the present invention, any nucleic acid can be used as the nucleic acid.
  • the nucleic acid can be single-stranded, double-stranded, or triple-stranded. It has already been mentioned that even if it is not single-stranded, it can be used as a nucleic acid without a denaturation step if appropriate conditions are given.
  • the nucleic acid to be used may be DA or RNA.
  • 4 complementary strand synthesis reaction can be used as RNA.
  • DNA polymerases having a reverse transcriptase activity with respect to RA and catalyzing a complementary strand synthesis reaction involving strand displacement are known.
  • ca DNA polymerase is a DNA polymerase having a strand displacement property and also having a reverse transcriptase activity.
  • the method for synthesizing a nucleic acid according to the present invention can be applied after synthesizing the second strand to obtain a complete double-stranded cDNA.
  • the origin of the nucleic acid is not limited. Normally, nucleic acids are derived from biological materials, but artificially synthesized nucleic acids can be used as ⁇ M.
  • the method for synthesizing a nucleic acid or the method for amplifying a nucleic acid according to the present invention is useful for synthesizing DNA to be immobilized on a DNA chip.
  • a process for preparing a DNA chip using the present invention will be exemplified.
  • the primer of the present invention is designed based on the base sequence of the nucleic acid to be immobilized on the DNA chip. Designing primers necessary for amplification of a desired nucleic acid based on the disclosure of the present invention can be usually performed by those skilled in the art. By incubating the designed primer together with the components necessary for the amplification reaction of the nucleic acid of the present invention under appropriate conditions, the reaction solution has a unique characteristic of the LAMP method in which the target base sequence is linked. A large amount of nucleic acid having a structure is produced. The obtained product is fixed on a substrate by a known method as it is or after an appropriate treatment. A glass slide / nylon membrane is used for the substrate. Methods for immobilizing nucleic acids on a substrate are known.
  • any nucleic acid can be used as a symbol. More specifically, cDNA clones derived from various cells are used as. Alternatively, an arbitrary region can be amplified according to the present invention with the genome as a type III, and immobilized on a DNA chip.
  • the structural feature of LAMP products is that they have an impartet repeat structure. That is, it has a structure in which complementary base sequences are alternately linked.
  • a nucleic acid having such a structure it is expected that intramolecular annealing occurs between regions each having a complementary base sequence, which may affect the efficiency of the hybrid.
  • problems due to intramolecular annealing can be easily solved by simple processing.
  • the LAMP product can be fragmented by restriction enzyme treatment, for example, using a restriction enzyme site introduced at a specific position. Respective fragmented nucleic acids no longer cause intramolecular annealing.
  • the nucleic acid synthesized by the LAMP method has a region of about 20-100 bp between the inner primers. With such a length, it is difficult to increase the reactivity with the probe.
  • ADVANTAGE OF THE INVENTION According to this invention, comparatively long DNA which has not been tried before can be amplified based on LAMP method. As a result, a DNA chip prepared using the synthetic product of the present invention can detect signals more efficiently than a chip using a nucleic acid synthesized by the known LAMP method.
  • the following method is generally used. First, labeled cDNA prepared from mRNA samples derived from two different cells is hybridized to a microarray. Next, immobilize it on a DNA chip The signal of the probe hybridized to the nucleic acid is measured. If the signal is measured for each nucleic acid and the signal intensity is analyzed, the relative expression levels of each mRNA are compared. At this time, if there is a variation in the hybridization efficiency between the nucleic acids immobilized on the chip, this may cause a loss of the reliability of the analysis result. Therefore, it is significant to immobilize longer nucleic acids and provide sufficient reactivity.
  • a polynucleotide comprising a target nucleotide sequence obtained by linking nucleotide sequences derived from two different genes can be synthesized and used as a probe in a DNA chip.
  • a probe must be prepared for each gene in a DNA chip.
  • polynucleotides synthesized by combining base sequences derived from different genes according to the present invention are used, one polynucleotide functions as a probe for two genes. Furthermore, there is no need to calibrate (normalize) the signal because the amount of probe for each gene is equal.
  • the method for synthesizing a nucleic acid of the present invention can be applied to gene closing. Cloning of a gene based on the PCR method is performed by digesting the reaction product of the PCR method with a restriction enzyme and inserting the product into a cloning vector. Similarly, the LAMP product can be used as a fragment for integration into a cloning vector by digesting the LAMP product with an appropriate restriction enzyme.
  • the LAMP product can be used not only for synthesizing a nucleic acid to be immobilized on a DNA chip, but also for preparing a labeled probe. That is, it is possible to prepare a labeled probe by primer extension using the LAMP product as ⁇ S. Since the LAMP method produces a large amount of DNA, the labeled probe can be easily prepared in a large amount. The labeled probe prepared in this way is useful as a probe for hybridization or analysis using a DM chip.
  • the nucleic acid amplification method of the present invention can be used for obtaining a gene for expression.
  • the amino acid sequence of the protein The average was predicted to be 352 amino acids. This corresponds to 1056 bp of DNA.
  • DNA of at least about 500 bp can be amplified. In other words, about half the area of the average human protein can be enhanced.
  • Functional analysis of proteins has been positioned as an important research theme for the bost genome generation. For that, it is necessary to express the protein. It is believed that the present invention can contribute to the amplification of the translation region of a gene.
  • nucleic acids can be detected or quantified using the product of the nucleic acid amplification method of the present invention as an index. That is, when a primer designed for the purpose of amplifying a specific nucleotide sequence is used, a large amount of product is generated, which means that the nucleotide sequence is present in the sample. Furthermore, the abundance of the nucleic acid containing the target base sequence in the sample can be compared using the signal intensity or the reaction time required to reach a certain signal intensity as an index.
  • Nucleic acids synthesized by the LAMP method are composed of complementary base sequences, although they are single-stranded, and most of them form base pair bonds. By utilizing this feature, it is possible to detect synthetic products. If the method for synthesizing a nucleic acid according to the present invention is carried out in the presence of a fluorescent dye which is a double-strand-specific interpolator such as ethidium promide, SYBR Green I, or Pico Green, an increase in the product A corresponding increase in the fluorescence intensity is observed.
  • a fluorescent dye which is a double-strand-specific interpolator such as ethidium promide, SYBR Green I, or Pico Green
  • mutations can be detected based on the nucleic acid amplification method of the present invention. ⁇ If the nucleic acid does not have the expected base sequence, any complementary strand synthesis reaction that constitutes the method for synthesizing or amplifying the nucleic acid of the present invention is inhibited so that the reaction is generated. Mutation can be detected using the amount or presence or absence of the substance as an index. The method for detecting a mutation according to the present invention is specifically described below.
  • the method for synthesizing or amplifying a nucleic acid of the present invention comprises a plurality of complementary strand synthesis reactions.
  • the complementary strand synthesis reaction starting from the 3 ′ of the insert primer or the complementary strand synthesis reaction starting from the 3 ′ end of the complementary strand synthesized using the insert primer is a unique feature of the present invention. It is a reaction.
  • at least one of the two types of complementary strand synthesis reactions related to these insert primers is controlled by the nucleotide sequence of the nucleic acid.
  • the base sequence at the 5' end of the insert primer does not become a complementary base sequence. It is desirable to do so.
  • the insert primer described as [[insert primer 2 for synthesizing a continuous target base sequence in S nucleic acid]] may be used.
  • the complementary strand synthesized using the insert primer as type II always anneals to the region consisting of the target base sequence, and the base sequence check mechanism operates.
  • the nucleic acid synthesized using the insert primer as the base sequence is based on the base sequence derived from the base sequence of the insert primer. Will be anneal. Unless the base sequence derived from the target base sequence is annealed, the base sequence cannot be checked.
  • the insert primer may be designed so that the region near the Z or 5 'end corresponds to the region where the mutation is to be detected. More specifically, if the design is designed such that the mutation to be detected is located in the region from 3 ′ ⁇ ® to 53 ⁇ 4S, more preferably 2 to 4 bases, the complementary strand synthesis is performed by changing the base sequence in the type III nucleic acid. The reaction can be adjusted. It is needless to say that the base sequence can be designed under the same conditions in the case where the synthesis of the complementary strand is adjusted with 3, of the complementary strand synthesized using the insert primer as the ⁇ ⁇ ⁇ ⁇ ⁇ .
  • the complementary strand synthesis reaction starting from the 3 'end of the inner primer or the 3' end of the complementary strand synthesized using the inner primer as ⁇ M is used. It can also be regulated by nucleic acid mutations.
  • the base sequence of each primer is designed so that the vicinity of 3 ′, which is the starting point of phase capture synthesis, corresponds to the mutation to be detected.
  • a mutation check mechanism is required since the 3 'of the complementary strand synthesized using the inner primer as a anneal many times to the region consisting of the base sequence derived from, and serves as a starting point for complementary strand synthesis.
  • the interval between the insert primer and the inner primer can be freely set, so that there is no problem even if the mutation to be detected is distant.
  • various analysis methods can be used in accordance with the type of mutation to be detected.
  • the analysis method according to the present invention will be described using an example in which two SNPs A and B are linked to a specific trait.
  • a and B are a combination of specific bases
  • design primers so that a complementary strand synthesis reaction is formed only for that combination.
  • the formation of the reaction product means that the bases A and B in the nucleic acid contained in the sample are both bases to be detected. I have.
  • any combination of bases can be detected for two SNPs.
  • B is not any of ate, that is, g. Therefore, the base of this nucleic acid can be determined to be A-aZB_g. That is, all combinations of two bases can be determined by using 12 combinations.
  • the method for detecting a mutation according to the present invention is excellent in specificity.
  • the primer is annealed to the nucleic acid copied with the primer as ⁇ M, and a complementary strand synthesis reaction is performed. Therefore, it is considered difficult to detect a single base mutation.
  • the complementary strand synthesis reaction is controlled by a region that anneals to a base sequence derived from waking. That is, the mutation can be detected more specifically.
  • reagents required for the nucleic acid synthesis method or amplification method according to the present invention can be packaged in advance and supplied as a kit.
  • various oligonucleotides required as insert primers, inner primers, outer primers, or loop primers, dNTPs as substrates for complementary strand synthesis, and complementary strand synthesis with strand displacement A kit comprising a DNA polymerase to be carried out, a buffer for providing suitable conditions for the enzymatic reaction, and, if necessary, reagents necessary for separating and cleaving the synthesis reaction product is provided.
  • kits of the present invention when the kit of the present invention is aimed at detecting a nucleic acid, a detecting agent for detecting a nucleic acid synthesized by the synthesis method of the present invention can be combined. Or In a kit for obtaining a DNA encoding a fusion protein based on the above description, a DNA encoding a fusion partner can be combined with the kit.
  • the addition of reagents during the reaction is unnecessary, so that the reagent necessary for one reaction is supplied in a dispensed state to the reaction vessel, and the reaction is performed only by adding the sample. Can be started. If the system is designed so that the required DNA can be prepared in the reaction container, the opening of the container after the reaction can be completely abolished. This is very desirable in preventing contamination.
  • FIG. 1 is a diagram showing a basic reaction principle of a nucleic acid synthesis method according to the present invention.
  • the 3 'end of each chain is indicated by an arrow.
  • FIG. 2 shows the basic reaction principle of the nucleic acid synthesis method according to the present invention (continuation of FIG. 1).
  • FIG. 3 is a diagram showing the basic reaction principle of the nucleic acid synthesis method according to the present invention (continuation of FIG. 2).
  • FIG. 4 is a diagram showing the basic reaction principle of the nucleic acid synthesis method according to the present invention (continuation of FIG. 3).
  • FIG. 5 shows the basic reaction principle of the nucleic acid synthesis method according to the present invention (continuation of FIG. 4).
  • FIG. 6 is a diagram schematically showing the positional relationship of the inner primer and the insert primer set in the example with respect to the nucleic acid to be activated.
  • FIG. 7 is a photograph showing the result of agarose electrophoresis of a reaction product of the LAMP method using an insert primer. Each lane shows the following result in order from the left! / Lane 1: 10 Obp DNA ladder
  • Lane 3 Insert primer 10 pmol / 25 / z L added
  • Lane 7 Insert primer 50 pmol / 25 / z L inlet
  • FIG. 8 is a photograph showing the results of agarose gel electrophoresis of a reaction product of the LAMP method using an insert primer, which was digested with a restriction enzyme BamHI. Each lane shows the following results from left to right
  • Lane 2 1: BamHr digest of product (when insert primer was not used)
  • Lane 3 +: BamHI digest of product (when insert primer was used) The figure which shows the positional relationship of a primer.
  • FIG. 10 is a photograph showing the result of agarose electrophoresis after digestion of the reaction product of the LAMP method using an insert primer that anneals to a distant position on type III with the restriction enzyme EcoRV.
  • FIG. 11 is a diagram showing a design position of an insert primer.
  • FIG. 12 is a photograph showing the result of electrophoresis of a LAMP product.
  • FIG. 13 is a photograph showing the result of digesting the LAMP product with the restriction enzyme BssHII and performing agarose gel electrophoresis.
  • Lane 5 InsF2 / R2, BssHII digest Best mode for carrying out the invention
  • nucleic acid synthesis reaction Using the 387b P DNA fragment inserted into the pBSTspRI vector as, a nucleic acid synthesis reaction according to the present invention was performed under the following reaction conditions.
  • the nucleotide sequence of the nucleic acid used as the amino acid sequence is shown in SEQ ID NO: 1 (base sequence of human 'interleukin 8 gene).
  • the nucleotide sequence of SEQ ID NO: 1 includes a region that recognizes each primer.
  • Various primers having the base sequences described below were synthesized and used for this nucleic acid.
  • Inner primer F / Inner F (SEQ ID NO: 2)
  • Insert primer / Insert R (SEQ ID NO: 7)
  • Lane 2 Insert primer 10 pmol / 25 / i L
  • Lane 3 Insert primer 20 pmol / 25 / z L added
  • Lane 4 Insert primer 30 pmol / 25 / L added
  • Lane 5 Insert primer 40 pmol / 25 / L added
  • the above reaction product was digested with BamHI to confirm that it had the desired structure. While the fragments produced according to the theory were generated by restriction enzyme digestion, if the high-sized smeared pattern and the unmigrated band observed in FIG. 7 disappeared, all of these fragments were synthesized according to the present invention. It can be confirmed that the nucleic acid has a complementary base sequence linked on the chain.
  • a reaction product using an insert primer of 40 pmol / 25 L was used.
  • the digest was run on a 2% agarose gel at 100 V for 30 minutes. After electrophoresis, the gel was stained with Etbr to detect nucleic acids. As the molecular weight marker, 10 Obp DNA ladder (NEW ENGLA D BioLabs) was used. The results are as shown in FIG. Each lane corresponds to the next sample.
  • Lane 2 BamHI digest of the product (when no insert primer is used)
  • Lane 3 +: BamHI digest of the product (when the insert primer is used) If the insert primer has not been added, this is the target. No pande could be confirmed, but the added one detected a pande around 400 bp. The region between the inner primers is 396 bp.
  • BssHII digestion was performed to confirm that the LAMP product had the desired structure.
  • the 25/251 reaction solution containing the reaction product was purified using a QIAquic PCR purification kit, and eluted with 50 / il Tris / HCl pH 8.0. Of these, 1 ⁇ 1 was digested with BssHII at 37 ° C for 1 hour. If the ladder-formed amplification product in FIG. 12 is digested with BssHII, the portion corresponding to the sequence sandwiched by BssHII in FIG. 11 should be produced as a main band of about 600 bp.
  • the digest was electrophoresed on a 2% agarose gel at 100 V for 30 minutes. After electrophoresis, the gel was stained with EtBr to detect nucleic acids. As a marker, a 100 bp DNA ladder (NEW ENGLAND Bio Labs) was used. As a result, a pan was confirmed at the expected position (Fig. 13). This time, the region between the inner primers is 536 bp. As described above, even when another mirror type was used, the use of the insert primer could easily amplify a relatively long primer. Further, in this example, two types of insert primers (InsF / R and InsF2 / R2) were designed, and it was possible to amplify either primer.
  • InsF / R and InsF2 / R2 two types of insert primers
  • oligonucleotides having the following nucleotide sequences were used as an inner primer, an insert primer, and an outer primer.
  • a sense or antisense of an arbitrary sequence CATCAAATAT was added as a linker sequence.
  • the insert primer F / Insert F and the insert primer: / Insert R each anneal to a distant region of IDA.
  • the part shown in [Deletion] located in the middle part was deleted as shown in Fig. 9, and the base connecting the two regions between the arrows was linked via the linker sequence CATCAAATAT.
  • the target base sequence consisting of the sequence should be synthesized.
  • the number of bases between F1 / R1 in Fig. 9 was 5 bp in type II (upper in Fig. 9), It is synthesized as a 6 bp target nucleotide sequence (lower in Fig. 9).
  • Example 1 prepare a DNA that does not undergo thermal transformation '
  • the following concentrations of the primers were added to the reaction solution having the composition, and reacted at 65 ° C. for 4 hours.
  • LDNA (1 ⁇ 10 6 molecules) as type I
  • a synthesis reaction of the nucleic acid of the present invention was performed under the conditions described above.
  • the reaction solution of 25 / ⁇ was purified using a 0 ⁇ ( ⁇ : 1 ⁇ 1 ⁇ PCR purification kit (QIAGEN) and eluted with Tris / HCl pH 8.0, of which 1 ⁇ L was extracted with EcoRV. Digested for 1 hour at ° C.
  • the nucleotide sequence of the above reaction product was determined.
  • the vector (pBlue script) previously digested with EcoRV and the reaction product digested with EcoRV were ligated to obtain pBS525 / EcoRV. Furthermore, this vector was transformed into a competent cell (DH5). After cloning and growing the transformed E. coli, The plasmid was recovered by the SDS method.
  • the sequencing reaction was performed using Cy5.5-labeled vector-specific primer, ThermoSequenas e Cycle Sequencing Kit (Amersham pharmacia biotech II), and the base sequence was determined using the TYPEFIMA method. It was determined using the (Fuarumashiane ring model S e q4x4).
  • Each insert primer has a 10-base linker sequence (CATCAAATAT), which is a complementary base sequence.
  • CAAATAT 10-base linker sequence
  • a sequence occurs through this sequence to initiate DNA synthesis. This can be expected to yield a sequence in which the region flanked by the insert primers ([deletion] in FIG. 9) has been deleted.
  • DM consisting of the predicted nucleotide sequence was synthesized.
  • a deletion mutation can be obtained by using an insert primer.
  • an insert primer to which a base sequence other than the target base sequence was added, a base could be inserted into the amplified sequence.
  • a new method for synthesizing a nucleic acid which was not known by the known LAMP method, is provided while utilizing the principle of the LAMP method. According to the nucleic acid synthesis method of the present invention, the following new advantages can be expected.
  • the nucleic acid synthesis method of the present invention can synthesize a long target base sequence in a base sequence-specific manner.
  • the LAMP method of ⁇ P in which primers are set at the end of the target nucleotide sequence, depending on the conditions, non-specific synthetic Sometimes a product was formed. Non-specific synthesis products imply reduced yields in nucleic acid synthesis.
  • the method of the present invention suppresses the production of nonspecific by-products even with a long target nucleotide sequence of, for example, more than 400 nucleotides, and can rapidly produce the target nucleic acid. Can be synthesized.
  • the nucleic acid synthesis method of the present invention is useful for synthesizing a probe to be immobilized on a DNA microarray. It has already been mentioned that DNA having a length of several hundred bases is required for a probe to be immobilized on a DNA microarray. According to the present invention, DNA having a length of 400 bases or more can be synthesized quickly and easily. Also, since the LAMP method is originally a method that can be expected to be highly specific to ⁇ M, high accuracy can be expected for the synthesized product.
  • a primer is set not only at the target base sequence but also at the middle. Using this feature, mutations can be introduced in the middle of the target base sequence. Even in the PCR method, which is now widely used as a method for synthesizing a gene, the primer is not set at a part of the target base sequence. Therefore, a technique capable of introducing a mutation into the central portion of a target nucleotide sequence as in the present invention is very useful as a technique for gene manipulation.
  • nucleic acid consisting of a base sequence obtained by linking a plurality of regions located at a distance in a nucleic acid can be obtained. This feature enables the present invention to freely synthesize mutants in which any region of a nucleic acid has been deleted.
  • nucleic acid having an arbitrary region selected from different nucleic acids as one continuous base sequence can be obtained by the nucleic acid synthesis method or the amplification method of the present invention. Due to this feature, the present invention regards freely synthesizing a gene encoding any fusion protein as g.

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Abstract

L'invention concerne un procédé permettant la synthèse d'un acide nucléique contenant une séquence de base cible, à partir d'au moins deux acides nucléiques possédant des séquences de base complémentaires l'une à l'autre à l'extrémité 3,' et de séquences de base complémentaires à des régions arbitraires des séquences de base qui se forment à l'extrémité 5'. Ce procédé peut être mis en oeuvre d'une manière simple au moyen d'une amorce destinée à la technique LAMP et d'une amorce d'insertion, et comprend l'incubation isotherme d'une ADN-polymérase qui catalyse la réaction de synthèse d'un brin complémentaire, conjointement à un processus de déplacement du brin complémentaire.
PCT/JP2002/004479 2001-05-08 2002-05-08 Procede de synthese d'acide nucleique Ceased WO2002090538A1 (fr)

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JP2002587599A JPWO2002090538A1 (ja) 2001-05-08 2002-05-08 核酸を合成する方法

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WO2006051988A1 (fr) * 2004-11-15 2006-05-18 Riken Procédé d'amplification d'acide nucléique
WO2010146349A1 (fr) * 2009-06-15 2010-12-23 Lumora Ltd Technique d'amplification isotherme d'acides nucléiques accélérée par des amorces en tige
JP2013039077A (ja) * 2011-08-16 2013-02-28 Univ Of Tokyo 食中毒原因大腸菌検出用プライマー及び検出用キット
CN107429292A (zh) * 2014-12-15 2017-12-01 塞弗德公司 底数大于2的指数核酸扩增
CN107893120A (zh) * 2017-11-28 2018-04-10 保定佑安生物科技有限公司 检测运动基因snp的引物组及其应用和产品以及检测运动基因snp的检测方法及应用
CN109825558A (zh) * 2018-09-06 2019-05-31 湖南融健基因生物科技有限公司 一种基于引物激活的碱基突变序列扩增检测方法
US11352622B2 (en) 2018-10-29 2022-06-07 Cepheid Exponential base-3 and greater nucleic acid amplification with reduced amplification time
CN114829625A (zh) * 2019-12-09 2022-07-29 凸版印刷株式会社 检测方法

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WO2000028082A1 (fr) * 1998-11-09 2000-05-18 Eiken Kagaku Kabushiki Kaisha Procede de synthese d'acide nucleique

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006051988A1 (fr) * 2004-11-15 2006-05-18 Riken Procédé d'amplification d'acide nucléique
WO2010146349A1 (fr) * 2009-06-15 2010-12-23 Lumora Ltd Technique d'amplification isotherme d'acides nucléiques accélérée par des amorces en tige
CN102652176A (zh) * 2009-06-15 2012-08-29 路玛拉有限公司 茎部加速的等温核酸扩增技术
JP2012529904A (ja) * 2009-06-15 2012-11-29 ルモラ・リミテッド ステム加速等温核酸増幅技術
AU2010261541B2 (en) * 2009-06-15 2016-03-10 Lumora Ltd Stem accelerated isothermal nucleic acid amplification technology
JP2016041070A (ja) * 2009-06-15 2016-03-31 ルモラ・リミテッドＬｕｍｏｒａ Ｌｉｍｉｔｅｄ ステム加速等温核酸増幅技術
US9410190B2 (en) 2009-06-15 2016-08-09 Lumora Ltd. Stem accelerated isothermal nucleic acid amplification technology
JP2013039077A (ja) * 2011-08-16 2013-02-28 Univ Of Tokyo 食中毒原因大腸菌検出用プライマー及び検出用キット
CN107429292A (zh) * 2014-12-15 2017-12-01 塞弗德公司 底数大于2的指数核酸扩增
US11028434B2 (en) 2014-12-15 2021-06-08 Cepheid Exponential base-greater-than-2 nucleic acid amplification
CN107429292B (zh) * 2014-12-15 2021-12-17 塞弗德公司 底数大于2的指数核酸扩增
US11952621B2 (en) 2014-12-15 2024-04-09 Cepheid Exponential base-greater-than-2 nucleic acid amplification
CN107893120A (zh) * 2017-11-28 2018-04-10 保定佑安生物科技有限公司 检测运动基因snp的引物组及其应用和产品以及检测运动基因snp的检测方法及应用
CN107893120B (zh) * 2017-11-28 2021-05-04 保定佑安生物科技有限公司 检测运动基因snp的引物组及其应用和产品以及检测运动基因snp的检测方法及应用
CN109825558A (zh) * 2018-09-06 2019-05-31 湖南融健基因生物科技有限公司 一种基于引物激活的碱基突变序列扩增检测方法
US11352622B2 (en) 2018-10-29 2022-06-07 Cepheid Exponential base-3 and greater nucleic acid amplification with reduced amplification time
US12098368B2 (en) 2018-10-29 2024-09-24 Cepheid Exponential base-3 and greater nucleic acid amplification with reduced amplification time
CN114829625A (zh) * 2019-12-09 2022-07-29 凸版印刷株式会社 检测方法

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