WO2009123216A1 - 高選択・高効率でpcr増幅が可能な新規dna - Google Patents
高選択・高効率でpcr増幅が可能な新規dna Download PDFInfo
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- WO2009123216A1 WO2009123216A1 PCT/JP2009/056718 JP2009056718W WO2009123216A1 WO 2009123216 A1 WO2009123216 A1 WO 2009123216A1 JP 2009056718 W JP2009056718 W JP 2009056718W WO 2009123216 A1 WO2009123216 A1 WO 2009123216A1
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- 0 C*NC(*(C)*)=O Chemical compound C*NC(*(C)*)=O 0.000 description 1
- VIYFTBCCDPWICR-UHFFFAOYSA-N C[n]1c(nccc2-c3ccc[s]3)c2nc1 Chemical compound C[n]1c(nccc2-c3ccc[s]3)c2nc1 VIYFTBCCDPWICR-UHFFFAOYSA-N 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- 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
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- 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
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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- 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
- C12Q1/6869—Methods for sequencing
Definitions
- the present invention relates to an artificial base pair capable of replication with high selectivity and high efficiency, and a method for replicating a nucleic acid containing the artificial base pair.
- the present invention also relates to a method for introducing an artificial base to which a functional substituent is bound into DNA by a nucleic acid replication reaction.
- the present invention further relates to a method for replicating and selectively recovering a nucleic acid containing an artificial base pair from a nucleic acid pool.
- the present invention further relates to a method for determining the sequence of a natural base in the vicinity of an artificial base in DNA for realizing highly efficient and highly selective replication of a nucleic acid containing the artificial base.
- Nucleic acids are amplified by the self-complementarity of AT (U) and GC base pairs, and function as catalysts and ligands.
- U AT
- GC base pairs compared to the 20 different amino acids in natural proteins, the limited number of natural nucleic acids formed by nucleotides consisting of only 4 different bases limits the function of DNA and RNA molecules.
- the non-natural base pair system is a solution to this problem because it can extend the genetic information by increasing the types of nucleic acid bases (Non-Patent Documents 1-5).
- Non-natural base pairs require high specific complementarity that allows site-specific introduction of specific nucleotide analogs into DNA and RNA by polymerase catalysis. If this is possible, current genetic engineering, which is limited by the number of naturally occurring bases, can be replaced by new techniques using non-natural base pairing systems.
- Non-patent Documents 6-7 The first attempt to create an unnatural base pair was performed by Benner et al. (Non-patent Documents 6-7). They have developed several unnatural base pairs, such as isoguanine-isocytosine (isoG-isoC) and xanthosine-diaminopyrimidine, with hydrogen bonding patterns that differ from those of natural base pairs. Recently, these non-natural base pairs have been applied to PCR amplification of DNA fragments containing the base pairs (Non-patent Documents 8-9) and sequence analysis (Non-patent Document 10). However, fidelity is not relatively high and / or a cumbersome method is required.
- Non-patent Documents 11-12 Synthesized a hydrophobic base having a form similar to that of a natural base but lacking the ability to form a hydrogen bond in base pairing. These hydrophobic bases were selectively recognized by DNA polymerase, suggesting that geometrical complementarity between base pairs is more important in replication than in hydrogen bonding interactions. . Recently, a series of hydrophobic base pairs were developed by Romesberg et al., And these base pairs were complementarily introduced into DNA by Klenow fragment of DNA polymerase I derived from E. coli (Non-patent Documents 13-15). . However, non-specific introduction of hydrophobic bases between hydrophobic bases occurred in replication without following shape complementarity (Non-patent Document 14).
- Non-patent Documents 19-21 Although this specific transcription can be put into practical use as a technique for developing functional RNA molecules (Non-patent Documents 19-21), the selectivity of sy and vy base pairs in replication is limited in transcription. Compared with selectivity, it is not so high (Non-Patent Documents 16 and 18).
- Ds-Pn base pair (Ds is 5-amino-7- (2-thienyl) -3H-imidazo [4,5-b] pyridin-3-yl group, Pa is 2-formyl-1H-pyrrole-1 -Yl group and Pn each mean 2-nitro-1H-pyrrol-1-yl group)
- Patent Document 4 and Non-Patent Documents 23 and 24
- the artificial base pairs of Benner et al. And EraGen have low selectivity in replication, the number of PCR cycles is limited, and it is difficult to detect trace amounts of DNA.
- Hirao et al.'S artificial base pair has high selectivity, but a special substrate must be used for replication, and PCR amplification efficiency is not so high.
- the conservation rate of artificial bases in DNA in one cycle of PCR amplification was 97.5% for Benner et al. PZ base pairs, ⁇ 96% for EraGen isoG-isoC base pairs, and the inventors previously The developed Ds-Pa and Ds-Pn base pairs are ⁇ 99% each.
- artificial base pairs do not exist. Therefore, PZ and isoG-isoC base pairs are difficult to apply to various techniques using nucleic acid replication / amplification reactions using a small amount of DNA. In addition, there is no report on the practical use level of DNA sequencing methods including these artificial base pairs.
- a specially modified substrate ⁇ -amide triphosphate derivative
- the position of the artificial base pair in the DNA can be confirmed by sequencing, but depending on the sequence of the natural base pair in the vicinity of the artificial base pair, the sequencing result may be disturbed. There was room for improvement.
- the present inventors have found that the combination of the 1-propynyl derivative of the artificial base Pn and the artificial base Ds is an artificial base pair Ds-Pa or the like developed so far.
- the inventors found that the present invention has high selectivity in a nucleic acid amplification reaction as compared with the combination of Ds-Pn and arrived at the present invention (Ds is 5-amino-7- (2-thienyl) -3H-imidazo [4 , 5-b] pyridin-3-yl group, Pn means 2-nitro-1H-pyrrol-1-yl group, and Pa means 2-formyl-1H-pyrrol-1-yl group).
- Ds and A modified substrates can achieve PCR amplification with high selectivity (I. Hirao, et al., Nature Methods, 3: 729-735 (2006) ).
- the artificial base Pa is changed to the artificial base Pn, it is found that the unfavorable base pair A-Pn is less likely to be formed than A-Pa, and without using the modified substrate of A ( ⁇ -amide triphosphate), PCR amplification became possible (I.
- a derivative was developed in which a propynyl group, which is one of the ⁇ -electron substituents for increasing the affinity between the substrate Pn and DNA polymerase, was bonded to the 4-position of Pn.
- a Ds modified substrate ⁇ -amidotriphosphate
- PCR amplification using triphosphate as a substrate was possible.
- an artificial base to which a functional compound (amino group, fluorescent dye, biotin, etc.) is added at the end of a ⁇ -electron substituent bonded to Pn via a linker portion having various lengths Similarly, the inventors have found that the nucleic acid amplification reaction has high selectivity. As a result, the present invention has been conceived.
- the present invention provides the following aspects 1-19.
- a method of replicating nucleic acid containing artificial base pairs wherein the following formula I:
- Embodiment 2 The method according to embodiment 1, wherein the template strand is DNA containing at least two nucleotides having a base Ds and / or a base represented by Formula II.
- Embodiment 3 The method according to Embodiment 1 or 2, wherein the replication substrate is not deoxyribonucleoside 5'-triphosphate substituted with a hydroxyl group of phosphoric acid at the ⁇ -position.
- Embodiment 4 The method according to any one of Embodiments 1 to 3, wherein the fluorescent dye is carboxyfluorescein (FAM).
- FAM carboxyfluorescein
- a method for introducing an artificial base to which a functional substituent is bound into DNA by a nucleic acid replication reaction The following formula I:
- a nucleic acid comprising a nucleotide having a base represented by (hereinafter referred to as Ds) as a template strand;
- Ds a base represented by (hereinafter referred to as Ds) as a template strand;
- Embodiment 6 The method according to embodiment 5, wherein the template strand is a nucleic acid containing at least two nucleotides having a base Ds.
- Embodiment 7 The method according to Embodiment 5 or 6, wherein the replication substrate is not deoxyribonucleoside 5'-triphosphate substituted with a hydroxyl group of phosphoric acid at the ⁇ -position.
- the template strand includes a sequence of 5′-N 1 N 2 N 3 (Ds) N 4 N 5 N 6 -3 ′ (SEQ ID NO: 1) as a sequence in the vicinity of the base Ds, where N 1 , N 2 , N 3 , N 4 , N 5 , N 6 are nucleotides having a natural base, and the following conditions: (A) N 1 is thymine (T) or cytosine (C); (B) N 3 is cytosine (C); (C) N 4 is thymine (T); (D) N 5 is thymine (T) or cytosine (C); and (e) N 6 is guanine (G); The method according to any one of aspects 5 to 7, wherein at least two conditions selected from the group consisting of:
- Embodiment 9 The method according to embodiment 8, wherein the fluorescent dye is carboxyfluorescein (FAM).
- FAM carboxyfluorescein
- a method for replicating and selectively recovering a nucleic acid containing an artificial base pair from a nucleic acid pool (1) The following formula I:
- Embodiment 11 The method according to embodiment 10, wherein the nucleic acid containing a nucleotide having a base Ds contains at least two nucleotides having a base Ds.
- Embodiment 12 The method according to embodiment 10 or 11, wherein the replication substrate is not deoxyribonucleoside 5'-triphosphate substituted with a hydroxyl group of phosphoric acid at the ⁇ -position.
- Aspect 13 In a nucleic acid comprising a nucleotide having base Ds, the sequence of 5′-N 1 N 2 N 3 (Ds) N 4 N 5 N 6 -3 ′ (SEQ ID NO: 1) is used as the sequence in the vicinity of base Ds.
- N 1 , N 2 , N 3 , N 4 , N 5 , N 6 are nucleotides having a natural base, and the following conditions: (A) N 1 is thymine (T) or cytosine (C); (B) N 3 is cytosine (C); (C) N 4 is thymine (T); (D) N 5 is thymine (T) or cytosine (C); and (e) N 6 is guanine (G); The method according to any one of Embodiments 10 to 12, wherein at least two conditions selected from the group consisting of:
- Embodiment 14 The method according to embodiment 13, wherein the fluorescent dye is carboxyfluorescein (FAM).
- FAM carboxyfluorescein
- a method for determining the sequence of a natural base in the vicinity of an artificial base in DNA for realizing highly efficient and highly selective replication of a nucleic acid containing the artificial base (1) A DNA library containing a random region represented by 5 ′-(N) n (N u1 ) (N) m -3 ′ (SEQ ID NO: 2) is prepared, wherein n and m are independent of each other.
- N u1 is the first artificial base; (2) a nucleoside having a second unnatural base N u2 which forms an N u1 and artificial base pair performs replication reaction of a nucleic acid to the DNA library using the replication substrate, wherein, N u2 is Contains functional functional groups; (3) recovering a nucleic acid into which a functional substituent has been introduced by the formation of an artificial base pair of N u1 and N u2 based on the nature of the functional substituent; (4) repeating steps (2) and (3) for the nucleic acid recovered in (3); and (5) determine the sequence of the nucleic acid obtained; Said method.
- a method for determining the sequence of a natural base in the vicinity of an artificial base in DNA for realizing highly efficient and highly selective replication of a nucleic acid containing the artificial base (1) The following formula I:
- Z is a function selected from the group consisting of fluorescent dyes, biotin, compounds that bind to antibodies, photo
- Embodiment 17 The method according to embodiment 16, wherein the replication substrate is not deoxyribonucleoside 5'-triphosphate in which the hydroxyl group of phosphoric acid at the ⁇ -position is substituted.
- Embodiment 18 A nucleic acid obtained by the method according to Embodiment 16 or 17.
- N 1 , N 2 , N 3 , N 4 , N 5 , N 6 are nucleotides having natural bases, and the following conditions: (A) N 1 is thymine (T) or cytosine (C); (B) N 3 is cytosine (C); (C) N 4 is thymine (T); (D) N 5 is thymine (T) or cytosine (C); and (e) N 6 is guanine (G);
- a derivative in which a substituent having a ⁇ -electron system was bonded to the 4-position of the artificial base Pn and an artificial base pair of the artificial base Ds were developed.
- all the bases (Ds, Pn derivatives, A, G, C, and T bases) in the nucleic acid replication reaction containing the artificial base are unmodified nucleotides 5′-triphosphorus. Acids can also be used as replication substrates.
- the conservation rate in the nucleic acid replication reaction of the artificial base pair of the present invention is very high, it can be applied to various nucleic acid replication / amplification techniques.
- a functional substituent can be added to the tip of a substituent having a ⁇ -electron system bonded to Pn via a linker moiety having various lengths, the function can be performed using the artificial base of the present invention. It has become possible to introduce sex substituents into DNA regioselectively and to replicate the introduced DNA itself.
- FIG. 1 is a diagram showing the structures of Ds, Pn, NH 2 -hx-Pn, and FAM-hx-Pn.
- FIG. 2 is a schematic diagram of a single nucleotide insertion experiment using Klenow fragment and a graph showing the results.
- FIG. 3 is a schematic diagram of an experiment for determining the sequence of a natural base in the vicinity of an artificial base in DNA for realizing highly efficient and highly selective PCR amplification of DNA containing the artificial base, and a table showing the results and It is a sequencing peak pattern.
- FIG. 4 is a sequencing peak pattern when ddPa′TP and dPa′TP are used in the sequencing reaction.
- FIG. 5A is a schematic diagram of an experiment for introducing FAM-hx-dPnTP into DNA by PCR amplification, and an electrophoretic photograph showing the results.
- FIG. 5-2 is a schematic diagram of an experiment in which NH 2 -hx-dPnTP is introduced into DNA by PCR amplification, and a sequencing peak pattern showing the result of analyzing the sequence of the amplified DNA.
- FIG. 6 is a schematic diagram of a PCR amplification experiment of DNA containing a plurality of artificial bases, and electrophoresis showing the result is a sequencing peak pattern showing the result of analyzing the sequence of new and amplified DNA.
- FIG. 7 is a schematic diagram of a PCR amplification and isolation experiment of a DNA fragment containing Ds under conditions where foreign DNA is mixed, and a sequencing peak pattern of the amplified nucleic acid at each stage of the experiment.
- FIG. 8 is a schematic diagram of a PCR amplification and isolation experiment of a DNA fragment containing Ds under a condition where foreign DNA is mixed, and a sequencing peak pattern of a nucleic acid containing an amplified and isolated artificial base pair.
- the artificial base pair of the present invention is a base pair formed by an artificial base Ds and a derivative of the artificial base Pn.
- the artificial base Pn derivative has the following formula II:
- X is selected from the group consisting of —C ⁇ C—CH 2 —, —C ⁇ C—, —C ⁇ C—, aryl, thienyl, imidazolyl, and thiazolyl, preferably —C ⁇ C—CH 2 — or —C ⁇ C—, more preferably —C ⁇ C—CH 2 —; Y represents -CH 3 , -C 2 H 5 , -NH 2 , -OH, -COOH, -CHO, -SH, substituted or unsubstituted aryl, -NHCO-Z, -CONH-Z, -
- a substituent having a ⁇ electron system is selected as the substituent X in order to increase the affinity with DNA polymerase or reverse transcriptase.
- the —NHCO— (CH 2 ) n — and —NHCO— (CH 2 ) m —NHCO— (CH 2 ) 1 — moieties of the substituent R are linker moieties.
- the substituent Y may be a functional substituent moiety.
- the functional substituent means a functional group that can undergo chemical modification, a reactive functional group that can participate in a chemical reaction, a labeling substance that enables detection, and a molecule that can be captured and separated. It refers to a substituent having a certain function such as a functional group.
- Examples of when the substituent Y includes a functional group capable of undergoing chemical modification include when the substituent Y is —NH 2 , —OH, —COOH, —CHO, —SH, and Examples include a case where Y contains an amino acid as a substituent Z.
- Examples of the case where the substituent Y includes a reactive functional group capable of participating in a chemical reaction include the case where the substituent Y includes a chelating agent as the substituent Z. Since the chelating agent can participate in the cleavage of a nucleic acid or protein chain existing in the vicinity thereof, a new function can be imparted to the nucleic acid having an artificial base.
- the substituent Y includes a labeling substance that enables detection or a functional group that enables capture and separation of molecules
- the substituent Y binds to a fluorescent dye, biotin, or antibody as the substituent Z.
- fluorescent dye biotin
- examples include compounds, photocrosslinking agents, chelating agents, amino acids or peptides.
- the fluorescent dye is not particularly limited as long as it is a molecule that emits fluorescence, but preferably 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5-carboxytetramethylrhodamine.
- a nucleotide or nucleoside having a base of the formula II having a fluorescent dye can detect a nucleic acid according to the type of the fluorescent dye.
- FAM has an absorption maximum wavelength of 493 nm and a fluorescence maximum wavelength of 522 nm.
- TAMRA has an absorption maximum wavelength of 553 nm and a fluorescence maximum wavelength of 578 nm.
- DANSYL has an absorption maximum wavelength of 335 nm and a fluorescence maximum wavelength of 518 nm.
- HEX has an absorption maximum wavelength of 535 nm and a fluorescence maximum wavelength of 556 nm.
- the absorption maximum wavelength of TET is 521 nm, and the fluorescence maximum wavelength is 536 nm.
- the absorption maximum wavelength of 5-ROX is 567 nm, and the fluorescence maximum wavelength is 591 nm.
- 6-ROX has an absorption maximum wavelength of 570 nm and a fluorescence maximum wavelength of 590 nm.
- Biotin is also called coenzyme R and is a kind of vitamin B group. Biotin is known to specifically bind to avidin, a glycoprotein contained in egg white, to form a complex. Thus, nucleotides and nucleosides that have biotin as a substituent specifically bind to avidin protein. For this reason, since a nucleic acid containing a nucleotide to which biotin is bound can be bound to a carrier to which avidin is bound, it can be used to capture and separate the nucleic acid.
- the compound that binds to the antibody is not particularly limited as long as it is a substance that binds to the antibody.
- Examples of the compound that binds to the antibody include digoxigenin, ascorbic acid, benzopyrene, and the like.
- the photocrosslinking agent is not particularly limited as long as it is a substance that causes a crosslinking reaction by light irradiation.
- Examples of the photocrosslinking agent include benzophenone, azide, trifluoromethyldiazilinyl, and the like.
- the amino acid may be 20 naturally occurring ⁇ -amino acids or non-natural amino acids.
- Peptide refers to a substance in which two or more amino acids are bonded through an amide bond.
- the number of amino acids constituting the peptide is not particularly limited, but may be preferably 2 to 15, more preferably 2 to 10, and further preferably 2 to 8.
- the chelating agent is not particularly limited as long as it coordinates with a metal ion, a radioactive substance, or the like as a ligand.
- chelating agents include nitrilotriacetic acid (NTA), trans-1,2-cyclohexadiamine-N, N, N ′, N′-tetraacetic acid (CyDTA), ethylenediaminetetraacetic acid (EDTA), and the like. It is done.
- the artificial base Pn derivative of the present invention comprises NH 2 -hx-Pn: 4- [3- (6-aminohexanamide) -1-propynyl] -2-nitropyrrol-1-yl group; or FAM-hx-Pn: 4- [3- [6- (Fluorescein-5-carboxamido) hexanamide] -1-propynyl] -2-nitropyrrol-1-yl group; It is.
- the present invention relates to a method of replicating nucleic acid containing artificial base pairs, which is a template strand that is a nucleic acid containing nucleotides having Ds and / or Pn derivatives as bases.
- nucleic acid replication reaction using a substituted or unsubstituted deoxyribonucleoside 5′-triphosphate having a base Ds, a base Pn derivative, and / or a natural base as a replication substrate, the base Ds and the above formula
- the above-described method is provided for replicating a nucleic acid containing an artificial base pair of a base represented by II.
- nucleoside means a glycoside compound in which a nucleobase and a reducing group of a sugar are bonded by a glycosidic bond.
- the “nucleobase” includes natural bases such as adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), derivatives thereof, and artificial bases. It is a concept.
- nucleotide refers to a compound in which the sugar moiety of the nucleoside is made of phosphoric acid and an ester. More preferred are mono-, di-, or triphosphates.
- the template strand is a nucleic acid containing a nucleotide having a Ds and / or Pn derivative as a base.
- the template strand nucleic acid may be DNA or RNA.
- the template strand may have only one Ds or Pn derivative, or may have at least two.
- the template strand may also have Ds and Pn derivatives on the same template strand.
- the upper limit of the number of Ds and / or Pn derivatives that the template chain may have is not particularly limited, and may be, for example, 20, 15, 10, or 5.
- the replication substrate used in the method of the present invention is Ds, a Pn derivative, and / or a substituted or unsubstituted deoxyribonucleoside 5'-triphosphate having a natural base.
- Ds deoxyribonucleoside 5'-triphosphate
- Pn derivative a Pn derivative
- Ds-Pn derivative since the conservation rate of base pair formation of the Ds-Pn derivative is high, it is possible to use unsubstituted deoxyribonucleoside 5'-triphosphate as a replication substrate. Substituted deoxyribonucleoside 5'-triphosphate may also be used.
- the hydroxyl group of the phosphoric acid at the ⁇ -position was substituted with a group selected from the group consisting of an amino group, a methylamino group, a dimethylamino group, a mercapto group and a fluoro group Derivatives, derivatives in which a fluorescent dye is bound to phosphoric acid at the ⁇ position, derivatives where the phosphoric acid at the ⁇ position is thiophosphoric acid, and the like.
- the substituted deoxyribonucleoside 5'-triphosphate does not include a derivative in which the hydroxyl group of phosphoric acid at the ⁇ -position is substituted with an amino group, that is, a ⁇ -amido triphosphate derivative.
- the nucleic acid replication reaction means a reaction that enzymatically generates a complementary strand to the template strand nucleic acid.
- the nucleic acid replication reaction includes a reaction with DNA polymerase or reverse transcriptase.
- DNA polymerase it is preferable to use a DNA polymerase having exonuclease activity in order to prevent unspecific base pairing which is not preferable in nucleic acid replication.
- the DNA polymerase having exonuclease activity is selected from the group consisting of Klenow fragment, T4 DNA polymerase, Vent DNA polymerase, and DeepVent DNA polymerase having 3 ' ⁇ 5' exonuclease activity.
- reverse transcriptase examples include AMV Reverse Transcriptase XL (AMV-RT) (Life Science), M-MLV Reverse Transcriptase (Promega), HIV Reverse Transcriptase (Applied Biosystems). .
- AMV-RT AMV Reverse Transcriptase XL
- M-MLV Reverse Transcriptase Promega
- HIV Reverse Transcriptase Applied Biosystems.
- the replication reaction in the method of the present invention can be performed according to a known method.
- the nucleic acid replication method of the present invention has a high conservation rate of base pair formation of Ds-Pn derivatives. Template strands present at the 10 ⁇ 20 mol level can also be amplified and detected in a manner that preserves the base pairing of the Ds-Pn derivative by the nucleic acid replication method of the present invention.
- the present invention provides a method for introducing an artificial base to which a functional substituent is bound into DNA using the nucleic acid replication method of the present invention. That is, the present invention is a method for introducing an artificial base to which a functional substituent is bound into DNA by a nucleic acid replication reaction, and a replication substrate for a template strand that is a nucleic acid containing a nucleotide having Ds as a base.
- a nucleic acid containing an artificial base pair is generated, whereby an artificial base to which a functional substituent is bound is introduced into DNA.
- the artificial base Pn derivative of the present invention has a functional substituent represented by substituent Y in the above structure.
- An artificial nucleic acid having a functional substituent attached thereto by generating a nucleic acid containing an artificial base pair of a Ds-Pn derivative by a nucleic acid replication method of the present invention on a template strand that is a nucleic acid containing a nucleotide having Ds as a base.
- Bases can be introduced site-specifically into DNA.
- the artificial base Pn derivative of the present invention is selected from the group consisting of fluorescent dyes, biotin, compounds that bind to antibodies, photocrosslinkers, chelating agents, amino acids, and peptides. May have a functional substituent.
- a Pn derivative has these functional substituents, a nucleic acid containing an artificial base pair of the Ds-Pn derivative generated by the nucleic acid replication method of the present invention is selectively detected based on the properties of these functional substituents. And / or can be recovered.
- examples of the artificial base having a functional substituent capable of detecting and / or recovering a nucleic acid containing an artificial base pair include a fluorescent dye, biotin as the substituent Z in the Pn derivative represented by the formula II And a compound having an antibody binding, a photocrosslinking agent, a chelating agent, an amino acid, or a peptide.
- a fluorescent dye When a fluorescent dye is used as a functional substituent, it is possible to detect a nucleic acid according to the type of the fluorescent dye. It can also be recovered by selectively capturing and separating nucleic acids containing artificial base pairs of Ds-Pn derivatives produced by the nucleic acid replication method of the present invention using an antibody that binds to a fluorescent dye. .
- a nucleic acid containing an artificial base pair of a Ds-Pn derivative generated by the nucleic acid replication method of the present invention is selectively captured and utilized using a specific biotin-avidin bond. It can collect
- the nucleic acid containing the artificial base pair of the Ds-Pn derivative produced by the nucleic acid replication method of the present invention is selectively captured and utilized by binding to the antibody. It can collect
- a nucleic acid having a compound that binds to the antibody as a functional substituent can be detected by a technique such as ELISA.
- the nucleic acid containing an artificial base pair of the Ds-Pn derivative produced by the nucleic acid replication method of the present invention by light irradiation is crosslinked with a carrier, etc. It can be recovered by capture and separation.
- the nucleic acid containing the artificial base pair obtained by the method of the present invention is selectively captured and separated by utilizing the binding with an antibody that binds to the amino acid, and recovered. be able to. Further, by utilizing the binding with an antibody, a nucleic acid having an amino acid as a functional substituent can be detected by a technique such as ELISA.
- a chelating agent When having a chelating agent as a functional functional group, it can be recovered by capturing and separating a nucleic acid containing an artificial base via an appropriate ligand. It is also possible to detect a nucleic acid containing an artificial base via an appropriate ligand.
- a substance that binds to the peptide is used to selectively capture and separate a nucleic acid containing an artificial base pair of a Ds-Pn derivative generated by the nucleic acid replication method of the present invention. By this, it can be recovered.
- the substance that binds to the peptide is not particularly limited, but may be an antibody.
- a nucleic acid having an artificial base pair can be detected by a technique such as ELISA.
- Specific examples of combinations of peptides and substances that bind to them include the following combinations.
- the substance that binds to the peptide can be Ni-NTA.
- the substance that binds to the peptide can be glutathione-S-transferase.
- the peptide is a FLAG tag (DYKDDDDK (SEQ ID NO: 58)) or MYC tag (EQKLISEEDL (SEQ ID NO: 59)
- the substance that binds to the peptide is an antibody that binds to the tag.
- the nucleic acid containing the artificial base pair of the generated Ds-Pn derivative is selectively detected and / Or can be recovered.
- the present invention relates to a method for replicating and selectively recovering a nucleic acid containing an artificial base pair from a nucleic acid pool, wherein the nucleic acid pool contains a nucleic acid containing a nucleotide having an artificial base Ds.
- the nucleic acid replication reaction is carried out by the nucleic acid replication method of the present invention; and the functionality of the Pn derivative having a nucleic acid containing an artificial base pair of an artificial base Ds and an artificial base Pn derivative among the generated nucleic acids. Selectively recovering based on the nature of the substituents.
- the present invention relates to a method for replicating and selectively detecting a nucleic acid containing an artificial base pair from a nucleic acid pool, wherein the nucleic acid pool contains a nucleic acid containing a nucleotide having an artificial base Ds.
- the nucleic acid replication reaction is carried out by the nucleic acid replication method of the present invention; and the functionality of the Pn derivative having a nucleic acid containing an artificial base pair of an artificial base Ds and an artificial base Pn derivative among the generated nucleic acids.
- the nucleic acid pool refers to an assembly of a plurality of types of nucleic acids. There are no limitations on the sequence of nucleic acids and their lengths contained in the nucleic acid pool, and they may have various sequences and various lengths.
- the nucleic acid pool contains nucleic acids comprising nucleotides having at least one artificial base Ds.
- the method of the present invention is capable of replicating and selectively detecting and / or recovering a nucleic acid containing an artificial base pair from a nucleic acid pool that is a collection of nucleic acids having various sequences and various lengths. Therefore, the present invention can be applied to authentication techniques using nucleic acids into which artificial bases are introduced. For example, as a proof that the product is genuine, a nucleic acid containing an artificial base is incorporated into a tag of the product together with a large amount of foreign DNA, and the nucleic acid containing the incorporated artificial base is amplified to a detectable level.
- the present invention can be applied to a technique for determining authenticity by detecting and / or recovering a nucleic acid containing a base and confirming the sequence.
- N 1 , N 2 , N 3 , N 4 , N 5 , N 6 are nucleotides having a natural base, and the following conditions: (A) N 1 is thymine (T) or cytosine (C); (B) N 3 is cytosine (C); (C) N 4 is thymine (T); (D) N 5 is thymine (T) or cytosine (C); and (e) N 6 is guanine (G); There is also provided a method further comprising using a template strand that satisfies at least two conditions selected from the group consisting of: Preferably, a template strand that satisfies the above conditions (a) to (e) of at least 3 or more, more preferably 4 or more, and even
- 5′-N 1 ′ N 2 ′ N 3 ′ (Pn derivative) N 4 ′ N 5 ′ N 6 ′ ⁇ is used as the sequence in the template strand in the vicinity of the artificial base Ds.
- N 1 ′ is cytosine (C);
- B N 2 ′ is adenine (A) or guanine (G);
- C N 3 ′ is adenine (A);
- D N 4 ′ is guanine (G);
- e N 6 ′ is adenine (A) or guanine (G);
- a method further comprising using a template strand that satisfies at least two conditions selected from the group consisting of:
- a template strand that satisfies the above conditions (a) to (e) of at least 3 or more, more preferably 4 or more, and even more preferably all 5 may be used.
- the present invention also relates to a nucleic acid containing a nucleotide having an artificial base Ds, and 5′-N 1 N 2 N 3 (Ds) N 4 N 5 N 6 ⁇ 3 ′ ( SEQ ID NO: 1), wherein N 1 , N 2 , N 3 , N 4 , N 5 , N 6 are nucleotides having a natural base, and the following conditions: (A) N 1 is thymine (T) or cytosine (C); (B) N 3 is cytosine (C); (C) N 4 is thymine (T); (D) N 5 is thymine (T) or cytosine (C); and (e) N 6 is guanine (G);
- it may be a nucleic acid that satisfies the above conditions (a) to (e) of at least 3 or more, more preferably 4 or more, and even more preferably all 5.
- the present invention provides a nucleic acid containing a nucleotide having an artificial base Pn derivative, which is a complementary strand of a nucleic acid containing a nucleotide having the artificial base Ds, and has a 5′-N as a sequence in the vicinity of the artificial base Pn derivative.
- N 1 ′ N 2 ′ N 3 ′ (Pn derivative) comprising the sequence N 4 ′ N 5 ′ N 6 ′ ⁇ 3 ′ (SEQ ID NO: 4), where N 1 ′ , N 2 ′ , N 3 ′ , N 4 ′ , N 5 ′ , and N 6 ′ are nucleotides having a natural base, and the following conditions: (A) N 1 ′ is cytosine (C); (B) N 2 ′ is adenine (A) or guanine (G); (C) N 3 ′ is adenine (A); (D) N 4 ′ is guanine (G); and (e) N 6 ′ is adenine (A) or guanine (G);
- it may be a nucleic acid that satisfies the above conditions (a) to (e) of at least 3 or more, more preferably 4 or more, and even more
- Hirao et al. is a substrate of a propynyl derivative of Pa which is a complementary base of Ds (dPa'TP: 1- (2-deoxy- ⁇ -D-ribofuranosyl) -4-
- dPa'TP 1- (2-deoxy- ⁇ -D-ribofuranosyl) -4-
- a method utilizing the difference in sequencing patterns with and without (1-propynyl) pyrrole-2-carbaldehyde 5′-triphosphate has been reported.
- a peak pattern is obtained in which the sequence reaction stops at the base portion complementary to Ds in the template DNA. .
- the didinoxy derivative substrate (ddPa′TP: 1- (2,3-dideoxy- ⁇ ) modified with propynyl group is used.
- ddPa′TP 1- (2,3-dideoxy- ⁇ ) modified with propynyl group.
- a method for determining a base sequence of a DNA fragment containing an artificial base Ds using -D-ribofuranosyl) -4- (1-propynyl) pyrrole-2-carbaldehyde (5'-triphosphate).
- the present invention relates to a method for determining the sequence of a nucleic acid containing an artificial base.
- a method for determining the sequence of a natural base in the vicinity of an artificial base in DNA for realizing efficient and highly selective replication is provided.
- the present invention is a method for determining the sequence of a natural base in the vicinity of an artificial base in DNA for realizing highly efficient and highly selective replication of a nucleic acid containing the artificial base, (1)
- a DNA library containing a random region represented by 5 ′-(N) n (N u1 ) (N) m -3 ′ (SEQ ID NO: 2) is prepared, wherein n and m are independent of each other.
- N u1 is the first artificial base; (2) a nucleoside having a second unnatural base N u2 which forms an N u1 and artificial base pair performs replication reaction of a nucleic acid to the DNA library using the replication substrate, wherein, N u2 is Contains functional functional groups; (3) recovering a nucleic acid into which a functional substituent has been introduced by the formation of an artificial base pair of N u1 and N u2 based on the nature of the functional substituent; (4) repeating steps (2) and (3) for the nucleic acid recovered in (3); and (5) determine the sequence of the nucleic acid obtained; Providing the method.
- the combination of N u1 and N u2 artificial base pairs is not particularly limited as long as it is a combination of artificial base pairs, isoG-isoC (Patent Document 3 and Non-Patent Document 9); PZ (P: 2-amino-imidazo [1,2-a] -1,3,5-triazin-4 (8H) -one, Z: 6-amino-5-nitro-2 (1H) -pyridone (Non-Patent Document 22); sy (s: 2-amino-6- (2-thienyl) purine, y: 2-oxopyridine) (Non-patent Documents 16-17); vy (v: 2-amino-6- (2-thiazolyl) purine, y: 2-oxopyridine) (Non-patent Document 18); Ds-Pa; Ds-Pn; and Ds-Pn derivatives; The combination of is mentioned. It will be easily understood by those skilled in the art that when the former of the above
- N u1 is Ds and N u2 is a Pn derivative represented by the formula II herein.
- the step of determining the sequence of the obtained nucleic acid can be performed using any method capable of determining the sequence of a nucleic acid containing an artificial base.
- the combination of the N u1 and N u2 artificial base pairs is a combination of Ds-Pa, Ds-Pn, or Ds-Pn derivative
- a nucleic acid obtained by preferably using the method using ddPa′TP described above The sequence may be determined.
- the present invention also provides a nucleic acid obtained by the method for determining the sequence of a natural base in the vicinity of an artificial base in DNA for realizing highly efficient and highly selective replication of the nucleic acid containing the artificial base of the present invention. provide.
- Example 1 Analysis of reaction rate constant of single nucleotide insertion reaction with Klenow fragment
- Nucleotide insertion experiment using Klenow fragment was performed and the reaction rate constant was analyzed.
- each substrate ie, nucleoside triphosphate or ⁇ -amide triphosphate solution (base is NH 2 -hx-Pn, Pn, Pa, Ds, A, G, C, or T) (1 ⁇ M-5 mM) was added, and the enzyme reaction was carried out at 37 ° C. (1-28.2 minutes). The reaction was stopped by adding 95% formamide solution (stop solution) containing 10 ⁇ l of 20 mM EDTA and heating at 75 ° C. for 3 minutes.
- base NH 2 -hx-Pn, Pn, Pa, Ds, A, G, C, or T
- reaction solution (10 ⁇ l) 1 ⁇ M or 5 ⁇ M primer / template duplex, 2-50 nM enzyme, and 0.3-1500 ⁇ M substrate were used.
- the reaction solution (10 ⁇ l) also contains 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM DTT and 0.05 mg / ml BSA.
- Run Module is GS Run 36C-2400. The electrophoresis time was about 1 hour, and the peak pattern of the reaction product was analyzed and quantified with an automatic ABI377 DNA sequencer equipped with GeneScan software (version 3.0).
- Vmax was obtained by standardizing the enzyme concentration to 20 nM and the double-stranded DNA concentration to 5 ⁇ M.
- the results are shown in FIG.
- the incorporation efficiency of Pn substrate with respect to Ds in the template strand was 3.7 ⁇ 10 5 .
- a substrate of a Pn derivative having a propynyl group NH 2 -hx-dPnTP
- the incorporation efficiency for Ds in the template chain becomes 7.4 ⁇ 10 5
- the Pn substrate for Ds in the template chain About 2 times higher than the uptake efficiency.
- the uptake efficiency of the Ds substrate with respect to Ds in the template strand was 2.0 ⁇ 10 5 , and the uptake efficiency with respect to Ds of the Pn derivative substrate having a propynyl group was higher. Furthermore, it was also found that the efficiency of incorporation of a Pn derivative substrate having a propynyl group into a natural base is lower than that of Ds. Therefore, artificial base pairs of Ds and Pn derivatives are formed more efficiently than unfavorable base pairs of Ds-Ds and A-Pn in replication.
- the substrate of the Pn derivative having the propynyl group which is a ⁇ -electron substituent can be selectively incorporated into Ds in the template strand DNA more efficiently than the substrate of the artificial base Pn.
- a ⁇ -amido triphosphate derivative of Ds was used to prevent the substrate Ds from being incorporated into the template Ds.
- the efficiency with which the substrate of the Pn derivative is incorporated into the template Ds was higher than the efficiency with which the substrate Ds was incorporated into the template Ds.
- Example 2 Formation of an artificial base pair between a template base and a substrate in sequencing replication of a natural base in the vicinity of an artificial base in DNA to realize highly efficient and highly selective PCR amplification of DNA containing the artificial base It is hypothesized that the efficiency and selectivity of DNA depends on the sequence of natural bases in the vicinity of artificial base pairs in DNA, and artificial base pairs efficiently amplified by PCR and the natural bases in the vicinity The sequence of was examined using in vitro evolution engineering (in vitro selection method).
- a DNA fragment containing Ds is a single-stranded DNA (55-mer; 5′-TTTCACACAGGAAACAGCTATGACGG-NNN-Ds-NNN-CCCTATAGTGAGTCGTATTATC-3 ′) in which 3 bases on both sides of the artificial base Ds are randomly selected.
- SEQ ID NO: 9 was chemically synthesized with a DNA synthesizer and purified by gel electrophoresis.
- PCR reaction scale 200 ⁇ l, and the composition of the reaction buffer is 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% TritonX-100. is there.
- the 5 ′ primer (5′-GATAATACGACTCACTATAG-3 ′ (SEQ ID NO: 10)) and the 3 ′ primer (5′-TTTCACACAGGAAACAGCTATGAC-3 ′ (SEQ ID NO: 11)) each had a concentration of 1 ⁇ M, and each of the natural bases Substrate dNTP (0.3 mM) and FAM-hx-dPnTP (2.5 ⁇ M) and dDsTP (50 ⁇ M) were used as substrates for the artificial base. PCR conditions were 94 ° C for 30 seconds, 45 ° C for 30 seconds, and 65 ° C for 4 minutes for one cycle. After 10 cycles of PCR amplification, the full-length PCR product was purified by electrophoresis through a 10% PAGE-7M urea gel, and the concentration of the recovered DNA was calculated from the absorbance at 260 nm.
- the amplified DNA fragment (corresponding to about 20 pmol for single-stranded DNA) and 20 ⁇ l of anti-FAM antibody (1 mg / ml, purchased from Invitrogen) were mixed in phosphate buffer (PBS, final volume 100 ⁇ l) on ice. Left for 1 hour.
- PBS phosphate buffer
- This solution was subjected to ultrafiltration using Millipore Microcon YM-100, and a DNA fragment amplified by incorporating FAM-hx-Pn bound to the anti-FAM antibody was isolated.
- This solution was treated with phenol / chloroform, and DNA was recovered by ethanol precipitation of the aqueous layer. About 1 pmol of the total amount of DNA thus obtained was used as a template for the next selection round PCR (200 ⁇ l scale, reaction conditions are the same as those described above).
- PCR amplification and subsequent isolation of the fragment incorporating FAM-hx-Pn was taken as one round, and a total of five rounds of selection were performed.
- PCR was performed using the DNA fragment obtained after 5 rounds, and the base sequence of the obtained DNA fragment was analyzed by the following method 1 and method 2.
- the DNA sequencing reaction was carried out in a total volume of 20 ⁇ l on a primer (4 pmol, 5′-CGTTGTAAAACGACGGCCAG-3 ′) (SEQ ID NO: 13) in 8 ⁇ l of Cycle Sequencing Mix of a commercially available BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems). And a PCR-amplified DNA fragment (approximately 0.3 pmol) and dPa′TP (40 pmol) were added, and 25 cycles of PCR (96 ° C. for 10 seconds, 50 ° C. for 5 seconds, 60 ° C. for 4 minutes) were performed.
- Method 2 After 5 rounds of PCR and selection, a portion of the obtained DNA was premixed using Premix Ex Taq (Takara), and 8 cycles of PCR (94 ° C. for 30 seconds, without adding an artificial base substrate) 45 ° C. for 30 seconds and 72 ° C. for 1 minute), and the PCR product was used for cloning with TOPO TA Cloning Kit Dual Promoter (Invitrogen). Individual clones were subjected to a normal sequencing reaction using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), and a random sequence region of 3 bases was analyzed with a DNA sequencer (Model 3100, Applied Biosystems). The sequence was obtained.
- PCR was performed for 15 cycles using a primer labeled with 32 P at the 5 ′ end, and the product was analyzed by gel electrophoresis.
- the PCR reaction scale is 25 ⁇ l or 50 ⁇ l, and the composition of the reaction solution is 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% TritonX-100.
- the 5′-primer (5′-CGTTGTAAAACGACGGCCAGGATAATACGACTCACTATAG-3 ′ (SEQ ID NO: 12)) and the 3′-primer (5′-TTTCACACAGGAAACAGCTATGAC-3 ′ (SEQ ID NO: 11)) were each used at 1 ⁇ M, and each natural base substrate dNTP ( 0.3 mM), dDsTP (50 ⁇ M) as well as FAM-hx-dPnTP (2.5 ⁇ M) and DeepVent DNA polymerase (0.02 units / ⁇ l) as substrates for the artificial base.
- the PCR cycle was 94 ° C. for 30 seconds, 45 ° C. for 30 seconds, and 65 ° C. for 4 minutes.
- the concentration of the DNA fragment used for the template is 0.6 nM.
- the PCR product after 15 cycles was electrophoresed on a 15% polyacrylamide-7M urea gel, and the amplified DNA band was detected and quantified with a bioimager FLA-7000 (Fuji Film).
- the primer was labeled with 32 P, its radioactivity was exposed to an imaging plate and analyzed.
- fluorescence derived from FAM-hx-Pn incorporated into DNA by PCR amplification was detected in a fluorescence analysis (laser: 473 nm, Y520 for filter blue excitation) mode by placing the gel directly on the stage. The results are shown in Table 1.
- the fluorescence intensity derived from the amplified DNA band was measured to examine the efficiency of incorporation of FAM-hx-dPnTP, and the intensity of 32 P derived from the amplified DNA band was measured. DNA amplification efficiency was examined. Each value was determined as a relative intensity based on the first library (Pool) used for selection.
- the former FAM fluorescence intensity measurement (Relative fluorescence intensity incorporated into DNA) depends on the abundance of artificial base pairs in the amplified DNA, and the latter radioisotope measurement (Relative efficiency of DNA fragments amplified by PCR). Shows the relative value of the total amount of amplified DNA. Therefore, the value obtained by dividing the former value by the latter value (Fluorescence intensity / Relative amplification efficiency) indicates the relative fidelity (selectivity) in PCR of the artificial base pair in each sequence.
- Ds 5′-YNC
- the PCR amplification efficiency of these DNAs was improved from 1.6 to 2.0 times.
- the uptake efficiency of FAM-hx-Pn was improved from 1.9 times to 2.9 times compared to the DNA of random sequence.
- the fidelity (selectivity) of the artificial base pair is also correlated with these values, and the selectivity and efficiency of the artificial base pair in PCR depends on the sequence of the natural base in the vicinity of the artificial base pair. I found out.
- Example 3 Method for Determining Base Sequence of DNA Containing Artificial Base
- a method for determining the base sequence of a DNA fragment containing artificial base Ds has already been reported by the present inventors (I. Hirao, et al., Nature Methods, 3 : 729-735 (2006).).
- the difference in the sequencing pattern depending on the presence or absence of a substrate (dPa′TP) of a propynyl derivative of Pa which is a complementary base of Ds was used.
- a peak pattern is obtained in which the sequence reaction stops at the base portion complementary to Ds in the template strand DNA.
- dPa′TP is added and sequencing is performed, the reaction proceeds, but the fluorescently labeled dideoxy dye terminator is not added, so only the position of the base complementary to Ds in the template strand DNA is obtained.
- a peak pattern is given in which the sequencing peaks disappear (FIG. 3b).
- the conventional method is a method for determining the sequence of DNA containing an artificial base by comparing these two sequencing patterns.
- a dideoxy derivative substrate (ddPa′TP) modified with propynyl group was synthesized according to the following scheme.
- 1- (2,3-dideoxy-5-O-trityl) - ⁇ -D-ribofuranosyl) -4- (1-propynyl) pyrrole-2-carbaldehyde (82 mg, 171 ⁇ mol) was dissolved in 80% acetic acid (10 ml) and stirred at room temperature for 3 hours and then at 50 ° C. for another 2 hours. did.
- the reaction solution was concentrated under reduced pressure and azeotroped with water.
- the obtained crude product was purified by silica gel column chromatography (0-1% CH 3 OH in CH 2 Cl 2 ) and RP-HPLC (35-80% CH 3 CN in water, 12 minutes) to obtain the desired product. (31 mg, 78%) was obtained.
- Example 4 A method of introducing an artificial base having a functional substituent bonded thereto into DNA by PCR amplification and a method for detecting and isolating the DNA ( 4-1: PCR amplification of DNA containing an artificial base ) Primers and artificial base substrates (dDsTP and FAM-hx-dPnTP or NH 2 -hx-dPnTP) were added to the Ds-containing fragment (55-mer), and PCR was carried out for 15 to 40 cycles.
- the PCR reaction scale is 25 ⁇ l or 50 ⁇ l, and the composition of the reaction solution is 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% TritonX-100. .
- 5′-side primer (5′-CGTTGTAAAACGACGGCCAGGATAATACGACTCACTATAG-3 ′ (SEQ ID NO: 12)) and 3′-side primer (5′-TTTCACACAGGAAACAGCTATGAC-3 ′ (SEQ ID NO: 11)) (each 1 ⁇ M), substrate dNTP of each natural base ( 0.3 mM), dDsTP (50 ⁇ M), FAM-hx-dPnTP (2.5 ⁇ M) or NH 2 -hx-dPnTP (50 ⁇ M), Deep Vent DNA polymerase (0.02 units / ⁇ l) was used for PCR. One cycle of PCR was performed at 94 ° C. for 30 seconds, 45 ° C. or 42 ° C.
- PCR using FAM-hx-dPnTP when the template concentration is 0.6 nM (25 ⁇ l scale, 15 fmol), the amplification product can be confirmed in 15 cycles (annealing temperature is 45 ° C.), and the template concentration is 6 fM (25 ⁇ l scale). In the case of 0.15 amol), the amplification product could be confirmed in 30 cycles (the annealing temperature was 42 ° C.). That is, in the technique using FAM-hx-dPnTP, a DNA fragment containing 15 fmol amount of Ds is amplified about 300 times by 15 cycles of PCR, and about 10 7 in 30 cycle PCR of 0.15 amol amount of DNA fragment. Amplified twice (Fig. 5-1).
- the template concentration was 0.6 fM (0.015 amol on a 25 ⁇ l scale), and amplification was performed by PCR with 40 cycles (annealing temperature: 45 ° C.) using NH 2 -hx-dPnTP. After purification of the product by gel electrophoresis, its sequence was confirmed with a DNA sequencer. For the sequencing method, see (Method 1) and Example 3 in Example 2-1. As a result, it was found that even after 40 cycles of PCR amplification, the artificial base Ds was almost completely preserved in the DNA.
- the DNA fragment containing two Ds used is shown below.
- 5′-side primer (5′-CGTTGTAAAACGACGGCCAGGATAATACGACTCACTATAG-3 ′ (SEQ ID NO: 12)) and 3′-side primer (5′-TTTCACACAGGAAACAGCTATGAC-3 ′ (SEQ ID NO: 11)) (each 1 ⁇ M), substrate dNTP of each natural base ( 0.3 mM), dDsTP (50 ⁇ M), FAM-hx-dPnTP (2.5 ⁇ M) or NH 2 -hx-dPnTP (50 ⁇ M), Deep Vent DNA polymerase (0.02 units / ⁇ l) was used for PCR. One cycle of PCR was performed at 94 ° C. for 30 seconds, 45 ° C.
- PCR products were separated by electrophoresis on a 15% polyacrylamide-7M urea gel, and the product bands were detected and quantified with Bioimager FLA-7000 (Fuji Film). Fluorescence derived from FAM-hx-Pn incorporated into DNA by PCR amplification was detected by fluorescence analysis (laser: 473 nm, Y520 for filter blue excitation) mode with the gel placed directly on the stage.
- the amplified product by PCR was purified by gel electrophoresis, followed by a sequencing reaction using a final concentration of 50 ⁇ M dPa′TP, and the sequence was confirmed by a DNA sequencer.
- a sequencing reaction using a final concentration of 50 ⁇ M dPa′TP, and the sequence was confirmed by a DNA sequencer.
- For the sequencing method see (Method 1) and Example 3 in Example 2-1.
- Example 5 PCR and sequencing of DNA fragments containing Ds under conditions where foreign DNA is mixed (1)
- FAM-hx-Pn can be incorporated into its complementary DNA, so that only a DNA fragment containing an artificial base pair can be isolated with an anti-FAM antibody.
- the primer region has the same sequence, but the other three sequences of different DNA fragments and the DNA fragment containing the artificial base Ds are present in equal amounts, The following experiment was conducted.
- DNA1 55-mer: 5'-TTTCACACAGGAAACAGCTATGACGGCCC (Ds) TTGCCCTATAGTGAGTCGTATTATC-3 '
- DNA2 55-mer: 5'-TTTCACACAGGAAACAGCTATGACACATGGAACTGCTATAGTGAGTCGTATTATC-3 '
- DNA4 55-mer
- the magnetic beads from which the solution had been removed were washed twice with a buffer solution (100 ⁇ l), and then 20 ⁇ l of 1 mM EDTA solution (pH 8.0) was added and heated at 75 ° C. for 30 seconds to elute the DNA bound to the beads.
- the sequencing reaction was performed using 10 ⁇ l of the eluted DNA solution in the presence of dPa′TP (final concentration 2 ⁇ M).
- dPa′TP final concentration 2 ⁇ M
- Example 6 PCR and sequencing of DNA fragments containing Ds under conditions where foreign DNA is present (2)
- a DNA fragment containing Ds 55-mer, 0.3 amol
- PCR amplification 30 cycles
- 10 7- fold amount of a 100-mer fragment consisting of a random sequence of natural bases 10 7- fold amount of a 100-mer fragment consisting of a random sequence of natural bases.
- An example is shown in which the base sequence of a DNA fragment containing Ds can be analyzed by isolating only a DNA fragment containing a base with an anti-FAM antibody.
- DNA fragment (55-mer) containing Ds is shown below (SEQ ID NO: 45): 5'-TTTCACACAGGAAACAGCTATGACGGCCC (Ds) TTGCCCTATAGTGAGTCGTATTATC-3 '
- This DNA fragment (final concentration: 6 fM) was mixed with 100-mer DNA (final concentration: 60 nM) consisting of a random sequence of natural bases, and FAM-hx-dPnTP (2.5 ⁇ M) and dDsTP (50 ⁇ M) were used as artificial base substrates. ), PCR (50 ⁇ l scale) for 30 cycles (94 ° C. for 30 seconds, 45 ° C. for 30 seconds, 65 ° C.
- an artificial base pair of a derivative (Pn derivative) and an artificial base Ds in which a substituent having a ⁇ -electron system is bonded to the 4-position of the artificial base Pn of the present invention For these bases (Ds, Pn derivatives, A, G, C, and T bases), an unmodified nucleotide 5′-triphosphate can also be used as a replication substrate.
- a functional substituent can be added to the Pn derivative, the functional substituent is regioselectively introduced into DNA using the artificial base of the present invention, and the introduced DNA itself is replicated. It is possible.
- the conservation rate in the nucleic acid replication reaction of the artificial base pair of the present invention is very high. Taking advantage of these features, it is expected to be applied to various nucleic acid replication / amplification technologies such as in-vitro selection method for handling a small amount of DNA and DNA authentication technology.
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Abstract
Description
nは、1ないし12から選択される整数であり;
mは、1ないし12から選択される整数であり;
lは、1ないし12から選択される整数であり;
Xは、-C≡C-CH2-、-C≡C-、-C=C-、アリール、チエニル、イミダゾリル、およびチアゾリルからなる群より選択され;
Yは、-CH3、-C2H5、-NH2、-OH、-COOH、-CHO、-SH、置換もしくは非置換アリール、-NHCO-Z、-CONH-Z、-NHCONH-Z、-O-Z、-COO-Z、-O-C(=O)-Z、-CO-Z、および、-S-Z、からなる群より選択され、ここでZは、蛍光色素、ビオチン、抗体に結合する化合物、光架橋剤、キレート剤、アミノ酸、およびペプチドからなる群より選択される]
で表される塩基を有するヌクレオチドを含む核酸である鋳型鎖に対し、複製基質として塩基Ds、前記式IIで表される塩基、および/または天然型の塩基、を有する置換または非置換デオキシリボヌクレオシド5’-三リン酸、を用いて核酸の複製反応を行うことにより、塩基Dsと前記式IIで表される塩基の人工塩基対を含む核酸を複製する、前記方法。
以下の式I:
nは、1ないし12から選択される整数であり;
mは、1ないし12から選択される整数であり;
lは、1ないし12から選択される整数であり;
Xは、-C≡C-CH2-、-C≡C-、-C=C-、アリール、チエニル、イミダゾリル、およびチアゾリルからなる群より選択され;
Yは、-CH3、-C2H5、-NH2、-OH、-COOH、-CHO、-SH、置換もしくは非置換アリール、-NHCO-Z、-CONH-Z、-NHCONH-Z、-O-Z、-COO-Z、-O-C(=O)-Z、-CO-Z、および、-S-Z、からなる群より選択され、ここでZは、蛍光色素、ビオチン、抗体に結合する化合物、光架橋剤、キレート剤、アミノ酸、およびペプチドからなる群より選択される]
で表される塩基、塩基Ds、および/または天然型の塩基、を有する置換または非置換デオキシリボヌクレオシド5’-三リン酸、を用いて核酸の複製反応を行い;
それにより、塩基Dsと前記式IIで表される塩基の人工塩基対を含む核酸が生成することにより、機能性置換基を結合した人工塩基がDNA中に導入される;
前記方法。
(a)N1がチミン(T)もしくはシトシン(C);
(b)N3がシトシン(C);
(c)N4がチミン(T);
(d)N5がチミン(T)もしくはシトシン(C);および
(e)N6がグアニン(G);
からなる群より選択される少なくとも2つ以上の条件を満たす、態様5~7のいずれか1項に記載の方法。
(1)以下の式I:
nは、1ないし12から選択される整数であり;
mは、1ないし12から選択される整数であり;
lは、1ないし12から選択される整数であり;
Xは、-C≡C-CH2-、-C≡C-、-C=C-、アリール、チエニル、イミダゾリル、およびチアゾリルからなる群より選択され;
Yは、Zで置換されたアリール、-NHCO-Z、-CONH-Z、-NHCONH-Z、-O-Z、-COO-Z、-O-C(=O)-Z、-CO-Z、および、-S-Z、からなる群より選択され、ここでZは、蛍光色素、ビオチン、抗体に結合する化合物、光架橋剤、キレート剤、アミノ酸、およびペプチドからなる群より選択される機能性置換基である]
で表される塩基、塩基Ds、および/または天然型の塩基、を有する置換または非置換デオキシリボヌクレオシド5’-三リン酸、を用いて核酸の複製反応を行い;
(2)生成した核酸の中から、塩基Dsと前記式IIで表される塩基の人工塩基対を含む核酸を、前記式IIで表される塩基が有する機能性置換基の性質に基づいて選択的に回収する;
ことを含む、前記方法。
(a)N1がチミン(T)もしくはシトシン(C);
(b)N3がシトシン(C);
(c)N4がチミン(T);
(d)N5がチミン(T)もしくはシトシン(C);および
(e)N6がグアニン(G);
からなる群より選択される少なくとも2つ以上の条件を満たす、態様10~12のいずれか1項に記載の方法。
(1)5’-(N)n(Nu1)(N)m-3’(配列番号2)、で示されるランダム領域を含むDNAライブラリーを調製し、ここでnおよびmはそれぞれ独立して1ないし10から選択される整数であり、Nu1は第一の人工塩基である;
(2)Nu1と人工塩基対を形成する第二の人工塩基Nu2を有するヌクレオシドを含む、複製基質を用いて前記DNAライブラリーに対して核酸の複製反応を行い、ここで、Nu2は機能性官能基を含んでいる;
(3)Nu1とNu2の人工塩基対の形成により機能性置換基が導入された核酸を、当該機能性置換基の性質に基づいて回収し;
(4)(3)で回収した核酸に対して、(2)および(3)の工程を繰り返し;そして、
(5)得られた核酸の配列を決定する;
ことを含む、前記方法。
(1)以下の式I:
(2)複製基質として、以下の式II:
nは、1ないし12から選択される整数であり;
mは、1ないし12から選択される整数であり;
lは、1ないし12から選択される整数であり;
Xは、-C≡C-CH2-、-C≡C-、-C=C-、アリール、チエニル、イミダゾリル、およびチアゾリルからなる群より選択され;
Yは、Zで置換されたアリール、-NHCO-Z、-CONH-Z、-NHCONH-Z、-O-Z、-COO-Z、-O-C(=O)-Z、-CO-Z、および、-S-Z、からなる群より選択され、ここでZは、蛍光色素、ビオチン、抗体に結合する化合物、光架橋剤、キレート剤、アミノ酸、およびペプチドからなる群より選択される機能性置換基である]
で表される塩基、塩基Ds、および/または天然型の塩基、を有する置換または非置換デオキシリボヌクレオシド5’-三リン酸、を用いて前記DNAライブラリーに対して核酸の複製反応を行い;
(3)塩基Dsと前記式IIで表される塩基の人工塩基対の形成により機能性置換基が導入された核酸を、当該機能性置換基の性質に基づいて回収し;
(4)(3)で回収した核酸に対して、(2)および(3)の工程を繰り返し;そして
(5)得られた核酸の配列を決定する;
ことを含む、前記方法。
本発明の人工塩基対は、人工塩基Dsと人工塩基Pnの誘導体とで形成される塩基対である。
nは、1ないし12、好ましくは1ないし10、より好ましくは1ないし8、さらに好ましくは1ないし5、から選択される整数であり;
mは、1ないし12、好ましくは1ないし10、より好ましくは1ないし8、さらに好ましくは1ないし5、から選択される整数であり;
lは、1ないし12、好ましくは1ないし10、より好ましくは1ないし8、さらに好ましくは1ないし5、から選択される整数であり;
Xは、-C≡C-CH2-、-C≡C-、-C=C-、アリール、チエニル、イミダゾリル、およびチアゾリルからなる群より選択され、好ましくは-C≡C-CH2-もしくは-C≡C-、より好ましくは-C≡C-CH2-であり;
Yは、-CH3、-C2H5、-NH2、-OH、-COOH、-CHO、-SH、置換もしくは非置換アリール、-NHCO-Z、-CONH-Z、-NHCONH-Z、-O-Z、-COO-Z、-O-C(=O)-Z、-CO-Z、および、-S-Z、からなる群より選択され、ここでZは、蛍光色素、ビオチン、抗体に結合する化合物、光架橋剤、キレート剤、アミノ酸、およびペプチドからなる群より選択される]
で表される。
特に好ましい態様において、本発明の人工塩基Pn誘導体は、
NH2-hx-Pn: 4-[3-(6-アミノヘキサンアミド)-1-プロピニル]-2-ニトロピロール-1-イル基;または
FAM-hx-Pn: 4-[3-[6-(フルオレセイン-5-カルボキサミド)ヘキサンアミド]-1-プロピニル]-2-ニトロピロール-1-イル基;
である。
一態様において本発明は、人工塩基対を含む核酸を複製する方法であって、Dsおよび/またはPn誘導体を塩基として有するヌクレオチドを含む核酸である鋳型鎖に対し、複製基質として塩基Ds、塩基Pn誘導体、および/または天然型の塩基、を有する置換または非置換デオキシリボヌクレオシド5’-三リン酸、を用いて核酸の複製反応を行うことにより、塩基Dsと前記式IIで表される塩基の人工塩基対を含む核酸を複製する、前記方法、を提供する。
本発明の人工塩基Pn誘導体は、蛍光色素、ビオチン、抗体に結合する化合物、光架橋剤、キレート剤、アミノ酸、およびペプチドからなる群より選択される機能性置換基を有していてもよい。これらの機能性置換基をPn誘導体が有する場合、これら機能性置換基の性質に基づいて、本発明の核酸の複製方法により生成したDs-Pn誘導体の人工塩基対を含む核酸を選択的に検出および/または回収することができる。本発明において、人工塩基対を含む核酸の検出および/または回収が可能な機能性置換基を有する人工塩基の例には、式IIで表されるPn誘導体において、置換基Zとして蛍光色素、ビオチン、抗体に結合する化合物、光架橋剤、キレート剤、アミノ酸、またはペプチド等を有する場合が挙げられる。
本発明者らは、複製における鋳型塩基と複製基質環での人工塩基対の形成の効率と選択性は、DNA中の人工塩基対近傍の天然型塩基の配列にも依存するとの仮説を立てた。そして、実施例2で後述するように、核酸複製反応において効率よく核酸が増幅する人工塩基対とその近傍の天然型塩基の配列についてin vitroセレクション法の手法を用いて調べた。その結果、核酸複製反応において効率よく核酸が増幅する人工塩基対とその近傍の天然型塩基の配列を見出した。
(a)N1がチミン(T)もしくはシトシン(C);
(b)N3がシトシン(C);
(c)N4がチミン(T);
(d)N5がチミン(T)もしくはシトシン(C);および
(e)N6がグアニン(G);
からなる群より選択される少なくとも2つ以上の条件を満たす、鋳型鎖を用いることをさらなる特徴とする方法をも提供する。好ましくは、上記(a)-(e)の条件を少なくとも3つ以上、より好ましくは4つ以上、さらに好ましくは5つすべて、の条件を満たす鋳型鎖を用いてもよい。あるいは、上記(b)、(c)および(e)からなる群より選択される少なくとも2つ以上の条件を満たす鋳型鎖を用いてもよい。
(a)N1'がシトシン(C);
(b)N2'がアデニン(A)もしくはグアニン(G);
(c)N3'がアデニン(A);
(d)N4'がグアニン(G);および
(e)N6'がアデニン(A)もしくはグアニン(G);
からなる群より選択される少なくとも2つ以上の条件を満たす、鋳型鎖を用いることをさらなる特徴とする方法をも提供する。好ましくは、上記(a)-(e)の条件を少なくとも3つ以上、より好ましくは4つ以上、さらに好ましくは5つすべて、の条件を満たす鋳型鎖を用いてもよい。あるいは、上記(a)、(c)および(d)からなる群より選択される少なくとも2つ以上の条件を満たす鋳型鎖を用いてもよい。
(a)N1がチミン(T)もしくはシトシン(C);
(b)N3がシトシン(C);
(c)N4がチミン(T);
(d)N5がチミン(T)もしくはシトシン(C);および
(e)N6がグアニン(G);
からなる群より選択される少なくとも2つ以上の条件を満たす前記核酸、を提供する。好ましくは、上記(a)-(e)の条件を少なくとも3つ以上、より好ましくは4つ以上、さらに好ましくは5つすべて、の条件を満たす核酸であってもよい。あるいは、上記(b)、(c)および(e)からなる群より選択される少なくとも2つ以上の条件を満たす核酸であってもよい。
(a)N1'がシトシン(C);
(b)N2'がアデニン(A)もしくはグアニン(G);
(c)N3'がアデニン(A);
(d)N4'がグアニン(G);および
(e)N6'がアデニン(A)もしくはグアニン(G);
からなる群より選択される少なくとも2つ以上の条件を満たす前記核酸を提供する。好ましくは、上記(a)-(e)の条件を少なくとも3つ以上、より好ましくは4つ以上、さらに好ましくは5つすべて、の条件を満たす核酸であってもよい。あるいは、上記(a)、(c)および(d)からなる群より選択される少なくとも2つ以上の条件を満たす核酸であってもよい。
人工塩基Dsを含むDNA断片の塩基配列決定は、Ds-Pa塩基対を用いておこなうことができる。
一態様において、本発明は、人工塩基を含む核酸の高効率かつ高選択的な複製を実現するためのDNA中の人工塩基近傍の天然型塩基の配列を決定する方法を提供する。すなわち、本発明は、人工塩基を含む核酸の高効率かつ高選択的な複製を実現するためのDNA中の人工塩基近傍の天然型塩基の配列を決定する方法であって、
(1)5’-(N)n(Nu1)(N)m-3’(配列番号2)、で示されるランダム領域を含むDNAライブラリーを調製し、ここでnおよびmはそれぞれ独立して1ないし10から選択される整数であり、Nu1は第一の人工塩基である;
(2)Nu1と人工塩基対を形成する第二の人工塩基Nu2を有するヌクレオシドを含む、複製基質を用いて前記DNAライブラリーに対して核酸の複製反応を行い、ここで、Nu2は機能性官能基を含んでいる;
(3)Nu1とNu2の人工塩基対の形成により機能性置換基が導入された核酸を、当該機能性置換基の性質に基づいて回収し;
(4)(3)で回収した核酸に対して、(2)および(3)の工程を繰り返し;そして、
(5)得られた核酸の配列を決定する;
ことを含む、前記方法、を提供する。
isoG-isoC(特許文献3、および非特許文献9);
P-Z(P:2-アミノ-イミダゾ[1,2-a]-1,3,5-トリアジン-4(8H)-オン、Z:6-アミノ-5-ニトロ-2(1H)-ピリドン)(非特許文献22);
s-y(s:2-アミノ-6-(2-チエニル)プリン、y:2-オキソピリジン)(非特許文献16-17);
v-y(v:2-アミノ-6-(2-チアゾリル)プリン、y:2-オキソピリジン)(非特許文献18);
Ds-Pa;
Ds-Pn;および
Ds-Pn誘導体;
の組み合わせが挙げられる。なお、上記組み合わせのうちの前者がNu1である場合は後者がNu2となり、一方で後者がNu1である場合は前者がNu2となることは当業者に容易に理解されるであろう。
Pn: 2-ニトロ-1H-ピロール-1-イル基
Pa: 2-ホルミル-1H-ピロール-1-イル基
NH2-hx-Pn: 4-[3-(6-アミノヘキサンアミド)-1-プロピニル]-2-ニトロピロール-1-イル基
FAM-hx-Pn: 4-[3-[6-(フルオレセイン-5-カルボキサミド)ヘキサンアミド]-1-プロピニル]-2-ニトロピロール-1-イル基
NH2-hx-dPnTP: 1-(2-デオキシ-β-D-リボフラノシル)-4-[3-(6-アミノヘキサンアミド)-1-プロピニル]-2-ニトロピロール 5’-三リン酸
FAM-hx-dPnTP: 1-(2-デオキシ-β-D-リボフラノシル)-4-[3-[6-(フルオレセイン-5-カルボキサミド)ヘキサンアミド]-1-プロピニル]-2-ニトロピロール 5’-三リン酸
dPa’TP: 1-(2-デオキシ-β-D-リボフラノシル)-4-(1-プロピニル)ピロール-2-カルボアルデヒド 5’-三リン酸
ddPa’TP: 1-(2,3-ジデオキシ-β-D-リボフラノシル)-4-(1-プロピニル)ピロール-2-カルボアルデヒド 5’-三リン酸
また、Ds、Pn、NH2-hx-Pn、FAM-hx-Pnについての構造を図1に示す。
複製における人工塩基Pnの1-プロピニル誘導体とDsによる塩基対形成の効率と選択性を調べるために、大腸菌由来のDNAポリメラーゼIのクレノウ断片によるヌクレオチド挿入実験を行い、反応速度定数を解析した。
複製における鋳型塩基と基質間での人工塩基対の形成の効率と選択性は、DNA中の人工塩基対近傍の天然型の塩基の配列にも依存するのではないかとの仮説を立て、PCRで効率よく増幅する人工塩基対とその近傍の天然型塩基の配列を試験管内進化工学(in vitroセレクション法)の手法を用いて調べた。
具体的には、ランダム配列のDNAライブラリーを用いる進化工学の手法を用いて、以下の手順で高効率かつ高選択でPCR増幅可能な人工塩基近傍の天然型塩基配列を決定するin vitroセレクションを行った。まず、Dsを含むDNA断片を、人工塩基Dsの両側の天然型塩基のそれぞれ3塩基をランダムにした一本鎖DNA(55-mer; 5’-TTTCACACAGGAAACAGCTATGACGG-NNN-Ds-NNN-CCCTATAGTGAGTCGTATTATC-3’(配列番号9))をDNA合成機により化学合成し、これをゲル電気泳動で精製して作成した。このDNA断片(2pmol)を鋳型にして、天然型塩基の基質(dNTPs、N=A、G、C、T)と人工塩基の基質(dDsTP、FAM-hx-dPnTP)、そしてDeepVent DNAポリメラーゼ(0.04ユニット/μl)を用いてPCRを行った。PCRの反応スケールは200 μlであり、反応緩衝液の組成は20mM Tris-HCl(pH8.8)、10mM KCl、10mM (NH4)2SO4、2mM MgSO4、0.1% TritonX-100である。5’側プライマー(5’-GATAATACGACTCACTATAG-3’(配列番号10))および3’側プライマー(5’-TTTCACACAGGAAACAGCTATGAC-3’(配列番号11))は各々1μMの濃度で、また各天然型塩基の基質dNTP(0.3mM)、人工塩基の基質としてFAM-hx-dPnTP(2.5 μM)とdDsTP(50 μM)を使用した。PCR条件は、94℃で30秒、45℃で30秒、65℃で4分を1サイクルとした。10サイクルのPCR増幅後、全長のPCR産物を10%PAGE-7M尿素ゲルによる電気泳動により精製し、260nmの吸光度から回収されたDNAの濃度を算出した。
方法1のシーケンシングパターンから、5’-CNA(Pa)GNG-3’(配列番号14)、すなわち5’-CNC(Ds)TNG-3’配列(配列番号15)(NはA、G、C、Tのどれかを示す)に収束していることがわかった(図3:方法1として示されるシーケンシングピークのパターン)。 また、方法2でクローニングして得られた配列中の各部位でのそれぞれの塩基の出現頻度を調べると、図3中の方法2の表に示したように、方法1と同様に、5’-CNC(Ds)TNG-3’配列(NはA、G、C、Tのどれかを示す)(配列番号15)が得られた。このように、人工塩基対に隣接する塩基のみならず、人工塩基から5’側と3’側の両側の3番目の塩基もPCRの効率と選択性に依存していることがわかった。
実施例2のセレクションにおいて、方法2の塩基配列解析法によりクローニングして得られた配列のDNA断片(S1-S8、(それぞれ、配列番号16-23))、および得られた配列を人為的に変えたDNA断片(N9-N12(それぞれ、配列番号24-27))をDNA合成機で作成し、それぞれのDNA断片のPCRにおける増幅効率と人工塩基の選択性を調べた。
)は、高い頻度で、5’-YNC(Ds)TYG-3’配列(Y=CまたはT、N=A、G、C、またはT)(配列番号29)を含み、ランダム配列のDNAと比較してこれらのDNAのPCR増幅効率は1.6倍から2.0倍向上した。また、FAM-hx-Pnの取り込み効率も、ランダム配列のDNAと比較して1.9倍から2.9倍向上した。さらに、人工塩基対の忠実度(選択性)も、これらの値と相関関係にあり、人工塩基対のPCRにおける選択性とその効率が人工塩基対の近傍の天然型塩基の配列に依存していることがわかった。
人工塩基Dsを含むDNA断片の塩基配列決定法は本発明者らがすでに報告している(I. Hirao, et al., Nature Methods, 3: 729-735 (2006).)。このDs-Pa塩基対を用いた従来法では、Dsの相補塩基であるPaのプロピニル誘導体の基質(dPa’TP)の有無によるシーケンシングパターンの違いを利用していた。たとえば、dPa’TPを加えずにDsを含むDNA断片のジデオキシダイミネーター法によるシーケンシングを行うと、鋳型鎖DNA中のDsに相補する塩基の部分でシーケンス反応が止まったピークパターンを与える。一方、dPa’TPを加えてシーケンシングをおこなうと、反応は進行するが、蛍光標識化したPa’のジデオキシダイターミネーターは加えていないため、鋳型鎖DNA中のDsに相補する塩基の位置のみ、シーケンシングのピークが消えたピークパターンを与える(図3b)。従来法は、これらの2つのシーケンシングパターンを比較することにより、人工塩基を含むDNAの配列を決定する方法である。
1-(2-デオキシ-β-D-リボフラノシル)-4-(1-プロピニル)ピロール-2-カルボアルデヒド(249mg,1.00mmol)(I. Hirao, et al., Nature Methods, 3: 729-735 (2006))を無水ピリジン (10ml) に溶解させ、塩化トリチル (1.18g,4.21mmol) およびN,N-ジイソプロピルアミン (1.11ml,6.40mmol) を加え、室温で29時間、その後50℃で1.5時間攪拌した。反応溶液を減圧下で濃縮し、残渣を酢酸エチルで希釈し、有機層を飽和炭酸水素ナトリウム水溶液で2回洗浄し、硫酸マグネシウム上で乾燥した。得られた粗生成物をシリカゲルカラムクロマトグラフィー(展開溶媒;ジクロロメタン:酢酸エチル=100:0→100:3、その後ジクロロメタン:メタノール=100:2)で精製し、目的物(395mg,80%)を得た。
HRMS (FAB, 3-NBA マトリクス) C32H29N1Na1O4 [M + Na]+ について: 計算値, 514.1994;実測値, 514.1992.
工程(b)および(c):1-(2,3-ジデオキシ-5-O-トリチル-β-D-リボフラノシル)-4-(1-プロピニル)ピロール-2-カルボアルデヒドの合成
1-(2-デオキシ-5-O-トリチル-β-D-リボフラノシル)-4-(1-プロピニル)ピロール-2-カルボアルデヒド(392mg,797μmol)のアセトニトリル溶液に、アルゴン雰囲気下、4-ジメチルアミノピリジン(975mg,7.98mmol)およびクロロチオギ酸フェニル(470μl,3.40mmol)を加え、室温で攪拌した。23時間後、反応溶液を減圧下で濃縮し、シリカゲルカラムクロマトグラフィーで精製し(ヘキサン中、50-67% CH2Cl2)、中間体を得た(193mg,若干の不純物を含む)。この中間体のトルエン溶液(10ml)に水素化トリ-n-ブチルスズ(165μl,612μmol)およびα,α’-アゾビスイソブチロニトリル(15mg,92μmol)を加え、アルゴン雰囲気下5時間還流させた。反応溶液を減圧濃縮し、得られた粗生成物をシリカゲルカラムクロマトグラフィー(展開溶媒;ヘキサン中、50-80% CH2Cl2)で精製し、目的物(84mg,58%)を得た。
HRMS (FAB, 3-NBA マトリクス) C32H29N1Na1O3 [M + Na]+ について:計算値, 498.2045; 実測値, 498.2072.
工程(d):1-(2,3-ジデオキシ-β-D-リボフラノシル)-4-(1-プロピニル)ピロール-2-カルボアルデヒドの合成
1-(2,3-ジデオキシ-5-O-トリチル-β-D-リボフラノシル)-4-(1-プロピニル)ピロール-2-カルボアルデヒド(82mg,171μmol)を80%酢酸(10ml)に溶解させ、室温で3時間、その後50℃でさらに2時間攪拌した。反応溶液を減圧下で濃縮し、水で共沸を行った。得られた粗生成物をシリカゲルカラムクロマトグラフィー(CH2Cl2中、0-1% CH3OH)およびRP-HPLC(水中、35-80% CH3CN,12分)で精製し、目的物(31mg,78%)を得た。
13C NMR (DMSO-d6) δ 179.94, 130.80, 130.56, 127.11, 105.87, 87.74, 85.38, 82.80, 73.91, 62.32, 34.73, 24.58, 4.30.
HRMS (FAB, 3-NBA マトリクス) C13H16NO3 (M + 1)について: 計算値, 234.1130; 実測値, 234.1137.
工程(e):1-(2,3-ジデオキシ-β-D-リボフラノシル)-4-(1-プロピニル)ピロール-2-カルボアルデヒド 5’-三リン酸(ddPa’TP)の合成
1-(2,3-ジデオキシ-β-D-リボフラノシル)-4-(1-プロピニル)ピロール-2-カルボアルデヒド(20.8mg,89μmol)に無水ピリジン(90μl)および無水ジオキサン(270μl)を加えて溶解し、2-クロロ-4H-1,2,3-ジオキサホスホリン-4-オンの1M ジオキサン溶液(100μl,100μmol) を加えて室温で10分間攪拌した後に、トリ-n-ブチルアミン(90μl)およびビス(トリ-n-ブチルアンモニウム)ピロホスフェートの0.5M DMF溶液(270μl)を加えて10分間攪拌した。1%ヨウ素/水/ピリジン溶液(1.8ml)を加え、室温で15分間攪拌し、5%亜硫酸水素ナトリウム水溶液(135μl)を加えた後に反応溶液を減圧濃縮した。得られた油状物質に水(5ml)を加え、室温で1時間攪拌した。これをDEAE Sephadex A-25カラムクロマトグラフィー(1.5×30cm,濃度直線勾配;TEABの50mM-1M溶液)およびC18-HPLC(濃度勾配;0%-15%アセトニトリルの0.1M 酢酸トリエチルアンモニウム緩衝液,pH7.0)で精製し、目的物を得た。
31P NMR (D2O) δ -23.28, -10.98, -10.98.
MS (ESI) C13H17N1O12P3 [M-H]- について:計算値, 472.00; 実測値, 471.94.
実施例4:機能性置換基を結合した人工塩基をPCR増幅でDNA中に導入する方法とその検出・単離方法
(4-1:人工塩基を含むDNAのPCR増幅)
Dsを含む断片(55-mer)にプライマーと人工塩基の基質(dDsTPとFAM-hx-dPnTPあるいはNH2-hx-dPnTP)を加えて、15から40サイクルのPCRを行った。
複数の人工塩基を含むDNA断片がPCRで増幅可能かどうかを調べるために、Dsを2つ含む断片(60-mer、62-mer、65-mer、68-mer)を鋳型にして、プライマーと人工塩基の基質(dDsTPとFAM-hx-dPnTPあるいはNH2-hx-dPnTP)を加えて、15サイクルのPCRを行った。
5’-TTTCACACAGGAAACAGCTATGACGGCCC(Ds)TTAC(Ds)GTGCCCTATAGTGAGTCGTATTATC-3’
Dsを2つ含むDNA断片 (62-mer)の配列(配列番号34):
5’-TTTCACACAGGAAACAGCTATGACGGCCC(Ds)TTGTAC(Ds)GTGCCCTATAGTGAGTCGTATTATC-3’
Dsを2つ含むDNA断片 (65-mer)の配列(配列番号35):
5’-TTTCACACAGGAAACAGCTATGACGGCCC(Ds)TTGTAATAC(Ds)GTGCCCTATAGTGAGTCGTATTATC-3’
Dsを2つ含むDNA断片 (68-mer)の配列(配列番号36):
5’-TTTCACACAGGAAACAGCTATGACGGCCC(Ds)TTGTAACGATAC(Ds)GTGCCCTATAGTGAGTCGTATTATC-3’
PCRの反応スケールは25μlで、反応液の組成は20mM Tris-HCl(pH8.8)、10mM KCl、10mM (NH4)2SO4、2mM MgSO4、0.1% TritonX-100である。5’側プライマー(5’-CGTTGTAAAACGACGGCCAGGATAATACGACTCACTATAG-3’(配列番号12))および3’側プライマー(5’-TTTCACACAGGAAACAGCTATGAC-3’(配列番号11))(各々1μM)、各天然型塩基の基質dNTP(0.3mM)、dDsTP(50μM)、FAM-hx-dPnTP(2.5μM)あるいはNH2-hx-dPnTP(50μM)、DeepVent DNAポリメラーゼ(0.02ユニット/μl)を用いてPCRを行った。PCRの1サイクルは、94℃30秒、45℃30秒、65℃4分で行った。鋳型に用いたDNA断片の濃度は0.6nMとした。PCR産物を15%ポリアクリルアミド-7M 尿素ゲルによる電気泳動で分離し、その産物のバンドをバイオイメージャーFLA-7000(富士フィルム)で検出・定量した。PCR増幅によりDNA中に取り込まれたFAM-hx-Pn由来の蛍光は、ゲルを直接ステージ上に載せ、蛍光解析(レーザー:473nm、フィルター青色励起用Y520)モードで検出した。また、塩基配列の解析では、PCRによる増幅産物をゲル電気泳動で精製した後に、最終濃度50μM dPa’TPを用いてシーケンシング反応を行い、その配列をDNAシーケンサーにより確認した。シーケンシングの方法は実施例2-1の(方法1)および実施例3を参照。
人工塩基Dsを含むDNA断片のPCR増幅では、その相補鎖DNA中にFAM-hx-Pnを取り込ませることができるので、抗FAM抗体で人工塩基対を含むDNA断片のみを単離することができる。このことを確認するために、本実施例では、プライマー領域は同じ配列であるが、それ以外の配列が異なる3種類のDNA断片と人工塩基Dsを含むDNA断片が等量ずつ存在する場合について、以下の実験を行った。
DNA1(55-mer)(配列番号45):
5’-TTTCACACAGGAAACAGCTATGACGGCCC(Ds)TTGCCCTATAGTGAGTCGTATTATC-3’
DNA2 (55-mer) (配列番号46):
5’-TTTCACACAGGAAACAGCTATGACACATGGAACTGCTATAGTGAGTCGTATTATC-3’
DNA3 (55-mer) (配列番号47):
5’-TTTCACACAGGAAACAGCTATGACCATGATGCAGACTATAGTGAGTCGTATTATC-3’
DNA4 (55-mer) (配列番号48):
5’-TTTCACACAGGAAACAGCTATGACTTGATCCGTATCTATAGTGAGTCGTATTATC-3’
これらのDNA断片(各断片の最終濃度0.15nM)の混合物を鋳型にして、FAM-hx-dPnTP(2.5μM)およびdDsTP(50μM)、各天然型塩基の基質:dNTP(300μM)を用いて、DeepVent DNAポリメラーゼで15サイクル(94℃30秒、45℃30秒、65℃4分)のPCR(50μlスケール)を行った。増幅後のPCR溶液からその20μlをマイクロコンYM-30(ミリポア)を通し、さらに緩衝液(20mM Tris-HCl pH7.6、0.5M NaCl、10mM MgCl2)で洗浄し、溶液中に含まれる未反応の基質を除いた。この溶液(30μl)を予めストレプトアビジン磁気ビーズ(4mg/ml溶液を10μl、New England Biolabs)に固定したビオチン修飾された抗FAM抗体(1mg/ml溶液を10μl、インビトロジェン)と混ぜ、氷上にて1時間インキュベートした。溶液を除いた磁気ビーズを緩衝液(100μl)で2回洗浄し、次いで1mM EDTA溶液(pH8.0)を20μl加えて75℃で30秒間加温し、ビーズに結合したDNAを溶出した。シークエンス反応は、溶出されたDNA溶液を10μl用いて、dPa’TP存在下(最終濃度2μM)で行った。また、コントロールとして、抗FAM抗体による精製前、即ちPCR直後のサンプルの一部をゲル電気泳動により精製し、その産物を用いてシークエンス反応を行った。
本実施例では、Dsを含むDNA断片(55-mer、0.3amol)に天然型塩基のランダム配列からなる100-merの断片を107倍量加えてPCR増幅(30サイクル)を行い、人工塩基を含むDNA断片のみを抗FAM抗体で単離することにより、Dsを含むDNA断片の塩基配列を解析することができる例を示す。
5’-TTTCACACAGGAAACAGCTATGACGGCCC(Ds)TTGCCCTATAGTGAGTCGTATTATC-3’
このDNA断片(最終濃度6fM)に天然型塩基のランダム配列からなる100-merのDNA(最終濃度60nM)を混在させ、人工塩基の基質にFAM-hx-dPnTP(2.5μM)およびdDsTP(50μM)、各天然型塩基の基質にdNTP(300μM)を用いて、DeepVent DNAポリメラーゼで30サイクル(94℃30秒、45℃30秒、65℃4分)のPCR(50μlスケール)を行った。この溶液から20 μl分をCentri-SepTMスピンカラムで処理して、過剰の基質を除いた。得られた溶液を最終濃度で20mM Tris-HCl pH7.6、0.5M NaCl、1mM EDTAを含む溶液(40-50μl)に調製し、この溶液を、予めストレプトアビジン磁気ビーズ(4mg/ml溶液を20μl、New England Biolabs)に固定したビオチン修飾された抗FAM抗体(1mg/ml溶液を5μl、インビトロジェン)と混ぜ、氷上にて30分インキュベートした。溶液を除いた磁気ビーズを緩衝液(100μl)で1回洗浄し、次いで1mM EDTA溶液(pH8.0)を20μl加えて75℃で30秒間加温し、ビーズに結合したDNAを溶出した。溶出されたDNA溶液10μlを用いて、dPa’TP存在下(最終濃度2μM)でシーケンシングを行った。図8に示したとおり、大過剰のランダムなDNA断片が混在する場合でも、PCR産物から人工塩基を含む断片を抗FAM抗体で単離することができ、そして、その後シーケンスを行うことにより、Dsを含むDNA断片の塩基配列を解析することができた。
Claims (19)
- 人工塩基対を含む核酸を複製する方法であって、以下の式I:
Rは、-X-Y、
nは、1ないし12から選択される整数であり;
mは、1ないし12から選択される整数であり;
lは、1ないし12から選択される整数であり;
Xは、-C≡C-CH2-、-C≡C-、-C=C-、アリール、チエニル、イミダゾリル、およびチアゾリルからなる群より選択され;
Yは、-CH3、-C2H5、-NH2、-OH、-COOH、-CHO、-SH、置換もしくは非置換アリール、-NHCO-Z、-CONH-Z、-NHCONH-Z、-O-Z、-COO-Z、-O-C(=O)-Z、-CO-Z、および、-S-Z、からなる群より選択され、ここでZは、蛍光色素、ビオチン、抗体に結合する化合物、光架橋剤、キレート剤、アミノ酸、およびペプチドからなる群より選択される]
で表される塩基を有するヌクレオチドを含む核酸である鋳型鎖に対し、複製基質として塩基Ds、前記式IIで表される塩基、および/または天然型の塩基、を有する置換または非置換デオキシリボヌクレオシド5’-三リン酸、を用いて核酸の複製反応を行うことにより、塩基Dsと前記式IIで表される塩基の人工塩基対を含む核酸を複製する、前記方法。 - 鋳型鎖が、塩基Dsおよび/または式IIで表される塩基を有するヌクレオチドを少なくとも2つ含むDNAである、請求項1に記載の方法。
- 複製基質が、γ位のリン酸の水酸基が置換されているデオキシリボヌクレオシド5’-三リン酸ではない、請求項1または2に記載の方法。
- 蛍光色素がカルボキシフルオレセイン(FAM)である、請求項1~3のいずれか1項に記載の方法。
- 機能性置換基を結合した人工塩基を、核酸の複製反応によりDNA中に導入する方法であって、
以下の式I:
複製基質として、以下の式II:
Rは、-X-Y、
nは、1ないし12から選択される整数であり;
mは、1ないし12から選択される整数であり;
lは、1ないし12から選択される整数であり;
Xは、-C≡C-CH2-、-C≡C-、-C=C-、アリール、チエニル、イミダゾリル、およびチアゾリルからなる群より選択され;
Yは、-CH3、-C2H5、-NH2、-OH、-COOH、-CHO、-SH、置換もしくは非置換アリール、-NHCO-Z、-CONH-Z、-NHCONH-Z、-O-Z、-COO-Z、-O-C(=O)-Z、-CO-Z、および、-S-Z、からなる群より選択され、ここでZは、蛍光色素、ビオチン、抗体に結合する化合物、光架橋剤、キレート剤、アミノ酸、およびペプチドからなる群より選択される]
で表される塩基、塩基Ds、および/または天然型の塩基、を有する置換または非置換デオキシリボヌクレオシド5’-三リン酸、を用いて核酸の複製反応を行い;
それにより、塩基Dsと前記式IIで表される塩基の人工塩基対を含む核酸が生成することにより、機能性置換基を結合した人工塩基がDNA中に導入される;
前記方法。 - 鋳型鎖が、塩基Dsを有するヌクレオチドを少なくとも2つ含む核酸である、請求項5に記載の方法。
- 複製基質が、γ位のリン酸の水酸基が置換されているデオキシリボヌクレオシド5’-三リン酸ではない、請求項5または6に記載の方法。
- 鋳型鎖において、塩基Dsの近傍の配列として、5’-N1N2N3(Ds)N4N5N6-3’(配列番号1)の配列を含み、ここで、N1、N2、N3、N4、N5、N6は天然型の塩基を有するヌクレオチドであって、以下の条件:
(a)N1がチミン(T)もしくはシトシン(C);
(b)N3がシトシン(C);
(c)N4がチミン(T);
(d)N5がチミン(T)もしくはシトシン(C);および
(e)N6がグアニン(G);
からなる群より選択される少なくとも2つ以上の条件を満たす、請求項5~7のいずれか1項に記載の方法。 - 蛍光色素がカルボキシフルオレセイン(FAM)である、請求項8に記載の方法。
- 核酸プールの中から人工塩基対を含む核酸を複製し、選択的に回収する方法であって、
(1)以下の式I:
Rは、-X-Y、
nは、1ないし12から選択される整数であり;
mは、1ないし12から選択される整数であり;
lは、1ないし12から選択される整数であり;
Xは、-C≡C-CH2-、-C≡C-、-C=C-、アリール、チエニル、イミダゾリル、およびチアゾリルからなる群より選択され;
Yは、Zで置換されたアリール、-NHCO-Z、-CONH-Z、-NHCONH-Z、-O-Z、-COO-Z、-O-C(=O)-Z、-CO-Z、および、-S-Z、からなる群より選択され、ここでZは、蛍光色素、ビオチン、抗体に結合する化合物、光架橋剤、キレート剤、アミノ酸、およびペプチドからなる群より選択される機能性置換基である]
で表される塩基、塩基Ds、および/または天然型の塩基、を有する置換または非置換デオキシリボヌクレオシド5’-三リン酸、を用いて核酸の複製反応を行い;
(2)生成した核酸の中から、塩基Dsと前記式IIで表される塩基の人工塩基対を含む核酸を、前記式IIで表される塩基が有する機能性置換基の性質に基づいて選択的に回収する;
ことを含む、前記方法。 - 塩基Dsを有するヌクレオチドを含む核酸が、塩基Dsを有するヌクレオチドを少なくとも2つ含む、請求項10に記載の方法。
- 複製基質が、γ位のリン酸の水酸基が置換されているデオキシリボヌクレオシド5’-三リン酸ではない、請求項10または11に記載の方法。
- 塩基Dsを有するヌクレオチドを含む核酸において、塩基Dsの近傍の配列として、5’-N1N2N3(Ds)N4N5N6-3’(配列番号1)の配列を含み、ここで、N1、N2、N3、N4、N5、N6は天然型の塩基を有するヌクレオチドであって、以下の条件:
(a)N1がチミン(T)もしくはシトシン(C);
(b)N3がシトシン(C);
(c)N4がチミン(T);
(d)N5がチミン(T)もしくはシトシン(C);および
(e)N6がグアニン(G);
からなる群より選択される少なくとも2つ以上の条件を満たす、請求項10~12のいずれか1項に記載の方法。 - 蛍光色素がカルボキシフルオレセイン(FAM)である、請求項13に記載の方法。
- 人工塩基を含む核酸の高効率かつ高選択的な複製を実現するためのDNA中の人工塩基近傍の天然型塩基の配列を決定する方法であって、
(1)5’-(N)n(Nu1)(N)m-3’(配列番号2)、で示されるランダム領域を含むDNAライブラリーを調製し、ここでnおよびmはそれぞれ独立して1ないし10から選択される整数であり、Nu1は第一の人工塩基である;
(2)Nu1と人工塩基対を形成する第二の人工塩基Nu2を有するヌクレオシドを含む、複製基質を用いて前記DNAライブラリーに対して核酸の複製反応を行い、ここで、Nu2は機能性官能基を含んでいる;
(3)Nu1とNu2の人工塩基対の形成により機能性置換基が導入された核酸を、当該機能性置換基の性質に基づいて回収し;
(4)(3)で回収した核酸に対して、(2)および(3)の工程を繰り返し;そして、
(5)得られた核酸の配列を決定する;
ことを含む、前記方法。 - 人工塩基を含む核酸の高効率かつ高選択的な複製を実現するためのDNA中の人工塩基近傍の天然型塩基の配列を決定する方法であって、
(1)以下の式I:
(2)複製基質として、以下の式II:
Rは、-X-Y、
nは、1ないし12から選択される整数であり;
mは、1ないし12から選択される整数であり;
lは、1ないし12から選択される整数であり;
Xは、-C≡C-CH2-、-C≡C-、-C=C-、アリール、チエニル、イミダゾリル、およびチアゾリルからなる群より選択され;
Yは、Zで置換されたアリール、-NHCO-Z、-CONH-Z、-NHCONH-Z、-O-Z、-COO-Z、-O-C(=O)-Z、-CO-Z、および、-S-Z、からなる群より選択され、ここでZは、蛍光色素、ビオチン、抗体に結合する化合物、光架橋剤、キレート剤、アミノ酸、およびペプチドからなる群より選択される機能性置換基である]
で表される塩基、塩基Ds、および/または天然型の塩基、を有する置換または非置換デオキシリボヌクレオシド5’-三リン酸、を用いて前記DNAライブラリーに対して核酸の複製反応を行い;
(3)塩基Dsと前記式IIで表される塩基の人工塩基対の形成により機能性置換基が導入された核酸を、当該機能性置換基の性質に基づいて回収し;
(4)(3)で回収した核酸に対して、(2)および(3)の工程を繰り返し;そして
(5)得られた核酸の配列を決定する;
ことを含む、前記方法。 - 複製基質が、γ位のリン酸の水酸基が置換されているデオキシリボヌクレオシド5’-三リン酸ではない、請求項16に記載の方法。
- 請求項16または17に記載の方法により得られる核酸。
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US20110053782A1 (en) | 2011-03-03 |
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AU2009232716A1 (en) | 2009-10-08 |
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US8426569B2 (en) | 2013-04-23 |
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