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WO2007105623A1 - Support solide a oligonucleotide immobilise - Google Patents

Support solide a oligonucleotide immobilise Download PDF

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Publication number
WO2007105623A1
WO2007105623A1 PCT/JP2007/054645 JP2007054645W WO2007105623A1 WO 2007105623 A1 WO2007105623 A1 WO 2007105623A1 JP 2007054645 W JP2007054645 W JP 2007054645W WO 2007105623 A1 WO2007105623 A1 WO 2007105623A1
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Prior art keywords
oligonucleotide
solid phase
group
phase carrier
complementary
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PCT/JP2007/054645
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English (en)
Japanese (ja)
Inventor
Mitsuo Sekine
Kohji Seio
Akihiro Ohkubo
Kunihiko Tanaka
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Tokyo Institute of Technology NUC
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Tokyo Institute of Technology NUC
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to an oligonucleotide-immobilized solid phase carrier.
  • the oligonucleotide-immobilized solid phase carrier of the present invention is useful for improving the sensitivity of long-chain DNA synthesis and SNPs analysis.
  • Non-patent Document 1 In genetic engineering, the cleavage and ligation of nucleic acid molecules such as DNA is one of the most important basic techniques. Therefore, with the development of genetic engineering, efficient technology for binding DNA is required. Conventionally, the ligation of double-stranded nucleic acids using restriction enzymes has been the main method for linking two types of nucleic acids. In recent years, chemical ligation, a new DNA binding technology, has been reported (Non-patent Document 1).
  • Chemical ligation is the complementary D-complementation of an oligonucleotide having a highly nucleophilic functional group such as phosphate at the 3 ′ end of a nucleic acid and an oligonucleotide having a leaving group such as a iodine at the 5 ′ end.
  • This is a technology that uses NA as a template to synthesize a single oligonucleotide by forming a covalent bond.
  • DNA chips and DNA microarrays are made by aligning a large number of DNA molecules on a substrate such as a glass slide, silicon, or plastic, and are useful for analyzing gene polymorphisms.
  • DNA is grown on microporous glass (CPG), which is the most suitable material for DNA synthesis.
  • CPG microporous glass
  • a probe-on-carrier method has been developed that can be used to detect SNPs for each CPG without separating the probe molecule from the CPG carrier force. This technique not only improves the efficiency of DNA probe strand extension, but also reduces the cost of the DNA chip and allows the probe to be arranged three-dimensionally, so it can be expected to improve detection sensitivity. However, it is desired to further improve the SN p s detection efficiency.
  • Oligonucleotides having a leaving group at the 5 'end are useful for genetic diagnosis, and many methods for their synthesis have been reported (for example, Non-Patent Document 2 and Non-Patent Document 3).
  • the chemical ligation technique as described above is useful for genetic diagnosis to examine gene polymorphism, but it binds an oligonucleotide having a leaving group at the 5 ′ end and forms a duplex.
  • a solid support having a function has not been reported yet.
  • Such a solid phase carrier is useful for genetic diagnosis as described above, and development is desired.
  • Non-Special Reference 1 Yanzheng Xu and Eric T, Kooi, Journal of the American hemical Soc iety, 2000, 122, 9040-9041
  • Non-Patent Document 2 Mathias K. Herrlein, Jeffrey S. Nelson and Robert L. Letsinger, Journal of the American Chemical Society, 1995, 117, 10151-10152
  • Non-Patent Document 3 Yanzheng Xu and Eric T, Kool, Tetrahedron Letters, 1997, 38, 5595
  • an object of the present invention is to provide a solid phase carrier which binds an oligonucleotide having a leaving group at the 5 ′ end and has a double bond forming ability.
  • the present inventors diligently studied, and as a result, have found that the above object can be achieved by a solid phase synthesis method using chemical ligation.
  • the present invention has been made based on the above knowledge, and provides an oligonucleotide-immobilized solid phase carrier bound to an oligonucleotide derivative-forced solid phase carrier represented by the general formula (1). .
  • R 1 represents hydrogen or alkoxy group, which may be the same or different
  • R 2 represents hydrogen, which may be the same or different, or Represents an alkoxy group
  • B represents a natural or non-natural nucleobase, which may be the same or different
  • X represents a leaving group
  • n represents an integer of 2 to 50.
  • the present invention also provides a method for producing an oligonucleotide-immobilized solid phase carrier, wherein the oligonucleotide derivative represented by the general formula (1) is bound to a solid phase carrier,
  • reaction accelerator in the solid phase synthesis of oligonucleotides by the phosphoramidide method 1-hydroxybenzotriazole is used, and a method for producing an oligonucleotide-immobilized solid phase carrier is provided.
  • R 1 represents hydrogen or an alkoxy group, which may be the same or different
  • R 2 represents hydrogen, which may be the same or different, or Represents an alkoxy group
  • B represents a natural or non-natural nucleobase, which may be the same or different
  • X represents a leaving group
  • n represents an integer of 2 to 50.
  • the present invention also relates to the oligonucleotide having a complementary base sequence portion complementary to a predetermined base sequence portion in a gene with a leaving group bonded to the 5 'end side and fixed to the 3' end side.
  • Complementary oligonucleotide-immobilized solid phase carrier formed by binding a phase carrier and a complementary base sequence portion complementary to a base sequence portion adjacent to a predetermined base sequence portion in the gene
  • An oligonucleotide solid-phase synthesis method which comprises a step of reacting.
  • the present invention also provides a method for binding a leaving group to the 5 'end side of an oligonucleotide having a complementary base sequence portion complementary to a predetermined base sequence portion in a normal gene, and Complementary oligonucleotide-immobilized solid phase carrier formed by binding a solid phase carrier and an oligonucleotide having a complementary base sequence portion complementary to the base sequence portion adjacent to the predetermined base sequence portion in the normal gene
  • a specific nucleotide sequence in a normal gene and a sample gene corresponding to the sample gene which has a step of reacting with a complementary oligonucleotide having a highly nucleophilic functional group on the 3 ′ end of A method for confirming the difference from the base sequence portion is provided.
  • the present invention also includes a step of chemically synthesizing an oligonucleotide having a predetermined base sequence by coupling base-part unprotected nucleotide phosphoramidides in a predetermined order.
  • Ligated oligonucleotide solid phase is a method of chemically synthesizing a solid phase carrier, which is a nucleotide phosphoramidide having a leaving group at the 5 ′ end as a 5 ′ terminal base unprotected nucleotide phosphoramidide
  • a method using a nucleotide phosphoramidite having a solid phase carrier bound to the 3 ′ end as a 3′-terminal base-unprotected nucleotide phosphoramidide is a method of chemically synthesizing a solid phase carrier, which is a nucleotide phosphoramidide having a leaving group at the 5 ′ end as a 5 ′ terminal base unprotected nucleot
  • the oligonucleotide-immobilized solid phase carrier of the present invention can improve the sensitivity of long-chain DNA synthesis and SNPs analysis.
  • the method for confirming the difference between a predetermined base sequence portion in a normal gene and a corresponding base sequence portion in a sample gene of the present invention has improved sensitivity.
  • the method for chemically synthesizing an oligonucleotide-immobilized solid phase carrier of the present invention can synthesize long-chain DNA and an oligonucleotide-immobilized solid phase carrier that can improve the sensitivity of SNPs analysis. .
  • the oligonucleotide-immobilized solid phase carrier of the present invention is obtained by binding an oligonucleotide derivative represented by the general formula (1) to a solid phase carrier.
  • R 1 and R 2 may be the same or different and each represents hydrogen or an alkoxy group.
  • the alkoxy group an alkoxy group having from 5 to 5 carbon atoms is preferable, for example, a methoxy group, an ethoxy group, a propoxy group, a 1 ptyloxy group, a 1 pentyloxy group, and the like.
  • an alkoxy group in which a part or all of the side chain thereof is cyclized such as a branched alkoxy group such as a group, an isopropyloxy group, a cyclopropyloxy group, a cycloptynoxy group, and a cyclopentyloxy group is also included.
  • B is a natural or non-natural nucleic acid salt which may be the same or different.
  • natural bases such as adenine, cytosine, guanine, thymine, and urashinore are artificial bases such as 7-dazaadenine, 7-dazaadenine, 3-dazaadenine, 6-thioguanine, 2 thiouracil, 2 thiotimine,
  • substituents alkyl, alkenyl, alkynyl, halogen, nitro group, acyl group, hydroxyl group, etc.
  • substituents alkyl, alkenyl, alkynyl, halogen, Nitro group, acyl group, hydroxyl group, etc.
  • 8_position various substituents (alkyl, halogen, nitro group, acyl group, hydroxyl group, etc.) introduced adenine, 8_position, various substituents (alkyl, halogen, nitro group, acyl group, hydroxyl group
  • X represents a leaving group.
  • the leaving group include a halogen atom, a tosyl group, a mesinole group, a trifluoromethanesulfonyl group, and a phosphate group.
  • the halogen atom include a chlorine atom, a bromine atom, and an iodine atom.
  • n represents an integer of 2 to 50, preferably an integer of 5 to 20. Less than n force, chemical ligation is not performed efficiently, while if it is larger than 50, base discrimination ability decreases. However, the accuracy of chemical ligation is reduced.
  • the oligonucleotide-immobilized solid phase carrier of the present invention is bound to a solid phase carrier.
  • a solid phase carrier those conventionally used for producing DNA chips and DNA microarrays can be used without particular limitation.
  • slide glass, porous glass, polystyrene beads, plastic examples thereof include gold particles, gold plates, silver particles and silver plates, glass such as microporous glass and porous glass, magnetic beads having polystyrene, metal and ferrite as the core and the surface covered with glycine methacrylate.
  • the shape of the carrier may be any shape such as a plate (substrate) or a bead.
  • a thermoplastic resin or a thermosetting resin can be used as the plastic.
  • thermoplastic resin examples include linear polyolefins such as polyethylene and polypropylene, cyclic polyolefins, and fluorine-containing resins.
  • linear polyolefins such as polyethylene and polypropylene
  • cyclic polyolefins such as polyethylene and polypropylene
  • fluorine-containing resins such as polyethylene and polypropylene
  • microporous glass a DNA chip that can be used in the probe-on-carrier method can be obtained.
  • the oligonucleotide-immobilized solid phase carrier of the present invention the hydroxyl group at the 3-position of the sugar moiety of the residue on the 3 ′ end side is bonded to the carrier. That is, the oligonucleotide-immobilized solid phase carrier of the present invention is represented by the general formula (2).
  • R 2 , B, X and n are the same as described in the general formula (1), and D represents a solid support.
  • the method for producing the oligonucleotide-immobilized solid phase carrier of the present invention is not particularly limited, but after producing the oligonucleotide derivative represented by the general formula (1), it may be immobilized or adsorbed on the carrier. Each nucleotide unit may be bound. In the latter case, the production can be performed in solid phase synthesis using 1-hydroxybenzotriazole as a reaction accelerator.
  • a method for producing an oligonucleotide-immobilized solid phase carrier in which an oligonucleotide derivative represented by the general formula (1) is bound to a solid phase carrier, As a reaction accelerator in solid phase synthesis of oligonucleotides by the dye method 1-hydroxybenzotriazole is used, and a method for producing an oligonucleotide-immobilized solid phase carrier is provided.
  • the first step is to use a material in which a base on the 3 'end side is fixed to a solid-phase support as a material, and to this, a phosphoroamidide for binding oligonucleotides that contact P
  • 1-hydroxybenzotriazole is used as a reaction accelerator.
  • 1-hydroxybenzotriazole even a compound having a substituent can be used without particular limitation.
  • 6-nitro-1-hydroxybenzotriazole can be used.
  • the amount used is not particularly limited, but is about 0.5 to 40 equivalents, preferably 5 to 35 equivalents, more preferably about 10 to 30 equivalents with respect to phosphoroamidide.
  • this reaction is preferable from the viewpoint that an oligonucleotide derivative having a leaving group can be synthesized with high purity since an oligonucleotide derivative can be produced without requiring a protecting group in the base moiety.
  • the extension reaction by solid-phase synthesis reaction includes steps of detrityl lysis with trichloroacetic acid, etc., washing with dichloromethane, oxidation with iodine, etc., washing with pyridine and the like. In order to improve the reaction yield, the coupling and subsequent washing steps are carried out several times, preferably twice.
  • a leaving group is bonded to the 5 ′ end side of an oligonucleotide having a complementary base sequence portion complementary to a predetermined base sequence portion in a gene, and the 3 ′ end side Complementary oligonucleotide-immobilized solid phase carrier (hereinafter, also simply referred to as “complementary oligonucleotide-immobilized solid phase carrier”), which is formed by binding a solid phase carrier to a specific nucleotide sequence in the above gene Complementary oligonucleotide (hereinafter simply referred to as “complementary oligonucleotide”) having a highly nucleophilic functional group on the 3 ′ end side of an oligonucleotide having a complementary base sequence portion complementary to the base sequence portion adjacent to the portion (Also called).
  • the above-described oligonucleotide-immobilized solid phase carrier of the present invention is used.
  • the oligo to be synthesized is synthesized.
  • a gene having a nucleotide sequence complementary to the nucleotide chain is prepared.
  • a predetermined (arbitrary site) base sequence portion of this gene is selected, and a leaving group is bound to the 5 ′ end side of the oligonucleotide having a complementary base sequence portion complementary to this base sequence portion.
  • the complementary oligonucleotide-immobilized solid phase carrier is reacted with the complementary oligonucleotide.
  • a functional group having high nucleophilicity is bonded to the 3 'terminal side of the complementary oligonucleotide.
  • Examples of the functional group having high nucleophilicity include a thiophosphate group, a phosphate group, an amino group, a carboxy group, and a thiol group.
  • the 5 ′ end side of the complementary oligonucleotide-immobilized solid phase carrier and the 3 ′ end side of the complementary oligonucleotide are linked by a nucleophilic reaction, and the chain length of the oligonucleotide is increased.
  • the reaction between the complementary oligonucleotide-immobilized solid phase carrier and the complementary oligonucleotide can be carried out in an appropriate buffer, and the reaction temperature and reaction time are 5 to 60 ° C, respectively: ⁇ 48 hours is sufficient.
  • This reaction is a reaction generally called chemical ligation, and the reaction conditions can be appropriately selected depending on the number of bases of the oligonucleotide and the type of base pair.
  • the treatment with ammonia is preferably a treatment for a short time, preferably 6 to 18 hours. By this ammonia treatment, the protecting group of the phosphate group is deprotected.
  • a new second complementary oligonucleotide is further linked by attaching a leaving group to the 5 ′ end side of the complementary oligonucleotide.
  • the second complementary oligonucleotide should also be complementary to the adjacent sequence of the gene that is complementary to the complementary oligonucleotide. As described above, by sequentially using new complementary oligonucleotides, it is possible to extend the chain length of the oligonucleotides, and it is possible to synthesize oligonucleotides of several hundred bases by chemical ligation.
  • the method for confirming the difference with the corresponding base sequence portion in the sample gene of the present invention is a complementary base sequence portion complementary to the predetermined base sequence portion in the normal gene.
  • a complementary oligonucleotide-immobilized solid phase carrier having a leaving group bonded to the 5 ′ end side of an oligonucleotide having a base and a solid phase carrier bonded to the 3 ′ end side, and a predetermined in the normal gene A step of reacting a complementary oligonucleotide having a highly nucleophilic functional group on the 3 ′ end side of an oligonucleotide having a complementary base sequence portion complementary to the base sequence portion adjacent to the base sequence portion. It is characterized by that.
  • a functional group having high nucleophilicity is bonded to the 3 'end of the complementary oligonucleotide.
  • the functional group having high nucleophilicity is as described above.
  • the 5 ′ end side of the complementary oligonucleotide-immobilized solid phase carrier and the 3 ′ end side of the oligonucleotide are linked by a nucleophilic reaction, and the oligonucleotide chain length is increased.
  • the reaction between the complementary oligonucleotide-immobilized solid phase carrier and the complementary oligonucleotide can be carried out in an appropriate buffer, and the reaction temperature and reaction time are 5 to 60 ° C and 1 to 5 respectively. 48 hours is sufficient.
  • This reaction is a reaction generally called chemical ligation, and the reaction conditions can be appropriately selected depending on the number of bases of the oligonucleotide and the type of base pair.
  • a labeled compound is bound to a complementary oligonucleotide or contains a radioisotope, and chemical ligation can be detected using this label. If the base sequence part adjacent to the normal base part of the normal gene and the complementary oligonucleotide used are not completely complementary, chemical ligation will not occur and ligation cannot be confirmed by the labeling substance. It is possible to detect the presence of a normal gene and base substitution, deletion or addition.
  • the method of chemically synthesizing an oligonucleotide-immobilized solid phase carrier of the present invention comprises chemically synthesizing an oligonucleotide consisting of a predetermined base sequence by coupling base unprotected nucleotide phosphoramidides in a predetermined order.
  • a method of chemically synthesizing an oligonucleotide-immobilized solid phase carrier comprising an oligonucleotide bound to a solid phase carrier, comprising 5′-terminal base-unprotected nucleotide phosphoramidite, Use a nucleotide phosphoramidite with a leaving group at the 5 'end, as a nucleotide unprotected nucleotide phosphoramidide at the 3' end, and a nucleotide phosphoramidite with a solid phase carrier bound to the 3 'end. Use a dido.
  • the base-unprotected nucleotide phosphoramidide used in this method can be produced, for example, as in the examples described later.
  • the base-unprotected nucleotide phosphoramidide it is commercially available.
  • nucleotide phosphoramidite having a leaving group at the 5 ′ end a commercially available phosphoramidite unit (3′—O— (2-cyanoethyl) represented by the formula (7) can be used.
  • 3′—O— (2-cyanoethyl) represented by the formula (7) can be used.
  • 1 N, N′-diisopropyl phosphoramidite) -5′-odothymidine can be synthesized as follows.
  • Thymidine (1.94 g, 8 mmol) was dissolved in anhydrous dimethylformamide (20 ml), followed by methyl triphenoxyphosphonium iodide (4.35 g, 9.6 mmol). After stirring at room temperature for 10 minutes, methanol (10 ml) was added. Methanol (30 ml) was added again to the mixture obtained by evaporating the solvent under reduced pressure to obtain the desired 5′-odothymidine as crystals.
  • nucleotide phosphoramidite having a solid phase carrier bound to the 3 'end is used as the 3' end base-unprotected nucleotide phosphoramidide.
  • Nucleotide phosphoramidides having a solid phase carrier bound to the terminal can be produced, for example, by binding a solid phase carrier into which 16-hydroxyhexadecanoic acid has been introduced and a base part unprotected nucleotide phosphoramidide. it can.
  • the conditions at this time are not particularly limited, but can be carried out under the conditions described in the examples described later.
  • the oligonucleotide-immobilized solid phase carrier is obtained by coupling base-unprotected nucleotide phosphoramidides in a predetermined order. Is obtained.
  • a conventional method for producing an oligonucleotide using phosphoramidide is used. It is preferable to use 1-hydroxybenzotriazole as a reaction accelerator during the reaction. By including such a reaction accelerator, a side reaction to the base amino group can be suppressed, and hydroxyl group-selective phosphorylation can be performed.
  • the machine synthesis is a microporous glass with 16-hydroxyhexadecanoic acid at the end.
  • CPG CPG
  • a solid phase carrier (10 mg, 10 / i mol / g) was used.
  • Each chain extension cycle was as shown in Table 1 below, and benzoimidazolium triflate (BIT) and 6-nitro-1-hydroxybenzotriazole (nHoBt) were used in the condensation reaction.
  • BIT benzoimidazolium triflate
  • nHoBt 6-nitro-1-hydroxybenzotriazole
  • the obtained probe has a leaving group at the 5 'end and a solid support bound to the 3' end.
  • the phosphoramidide units represented by the formulas (3) to (5) were synthesized by the following method.
  • the phosphoramidite units represented by formulas (6) and (7) were purchased from Spotify Research.
  • the oligonucleotide-immobilized solid phase carrier having the base sequences of SEQ ID NOs: 2 to 4 shown below was synthesized in the same manner as in Example 1. All sequences have a 5 'terminal side and a 3' terminal side with CPG.
  • an oligonucleotide having the base sequence of SEQ ID NO: 5 shown below was synthesized in the same manner as in Example 1.
  • fluorescein is bonded to the 5 ′ end and a thiophosphate group is bonded to the 3 ′ end.
  • an oligonucleotide having the base sequence of SEQ ID NO: 6 was synthesized in the same manner as in Example 1.
  • SEQ ID NO: 6 (ATGAACCAGAGGCCCAT)
  • Oligonucleotide (0.25 nmol) was immersed in an oligonucleotide solution (0.5 mL, 30 mM phosphate buffer (pH 7.2), 1 M NaCl, 50 ⁇ M DTT), and stirred at 40 ° C. for 14 hours. After completion of the stirring, the solid support was washed with water and dried. After drying, the fluorescence brightness of the solid phase carrier was measured at a wavelength of 470 to 490 nm using a fluorescence microscope (manufactured by Olympus Corporation, BX51WI).
  • the fluorescence intensity is about as compared to when using an oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 3. It was 10 times.
  • the base sequence of SEQ ID NO: 2 is complementary to a part of the base sequence of SEQ ID NO: 6, and the base sequence of SEQ ID NO: 3 is different by only one base.
  • a chemical ligation reaction occurs, and when the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 3 is used.
  • the chemical ligation reaction did not occur. Therefore, it was found that a single base mismatch (A-C mismatch) can be detected by this example.
  • the oligonucleotide-immobilized solid phase carrier of the present invention has a double-strand forming ability.
  • the fluorescence intensity was measured in the same manner using the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 4 instead of the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 2. .
  • the base sequence of SEQ ID NO: 2 is complementary to a part of the base sequence of SEQ ID NO: 6, and the base sequence of SEQ ID NO: 3 differs by only two bases.
  • a chemical ligation reaction occurs, and when the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 4 is used.
  • the chemical ligation reaction did not occur. Therefore, it was found that a mismatch of two bases (A-C mismatch, C-A mismatch) can be detected by this example.
  • the ratio of match to single-base mismatch is about 1.9 times. Considering this, it can be seen that the method using the oligonucleotide-immobilized solid phase carrier of the present invention has an accuracy of 5 times or more that of the conventional method.

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Abstract

La présente invention concerne un support solide auquel un oligonucléotide possédant un groupement partant au niveau de l'extrémité 5'-terminale est lié et qui est susceptible de former une double liaison. Le support solide à oligonucléotide immobilisé comprend un support solide et un dérivé oligonucléotidique représenté par la formule générale (1) lié au support solide : où les radicaux R1 peuvent être différents les uns des autres ou identiques les uns aux autres et représentent indépendamment un hydrogène ou un groupement alcoxy ; les radicaux R2 peuvent être différents les uns des autres ou identiques les uns aux autres et représentent indépendamment un hydrogène ou un groupement alcoxy ; les B peuvent être différents les uns des autres ou identiques les uns aux autres et représentent indépendamment un nucléotide naturel ou non naturel ; X représente un groupement partant ; et n est un entier de 2 à 50.
PCT/JP2007/054645 2006-03-10 2007-03-09 Support solide a oligonucleotide immobilise Ceased WO2007105623A1 (fr)

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JP2006-066396 2006-03-10
JP2006066396A JP2009153381A (ja) 2006-03-10 2006-03-10 オリゴヌクレオチド固定化固相担体

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050202459A1 (en) * 2003-09-23 2005-09-15 Hud Nicholas V. Intercalation-mediated synthesis of polymers

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050202459A1 (en) * 2003-09-23 2005-09-15 Hud Nicholas V. Intercalation-mediated synthesis of polymers

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MILLER G. ET AL.: "A Simple Method for Electrophilic Functionalization of DNA", ORG. LETT., vol. 4, no. 21, 2002, pages 3599 - 3601, XP003017788 *
OHKUBO A. ET AL.: "DNA Oligomer o Shukugo Block ni Mochiita Koso Goseiho no Kaihatsu", CSJ: THE CHEMICAL SOCIETY OF JAPAN DAI 86 SHUNKI NENKAI (2006) KOEN YOKOSHU II, 13 March 2006 (2006-03-13), pages 858 + ABSTR. NO. 2 G3-20, XP003017790 *
OHKUBO A. ET AL.: "Hosuhoroamidaito-ho o Mochiita Suisanki Sentakuteki na Rinsanka Hanno no Kaihatsu to DNA Gosei eno Oyo", CSJ: THE CHEMICAL SOCIETY OF JAPAN DAI 84 SHUNKI NENKAI (2004) KOEN YOKOSHU II, 11 March 2004 (2004-03-11), pages 1042 + ABSTR. NO. 1 J3-09, XP003017789 *
XU Y. ET AL.: "Rapid and Selective Selenium-Mediated Autoligation of DNA Strands", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 122, no. 37, 2000, pages 9040 - 9041, XP002297261 *

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