WO2004087908A1 - Methode et trousse de reactifs permettant de regenerer un jeu ordonne de microechantillons - Google Patents

Methode et trousse de reactifs permettant de regenerer un jeu ordonne de microechantillons Download PDF

Info

Publication number
WO2004087908A1
WO2004087908A1 PCT/JP2003/003984 JP0303984W WO2004087908A1 WO 2004087908 A1 WO2004087908 A1 WO 2004087908A1 JP 0303984 W JP0303984 W JP 0303984W WO 2004087908 A1 WO2004087908 A1 WO 2004087908A1
Authority
WO
WIPO (PCT)
Prior art keywords
microarray
reagent
dna
oligonucleotide
dna probe
Prior art date
Application number
PCT/JP2003/003984
Other languages
English (en)
Japanese (ja)
Inventor
Takuya Matsui
Yuji Miyahara
Original Assignee
Hitachi High-Technologies Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hitachi High-Technologies Corporation filed Critical Hitachi High-Technologies Corporation
Priority to PCT/JP2003/003984 priority Critical patent/WO2004087908A1/fr
Priority to JP2004570128A priority patent/JP4101810B2/ja
Publication of WO2004087908A1 publication Critical patent/WO2004087908A1/fr

Links

Classifications

    • 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 a method and a reagent kit for regenerating a microarray used in the field of biotechnology such as gene diagnosis, DNA sequence analysis, or gene polymorphism analysis, particularly in the field of genetic testing.
  • a DNA chip / microarray (hereinafter collectively referred to as a microarray) is an array of hundreds to tens of thousands of spots of DNA serving as probes on a substrate such as slide glass silicon. According to the microarray, it is possible to provide a technology that can collectively and parallelly perform operations that required hundreds to tens of thousands of experiments in the conventional method. Therefore, microarrays are an important technology especially in fields requiring high throughput, such as searching for disease-related genes, such as in the field of life sciences such as pharmaceuticals.
  • genetic diagnosis is performed by flowing a solution sample containing nucleic acid increased by PCR or the like onto the microarray and detecting the hybridization between the probe on the microarray and the amplified nucleic acid. , Genetic polymorphism analysis, DNA sequence analysis, etc.
  • a microarray using the extension of a probe on a solid phase can simultaneously perform hybridization with a probe and nucleic acid amplification, and thus can detect in real time.
  • microarrays amplify nucleic acids from probes
  • the length of the probes changes before and after use, and cannot be reused.
  • these microarrays are expensive despite single use. Repeated use is useful for making low-cost microarrays, especially the ability to use a nucleic acid amplification-detection integrated microarray repeatedly. Can be said to be very useful in terms of a significant reduction in detection time.
  • the immobilized probe elongates, so the next time the probe is used, the step of returning the elongated probe to its original length is controlled by the hybridization temperature. Indispensable from such points.
  • PNA Peptide Nucleic Acid
  • Peptide nucleic acid is a substance whose basic skeleton is a peptide bond in which the sugar monophosphate moiety, which is the skeleton structure of DNA, is substituted with (2-aminoethyl) glycine, and has a similar steric structure and chemical properties Therefore, it can be a substitute for nucleic acids.
  • PNA is chemically synthesized by applying the solid-phase peptide synthesis method, and can be used as a probe to form a strong phase capture bond with DNA.
  • PNA molecules have no asymmetric centers, are highly soluble, neutral substances, and are resistant to nucleases. Therefore, in a microarray using a PNA as a probe, after performing an extension reaction from the probe using dNTP as a substrate, nuclease is passed over the microarray to cut and remove only the extended region (dNTP portion). However, it is possible to hold the probe PNA on the substrate.
  • microarrays using PNA as a probe are known to cause agglutination when the purine content (adenine and guanine) of the PNA probe exceeds 60%. Therefore, in a microarray using a PNA as a probe, there is a problem that the probe sequence is limited, and the range that can be detected is limited.
  • an object of the present invention is to provide a microarray regenerating method and a microarray regenerating reagent kit that can excise an extended chain amplified by an extension reaction from a DNA probe at low cost. Disclosure of the invention
  • the microarray regeneration method comprises performing an extension reaction from the 3 ′ end of the DNA probe on a microarray having a DNA probe having a 5 ′ end immobilized on a substrate, and then performing the extension reaction. Reacting an oligonucleotide having a thymidine bound to EDTA (ethylenediaminetetraacetic acid) with a sequence complementary to the extended strand amplified by the above, a divalent metal, and a Cleland reagent, Cleaving both the hybridized oligonucleotide and the extended strand with a metal and the Cleland reagent.
  • EDTA ethylenediaminetetraacetic acid
  • the microarray regeneration reagent kit is a microarray having a DNA probe having a 5 ′ end immobilized on a substrate, which is complementary to an extended strand amplified by an extension reaction from the 3 ′ end of the DNA probe. It includes an oligonucleotide having a thymidine sequence attached to EDTA, a divalent metal, and a Cleland reagent. According to the present invention, a divalent metal and a Cleland reagent are allowed to act in a state where the above-mentioned oligonucleotides are hybridized to the extended strand amplified by the extension reaction from the 3 ′ end of the DNA probe.
  • the Cleland reagent cleaves double-stranded and / or single-stranded DNA containing EDTA-bound thymidine in the presence of the divalent metal near the EDTA-bound thymidine.
  • the extended strand can be excised from the DNA probe.
  • the microarray from which the extended strand has been excised can be used again.
  • FIG. 1 is a schematic diagram showing a solid-phase amplification type microarray.
  • FIG. 2 is a schematic diagram showing a state of nucleic acid probe extension of a microarray.
  • FIG. 3 is a schematic view showing a state of cutting a nucleic acid probe of a microarray.
  • FIG. 4 is a schematic diagram showing a state of nucleic acid amplification of a microarray.
  • FIG. 5 is a schematic diagram showing a state of hybridization of DNA probes and oligonucleotides of a microarray.
  • FIG. 6 is a schematic diagram showing a state of regeneration of a nucleic acid probe of a microarray.
  • the microarray applied to the present invention includes a substrate 1 and a plurality of DNA probes 2 immobilized on the substrate 1, as shown in FIG.
  • the substrate 1 is not particularly limited, and is made of, for example, slide glass, metal, nylon, or nitrocellulose membrane.
  • the substrate 1 may have a part or all of its surface that has been subjected to a surface treatment, or may have a film made of aminosilane formed thereon.
  • the DNA probe is composed of a nucleic acid fragment, and is formed by immobilizing the nucleic acid fragment on one main surface of the substrate 1 at a predetermined density.
  • immobilization means not only a covalent bond but also any form of bonding such as an ionic bond, and a meaning including both a bond via a linker and a bond via a linker.
  • the nucleic acid fragment to be immobilized is not particularly limited, and examples thereof include a DNA fragment extracted from a sample and prepared by processes such as amplification and purification, and a synthesized oligo DNA. In immobilizing the nucleic acid fragment, a so-called spotter (arrayer) can be used.
  • the 5 ′ end of the nucleic acid fragment is immobilized on the substrate 1, and the 3 ′ end has regions 3 and 4 that can hybridize with the DNA to be detected.
  • region 3 and region 4 can be designed so that they can hybridize with different DNAs.
  • a region 3 corresponding to one SNP and a region 4 corresponding to the other SNP are designed and immobilized at different sites.
  • the DNA 6 hybridized to the immobilized DNA probe 2 is formed into a ⁇ shape, and a nucleic acid is extended from the 3 ′ terminal of the DNA probe.
  • the DNA hybridized under normal temperature conditions can be sequentially subjected to the extension reaction as type III, and the desired DNA can be amplified (FIG. 1 to FIG. 1).
  • Figure 4 Specifically, first, as shown in FIG. 1, a microarray is placed on a flow cell, and then a reaction reagent is applied to the microarray from a reagent inlet 7.
  • the reaction reagent is a sample containing the DNA 6 to be detected, a solution containing a DNA elongation enzyme (for example, a strand displacement enzyme), dNTP, and a DNA cleavage enzyme.
  • Making the reaction reagent act on the microarray means bringing the reaction reagent into contact with the surface of the substrate 1 on which the DNA probes 2 are immobilized. By bringing the reaction reagent into contact with the surface of the substrate 1, as shown in FIG. 1, the DNA 6 and the DNA probe 2 contained in the reaction reagent specifically hybridize.
  • regions 3 and 4 are designed to detect a predetermined SNP, one or both of region 3 and region 4 may be used in accordance with the SNP in DNA 6 to be detected contained in the sample. , The DNA to be detected hybridizes.
  • the DNA elongation enzyme contained in the reaction reagent formed DNA 6 hybridized to DNA probe 2 into type III, and used dNTP as a substrate to obtain a DNA elongation enzyme.
  • dNTP used dNTP as a substrate to obtain a DNA elongation enzyme.
  • W commercially available Taq polymerase, Bst polymerase ⁇ Bac polymerase and the like can be used as a DNA elongation enzyme.
  • a DNA-cleaving enzyme is an enzyme capable of inserting DNA into a DNA chain extended by a DNA elongase.
  • a restriction enzyme capable of recognizing a specific sequence and nicking the extended chain side can be used as a DNA cleavage enzyme.
  • commercially available EcoRI polymerase, Hindlll polymerase and the like can be used.
  • the DNA elongation reaction was started again from the nick formed by the DNA cleavage enzyme, as shown in FIG. While peeling off the formed complementary strand from the DNA New extended chains can be synthesized.
  • an extension reaction can be performed by a so-called strand displacement enzyme, and the DNA hybridized under normal temperature conditions can be subjected to the extension reaction shown in FIGS.
  • the desired DNA can be amplified.
  • the microarray regeneration reagent is injected from the reagent injection port 7.
  • the microarray regeneration reagent is an oligonucleotide having a sequence complementary to the extended strand amplified by the extension reaction from the 3, terminus of DNA probe 2 and having thymidine bound to EDTA (ethylenediamine tetraacetic acid); It contains a divalent metal and a cleanland reagent.
  • EDTA ethylenediamine tetraacetic acid
  • the oligonucleotide is designed to specifically hybridize to the extended strand remaining on the 3 ′ side of the DNA probe 2, and contains at least one thymidine to which EDTA is bound.
  • divalent iron [Fe (II)] is preferably used, but not limited thereto, and for example, Mg 2+ or Mn 2+ may be used.
  • the tallyland reagent for example, dithiothreitol can be used, but not limited thereto, and dithioerythritol or raercaptoethanol can be used.
  • the reagent When a microarray regeneration reagent is injected from the sample injection port 7, the reagent can be brought into contact with the surface of the substrate 1 on which the DNA probe 2 is immobilized. As a result, the oligonucleotides 10 and 11 contained in the microarray regeneration reagent were specifically hybridized to the extended strand remaining on the 3 'side of DNA probe 2, as shown in Fig. 5. Thus, the Cleland reagent can cleave DNA on the side of the extended strand in the vicinity of the EDTA-bound idine in the presence of a divalent metal.
  • the denatured substance was thermally denatured by heat treatment at 94 to 95 ° C, and then the microarray regeneration reagent containing oligonucleotides 10 and 11 was removed from the surface of the substrate 1 of the microarray. As shown in the figure, the DNA probe 2 instead of the microarray can be regenerated.
  • the oligonucleotide, the divalent metal, and the Cleland reagent may be simultaneously applied to the microarray, but first, only the oligonucleotides 10 and 11 are applied and the DNA probe 2 and the oligonucleotide 1 After 0 and 11 are hybridized, a divalent metal and a Cleland reagent may be allowed to act. Alternatively, oligonucleotides 10 and 11 may be acted on simultaneously with one of the divalent metal and the Tarryland reagent, and then the other one of the divalent metal and the Cleland reagent may be acted on .
  • the oligonucleotides 10 and 11 have a complementary sequence to the extended strand at a position separated from the 3, 3 end of the DNA probe 2 by 5 bases or more.
  • the Clearland reagent up to 5 bases may be excised from the EDTA-bound thymidine in order to cut the DNA on the side of the extended strand near the EDTA-bound thymidine. Therefore, by setting the oligonucleotides 10 and 11 to a complementary sequence at a position at least 5 bases away from the 3 'end of DNA probe 2 in the extended strand, cleavage of the extended strand DNA by the cleanland reagent In this case, the excision of the DNA probe 2 can be prevented.
  • the microarray to which the present invention is applied is not limited to a type in which an elongation reaction from a DNA probe is performed by a strand displacement type DNA elongation enzyme as described above.
  • An extension reaction from a DNA probe may be performed.
  • the present invention can provide a microarray regenerating method and a regenerating reagent kit that can repeatedly use a microarray utilizing the extension of a probe on a solid phase.
  • a microarray regenerating method and a regenerating reagent kit that can repeatedly use a microarray utilizing the extension of a probe on a solid phase.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Selon l'invention, un brin étendu ayant été amplifié au moyen d'une réaction d'extension à partir d'une sonde d'ADN est excisé à un prix réduit. L'invention concerne une méthode permettant de régénérer un jeu ordonné de microéchantillons qui consiste : à procéder à une réaction d'extension sur un jeu ordonné de microéchantillons comprenant une sonde d'ADN immobilisée à l'extrémité 5' sur une plaque de base à partir de l'extrémité 3' de la sonde d'ADN, puis à procéder à un traitement au moyen d'un oligonucléotide qui présente une séquence complémentaire du brin étendu ayant été amplifié par ladite réaction d'extension et une thymidine liée à EDTA et au moyen d'un réactif contenant un métal divalent et un réactif de Cleland. La méthode selon l'invention consiste également à exciser ledit oligonucléotide et le brin étendu ayant été hybridé au moyen dudit métal divalent et dudit réactif de Cleland.
PCT/JP2003/003984 2003-03-28 2003-03-28 Methode et trousse de reactifs permettant de regenerer un jeu ordonne de microechantillons WO2004087908A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/JP2003/003984 WO2004087908A1 (fr) 2003-03-28 2003-03-28 Methode et trousse de reactifs permettant de regenerer un jeu ordonne de microechantillons
JP2004570128A JP4101810B2 (ja) 2003-03-28 2003-03-28 マイクロアレイ再生方法及びマイクロアレイ再生試薬キット

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2003/003984 WO2004087908A1 (fr) 2003-03-28 2003-03-28 Methode et trousse de reactifs permettant de regenerer un jeu ordonne de microechantillons

Publications (1)

Publication Number Publication Date
WO2004087908A1 true WO2004087908A1 (fr) 2004-10-14

Family

ID=33105309

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/003984 WO2004087908A1 (fr) 2003-03-28 2003-03-28 Methode et trousse de reactifs permettant de regenerer un jeu ordonne de microechantillons

Country Status (2)

Country Link
JP (1) JP4101810B2 (fr)
WO (1) WO2004087908A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006187270A (ja) * 2004-12-09 2006-07-20 Sumitomo Bakelite Co Ltd Ｄｎａ鎖伸長方法、ｄｎａ鎖増幅方法およびｄｎａ鎖伸長用マイクロアレイ

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0870898A (ja) * 1994-09-07 1996-03-19 Hitachi Ltd Ｄｎａの分離・分取法及びその解析法
EP1207209A2 (fr) * 2000-11-09 2002-05-22 Agilent Technologies Inc. (a Delaware Corporation) Procédés de détection de polymorphismes mononucléotidiques utilisant des réseaux

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0870898A (ja) * 1994-09-07 1996-03-19 Hitachi Ltd Ｄｎａの分離・分取法及びその解析法
EP1207209A2 (fr) * 2000-11-09 2002-05-22 Agilent Technologies Inc. (a Delaware Corporation) Procédés de détection de polymorphismes mononucléotidiques utilisant des réseaux

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006187270A (ja) * 2004-12-09 2006-07-20 Sumitomo Bakelite Co Ltd Ｄｎａ鎖伸長方法、ｄｎａ鎖増幅方法およびｄｎａ鎖伸長用マイクロアレイ

Also Published As

Publication number Publication date
JPWO2004087908A1 (ja) 2006-07-06
JP4101810B2 (ja) 2008-06-18

Similar Documents

Publication Publication Date Title
US6280950B1 (en) Nucleic acid affinity columns
CA2810931C (fr) Capture directe, amplification et sequencage d'adn cible a l'aide d'amorces immobilisees
US20080274904A1 (en) Method of target enrichment
US20070141604A1 (en) Method of target enrichment
EP2647426A1 (fr) Réplication de molécules d'acide nucléique distribuées avec conservation de leur distribution relative par liaison à base d'hybridation
JP2003527867A (ja) ポリヌクレオチド配列改変のマイクロアレイベースの分析
CA2492032A1 (fr) Analyse d'expression genique au moyen d'agents de croisement
EP3527672A1 (fr) Matrices aux oligonucleotides pour la séquençage d'acides nucléiques
EP3971282A1 (fr) Réseau et procédé de détection d'informations spatiales d'acides nucléiques
US6638760B1 (en) Method and apparatus for flow-through hybridization
WO2017096394A1 (fr) Compositions de capture par hybridation à haut rendement et procédés
JP2004536317A (ja) 合成結合システムを用いる核酸のための選別および固定化システム
WO2000032824A9 (fr) Evaluation de la longueur de sequences nucleotidiques repetitives par extension discontinue d'amorce
US6852494B2 (en) Nucleic acid amplification
US20050064432A1 (en) Nucleic acid amplification and detection
NZ501775A (en) Hybridization analysis using low density nucleic acid arrays
EP1516933A1 (fr) Des hybrides stables en sélectionant la position de la sonde relativement à la cible
JPWO2019157445A5 (fr)
US20030099937A1 (en) Nucleic acid amplification
WO2004087908A1 (fr) Methode et trousse de reactifs permettant de regenerer un jeu ordonne de microechantillons
CN115109846A (zh) 用于准确的平行定量稀释或未纯化样品中的核酸的方法
US20060240431A1 (en) Oligonucletide guided analysis of gene expression
US20070184478A1 (en) Process for detecting a plurality of target nucleic acids
WO1999034014A2 (fr) Technique de determination d'un acide nucleique
JP4508632B2 (ja) 核酸検出方法及び液組成物

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CN JP KR US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2004570128

Country of ref document: JP

122 Ep: pct application non-entry in european phase