WO2003052139A1 - Procede de distinction entre des sondes hybridees et des sondes non hydridees avec des nucleotides contenus dans un echantillon sur une puce a adn au moyen d'une reaction enzymatique - Google Patents

Procede de distinction entre des sondes hybridees et des sondes non hydridees avec des nucleotides contenus dans un echantillon sur une puce a adn au moyen d'une reaction enzymatique Download PDF

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Publication number
WO2003052139A1
WO2003052139A1 PCT/KR2002/002286 KR0202286W WO03052139A1 WO 2003052139 A1 WO2003052139 A1 WO 2003052139A1 KR 0202286 W KR0202286 W KR 0202286W WO 03052139 A1 WO03052139 A1 WO 03052139A1
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Prior art keywords
probes
nucleotides
sample
hybridization
enzyme
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PCT/KR2002/002286
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English (en)
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Yun-Jung Kwon
Kyung-Hee Han
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Yun-Jung Kwon
Kyung-Hee Han
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Application filed by Yun-Jung Kwon, Kyung-Hee Han filed Critical Yun-Jung Kwon
Priority to AU2002366479A priority Critical patent/AU2002366479A1/en
Publication of WO2003052139A1 publication Critical patent/WO2003052139A1/fr

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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 bio chip is a biological micro chip which may be formed by microarrying and attaching hundreds to hundreds of thousands, at most, of nucleotides including, for example, DNA, DNA fragment, cDNA, oligonucleotide, RNA or RNA fragment, of which sequences are known, at regular intervals on a small solid substrate made of glass, silicone or nylon so as to analyze the expression pattern, distribution mode and mutation of genes.
  • nucleotides including, for example, DNA, DNA fragment, cDNA, oligonucleotide, RNA or RNA fragment, of which sequences are known, at regular intervals on a small solid substrate made of glass, silicone or nylon so as to analyze the expression pattern, distribution mode and mutation of genes.
  • Hyseq and Incyte possess a technology of transferring cDNA onto a glass plate treated with poly-L-lysine using a pin, followed by integration.
  • Incyte has developed a new technology by incorporating the principle of a cartridge used in an ink jet printer instead of a pin.
  • Affymetrix has developed a method for synthesizing tens of thousands of different base sequences by applying photolithography which is used in the semiconductor process and Nanogen has developed a method for electrically fixing genes using electrical properties of DNA which has negative charge.
  • the information processing technology which allows to establish a database with information gathered from a bio chip and renders it to be utilized in research has already consolidated its firm foundation owing to the highly advanced information and communication technology.
  • the signal detection technology that is a method for detecting an interaction between probes and nucleotides in a sample to discriminate hybridized or un-hybridized probes with the sample nucleotides makes a relatively slow progress.
  • Signal detection methods which are used at present include, for example, laser-induced fluorescence detection, electrochemical detection and mass detection.
  • the laser-induced fluorescent detection is a method for optically discriminating hybridization between probes and nucleotides, in which a fluorescent material is bonded to the nucleotides in a sample and the hybridization is detected by a fluorescence detector and is currently the most commonly used method.
  • this method requires pretreatment reaction to bond a fluorescent material to a sample prior to hybridization, causing loss or contamination of the sample (Conner, B. Proc.
  • the electrochemical detection is a method for detecting hybridization of probes using an electrochemical reaction of other chemicals, that is oxidation- reduction reaction, on an electrode where the probes couple with a sample.
  • This method is not yet developed as a general detection method. Since it has a recognition level inferior to the fluorescence detection method, it cannot provide a satisfactory result in terms of accuracy. Thus, it is hard to discriminate hybridization of probes only by the electrochemical detection and a separate method for electrochemically discriminating hybridization of probes is always required.
  • the mass detection is a detection method by electrically signalizing the interaction between probes and nucleotides in a sample.
  • Its representative example is Electrochemical Quartz Crystal Microbalance (QCM) detection, in which the change in frequency according to the mass of probes immobilized on a quartz plate vibrating at a high frequency is measured.
  • QCM Electrochemical Quartz Crystal Microbalance
  • SPR surface plasmon resonance
  • the coupling of probes with nucleotides in a sample can accomplished when their sequences are complementary to each other for the most part but not all. In other words, they can bond to each other even when their sequences are not complementary to each other for a base or a part of the sequences, that is, they are not perfectly complementary to each other.
  • the nucleotides with a base or a part uncomplementary to probes can hardly be discriminated from the nucleotides having perfect complementarity to probes. It is not ready to assay SNP (single nucleotide polymorphism) having a difference of about a base by the existing methods.
  • discrimination of hybridization between probes and nucleotides in a sample from a reaction using a bio chip is carried out by hybridizing probes with nucleotides in a sample, followed by treatment with an enzyme which can recognize and cleave any one of the probes hybridized with the nucleotides and the probes unhybridized with the nucleotides between proves and nucleotides in a sample hybridization.
  • the nucleotides refers to a substance which can complementarily bond to a specific probe immobilized on a substrate, that is, DNA, DNA fragment, cDNA, oligonucleotide, RNA, RNA fragment, etc.
  • the enzyme reaction refers to a reaction using an enzyme which can recognize and cleave probes unhybridized with the nucleotides after hybridization. Since the unhybridized probes are present in the form of a single strand after hybridization, S 1 nuclease, mung bean nuclease, RNase
  • a and RNase H which can recognize and cleave only a single-stranded nucleic acid can be used.
  • the SI nuclease and mung bean nuclease are usefully used in a reaction using a DNA chip, since they can recognize and cleave only single-stranded DNA.
  • RNase A is usefully used in a reaction using a RNA chip, since it can recognize and cleave only single-stranded RAN.
  • RNase H is usefully used in an analysis of a DNA sample using RNA as a probe, since it can selectively cleave single-stranded RNA in the hybridization of
  • the enzyme reaction refers to a reaction using an enzyme which can recognize and cleave probes hybridized with the nucleotides after hybridization. Since the hybridized probes are present in the form of a double strand after hybridization, DNase which can recognize and cleave only a double-stranded nucleic acid can be used.
  • a method for detecting interactions between the probes and the nucleotides can be carried out by a conventional method such as laser-induced fluorescence detection, electrochemical detection and mass detection.
  • Fig. 5 shows the results of the electrochemical detection of probes unhybridized or hybridized with nucleotides in a sample using the discrimination method according to the present invention.
  • probes 1, 2 are immobilized onto a substrate 3.
  • the probe 1 is hybridized with the nucleic acid to form a double-stranded nucleic acid 11 , as shown in Fig. lb, while the probe 2 which is not hybridized with the nucleic acid remains in the form of a single strand.
  • the substrate 3 is treated with an enzyme which can recognize and cleave only the single-stranded nucleic acids, as shown in Fig. lc, followed by washing.
  • the unhybridized probe 2 is removed remaining a part 22 attached to the substrate 3, as shown in Fig. Id. Therefore, after the treatment with an enzyme, only the probe 1 which is hybridized with the nucleic acid is present on the substrate 3.
  • a known detection method it is possible to precisely discriminate the probes hybridized with the nucleic acid from the probes unhybridized with the nucleic acid.
  • the production process of the bio chip used in the present invention varies according to a detection method of interaction between the probe and the nucleic acid in the sample after the enzyme reaction.
  • An instant case where laser-induced fluorescence detection is used as a detection method of interaction between the probe and the nucleic acid after the discrimination according to the present invention is explained in detail with reference to Fig. 2.
  • probes 5, 6 with fluorescent materials 4 of one type or different types attached are immobilized on a substrate 7 upon preparation of a chip, as shown in Fig. 2a, unlike the conventional fluorescence detection methods in which a fluorescent material is attached to a sample.
  • a sample containing nucleic acids is applied to the probes.
  • the probe 5 is hybridized with the nucleic acid to form a double-stranded nucleic acid 55 while the probe 6 remains in the form of a single strand without hybridization with the nucleic acid, as shown in Fig. 2b.
  • probes 9, 10 with thiol group 8 attached on one end so as to form a chemical bond with the gold surface are immobilized on a gold electrode 11, as shown in Fig. 3a.
  • the thiol group has properties of being readily attached to the gold surface and thus, is widely used to attach chemicals including
  • the electrode After hybridization, the electrode is treated with an enzyme which can recognize and cleave single-stranded nucleic acids. As a result, the unhybridized probes are removed, leaving a part which is attached to the electrode, as shown in Fig. 3c.
  • the electrode with only the hybridized probed attached was subject to a cyclic voltammetry.
  • the discrimination method according to the present invention can effectively remove false-positive reactions which may occur due to the co-presence of the hybridized probes and the unhybridized probes to the final result interpretation by removing either one of the probes hybridized or unhybridized with the nucleotides in the sample.
  • the discrimination method according to the present invention can provide a more precise result by making various chips and put the results therefrom together according to how to detect the interaction between the probes and nucleotides in the sample after the enzyme reaction.
  • probes hybridized or unhybridized with nucleotides in a sample were discriminated and optically detected.
  • oligonucleotides of 15 nucleotides were synthesized using a DNA synthesizer and were used as probes.
  • Example 2 Discrimination of probes hybridized or unhybridized with nucleotides in a sample according to the present invention and electrochemical detection thereof
  • a sample solution of 5.0 ⁇ M oligonucleotide having a base sequence complementary to the probes dissolved in 10 mM Tris buffer containing 0.1 M sodium chloride and 1 mM EDTA was applied on the electrode with the probes immobilized thereon and incubated 35 ° C for 2 hours.
  • the electrode was washed with 5 mM Tris buffer containing 10 mM sodium chloride and subjected to CV (Fig. 5c).
  • both electrodes generated electric currents at a similar level by negative charges of the nucleic acids.
  • the electrode without hybridization showed CV similar to that of the electrode with nothing immobilized thereon (Figs. 5a and 5d). Since the probes on the electrode without hybridization had been removed by SI nuclease, the electrode became the same with the empty electrode. Therefore, the electrochemical reaction occurs freely, regardless of negative charges of the nucleotides.
  • the electrode with hybridization showed the same CV before and after the enzyme reaction (Fig. 5e). It is because SI nuclease could not remove the nucleotides which had become the double-stranded form by hybridization, whereby there were nucleotides on the electrode even after the enzyme reaction.
  • the discrimination method according to the present invention there is no need of a pretreatment of a sample to discriminate hybridization between proves and nucleotides in a sample in an experiment using a bio chip. Also, since the probes hybridized or unhybridized with the nucleotides in a sample are removed during an experimental process prior to detection, it is possible to considerably reduce errors and false-positive reactions during inte ⁇ retation of results which may occur in the whole treatment process of the sample.
  • the method according to the present invention can be usefully applied to studies using a bio chip and their applied fields, including SNP analysis which is important in the studies of genetic diversity.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de distinction entre des sondes hybridées et des sondes non hybridées avec des nucléotides contenus dans un échantillon sur une puce à ADN au moyen d'une réaction enzymatique. Selon l'invention, la distinction entre des sondes hybridées et des sondes non hybridées avec des nucléotides contenus dans l'échantillon fait appel à une enzyme pouvant reconnaître et couper une quelconque sonde hybridée ou non avec les nucléotides contenus dans l'échantillon pour retirer une quelconque sonde située sur la puce à ADN. Ledit procédé comporte ensuite une étape de détection. Le procédé de distinction selon l'invention permet d'obtenir un résultat plus précis que les procédés traditionnels dans la mesure où les réactions faussement positives sont éliminées, de résoudre des problèmes que pose le prétraitement d'un échantillon et d'effectuer l'analyse de polymorphisme nucléotidique simple, qui s'avère importante dans le cadre d'études sur la diversité génétique. Ainsi, cette invention peut être utile pour des études faisant appel à une puce à ADN et leurs domaines d'application.
PCT/KR2002/002286 2001-12-17 2002-12-06 Procede de distinction entre des sondes hybridees et des sondes non hydridees avec des nucleotides contenus dans un echantillon sur une puce a adn au moyen d'une reaction enzymatique WO2003052139A1 (fr)

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AU2002366479A AU2002366479A1 (en) 2001-12-17 2002-12-06 Discrimination method of hybridization between probes and nucleotides in sample on the bio chip using enzyme reaction

Applications Claiming Priority (2)

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KR10-2001-0080121 2001-12-17
KR1020010080121A KR20030049802A (ko) 2001-12-17 2001-12-17 바이오 칩에서 프로브와 시료 핵산의 혼성화 여부를 효소반응으로 구별하는 방법

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2010026450A1 (fr) * 2008-09-03 2010-03-11 Quantumdx Group Limited Stratégies de détection et procédés destinés à la détection d'acides nucléiques au moyen de biocapteurs
US8871921B2 (en) 2008-09-03 2014-10-28 Quantumdx Group Ltd. Design, synthesis and use of synthetic nucleotides comprising charge mass tags
US9410196B2 (en) 2008-09-03 2016-08-09 Quantumdx Group Limited Methods and kits for nucleic acid sequencing
US10759824B2 (en) 2008-09-03 2020-09-01 Quantumdx Group Limited Design, synthesis and use of synthetic nucleotides comprising charge mass tags
US11180523B2 (en) 2016-01-04 2021-11-23 Quantumdx Group Limited Design, synthesis and use of synthetic nucleotides comprising charge mass tags

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US4775619A (en) * 1984-10-16 1988-10-04 Chiron Corporation Polynucleotide determination with selectable cleavage sites
US5102784A (en) * 1990-05-04 1992-04-07 Oncor, Inc. Restriction amplification assay
US6228580B1 (en) * 1995-07-31 2001-05-08 Genset Nucleic acid detection method using nucleotide probes enabling both specific capture and detection
WO2002059364A1 (fr) * 2001-01-27 2002-08-01 Electron-Bio, Inc. Procede et dispositif d'analyses d'hybridation nucleique faisant appel a une technique de clivage reagissant au double brin ou au simple brin complementaire d'acides nucleiques ou d'oligonucleotides

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Publication number Priority date Publication date Assignee Title
US4775619A (en) * 1984-10-16 1988-10-04 Chiron Corporation Polynucleotide determination with selectable cleavage sites
US5102784A (en) * 1990-05-04 1992-04-07 Oncor, Inc. Restriction amplification assay
US6228580B1 (en) * 1995-07-31 2001-05-08 Genset Nucleic acid detection method using nucleotide probes enabling both specific capture and detection
WO2002059364A1 (fr) * 2001-01-27 2002-08-01 Electron-Bio, Inc. Procede et dispositif d'analyses d'hybridation nucleique faisant appel a une technique de clivage reagissant au double brin ou au simple brin complementaire d'acides nucleiques ou d'oligonucleotides

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010026450A1 (fr) * 2008-09-03 2010-03-11 Quantumdx Group Limited Stratégies de détection et procédés destinés à la détection d'acides nucléiques au moyen de biocapteurs
US8871921B2 (en) 2008-09-03 2014-10-28 Quantumdx Group Ltd. Design, synthesis and use of synthetic nucleotides comprising charge mass tags
JP2015130882A (ja) * 2008-09-03 2015-07-23 クワンタムディーエックス・グループ・リミテッド バイオセンサを用いる核酸検出のための検知方策および方法
US9410196B2 (en) 2008-09-03 2016-08-09 Quantumdx Group Limited Methods and kits for nucleic acid sequencing
EP3075867A1 (fr) * 2008-09-03 2016-10-05 Quantumdx Group Limited Stratégies de détection et procédés destinés à la détection d'acides nucléiques au moyen de biocapteurs
US9605302B2 (en) 2008-09-03 2017-03-28 Quantumdx Group Limited Sensing strategies and methods for nucleic acid detection using biosensors
US9938573B2 (en) 2008-09-03 2018-04-10 Quantumdx Group Limited Methods and kits for nucleic acid sequencing
US10759824B2 (en) 2008-09-03 2020-09-01 Quantumdx Group Limited Design, synthesis and use of synthetic nucleotides comprising charge mass tags
US11180523B2 (en) 2016-01-04 2021-11-23 Quantumdx Group Limited Design, synthesis and use of synthetic nucleotides comprising charge mass tags

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AU2002366479A1 (en) 2003-06-30
KR20030049802A (ko) 2003-06-25

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