WO2002077282A1 - Methode pour detecter un gene - Google Patents

Methode pour detecter un gene Download PDF

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Publication number
WO2002077282A1
WO2002077282A1 PCT/JP2002/002893 JP0202893W WO02077282A1 WO 2002077282 A1 WO2002077282 A1 WO 2002077282A1 JP 0202893 W JP0202893 W JP 0202893W WO 02077282 A1 WO02077282 A1 WO 02077282A1
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WO
WIPO (PCT)
Prior art keywords
signal
nucleic acid
exonuclease
probe
dna
Prior art date
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PCT/JP2002/002893
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English (en)
Japanese (ja)
Inventor
Takeshi Yamamoto
Masafumi Ikeda
Original Assignee
International Reagents 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.)
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Publication date
Application filed by International Reagents Corporation filed Critical International Reagents Corporation
Priority to JP2002575322A priority Critical patent/JPWO2002077282A1/ja
Publication of WO2002077282A1 publication Critical patent/WO2002077282A1/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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

  • the present invention relates to a method for analyzing genes mainly in the fields of clinical chemistry, drug chemistry, biochemistry, and food chemistry, and includes methods for detecting, quantifying, and mutating genomic genes, viruses, bacteria, and other genes. Used for detection of Background art
  • the southern blot hybridization method and the northern blot hybridization method have been widely used for a long time.
  • the operation was complicated, took a long time, and had to deal with radioactive materials.
  • Hybridization Protection Assay was used as a homogeneous analysis method. Law (Clinical Chemistry 35, 1588-1594, 1984) Force S Reported. In this method, after a DNA nucleic acid labeled with an ata-dimeric ester is hybridized to a target nucleic acid and subjected to a hydrolysis treatment, the hybridized acryl-dimethyl ester is converted into a double-stranded nucleic acid. Therefore, it utilizes the fact that only the acridinium ester of the unhybridized probe is hydrolyzed and loses chemiluminescence. However, the hydrolysis operation is complicated and time-consuming.
  • An object of the present invention is to provide a practical and simple homogeneous measurement method for a nucleic acid detection method using exonuclease.
  • the present inventors have conducted intensive studies and found that a signal-producing label substance and a pair of label substances arranged so as to effectively attenuate the generation of the signal are each bound to a capture probe to obtain a target nucleic acid.
  • a nucleotide that has a function of effectively attenuating the signal of the signal-labeling substance and / or the signal of the signal-generating substance by the action of a certain exonuclease.
  • the inventors have found that a signal can be obtained by releasing from the probe, and have completed a measurement technique capable of judging the presence / absence of a nucleic acid sequence and quantification using a homogeneous system using this mechanism.
  • a homogeneous measurement system for detecting a nucleic acid sequence using an oligonucleotide probe having a signal-generating label substance and a pair of label substances arranged to effectively attenuate the generation of the signal After the hybridization between the target nucleic acid and the probe DNA, the probe DNA is moved from the 5 ′ or 3 ′ end by the action of 5 ′ ⁇ 3, exonuclease or 3 ′ ⁇ 5, exonuclease.
  • a signal is obtained by digesting and releasing a signal-generating substance-labeled nucleotide inserted into the probe DNA and / or a nucleotide labeled with a substance having a function of effectively attenuating the signal of the signal-generating substance.
  • FIG. 1 is an explanatory diagram showing a range of a quantifiable amount of DNA (Example 3).
  • FIG. 2 is an explanatory diagram showing the minimum detection sensitivity (Example 3).
  • the present invention relates to a method for detecting a target nucleic acid (hereinafter, referred to as “target DNA”) and a method for detecting a gene mutation, which are intended to effectively attenuate the generation of a signal and a signal.
  • target DNA a target nucleic acid
  • probe DNA a capture probe
  • the signal-generating labeling substance is acted upon by the action of exonuclease.
  • the combination of the signal-generating label substance and the pair of label substances arranged so as to effectively attenuate the generation of the signal in the present invention may be any combination having such a function.
  • those corresponding to optically detected signals are easy to handle and suitable.
  • the optically detected signal is fluorescent, It does not need to be fluorescent and may not be particularly limited.
  • the probe substances of probe DNA are Fluorescein and Tetramethylrhodamine, FAM and TAMRA, EDANS and DABCYL, FITC and DABCYL, Fluorescein and DABCYL, Fluorescein and Cy3, FITC and Cy3, Fluorescein and QSY. 7dye, but is not limited to these.
  • This signal generation labeling substance (hereinafter, also referred to as “reporter group”) is paired with a labeling substance having a function of effectively attenuating the generation of the signal (hereinafter, this is also referred to as “taenching group”).
  • the reporter group has a function of effectively attenuating the signal generated by the reporter group when the distance from the quenching group is shorter by + minutes.
  • the combination of labeling substances having such a function For example, in the case of a fluorescent substance, when the fluorescence wavelength of the reporter group and the absorption wavelength of the quenching group have a common spectrum. It can be said that it has a function as such a pair of labeling substances.
  • FRET fluorescence resonance energy transfer
  • a probe DNA having a pair of quenching groups and a reporter group is used, but if the labeled probe DNA has not been digested with exonuclease, no signal is detected or exonuclease is not detected.
  • the pair of labeling substances must be arranged so that only a significantly lower signal is generated as compared with the case where the reporter group or the quenching group is released by the action of the above.
  • One such example is the 5 'end of the probe DNA.
  • the distance between the quenching group and the reporter group only needs to be able to effectively attenuate the signal generation of one reporter group, but is preferably within 20 bases, more preferably within 10 bases.
  • the present invention uses a mechanism in which a signal is obtained from a reporter group when a probe DNA is digested by the action of exonuclease and a nucleotide labeled with either a reporter group or a quenching group is released. It is also characterized by detecting a nucleic acid sequence or detecting a gene mutation.
  • the mechanism by which this signal can be obtained is as follows. That is, in the method for detecting a nucleic acid sequence, quenching is performed by hybridizing the probe DNA with the target DNA in a complementary manner and then digesting the probe from the 5 or 3 ′ end by the action of exonuclease.
  • Signal is released by releasing the nucleotide labeled with the quenching group and the Z or reporter group, thereby increasing the distance between the reporter group and the quenching group and weakening the quenching group. It refers to the mechanism that produces it.
  • the probe DNA is present as a single strand, so it is not digested by the double-strand-specific exonuclease, or is minimally digested. As a result, the signal generation does not occur, and the signal generation is significantly lower than when the target DNA is present.
  • a probe DNA is hybridized with a target DNA to form a heteroduplex, and a base mutation, insertion, or deletion is present in the target DNA. Therefore, a protein having exonuclease activity recognizes a mismatch, bubble, or loop structure generated in the heteroduplex, and a probe is formed only when the mismatch, bubble, or loop structure is present. Digestion of DNA from its ends releases nucleotides labeled with a quenching group or reporter group, resulting in a greater distance between the reporter group and the quenching group, resulting in attenuation by the quenching group. A weak signal causes a signal to be generated. In the absence of mismatches, bubbles, or loop structures, is the digestion by exonucleases minimal, or even minimal, resulting in no signal generation? Signal generation is significantly lower than when a mismatch, bubble or loop structure is present.
  • the exonuclease used in the nucleic acid sequence detection method can release a nucleotide labeled with a quenching group or a reporter group in a probe DNA from a polynucleotide chain.
  • Any 5 'exonuclease may be used, but examples thereof include 5'exonuclease; Escherichia coli exonuclease III as exonuclease and 3, exonuclease. it can.
  • the protein having exonuclease activity used in the method for detecting a gene mutation recognizes a mismatch, bubble or loop structure in a heteroduplex, and has a mismatch, bubble or loop structure.
  • Hybridization of the target DNA and the labeled binding probe DNA in the present invention is carried out after the target DNA is denatured into a single strand by denaturation. If the target DNA is single-stranded, there is no need for denaturation and hybridization with the probe DNA can be performed as it is, but hybridization due to the higher-order structure of the single-stranded target DNA is possible. In order to eliminate the possibility of inhibition of the enzyme, it is preferable to carry out a denaturation operation.
  • the modification method in the present invention may be in accordance with a commonly used method, and examples thereof include a method by thermal denaturation and a method by chemical modification.
  • the method by thermal denaturation is usually performed by heating to 90 ° C or more.
  • denaturation is performed by heating at 94-97 ° C for about 5 minutes.
  • the method by chemical denaturation is performed by the action of a denaturant such as urea or formaldehyde, or by increasing the pH.
  • the pH can be increased by using 0.4 to 1. Omol / L NaOH. From the viewpoint of operability and the like, a method based on heat denaturation is preferably used.
  • the hybridization may be performed according to a commonly used method, but the conditions differ depending on the length of the probe or denatured double-stranded target DNA, base sequence, and Tm (melting temperature). Generally, a pH of 6.4 to 9.0 and a pH of 10 to 1000 mmol / L Hybridization is performed at room temperature to 70 ° C in the presence of stream ions.
  • fluorescence detection conditions include, for example, 5'-end of probe DNA with Dabcyl modification and Fluorescein modification in the chain, 3 ' ⁇ 5, degraded from 3' end by the action of exonuclease
  • the emitted fluorescence is detected using a fluorimeter under the conditions of a maximum excitation wavelength of 49.4 nm and a maximum fluorescence wavelength of 51.8 nm.
  • the signal generated by the degradation of the probe DNA by exonuclease for example, the fluorescence signal in the case of FRET, increases with the degradation reaction of the probe DNA, and the signal can be monitored during the reaction.
  • the signal generated by the degradation of the probe DNA by exonuclease increases with the degradation reaction of the probe DNA, and the signal can be monitored during the reaction.
  • reproducibility and quantitativeness can be improved and the measurement time can be shortened.
  • DNA extracted from a biological sample can be used, but the amount is often insufficient for analysis.
  • the nucleic acid extracted from the biological sample can be amplified by PCR or the like, and the amplified DNA product can be used for the gene mutation analysis according to the present invention. .
  • the probe DNA can be prepared by, for example, a chemical method using a general DNA synthesizer.
  • PCR primer As the target DNA, a double-stranded DNA whose human K-ras region was amplified by PCR was used.
  • the sequence of the PCR primer is as follows.
  • Nucleotide sequence 1 5'-GAGAGGCCTGCTGAAAATG
  • Nucleotide sequence 2 5 '-CCTCTATTGTTGGATCATATTC
  • a model system for detecting nucleic acid sequences was constructed using exonuclease III, which is a 3 'exonuclease, and a probe labeled with Dabcyl at the 5' end and Fluorescein in the chain.
  • exonuclease III which is a 3 'exonuclease
  • a probe labeled with Dabcyl at the 5' end and Fluorescein in the chain As the target DNA, a PCR product obtained by amplifying a partial sequence of the human K-ras gene was used. Oligonucleotide having the base sequence shown below as the probe DNA Leotide was chemically synthesized and used. Dabcyl is labeled at the 5 'end of the probe DNA and fluorescein is labeled in the strand. "Z" in the sequence represents Fluorescein-dT.
  • Nucleotide sequence 3 5 '-GCTCCAACZACCACAAGTTTATATTCAGTC
  • the optimal amount of enzyme, probe DNA amount, NaCl concentration, reaction temperature, and hybridization conditions were examined. Based on the results, the operation method of this method was as follows. Determined. That is,
  • the range of the amount of DNA that can be quantified by the method of the present invention was verified. As shown in FIG. 1, a good calibration curve was drawn until the PCR product amount was 300 fmol, indicating that the quantification range of the present method was wide.
  • the mean value of the relative fluorescence intensity (RFU) and twice the standard deviation (2SD) of each four simultaneous measurements is the minimum target DNA amount that does not fall below the value obtained by adding 2SD to the average RFU value at the target DNA force S0 fraol.
  • the minimum detection sensitivity of this method was 1.45 fmol, indicating that it was extremely sensitive (Fig. 2).
  • nucleic acid sequence can be performed by a simple operation, so that automation is also easy. Further, the signal can be measured while the reaction is in progress, and the analysis result can be obtained quickly.
  • exonuclease used in the present invention any exonuclease can be used as long as it can release a nucleotide having a modified reporter group or signal group.

Abstract

L'invention concerne une méthode par laquelle une séquence d'acide nucléique spécifique peut être détectée de façon rapide et pratique dans un système homogène. Un marqueur générateur de signal et une paire de marqueurs servant à atténuer la génération de signal sont reliées respectivement à l'extrémité 5' d'un ADN sonde et l'intérieur de son brin, à l'extrémité 3' et l'intérieur du brin, ou tous deux à l'intérieur du brin. La sonde ainsi marquée est hybridée avec un acide nucléique cible et traitée à l'exonucléase, de manière à digérer l'ADN sonde de l'extrémité 5' ou 3'. Un nucléotide ainsi marqué avec un groupe rapporteur ou un groupe extincteur à l'intérieur du brin et/ou un nucléotide marqué avec une substance capable d'atténuer de manière effective le signal du générateur de signal sont libérés de l'ADN sonde. Ainsi, l'effet d'atténuation du signal du groupe rapporteur par le groupe extincteur est supprimé ou diminué. Un signal détectable, qui permet de détecter et de quantifier un acide nucléique d'une séquence spécifique, est également généré.
PCT/JP2002/002893 2001-03-27 2002-03-26 Methode pour detecter un gene WO2002077282A1 (fr)

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JP2002575322A JPWO2002077282A1 (ja) 2001-03-27 2002-03-26 遺伝子検出方法

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JP2001090134 2001-03-27
JP2001-90134 2001-03-27

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005052143A (ja) * 2003-08-01 2005-03-03 F Hoffmann La Roche Ag ホットスタートリアルタイムポリメラーゼ連鎖反応のための新しい検出形式
JP2013538063A (ja) * 2010-09-20 2013-10-10 シージーン アイエヌシー 単一標識固定化プローブ及びエキソ核酸切断活性を利用した固相におけるターゲット核酸配列検出

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06327499A (ja) * 1993-05-21 1994-11-29 Eiken Chem Co Ltd 塩基配列検出方法
WO1996015270A1 (fr) * 1994-11-16 1996-05-23 Perkin-Elmer Corporation Sonde en fluorescence a auto-extinction et methode de fonctionnement
WO1997041256A2 (fr) * 1996-04-26 1997-11-06 Abbott Laboratories Procede et reactif de detection de sequences nucleotidiques multiples dans un echantillon d'essai
WO2001061034A1 (fr) * 2000-02-16 2001-08-23 Jussi Nurmi Procede homogene de detection d'un polynucleotide, notamment au moyen d'un marqueur chelate de lanthanide clivable

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06327499A (ja) * 1993-05-21 1994-11-29 Eiken Chem Co Ltd 塩基配列検出方法
WO1996015270A1 (fr) * 1994-11-16 1996-05-23 Perkin-Elmer Corporation Sonde en fluorescence a auto-extinction et methode de fonctionnement
WO1997041256A2 (fr) * 1996-04-26 1997-11-06 Abbott Laboratories Procede et reactif de detection de sequences nucleotidiques multiples dans un echantillon d'essai
WO2001061034A1 (fr) * 2000-02-16 2001-08-23 Jussi Nurmi Procede homogene de detection d'un polynucleotide, notamment au moyen d'un marqueur chelate de lanthanide clivable

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
COPLEY C.G. ET AL.: "Exonuclease cycling assay: an amplified assay for the detection of specific DNA sequences", BIOTECHNIQUES, vol. 13, no. 6, 1992, pages 888 - 892, XP002060879 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005052143A (ja) * 2003-08-01 2005-03-03 F Hoffmann La Roche Ag ホットスタートリアルタイムポリメラーゼ連鎖反応のための新しい検出形式
US20090181401A1 (en) * 2003-08-01 2009-07-16 Dieter Heindl Detection format for hot start real time polymerase chain reaction
JP2013538063A (ja) * 2010-09-20 2013-10-10 シージーン アイエヌシー 単一標識固定化プローブ及びエキソ核酸切断活性を利用した固相におけるターゲット核酸配列検出

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