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WO1990011374A1 - Produits de reaction de chaîne de polymerase incorporant des fractions rapporteuses et separation par affinite - Google Patents

Produits de reaction de chaîne de polymerase incorporant des fractions rapporteuses et separation par affinite Download PDF

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Publication number
WO1990011374A1
WO1990011374A1 PCT/US1990/001534 US9001534W WO9011374A1 WO 1990011374 A1 WO1990011374 A1 WO 1990011374A1 US 9001534 W US9001534 W US 9001534W WO 9011374 A1 WO9011374 A1 WO 9011374A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
primer
extension product
assay
sequence
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US1990/001534
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English (en)
Inventor
Frank Worden Hobbs, Jr.
Gerald Joseph Litt
Jeffrey Allan Miller
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
EIDP Inc
Original Assignee
EI Du Pont de Nemours and Co
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 EI Du Pont de Nemours and Co filed Critical EI Du Pont de Nemours and Co
Publication of WO1990011374A1 publication Critical patent/WO1990011374A1/fr
Priority to NO91913792A priority Critical patent/NO913792L/no
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • This invention relates to the detection of nucleic acid sequences and more specifically to a process of combining amplification of target nucleic acid sequences with detection of a reporter group specifically incorporated into the target nucleic acid sequence .
  • nucleic acid hybridization methods which can be used for detecting nucleic acid sequences of interest has been limited by several factors. These include lack of sensitivity, complexity of procedure, and the desire.to convert from radiometric to nonradiometric detection methods.
  • a variety of methods have been investigated for the purpose of increasing the sensitivity nonradiometric procedures. In one general approach, improvements in the total assay procedure have been examined, with concomitant effects on the issues of complexity and nonradiometric detection. In another approach, methods which increase the amount of nucleic acid to be detected by such assays have been pursued.
  • Patent 4,358,535, issued to Falko describes a method of culturing cells to increase their number and thus the amount of nucleic acid of the organism suspected to be present, depositing the sample onto fixed support, and then contacting the sample with a labeled probe, followed by washing the support and detecting the label.
  • One drawback to this method is that without culturing the organism first, the assay does not have adequate sensitivity. Adding a culture step, however, is time consuming and not always successful.
  • Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, pp.390-401 (1982) describe a method in which a nucleic acid of interest is amplified by cloning it into an appropriate host system. Then, when the host organism replicates in culture, the nucleic acid of interest is also replicated. This method also suffers from the requirement to perform a culture step and thus provides for a procedure that is time consuming and complicated.
  • An advantage of this method is that it can rapidly produce large quantities of a small portion of the sequence of the nucleic acid of an organism of interest.
  • a disadvantage of this method is that the detection of the nucleic acids produced, using a direct assay method, is complicated in that the amplification process can produce nucleic acid sequences w c are no a u cop es o e original nucleic acid which was to be copied. These erroneous nucleic acid sequences can provide false positives in the assay which increase the background noise and thus decrease the sensitivity of the entire method.
  • any unhybridized reporter probe is washed away followed by the detection of the label incorporated into the complex bound to the solid support.
  • An advantage of this technique over that disclosed by Ranki et al. is that the hybridization, which takes place in solution, is favored kinetically.
  • Some disadvantages are that the length of the target nucleic acid affects the overall efficiency of the reaction which decreases with increasing target nucleic acid length.
  • sandwich nucleic acid probe assays whether heterogeneous two-step or one-step, or utilizing solution hybridization, are not as sensitive as the direct assay method.
  • the nucleic acid assay of this invention for the detection and/or measurement of a preselected nucleic acid sequence in a sample suspected of including a nucleic acid containing said preselected sequence comprises the steps of:
  • step (D) contacting the product of step (C) with an oligonucleotide which is attached to a solid support and which is complementary to a portion of a primer extension product not including the nucleic acid sequences defined by both primers;
  • the nucleic acid assay of this invention comprises the following overall process for the detection of target nucleic acids of a preselected sequence: a) Using the polymerase chain reaction (PCR) nucleic acid amplification method described in U.S. 4,683,202, incorporated herein by reference, specific nucleic acid sequences are amplified by annealing the denatured target nucleic acid present in the sample w pr r r rm ng extension products.
  • a deoxyribonucleotide triphosphate containing a reporter group (moiety) dNTP-R
  • dNTP deoxyribonucleotide triphosphate containing a reporter group (moiety)
  • Each extension product formed is complementary to a portion of the preselected nucleic acid sequence contained within the target nucleic acid and becomes a template for further primer binding. This process is then repeated as necessary in order to produce the desired amount of primer extension product for detection and/or measurement.
  • the resulting nucleic acid is rendered single-stranded by known methods, such as treatment with heat, chaotropic agents, or by raising or lowering the pH.
  • the single-stranded nucleic acid so produced is then contacted with a capture probe which is attached to a solid support and allowed to hybridize with it.
  • the amplified nucleic acid - capture probe complex is washed with appropriate buffers to remove unhybridized product from above and any unincorporated dNTP-R.
  • the presence and quantity of the reporter group is detected and/or measured and is proportional to the amount of amplified target nucleic acid.
  • the amount of amplified target nucleic acid present is proportional to the unamplified target nucleic acid originally present in the sample.
  • a labeled antibody to the reporter group incorporated during the amplification process is employed. It is brought into contact with the amplified product before or subsequent to capture of the amplified product. The label on the antibody can then be used to detect the presence of the amplified product.
  • PCR as used herein in referring to the process of amplifying target nucleic acid sequences employing primer oligonucleotides to produce by enzymatic means a greatly increased number of copies of a small portion of the target nucleic acid is described in U.S. patent 4,683,202.
  • capture probe refers to an oligonucleotide which is complementary to a portion of a preselected sequence of the target nucleic acid and which is attached to a solid support.
  • the capture probe cannot be complementary to either primer or to those portions of a primer extension product whose nucleic acid sequences are defined by the primers.
  • Reporter group-containing deoxyribonucleotide triphosphates are known materials. Reporter groups of interest include fluorescent compounds such as fluorescein. In the alternative detection method, the reporter group has to be a moiety capable of forming a stable complex with an antibody.
  • Useful reporter groups in this invention include: fluorescein, rhodamine, and other chromogenic or fluorogenic compounds.
  • the PCR target amplification reaction requires approximately 20 to 30 repeat cycles in order to produce a sufficient quantity of the amplified target nucleic acid for further hybridization. Denaturation of the amplified nucleic acid can be accomplished by treatment with alkali, acid, chaotropic agents, or heat, although the preferred method is to place the amplified target nucleic acid in a boiling water bath for at least 10 minutes followed by a chilled water bath (4°C) for at least two minutes.
  • the hybridization step can be accomplished by contacting the amplified single-stranded target nucleic acid containing the reporter group(s) in solution with the capture probe, which is attached to a solid support, in an appropriate buffer, for a period of from 1 to 30 minutes.
  • the length of the capture probe is determined by the ease of its synthesis, by the desired reaction kinetics, and by the identity of the primers, and preferably is an oligonucleotide of approximately 20 to 30 bases.
  • the capture probe can be attached to the solid support by known means through sugar groups, preferably through either the 5'-terminal or the 3'-terminal sugar groups; or by attachment through a modified nucleotide base group.
  • solid supports can be utilized.
  • solid supports include magnetic particles, such as the chromium dioxide particles disclosed by Lau et al. , U.S. Patent 4,661,408, incorporated herein by reference, microtiter plates, and membranes.
  • the immobilized target nucleic acid on the solid support can then be washed several times, for example, in the temperature range of 25°C - 37°C, for approximately 5 to 10 minutes per wash cycle.
  • a variety of known detection methods can be utilized in the assay of this invention depending on the type of the reporter groups incorporated into the amplified product.
  • the reporter group is a chromophor or fluorophor
  • the incorporated reporter group can be detected by known spectroscopic techniques.
  • a labeled antibody to the reporter group, incorporated during the amplification process is brought into contact with the amplified product before or subsequent to capture of the amplified product by hybridization to the capture probe. The label on the antibody can then be used to detect the presence of the amplified product subsequent to the washing step.
  • Aliquots of serial dilutions (lxl0 +7 , lxl0 +6 , lxl0 +5 , lxlO ⁇ 4 , 1x10+ 3 , l ⁇ l0+ 2 , lxl0 +1 , and zero copies) of plasmid pBH10-R3 can be amplified using PCR.
  • Each aliquot can be combined with a buffer 200 ⁇ M in each of dATP, dTTP, dCTP, and dGTP and 10 ⁇ M in succinyl-fluorescein dTTP, 1.0 ⁇ M in each of Primer A and Primer B, and containing 1 ⁇ g of human placental DNA/reaction and 2.5 units of a DNA polymerase enzyme in a total reaction volume of 100 ⁇ l.
  • Each reaction mixture can then be temperature cycled as described in the product bulletin thirty (30) times.
  • This process is expected to result in the estimated increase in the number of target molecules by lxl0 +5 to lxl0 +6 .
  • Hybridization buffer can be prepared by combining: 3 ml of 20X SSC, pH 7.0, 0.1 ml of Triton X-100, an alkylaryl polyether alcohol having 9-10 ethoxy units, 1.0 ml of deionized formamide, 6.375 ml of H2O, and 25 ⁇ l of 1.0 N HC1.
  • 12 ⁇ l of capture oligonucleotide bound to chromium dioxide particles (12 ⁇ g) can then be added to the samples, incubated for 10 minutes at 37°C, centrifuged in a microfuge for 5 seconds, and placed in a Corning magnetic rack for two minutes at 25°C.
  • the pellets can then be washed three times at 25°C by adding 200 ⁇ l of wash buffer containing IX SSC, pH 7.0, and 0.17% Triton X-100, mixing, placing the samples in a magnetic rack for 2 minutes, and removing the wash buffer.
  • Detection can be accomplished by adding 200 ⁇ l of 10 mM Tris, pH 7.0, to each sample and placing them in boiling water bath for 10 minutes. The tubes can then be transferred to a chilled water bath (4°C) for two minutes and centrifuged in a microcentrifuge for 10 seconds. 200 ⁇ l portions can then be removed from each tube and the amplified nucleic acid product detected by direct fluorescence visualization.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • 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 une analyse de détection d'acide nucléiquepar incorporation d'un triphosphate de désoxyribonucléotide contenant une fraction rapporteuse dans un procédé d'amplification d'acide nucléique suivie de la détection de la fraction rapporteuse.
PCT/US1990/001534 1989-03-27 1990-03-26 Produits de reaction de chaîne de polymerase incorporant des fractions rapporteuses et separation par affinite Ceased WO1990011374A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
NO91913792A NO913792L (no) 1989-03-27 1991-09-26 Polymerasekjedereaksjonsprodukter som omfatter rapportoerrester, og affinitetsseparasjon.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US32912889A 1989-03-27 1989-03-27
US329,128 1989-03-27

Publications (1)

Publication Number Publication Date
WO1990011374A1 true WO1990011374A1 (fr) 1990-10-04

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PCT/US1990/001534 Ceased WO1990011374A1 (fr) 1989-03-27 1990-03-26 Produits de reaction de chaîne de polymerase incorporant des fractions rapporteuses et separation par affinite

Country Status (4)

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JP (1) JPH04504203A (fr)
AU (1) AU5359690A (fr)
CA (1) CA2012982A1 (fr)
WO (1) WO1990011374A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0469755A1 (fr) * 1990-07-19 1992-02-05 BEHRINGWERKE Aktiengesellschaft Procédé de production d'un polynucléotide pour utilisation dans une amplification à l'aide d'une seule amorce
EP0480289A1 (fr) * 1990-10-09 1992-04-15 Roche Diagnostics GmbH Méthode de détection spécifique de genre et d'espèce de bactéries dans un liquide a prober
WO1992006216A1 (fr) * 1990-10-09 1992-04-16 Boehringer Mannheim Gmbh Procede de depistage sensible d'acides nucleiques
EP0501356A1 (fr) * 1991-02-28 1992-09-02 Roche Diagnostics GmbH Détection de bactéries avec amplification d'acides nucléiques
WO1994009156A1 (fr) * 1992-10-08 1994-04-28 The Regents Of The University Of California Dosages pcr permettant de determiner la presence et la concentration d'une cible
US5439793A (en) * 1990-07-19 1995-08-08 Syntex (U.S.A.) Inc. Method for producing a polynucleotide having an intramolecularly base-paired structure
US5753433A (en) * 1909-12-05 1998-05-19 Boehringer Mannheim Gmbh Method for the sensitive detection of nucleic acids
US5985548A (en) * 1993-02-04 1999-11-16 E. I. Du Pont De Nemours And Company Amplification of assay reporters by nucleic acid replication
USH1985H1 (en) 1992-01-09 2001-08-07 The United States Of America As Represented By The Secretary Of The Navy Method for detecting biological toxins

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4605735A (en) * 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4775619A (en) * 1984-10-16 1988-10-04 Chiron Corporation Polynucleotide determination with selectable cleavage sites
EP0097373B1 (fr) * 1982-06-23 1992-10-07 Enzo Biochem, Inc. Nucléotides modifiés marqués et polynucléotides ainsi que leurs méthodes de préparation, d'utilisation et de détection

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0097373B1 (fr) * 1982-06-23 1992-10-07 Enzo Biochem, Inc. Nucléotides modifiés marqués et polynucléotides ainsi que leurs méthodes de préparation, d'utilisation et de détection
US4605735A (en) * 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4775619A (en) * 1984-10-16 1988-10-04 Chiron Corporation Polynucleotide determination with selectable cleavage sites
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (fr) * 1986-01-30 1990-11-27 Cetus Corp

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Volume 16, No. 7, 1977, pages 1364-1370, (Baltimore, MD, USA), MANNING et al.: "A method for gene enrichment based on the Avidin-Biotin Interaction. Application to the Drosphila Ribosomal RNA genes". See entire document. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5753433A (en) * 1909-12-05 1998-05-19 Boehringer Mannheim Gmbh Method for the sensitive detection of nucleic acids
EP0469755A1 (fr) * 1990-07-19 1992-02-05 BEHRINGWERKE Aktiengesellschaft Procédé de production d'un polynucléotide pour utilisation dans une amplification à l'aide d'une seule amorce
US5439793A (en) * 1990-07-19 1995-08-08 Syntex (U.S.A.) Inc. Method for producing a polynucleotide having an intramolecularly base-paired structure
US5595891A (en) * 1990-07-19 1997-01-21 Behringwerke Ag Method for producing a polynucleotide for use in single primer amplification
EP0480289A1 (fr) * 1990-10-09 1992-04-15 Roche Diagnostics GmbH Méthode de détection spécifique de genre et d'espèce de bactéries dans un liquide a prober
WO1992006216A1 (fr) * 1990-10-09 1992-04-16 Boehringer Mannheim Gmbh Procede de depistage sensible d'acides nucleiques
US6225094B1 (en) 1990-10-09 2001-05-01 Roche Diagnostics Gmbh Method for the genus-specific or/and species-specific detection of bacteria in a sample liquid
EP0501356A1 (fr) * 1991-02-28 1992-09-02 Roche Diagnostics GmbH Détection de bactéries avec amplification d'acides nucléiques
USH1985H1 (en) 1992-01-09 2001-08-07 The United States Of America As Represented By The Secretary Of The Navy Method for detecting biological toxins
WO1994009156A1 (fr) * 1992-10-08 1994-04-28 The Regents Of The University Of California Dosages pcr permettant de determiner la presence et la concentration d'une cible
US5747251A (en) * 1992-10-08 1998-05-05 The Regents Of The University Of California Polymerase chain reaction assays to determine the presence and concentration of a target nucleic acid in a sample
US5985548A (en) * 1993-02-04 1999-11-16 E. I. Du Pont De Nemours And Company Amplification of assay reporters by nucleic acid replication

Also Published As

Publication number Publication date
JPH04504203A (ja) 1992-07-30
CA2012982A1 (fr) 1990-09-27
AU5359690A (en) 1990-10-22

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