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WO1990011373A1 - Procede de detection rapide d'acide nucleique - Google Patents

Procede de detection rapide d'acide nucleique Download PDF

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Publication number
WO1990011373A1
WO1990011373A1 PCT/US1990/001533 US9001533W WO9011373A1 WO 1990011373 A1 WO1990011373 A1 WO 1990011373A1 US 9001533 W US9001533 W US 9001533W WO 9011373 A1 WO9011373 A1 WO 9011373A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
assay
primer
sequence
extension product
Prior art date
Application number
PCT/US1990/001533
Other languages
English (en)
Inventor
Frank Worden Hobbs, Jr.
Gerald Joseph Litt
Jeffrey Allan Miller
Original Assignee
E.I. Du Pont De Nemours And Company
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 E.I. Du Pont De Nemours And Company filed Critical E.I. Du Pont De Nemours And Company
Publication of WO1990011373A1 publication Critical patent/WO1990011373A1/fr
Priority to NO913793A priority Critical patent/NO913793D0/no

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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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • 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 sequence followed by affinity capture.
  • 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 which are not faithful copies of the 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: (A) rendering the target nucleic acid single- stranded; (B) amplifying at least one specific nucleic acid sequence contained within the preselected nucleic acid sequence in the presence of at least one deoxyribonucleotide triphosphate containing a reporter moiety in an amount up to the total replacement of the corresponding dNTP, by (1) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be sufficiently complementary to the different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it
  • step (3) treating the single-stranded molecules generated from step (2) with the primers of step (1) under conditions that a primer extension product is synthesized using each of the single strands produced in step (2) as a template;
  • step (C) contacting the product of step (B) with a solid affinity support matrix
  • 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 with primers specific for the target and forming extension products.
  • a deoxyribonucleotide triphosphate containing a reporter group (moiety) dNTP-R, is used to replace some or all of at least one of the corresponding deoxyribonucleotide triphosphates employed.
  • 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 brought in contact with an affinity support and allowed to bind to the affinity support for a period of from 1 to 30 minutes .
  • the affinity support is then rinsed with an appropriate buffer in order to remove non-incorporated reporter group modified deoxyribonucleotide triphosphate.
  • 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.
  • 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.
  • affinity supports can be utilized.
  • affinity membranes such as Gene Screen® hybridization transfer membrane (a registered trademark of E. I . du Pont de Nemours and Company; a nylon-based membrane) , nitrocellulose, and Gene Screen PlusTM, a positively charged and supported nylon 66 membrane.
  • Gene Screen® hybridization transfer membrane a registered trademark of E. I . du Pont de Nemours and Company; a nylon-based membrane
  • nitrocellulose nitrocellulose
  • Gene Screen PlusTM Gene Screen PlusTM
  • a variety of known detection methods can be utilized in the assay of this invention, depending upon the reporter group 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 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.
  • Aliquots of serial dilutions (lxl0 +7 , lxl0 +6 , lxl0 +5 , lxl0 +4 f lxlO +3 , lxl0 +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 .
  • the product of Step A can be adsorbed onto a microtiter plate having a bottom composed of Gene Screen Plus for 30 minutes at 25°C.
  • the microtiter plate wells can then be washed three times for 5 minutes at 25°C by adding 200 ⁇ l of wash buffer containing IX SSC, pH 7.0, and 0.17% Triton X-100, and aspirating between washes.
  • Detention Detection can be accomplished by adding 200 ⁇ l of 10 mM Tris, pH 7.00 to each sample and detecting the amplified nucleic acid product detected by reflectance fluorescence visualization.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • AIDS & HIV (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne une analyse de détermination d'acide nucléique par incorporation d'un triphosphate de désoxyribonucléotide contenant une moitié rapporteuse dans un acide nucléique amplifié, suivie de la capture par affinité et de la détection de la moitiéou fraction rapporteuse.
PCT/US1990/001533 1989-03-27 1990-03-26 Procede de detection rapide d'acide nucleique WO1990011373A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
NO913793A NO913793D0 (no) 1989-03-27 1991-09-26 Fremgangsmaate for hurtig nukleinsyrepaavisning.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US329,142 1981-12-10
US32914289A 1989-03-27 1989-03-27

Publications (1)

Publication Number Publication Date
WO1990011373A1 true WO1990011373A1 (fr) 1990-10-04

Family

ID=23284035

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1990/001533 WO1990011373A1 (fr) 1989-03-27 1990-03-26 Procede de detection rapide d'acide nucleique

Country Status (5)

Country Link
EP (1) EP0465557A1 (fr)
JP (1) JPH04504202A (fr)
AU (1) AU5354790A (fr)
CA (1) CA2012984A1 (fr)
WO (1) WO1990011373A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
WO1988006633A1 (fr) * 1987-03-02 1988-09-07 Arnold Lyle John Jr Supports polycationiques pour la purification, la separation et l'hybridation d'acides nucleiques
EP0297379A2 (fr) * 1987-06-30 1989-01-04 Miles Inc. Procédé pour l'amplification des gènes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) * 1985-03-28 1990-11-27 Cetus Corp
WO1988006633A1 (fr) * 1987-03-02 1988-09-07 Arnold Lyle John Jr Supports polycationiques pour la purification, la separation et l'hybridation d'acides nucleiques
EP0297379A2 (fr) * 1987-06-30 1989-01-04 Miles Inc. Procédé pour l'amplification des gènes

Also Published As

Publication number Publication date
AU5354790A (en) 1990-10-22
CA2012984A1 (fr) 1990-09-27
EP0465557A1 (fr) 1992-01-15
JPH04504202A (ja) 1992-07-30

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