EP0708840A1 - Procedes ameliores pour detecter des sequences d'acides nucleiques - Google Patents

Procedes ameliores pour detecter des sequences d'acides nucleiques

Info

Publication number
EP0708840A1
EP0708840A1 EP94921315A EP94921315A EP0708840A1 EP 0708840 A1 EP0708840 A1 EP 0708840A1 EP 94921315 A EP94921315 A EP 94921315A EP 94921315 A EP94921315 A EP 94921315A EP 0708840 A1 EP0708840 A1 EP 0708840A1
Authority
EP
European Patent Office
Prior art keywords
target
probe
rnase
concentration
reaction
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.)
Withdrawn
Application number
EP94921315A
Other languages
German (de)
English (en)
Inventor
Michael J. Lane
Albert S. Benight
Brian D. Faldasz
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.)
ID Biomedical Corp
Original Assignee
ID Biomedical Corp of Quebec
Research Foundation of State University of New York
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 ID Biomedical Corp of Quebec, Research Foundation of State University of New York filed Critical ID Biomedical Corp of Quebec
Publication of EP0708840A1 publication Critical patent/EP0708840A1/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the invention relates to the use of cycling probe reactions to detect the presence of nucleic acid sequences.
  • CPR Cycling Probe Reaction
  • a probe specific for a sequence to be identified hybridizes to a target single strand nucleic acid to form a duplex nucleic acid.
  • the probe includes a scissile link which becomes susceptible to cleavage upon formation of the duplex.
  • a DNA probe can include an RNA sequence segment. Formation of a duplex between such a probe and a single strand DNA molecule results in an RNA:DNA hybrid duplex.
  • RNase H which cleaves the RNA strand of an RNA:DNA hybrid duplex, is used to cleave the target-bound probe. Detection is based on this hybridization-specific cleavage.
  • the cleaved probe disassociates from the target and the target can enter the cycle with a new, uncleaved, probe.
  • the invention features a method for detecting a single-stranded target nucleic acid.
  • the method includes: a. providing a reaction mixture which includes the target nucleic acid, a complementary single-stranded nucleic acid probe, the probe being present in molar excess relative to the target and having the structure [NAj -R-NA2]n wherein NAj and NA2 are
  • DNA sequences wherein R is a scissile nucleic acid linkage, and wherein n is an integer from 1 to 10, and RNase H, the RNase H being present at a chemical potential sufficient to substantially increase the rate of duplex formation over what would be formed in the absence of the RNase H, and allowing target-probe duplex to form; b. treating the target-probe duplex from step (a) so as to cleave the probe within a predetermined sequence of the scissile nucleic acid linkage and thereby form at least one intact DNA-containing oligonucleotide fragment from the probe, such fragment being, or being treated so as to be, no longer capable of remaining hybridized to the target nucleic acid; c. repeating the cycle of steps (a) and (b); and d. detecting the intact DNA-containing fragments so formed and thereby detecting the single-stranded target nucleic acid.
  • reaction mixture which includes: a single-stranded target nucleic acid; a complementary single-stranded nucleic acid probe, the probe being present in molar excess relative to the target and having the structure [NA ⁇ -R-NA2] n wherein NAj and NA2 are DNA sequences, wherein R is a scissile nucleic acid linkage, and wherein n is an integer a RNase H, the RNase H being present at a chemical potential sufficient to substantially increase the rate of duplex formation over what would be formed in the absence of the RNase H.
  • Fig. 1 is a diagram of an improved CPR.
  • Fig. 2 is a depiction of a gel showing the products of an improved CPR.
  • Tm refers to the midpoint of the duplex to single strand melting transition.
  • the chemical potential of a reagent refers to the free energy change of a reaction mix when the reagent is added to the reaction mixture.
  • Chemical potential is a more exact measure of the activity of a species in a given reaction under a given set of conditions and takes into account considerations such as the number a sites a species can react with and whether all molecules of a species are available for reactions.
  • the chemical potential of the species can usually be most directly manipulated by changing the concentrations of a species.
  • the unit of chemical potential is free-energy, e.g., of cal/mole or J/mol, but in the methods described herein determination of an absolute chemical potential is not required.
  • Most of the methods disclosed herein require reaction mixtures in which the ratio of chemical potential between two species, or the difference between the chemical potential of two species, is required to be such that the chemical potential of the RNase H is sufficient to drive the reaction towards the duplex.
  • Purified nucleic acid refers to a purified DNA or RNA.
  • Purified DNA refers to DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring genome of the organism from which the DNA is derived.
  • RNA refers to an RNA which is substantially free of another RNA sequence with which it is found in a cell which produces the RNA. A non- naturally occurring nucleic acid sequence is purified when it is substantially free of other sequences.
  • Purified natural product refers to a product which is produced by an organism and which is substantially free of a macromolecule, e.g., a protein or a nucleic acid, with which it occurs in an organism from which it is derived.
  • Product which does not naturally occur in living cells refers to a product which is not synthesized or produced by living cells or organisms.
  • Methods of the invention allow CPR reactions to be driven by RNase H. As such, the rate of the reaction is controlled by the concentration or chemical potential of the RNase and not the probe or target nucleic acids.
  • CPR probes The making and using of CPR probes is known to the art, see e.g., Duck et al. U.S. Patent No. 4, 876,187 or Duck et al. U.S. Patent No. 5,011,069.
  • the nucleic acid probe which is useful in the practice of this invention comprises the structure:
  • NA ⁇ and NA2 are nucleic acid sequences, wherein R is an RNA sequence; and wherein n is an integer from about 1 to about 10.
  • NAj and NA2 in the nucleic acid probe independently comprise from about 0 to about 20 nucleotides, R comprises from about 1 to about 100 ribonucleotides, or more, and n is an integer from about 1 to about 10.
  • NAj and NA2 in the nucleic acid probe are DNA sequences.
  • NAj and NA2 in the nucleic acid probe are RNA sequences.
  • the nucleic acid probe comprises a structure wherein NAj is either an RNA or DNA sequence.
  • nicking the hybridized probe at predetermined RNA sequences is carried out with a double-stranded ribonuclease.
  • ribonucleases nick or excise ribonucleic acid sequences from double-stranded DNA-RNA hybridized stands.
  • An example of a ribonuclease useful in the practice of this invention is RNase H.
  • Other ribonucleases and enzymes may be suitable to nick or excise RNA from RNA-DNA strands, such as Exo III and reverse transcriptase.
  • the molecules of the present invention may have a detectable marker attached to one or more of the nucleic acid sequences, NAj or NA2.
  • This marker is contemplated to be any molecule or reagent which is capable of being detected. Examples of such detectable molecules are radioisotopes, radiolabelled molecules, fluorescent molecules, fluorescent antibodies, enzymes, or chemiluminescent catalysts.
  • Another suitable marker is a ligand capable of binding to specific proteins which have been tagged with an enzyme, fluorescent molecule or other detectable molecule.
  • a suitable ligand is biotin, which will bind to avidin or streptavidin.
  • the nucleic acid probe is. immobilized on a solid support.
  • the nucleic acid probe is labeled with a detectable marker.
  • the R portion described above may also be properly termed a scissile linkage in language consistent with usage in U.S. Patent No. 4,876,187. Such a linkage is capable of being cleaved or disrupted without cleaving or disrupting any nucleic acid sequence of the molecule itself or of the target nucleic acid molecule.
  • such a scissile linkage i.e., R
  • R is any connecting chemical structure which joins two nucleic acid sequences and which is capable of being selectively cleaved without cleavage of the nucleic acid sequences to which it is joined.
  • the scissile linkage may be single bond or a multiple unit sequence.
  • R denotes a ribonucleic acid (RNA) sequence.
  • Probe Reaction In this reaction a probe hybridizes to a target to form a duplex.
  • the probe includes a scissile link, which becomes susceptible to cleavage upon formation of a duplex.
  • the probe can have an RNase sensitive segment. Formation of a duplex between such a probe and a single strand DNA molecule results in an RNA:DNA hybrid duplex.
  • An enzyme which cleaves RNA:DNA hybrids, e.g., RNase H cleaves the probe only when it is bound to the target. Detection is based on this hybridization-specific cleavage.
  • RNase binds the duplex to form a duplex: RNase complex. Cleavage results in a cleaved probe:target complex.
  • the cleaved probe disassociates from the target and the target can enter the cycle with a new, uncleaved, probe.
  • Methods of the invention can be used to improve the performance of this reaction. As often used, this scheme is inappropriate for detecting a target sequence when insufficient RNase H is present.
  • probe concentration has been increased in attempts to drive the basic reaction to the right.
  • increasing the chemical potential of RNase H in the reaction mix will also favor the formation of duplex. Since RNaseH dictates duplex:RNaseH complex formation increasing the potential of this species in the reaction mix will favor generation of duplex substrate and therefore of cleaved-probe (signal).
  • the addition of RNase H can be used to increase sensitivity.
  • the method can be used for detecting a single-stranded target nucleic acid.
  • a reaction mixture is provided which includes the target nucleic acid, a complementary single-stranded nucleic acid probe, the probe being present in molar excess relative to the target and having the structure
  • NAj and NA2 are DNA sequences, wherein R is a scissile nucleic acid linkage, and wherein n is an integer from 1 to 10, and RNase H.
  • the RNase H is present at a chemical potential sufficient to substantially increase the rate of duplex formation over what would be formed in the absence of the RNase H. The mixture is maintained under conditions which allow target-probe duplex to form.
  • the target-probe duplex from step (a) is treated so as to cleave the probe within a predetermined sequence of the scissile nucleic acid linkage and thereby form at least one intact DNA-containing oligonucleotide fragment from the probe, such fragment being, or being treated so as to be, no longer capable of remaining hybridized to the target nucleic acid.
  • the cycle of steps (a) and (b) are then repeated and the intact DNA containing fragments so formed are detected to thereby detect the single-stranded target nucleic acid.
  • the method is performed at a temperature above the Tm of the duplex.
  • the temperature can be at least x C°, wherein x is 5, 10, 20, 30, 40, 50, 60, 70, or 80, above said Tm.
  • the rate of duplex formation is increased by at least y fold, wherein y is 2, 5, 10, 50, 100, 500, 10 3 , 10 4 , 10 5 , 10 6 , by the addition of RNase H.
  • the RNase H can be used to drive formation of duplex under conditions where the rate of duplex formation in the absence of RNase H would be substantially zero.
  • the concentration, number of molecules of, or the chemical potential of, the RNase H is greater than the concentration, number of molecules present, or chemical potential of the probe, the target, both the probe and the target, the duplex, or the combination of the probe, the target, and the duplex, e.g., at least z fold greater, wherein z is at least 1, 2, 5, 10, 25, 50, 100, 500, 10 3 , 10 4 , 10 5 or, 10 6 .
  • the concentration, number of molecules of, or the chemical potential of, the RNase H is sufficient to allow: detection of one target molecule in a biological sample, preferably with a CPR probe labeled with an activity of 40,000 cpm/ ⁇ l, using 20,000 cpm per reaction, detection of a target strand at a concentration of 10 ⁇ 5 pMole or less, preferably the detection being carried out with a CPR probe labeled with an activity of 40,000 cpm/ ⁇ l, using 20,000 cpm per reaction; detection of a target strand at a concentration of 10 ⁇ 6 pMole or less, preferably the detection being carried out with a CPR probe labeled with an activity of 40,000 cpm/ ⁇ l, using 20,000 cpm per reaction; detection of a target strand at a concentration of 10" ⁇ pMole or less, preferably the detection being carried out with a CPR probe labeled with an activity of 40,000 cpm/ ⁇ l, using 20,000 cpm per reaction
  • the probe and the target can be present in substantially equal amount, concentration, number, or chemical potentials in said reaction mix.
  • the ratio by weight, molarity, number, concentration, or chemical potential of the probe to the target can be less than or equal to 1:1, 2:1, 5:1, 10:1, 25:1, 50:1, 100:1, or 10 m :l, wherein m is an integer between 3 and 10, inclusive.
  • the ratio by weight, molarity, number, concentration, or chemical potential of the RNase H to the single strand in the highest concentration can be greater than 1:1, 2:1, 5:1, 10:1, 25:1, 50:1, 100:1, or 10 m :l, wherein m is an integer between 3 and 10, inclusive.
  • the RNase H will accelerate the rate of duplex formation at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 10 3 , 10 4 , 10 5 , 10 ⁇ .
  • the chemical potential driving duplex formation is provided by a high concentration of RNase H.
  • the RNAse H enzymatic activity cleaves the probe so as to produce a gapped duplex in which both double stranded regions are more unstable than the original probe: target duplex. Under appropriate experimental conditions this allows the reaction to proceed and cycle even though the reaction is performed at a temperature (65°C) well in excess of the Tm for the probe:target complex.
  • reaction conditions The following reaction scheme was employed: 1 ⁇ L ofa 5' 3 2p labeled probe sequence (the probe is a composite DNA-RNA molecule having a central RNA region disposed between two terminal DNA regions, is 28 bases in length, and is complementary to the target sequence) was added to tubes containing 1 ⁇ l of target homologous DNA and 0.5 ⁇ l of a buffer composed of 50mM Tris-Cl/lOmM MgCl2 (pH7.5) and 2.5 ⁇ L of ddl-PjO. A lul aliquot of highly concentrated RNAse H was then added and samples placed at 65°C.
  • RNAse H concentration was high enough to both facilitate formation of the duplex and to cleave the duplexes formed so as to create a situation where "cycling" occurred driven by the forward chemical potential for enzyme driven duplex formation and the backward potential for product dissociation (e.g., more than one probe could react with the same target).
  • target concentration number of molecules was serially diluted in tenfold increments.

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

Abstract

On décrit un cycle amélioré de réactions utilisant une sonde.
EP94921315A 1993-06-17 1994-06-16 Procedes ameliores pour detecter des sequences d'acides nucleiques Withdrawn EP0708840A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US7875993A 1993-06-17 1993-06-17
US15353693A 1993-11-17 1993-11-17
US153536 1993-11-17
PCT/US1994/006855 WO1995000667A1 (fr) 1993-06-17 1994-06-16 Procedes ameliores pour detecter des sequences d'acides nucleiques
US78759 2002-02-19

Publications (1)

Publication Number Publication Date
EP0708840A1 true EP0708840A1 (fr) 1996-05-01

Family

ID=26760905

Family Applications (1)

Application Number Title Priority Date Filing Date
EP94921315A Withdrawn EP0708840A1 (fr) 1993-06-17 1994-06-16 Procedes ameliores pour detecter des sequences d'acides nucleiques

Country Status (8)

Country Link
EP (1) EP0708840A1 (fr)
JP (1) JPH09502084A (fr)
KR (1) KR960703175A (fr)
CN (1) CN1129461A (fr)
AU (1) AU7208894A (fr)
BR (1) BR9406898A (fr)
CA (1) CA2165545A1 (fr)
WO (1) WO1995000667A1 (fr)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995014106A2 (fr) * 1993-11-17 1995-05-26 Id Biomedical Corporation Detection cyclique par scission de sondes de sequences d'acides nucleiques
WO1997011199A1 (fr) * 1995-09-22 1997-03-27 Lane Michael J Reactions d'acide nucleique
US7381525B1 (en) 1997-03-07 2008-06-03 Clinical Micro Sensors, Inc. AC/DC voltage apparatus for detection of nucleic acids
US6274316B1 (en) * 1997-07-03 2001-08-14 Id Biomedical Corporation Compositions and methods for detecting vancomycin resistant enterococci by cycling probe reactions
US6503709B1 (en) 1997-07-03 2003-01-07 Id Biomedical Corporation Methods for rapidly detecting methicillin resistant staphylococci
US20060275782A1 (en) 1999-04-20 2006-12-07 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
ATE553219T1 (de) 1999-04-20 2012-04-15 Illumina Inc Erkennung von nukleinsäurereaktionen auf bead- arrays
US8481268B2 (en) 1999-05-21 2013-07-09 Illumina, Inc. Use of microfluidic systems in the detection of target analytes using microsphere arrays
WO2001007665A2 (fr) 1999-07-26 2001-02-01 Clinical Micro Sensors, Inc. Determination de sequences d'acides nucleiques par detection electronique
US7582420B2 (en) 2001-07-12 2009-09-01 Illumina, Inc. Multiplex nucleic acid reactions
JP4287652B2 (ja) 2000-10-24 2009-07-01 ザ・ボード・オブ・トラスティーズ・オブ・ザ・レランド・スタンフォード・ジュニア・ユニバーシティ ゲノムｄｎａの直接多重処理による性状分析
EP2246438B1 (fr) 2001-07-12 2019-11-27 Illumina, Inc. Reactions multiplex d'acides nucléiques
AU2003208959A1 (en) 2002-01-30 2003-09-02 Id Biomedical Corporation Methods for detecting vancomycin-resistant microorganisms and compositions therefor
US20040259100A1 (en) 2003-06-20 2004-12-23 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
CN101225434B (zh) * 2007-01-18 2011-09-14 上海生物芯片有限公司 Cpt基因芯片及其检测方法和应用
CN102002522B (zh) * 2009-11-10 2014-07-09 复旦大学附属华山医院 一种检测肺炎支原体耐药突变的方法
US20190127783A1 (en) * 2016-04-27 2019-05-02 Prominex, Inc. Compositions and methods for the detection of nucleic acids
US10995104B2 (en) 2017-05-30 2021-05-04 Roche Molecular System, Inc. Catalysts for reversing formaldehyde adducts and crosslinks

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Publication number Priority date Publication date Assignee Title
US5011769A (en) * 1985-12-05 1991-04-30 Meiogenics U.S. Limited Partnership Methods for detecting nucleic acid sequences

Non-Patent Citations (1)

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Title
See references of WO9500667A1 *

Also Published As

Publication number Publication date
WO1995000667A1 (fr) 1995-01-05
JPH09502084A (ja) 1997-03-04
BR9406898A (pt) 1996-03-26
CA2165545A1 (fr) 1995-01-05
CN1129461A (zh) 1996-08-21
AU7208894A (en) 1995-01-17
KR960703175A (ko) 1996-06-19

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