EP0988400A1 - Procedes servant a determiner des quantites d'acides nucleiques - Google Patents

Procedes servant a determiner des quantites d'acides nucleiques

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Publication number
EP0988400A1
EP0988400A1 EP99934310A EP99934310A EP0988400A1 EP 0988400 A1 EP0988400 A1 EP 0988400A1 EP 99934310 A EP99934310 A EP 99934310A EP 99934310 A EP99934310 A EP 99934310A EP 0988400 A1 EP0988400 A1 EP 0988400A1
Authority
EP
European Patent Office
Prior art keywords
probe
sequence
amplification
hybridizing
target polynucleotide
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Withdrawn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
EP99934310A
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German (de)
English (en)
Inventor
Rajesh Patel
Edwin F. Ullman
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.)
Dade International Inc
Dade Behring Inc
Original Assignee
Dade International Inc
Dade Behring Inc
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Filing date
Publication date
Application filed by Dade International Inc, Dade Behring Inc filed Critical Dade International Inc
Publication of EP0988400A1 publication Critical patent/EP0988400A1/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Withdrawn legal-status Critical Current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
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research.
  • ssDNA single stranded deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories.
  • nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.
  • One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose -2-
  • the support is contacted with a suitably labeled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence based amplification
  • Q-beta-replicase method the amplified product must be detected.
  • a separate step is carried out prior to detecting amplified material.
  • One method for detecting nucleic acids is to employ nucleic acid probes that have sequences complementary to sequences in the amplified nucleic acid.
  • One method utilizing such probes is described in U.S. Patent No. 4,868,104.
  • a nucleic acid probe may be, or may be capable of being, labeled with a reporter group or may be, or may be capable of becoming, bound to a -3-
  • Detection of signal depends upon the nature of the label or reporter group. If the label or reporter group is an enzyme, additional members of the signal producing system include enzyme substrates and so forth.
  • the probe is comprised of natural nucleotides such as ribonucleotides and deoxyribonucleotides and their derivatives although unnatural nucleotide mimetics such as peptide nucleic acids (PNA) and oligomeric nucleoside phosphonates are also used.
  • binding of the probes to the target is detected by means of a label incorporated into the probe. Binding can be detected by separating the bound probe from the free probe and detecting the label. For this purpose it is usually necessary to form a sandwich comprised of the labeled probe, the target and a probe that is or can become bound to a surface. Alternatively, binding can be detected by a change in the signal-producing properties of the label upon binding, such as a change in the emission efficiency of a fluorescent or chemiluminescent label. This permits detection to be carried out without a separation step.
  • Homogeneous methods include the Taqman method used by Roche Molecular Diagnostics.
  • a probe is used that is labeled with a fluorescer and a quencher.
  • the polymerase used in PCR is capable of cutting the probe when it is bound to the target DNA and causing separation of these labels.
  • Changes in the polarization of fluorescence upon binding of a fluorescer-labeled probe to target DNA are used by Becton Dickenson to detect the formation of DNA in Strand Displacement Amplification (SDA). Binding of two probes, one with a chemiluminescer bead and one with a sensitizer bead has been used by Behring Diagnostics Inc. for detection of DNA produced by PCR and single primer amplification.
  • Binding of an electroluminescent ruthenium labeled probe to a biotinylated target RNA and capture of the complex on magnetic beads has been used by Organon Teknika for detection of RNA produced in NASBA.
  • GenProbe has carried out detection of RNA by means of an acridinium labeled probe that changes chemiluminescence efficiency when the probe is bound to target RNA.
  • a displacement polynucleotide assay method and polynucleotide complex reagent therefor is disclosed by Diamond, et al., in U.S. Patent No. 4,766,062.
  • a probe is used that is complementary to a portion of the target nucleic acid.
  • a labeled oligonucleotide is bound to a portion of the complementary sequence on the probe.
  • branch migration occurs and the displaced labeled oligonucleotide or the oligonucleotide that has not been displaced is measured and related to the presence of the target.
  • PCR-based assay that utilizes the inherent 5' nuclease of rTth DNA polymerase for the quantitative detection of HCV RNA is disclosed by Tsang, et a]., (94th General Meeting of the American Society for Microbiology, Las Vegas NE 5/94, Poster No. C376).
  • German patent application DE 4234086-A1 (92.02.05) discusses the determination of nucleic acid sequences amplified in vitro in enclosed reaction zone where probe(s) capable of interacting with target sequence is present during or after amplification and spectroscopically measurable parameters of probe undergo change thereby generating signal.
  • U.S. Patent No. 5,232,829 discloses detection of Chlamydia trachomatis by polymerase chain reaction using biotin labeled DNA primers and capture probes. A similar disclosure is made by Loeffelholz, et al. (1992) Journal of Clinical Microbiology, 30(1 1 ):2847-2851. A process for amplifying, detecting and/or cloning nucleic acid sequences is disclosed in U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 and 5,008,182.
  • One embodiment of the present invention is a method for detecting a single stranded target polynucleotide.
  • a combination which comprises (i) a medium suspected of containing the single stranded target polynucleotide, (ii) a first oligonucleotide probe and (iii) a second oligonucleotide probe.
  • the first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of the second probe.
  • the single stranded target polynucleotide has a sequence S1 that is capable of hybridizing to PP1 and a sequence S2 that is capable of hybridizing to a sequence PP2 of the second probe.
  • the first probe is incapable of hybridizing to PP2 or the single stranded target polynucleotide.
  • S1 and S2 or PP1 and PP2 are noncontiguous and one or both of the first probe and the second probe comprise members of a signal producing system.
  • the binding of the single stranded target polynucleotide, if present, to the second probe alters a signal generated by the signal producing system.
  • the combination is subjected to conditions under which the single stranded target polynucleotide, if present, hybridizes to the second probe and displaces the first probe. The signal is then detected.
  • Another embodiment of the present invention is a method for amplifying and detecting a target polynucleotide.
  • a combination comprising (i) a medium suspected of containing the target polynucleotide, (ii) all reagents required for conducting an amplification of the target polynucleotide to produce an amplification product, and (iii) first and second oligonucleotide probes.
  • the first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of the second probe.
  • the amplification product in single stranded form has a sequence S1 that is capable of hybridizing to PP1 and a sequence S2 that is capable of -7-
  • the first probe is incapable of hybridizing to the PP2 or the single strand.
  • S1 and S2 or PP1 and PP2 are noncontiguous.
  • One or both of the first probe and the second probe comprise members of a signal producing system.
  • the binding of the single stranded amplification product, if present, to the second probe alters a signal generated by the signal producing system.
  • the combination is subjected to conditions for amplifying the target polynucleotide and to conditions under which the single stranded amplification product, if present, hybridizes to the second probe and displaces the first probe from the second probe. The signal is then detected.
  • Another embodiment of the present invention is a method for amplifying and detecting a plurality of different polynucleotides.
  • a combination which comprises (i) a medium containing a plurality of different polynucleotides, (ii) all reagents required for conducting an amplification of the polynucleotides to produce a different amplification product for each different polynucleotide, and (iii) a plurality of sets of first and second oligonucleotide probes.
  • a different set is employed for each of the different amplification products and each is characterized as follows.
  • the first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of the second probe.
  • the amplification product in single stranded form has a sequence S1 that is capable of hybridizing to PP1 and a sequence S2 that is capable of hybridizing to a sequence PP2 of the second probe.
  • the first probe is incapable of hybridizing to the PP2 or the single strand.
  • S1 and S2 or PP1 and PP2 are noncontiguous.
  • One or both of the first probe and the second probe comprise members of a signal producing system unique to each of the sets such that the binding of the single stranded amplification product to the second probe alters a signal generated by the signal producing system.
  • the combination of the sequence pair S1 and PP1 and the sequence pair S2 and PP2 is unique in each of the sets and the signals generated by each of the signal producing systems are differentially detectable. The combination is subjected to conditions for amplifying the polynucleotides and to conditions under which each of the single stranded -8-
  • amplification products hybridizes to its respective second probe and displaces its respective first probe.
  • the detectable signals are differentially detected.
  • a combination is provided, which comprises (i) a medium containing a plurality of different RNA's, (ii) all reagents required for conducting an amplification of the RNA's to produce a different amplification product for each different RNA, and (iii) a plurality of sets of first and second oligonucleotide probes.
  • a different set is employed for each of the different amplification products and each set has the following characteristics.
  • the first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of the second probe.
  • the amplification product has a sequence S1 that is capable of hybridizing to PP1 and a sequence S2 that is capable of hybridizing to a sequence PP2 of the second probe.
  • the first probe is incapable of hybridizing to the PP2 or the RNA strand.
  • S1 and S2 or PP1 and PP2 are noncontiguous.
  • One of the probes is labeled with a sensitizer and the other of the probes is labeled with a chemiluminescer.
  • the binding of the amplification product to the second probe alters the chemiluminescence generated by the chemiluminescer.
  • the combination of the sequence pair S1 and PP1 and the sequence pair S2 and PP2 is unique in each of the sets and respective labels for each of the sets are differentially detectable.
  • the combination is subjected to isothermal conditions for amplifying the RNA's.
  • the combination is further subjected to conditions under which each of the amplification products hybridizes to its respective second probe and displaces its respective first probe.
  • the detectable signals are then each differentially detected.
  • kits for use in amplification and detection of a target polynucleotide is a packaged combination and comprises reagents for conducting an amplification of the target polynucleotide and first and second oligonucleotide probes.
  • the first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of the second probe.
  • the amplification product in single stranded form has a sequence S1 that is capable of hybridizing to -9-
  • first probe is incapable of hybridizing to the PP2 or the single strand.
  • S1 and S2 or PP1 and PP2 are noncontiguous.
  • One or both of the first probe and the second probe comprise members of a signal producing system such that, in use, the binding of the single stranded amplification product, if present, to the second probe alters a signal generated by the system.
  • kits for use in an amplification and quantitation of a specific RNA comprises in packaged combination one or more reference RNA's, a promoter, an enzyme and a plurality of sets of first and second oligonucleotide probes, one set for each different RNA to be analyzed with the present kit.
  • the first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of the second probe
  • the amplification product in single stranded form has a sequence S1 that is capable of hybridizing to PP1 and a sequence S2 that is capable of hybridizing to a sequence PP2 of the second probe
  • the first probe is incapable of hybridizing to the PP2 or the single strand
  • S1 and S2 or PP1 and PP2 are noncontiguous
  • one of the probes is labeled with a sensitizer and the other of the probes is labeled with a chemiluminescer.
  • the combination of the sequence pair S1 and PP1 and the sequence pair S2 and PP2 is unique in each of the sets and the respective labels for each of the sets are differentially detectable.
  • FIG. 1 is a schematic diagram depicting an embodiment in accordance with the present invention.
  • Fig. 2 is a schematic diagram depicting an alternate embodiment in accordance with the present invention.
  • Fig. 3 is a schematic diagram depicting an alternate embodiment in accordance with the present invention.
  • Fig. 4 is a schematic diagram depicting the experiments described in Example 1. -10-
  • the present invention provides for detection of nucleic acid sequences, particularly, the products of nucleic acid amplification reactions.
  • the invention is especially useful for monitoring the formation of a target nucleic acid produced during amplification under isothermal conditions such as that found in NASBA, 3SR, SDA or amplifications using Q- ⁇ -replicase.
  • the present invention utilizes a set of oligonucleotide probes.
  • a second oligonucleotide probe has a first region that is hybridizable with, preferably complementary with, a sequence in a first oligonucleotide probe and that is also hybridizable with, preferably complementary with, a first sequence in the target polynucleotide.
  • the second probe also has a second region, non-contiguous with the first region that is hybridizable with, preferably complementary to, a second sequence in the target polynucleotide that may or may not be contiguous with the first sequence of the target polynucleotide.
  • the method is carried out by combining the sample suspected of containing the target polynucleotide, the first oligonucleotide probe and the second oligonucleotide probe and, where applicable, all of the reagents necessary for carrying out an amplification.
  • the first probe and the second oligonucleotide probe are allowed to bind to each other prior to combining them with the other assay components.
  • the target polynucleotide if present displaces the first oligonucleotide probe from its complex with the second oligonucleotide probe as the result of branch migration. Then, either the displaced first oligonucleotide probe or the complex of the second oligonucleotide probe with the target polynucleotide is detected and related to the presence of the target polynucleotide.
  • At least the first oligonucleotide probe has a label capable of generating a signal.
  • the signal changes as a result of the binding of the second sequence of the target -11-
  • polynucleotide to the second region of the second oligonucleotide probe.
  • the proximity of these regions enables displacement of the labeled first oligonucleotide by the target polynucleotide.
  • the present method is particularly useful when it is desired to determine the relative amounts of two target polynucleotides that are identical except for a sequence of at least 8 bases.
  • the present invention differs from that of Diamond, supra, who discloses a process wherein binding of a target sequence adjacent to a signal oligonucleotide binding site leads to the well known strand displacement process wherein the junction between two polynucleotide strands bound to contiguous sites on a polynucleotide template, each having an unhybridized tail extending from the junction, can migrate with eventual displacement of one of the polynucleotides from the template.
  • the sites of binding are not contiguous and, therefore, it is not apparent that strand displacement would occur.
  • the present invention relates to a method for detecting a single stranded target polynucleotide. More particularly, the present invention relates to a method for amplifying and detecting a target polynucleotide.
  • Polynucleotide analyte - a compound or composition to be measured that is a polymeric nucleotide, which in the intact natural state can have about 30 to 5,000,000 or more nucleotides and in an isolated state can have about 20 to 50,000 or more nucleotides, usually about 100 to 20,000 nucleotides, more frequently 500 to 10,000 nucleotides. It is thus obvious that isolation of the analyte from the natural state often results in fragmentation.
  • the polynucleotide anaiytes include nucleic acids, and fragments thereof, from any source in purified or unpurified form including DNA (dsDNA and ssDNA) and RNA, including t-RNA, m-RNA, r-RNA, mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA-RNA hybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomes of biological material such as -12-
  • the polynucleotide analyte can be only a minor fraction of a complex mixture such as a biological sample.
  • the analyte can be obtained from various biological materials by procedures well known in the art. Some examples of such biological material by way of illustration and not limitation are disclosed in the following Table 1 :
  • Table 1 Microorganisms of interest include:
  • Aerobacter aerogenes The colliform
  • Salmonella typhosa Salmonella choleraesuis The Salmonellae
  • Proteus mirabilis Proteus species
  • Pasteurella pestis Cladosporium carrionii Pasteurella tulareusis Phialophora verrucosa
  • Clostridium perfringens Adenoviruses Clostridium novyi Herpes Viruses
  • Mycobacterium tuberculosis Variola (smallpox) hominis Mycobacterium bovis Vaccinia Mycobacterium avium Poxvirus bovis Mycobacterium leprae Paravaccinia Mycobacterium paratuberculosis Molluscum contagiosum -14-
  • the Spirochetes Influenza (A, B, and C)
  • Rickettsiae bacteria-like St. Louis Encephalitis Virus parasites
  • Paracoccidioides brasiliensis Candida albicans Aspergillus fumigatus
  • genes such as hemoglobin gene for sickle-cell anemia, cystic fibrosis gene, oncogenes, cDNA, and the like.
  • the polynucleotide analyte may be cleaved to obtain a fragment that contains a target polynucleotide sequence, for example, by shearing or by treatment with a restriction endonuclease or other site specific chemical cleavage method.
  • the polynucleotide analyte, or a cleaved fragment obtained from the polynucleotide analyte will usually be at least partially denatured or single stranded or treated to render it denatured or single stranded.
  • treatments are well-known in the art and include, for instance, heat or alkali treatment.
  • double stranded DNA can be heated at 90-100° C. for a period of about 1 to 10 minutes to produce denatured material.
  • Amplification of nucleic acids or polynucleotides any method that results in the formation of one or more copies of a nucleic acid or polynucleotide molecule or in the formation of one or more copies of the complement of a nucleic acid or polynucleotide molecule.
  • Exponential amplification of nucleic acids or polynucleotides any method that depends on the product catalyzed formation of multiple copies of a nucleic acid or polynucleotide molecule or its complement.
  • the amplification products are sometimes referred to as "amplicons.”
  • One such method for the enzymatic amplification of specific double stranded sequences of DNA is known as the polymerase chain reaction (PCR), as described above.
  • PCR polymerase chain reaction
  • This jn vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic template dependent polynucleotide polymerase, resulting in the exponential increase in copies of the desired sequence -16-
  • the two different PCR primers which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete double stranded fragment whose length is defined by the distance between the 5' ends of the oligonucleotide primers.
  • Another method for amplification is mentioned above and involves amplification of a single stranded polynucleotide using a single oligonucleotide primer.
  • the single stranded polynucleotide that is to be amplified contains two noncontiguous sequences that are complementary to one another and, thus, are capable of hybridizing together to form a stem-loop structure.
  • This single stranded polynucleotide already may be part of a polynucleotide analyte or may be created as the result of the presence of a polynucleotide analyte.
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence based amplification
  • the reagents for conducting NASBA include a first DNA primer with a 5' tail comprising a promoter, a second DNA primer, reverse transcriptase, RNAse-H, T7 RNA polymerase, NTP's and dNTP's.
  • Another method for amplifying a specific group of nucleic acids is the Q-beta- replicase method, which relies on the ability of Q-beta-replicase to amplify its RNA substrate exponentially.
  • the reagents for conducting such an amplification include "midi-variant RNA" (amplifiable hybridization probe), NTP's, and Q-beta-replicase. -17-
  • 3SR Another method for amplifying nucleic acids is known as 3SR and is similar to NASBA except that the RNAse-H activity is present in the reverse transcriptase.
  • Linear amplification of nucleic acids or polynucleotides any method that depends on the self catalyzed formation of one or more copies of the complement of only one strand of a nucleic acid or polynucleotide molecule, usually a nucleic acid or polynucleotide analyte.
  • linear amplification the primary difference between linear amplification and exponential amplification is that the latter is autocatalyzed, that is, the product serves to catalyze the formation of more product, whereas in the former process the starting sequence catalyzes the formation of product but is not itself replicated.
  • linear amplification the amount of product formed increases as a linear function of time as opposed to exponential amplification where the amount of product formed is an exponential function of time.
  • Target polynucleotide a sequence of nucleotides to be identified, usually existing within a portion or all of a polynucleotide analyte, the identity of which is known to an extent sufficient to allow preparation of various oligonucleotides, such as probes and primers, and other molecules necessary for conducting an amplification of the target polynucleotide.
  • primer extension amplification primers hybridize to, and are extended along (chain extended), at least the target sequence within the target polynucleotide and, thus, the target sequence acts as a template.
  • the extended primers are chain "extension products.”
  • the target sequence usually lies between two defined sequences but need not.
  • the primers hybridize with the defined sequences or with at least a portion of such target polynucleotide, usually at least a ten nucleotide segment at the 3'-end thereof and preferably at least 15, frequently 20 to 50 nucleotide segment thereof.
  • the target sequence usually contains from about 30 to 5,000 or more nucleotides, preferably 50 to 1 ,000 nucleotides.
  • the target polynucleotide is generally a fraction of a larger molecule or it may be substantially the entire molecule (polynucleotide analyte). The minimum -18-
  • the number of nucleotides in the target polynucleotide sequence is selected to assure that the presence of target polynucleotide in a sample is a specific indicator of the presence of polynucleotide analyte in a sample.
  • the sequence length is usually greater than about 1.6 log L nucleotides where L is the number of base pairs in the genome of the biologic source of the sample.
  • the maximum number of nucleotides in the target polynucleotide is normally governed by the length of the polynucleotide analyte and its tendency to be broken by shearing, or other processes during isolation and any procedures required to prepare the sample for assay and the efficiency of detection and/or amplification of the sequence.
  • Oligonucleotide - a polynucleotide, usually single stranded, usually a synthetic polynucleotide but may be a naturally occurring polynucleotide.
  • the oligonucleotide(s) are usually comprised of a sequence of at least 5 nucleotides, preferably, 10 to 100 nucleotides, more preferably, 20 to 50 nucleotides, and usually 10 to 30 nucleotides in length.
  • Various techniques can be employed for preparing an oligonucleotide utilized in the present invention. Such oligonucleotide can be obtained by biological synthesis or by chemical synthesis.
  • chemical synthesis will frequently be more economical as compared to the biological synthesis.
  • chemical synthesis provides a convenient way of incorporating low molecular weight compounds and/or modified bases during the synthesis step.
  • chemical synthesis is very flexible in the choice of length and region of the target polynucleotide binding sequence.
  • the oligonucleotide can be synthesized by standard methods such as those used in commercial automated nucleic acid synthesizers.
  • Chemical synthesis of DNA on a suitably modified glass or resin can result in DNA covalently attached to the surface. This may offer advantages in washing and sample handling.
  • standard replication methods employed in molecular biology can be used such as the use of M13 for single stranded DNA as described by J. Messing (1983) Methods Enzymol, 101 , 20-78. -19-
  • oligonucleotide synthesis include phosphotriester and phosphodiester methods (Narang, et aj. (1979) Meth. Enzymol 68: 90) and synthesis on a support (Beaucage, et al. (1981) Tetrahedron Letters 22: 1859-1862) as well as phosphoramidate technique, Caruthers, M. H., et aL, "Methods in Enzymology," Vol. 154, pp. 287-314 (1988), and others described in “Synthesis and Applications of DNA and RNA,” S.A. Narang, editor, Academic Press, New York, 1987, and the references contained therein.
  • Oligonucleotide probe - an oligonucleotide employed in the present invention to bind to a portion of a polynucleotide such as an oligonucleotide probe or a target polynucleotide.
  • the design and preparation of the oligonucleotide probes are important in performing the methods of this invention. A more detailed description of oligonucleotide probes in accordance with the present invention is found hereinbelow.
  • the oligonucleotide primer is usually a synthetic nucleotide that is single stranded, containing a sequence at its 3'-end that is capable of hybridizing with a defined sequence of the target polynucleotide.
  • an oligonucleotide primer has at least 80%, preferably 90%, more preferably 95%, most preferably 100%, complementarity to a defined sequence or primer binding site.
  • the number of nucleotides in the hybridizable sequence of an oligonucleotide primer should be such that stringency conditions used to hybridize the oligonucleotide primer will prevent excessive random non-specific hybridization.
  • the number of nucleotides in the oligonucleotide primer will be at least as great as the defined sequence of the target polynucleotide, namely, at least ten nucleotides, preferably at least 15 nucleotides and generally from about 10 to 200, preferably 20 to 50, nucleotides. -20-
  • Nucleoside triphosphates nucleosides having a 5'-triphosphate substituent.
  • the nucleosides are pentose sugar derivatives of nitrogenous bases of either purine or pyrimidine derivation, covalently bonded to the 1'-carbon of the pentose sugar, which is usually a deoxyribose or a ribose.
  • the purine bases include adenine(A), guanine (G), inosine (I), and derivatives and analogs thereof
  • the pyrimidine bases include cytosine (C), thymine (T), uracil (U), and derivatives and analogs thereof.
  • Nucleoside triphosphates include deoxyribonucleoside triphosphates such as the four common triphosphates dATP, dCTP, dGTP and dTTP and ribonucleoside triphosphates such as the four common triphosphates rATP, rCTP, rGTP and rUTP.
  • the term "nucleoside triphosphates" also includes derivatives and analogs thereof, which are exemplified by those derivatives that are recognized in a similar manner to the underivatized nucleoside triphosphates.
  • Such derivatives or analogs are those which are biotinylated, amine modified, alkylated, and the like and also include phosphorothioate, phosphite, ring atom modified derivatives, and the like.
  • Nucleotide - a base-sugar-phosphate combination that is the monomeric unit of nucleic acid polymers, i.e., DNA and RNA.
  • Modified nucleotide - is the unit in a nucleic acid polymer that results from the incorporation of a modified nucleoside triphosphate during an amplification reaction and therefore becoming part of the nucleic acid polymer.
  • Nucleoside - is a base-sugar combination or a nucleotide lacking a phosphate moiety.
  • Nucleotide polymerase - a catalyst usually an enzyme, for forming an extension of a polynucleotide along a DNA or RNA template where the extension is complementary thereto.
  • the nucleotide polymerase is a template dependent polynucleotide polymerase and utilizes nucleoside triphosphates as building blocks for extending the 3'-end of a polynucleotide to provide a sequence complementary with the polynucleotide template.
  • the catalysts are enzymes, such as DNA polymerases, for example, prokaryotic DNA polymerase (I, II, or III), T4 DNA -21-
  • RNA polymerase T7 DNA polymerase, Klenow fragment, reverse transcriptase, Vent DNA polymerase, Pfu DNA polymerase, Jag DNA polymerase, and the like, derived from any source such as cells, bacteria, such as E. coH, plants, animals, virus, thermophilic bacteria, and so forth.
  • RNA polymerases include T7 RNA polymerase, Q-beta-replicase, and so forth.
  • one or more may be combined with one or more of the remaining agents to form a subcombination. Subcombination and remaining agents can then be combined and can be subjected to the present method.
  • Hybridization and binding - in the context of nucleotide sequences these terms are used interchangeably herein.
  • the ability of two nucleotide sequences to hybridize with each other is based on the degree of complementarity of the two nucleotide sequences, which in turn is based on the fraction of matched complementary nucleotide pairs.
  • the more nucleotides in a given sequence that are complementary to another sequence the more stringent the conditions can be for hybridization and the more specific will be the binding of the two sequences.
  • Increased stringency is achieved by elevating the temperature, increasing the ratio of cosolvents, lowering the salt concentration, and the like.
  • homologous or substantially identical polynucleotides In general, two polynucleotide sequences that are identical or can each hybridize to the same polynucleotide sequence are homologous. The two sequences are homologous or substantially identical where the sequences each have at least 90%, preferably 100%, of the same or analogous base sequence where thymine (T) and uracil (U) are considered the same. Thus, the ribonucleotides A, U, C and G are taken as analogous to the deoxynucleotides dA, dT, dC, and dG, respectively.
  • Homologous sequences can comprise DNA, RNA or modified polynucleotides and may be homoduplexes, e.g., RNA:RNA and DNA:DNA or heteroduplexes, e.g., RNA:DNA.
  • RNA:RNA and DNA:DNA e.g., DNA:DNA
  • heteroduplexes e.g., RNA:DNA.
  • Complementary - Two sequences are complementary when the sequence of -22-
  • Non-contiguous - two sequences within a first single polynucleotide sequence are non-contiguous when the 5' end of one sequence is joined to the 3' end of the other sequence by more than a bond, usually by a chain of one or more nucleotides that are not hybridized to a second single polynucleotide strand to which the two sequences of the first single strand are hybridized to form a duplex.
  • Contiguous - two sequences within a first single polynucleotide strand are contiguous when the 5' end of one sequence is joined by a covalent bond directly to the 3' end of the other sequence without any intervening atoms or chain of nucleotides that are not hybridized to a second single polynucleotide strand to which the two sequences of the first single strand are hybridized to form a duplex.
  • Copy of a sequence - a sequence that was copied from, and has the same base sequence as, a single stranded polynucleotide sequence as differentiated from a sequence that is copied from and has a complementary base sequence to the sequence of such single stranded polynucleotide.
  • Means for extending a primer also includes nucleoside triphosphates or analogs thereof capable of acting as substrates for the enzyme and other materials and conditions required for enzyme activity such as a divalent metal ion (usually magnesium), pH, ionic strength, organic solvent (such as formamide), and the like.
  • sbp member Member of a specific binding pair
  • the members of the specific binding pair are -23-
  • ligand and receptor may be members of an immunological pair such as antigen-antibody, or may be operator-repressor, nuclease-nucleotide, biotin-avidin, hormones-hormone receptors, nucleic acid duplexes, IgG-protein A, DNA-DNA, DNA-RNA, and the like.
  • Receptor any compound or composition capable of recognizing a particular spatial and polar organization of a molecule, e.g., epitopic or determinant site.
  • Illustrative receptors include naturally occurring receptors, e.g., thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, repressors, protection enzymes, protein A, complement component C1q, DNA binding proteins or ligands and the like.
  • Small organic molecule a compound of molecular weight less than 1500, preferably 100 to 1000, more preferably 300 to 600 such as biotin, fluorescein, rhodamine and other dyes, tetracycline and other protein binding molecules, and haptens, etc.
  • the small organic molecule can provide a means for attachment of a nucleotide sequence to a label or to a support.
  • the support can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), etc.; either used by themselves or in conjunction with other materials; glass available as Bioglass, ceramics, metals, and the like. Natural or synthetic assemblies such as liposomes, phospholipid vesicles, and cells can also be employed
  • Binding of sbp members to a support or surface may be accomplished by well-known techniques, commonly available in the literature. See, for example, “Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970).
  • the surface can have any one of a number of shapes, such as strip, rod, particle, including bead, and the like.
  • Label a member of a signal producing system.
  • the label is part of an oligonucleotide probe either being conjugated thereto or otherwise bound thereto or associated therewith and is capable of being detected directly or indirectly.
  • the label may be part of the oligonucleotide primer.
  • Labels include reporter molecules that can be detected directly by virtue of generating a signal, specific binding pair members that may be detected indirectly by subsequent binding to a cognate that contains a reporter molecule, oligonucleotide primers that can provide a template for amplification or ligation or a specific polynucleotide sequence or recognition sequence that can act as a ligand such as for a repressor protein, wherein in the latter two instances the oligonucleotide primer or repressor protein will have, or be capable of having, a reporter molecule.
  • any reporter molecule that is detectable can be used.
  • the reporter molecule can be isotopic or nonisotopic, usually non-isotopic, and can be a catalyst, such as an enzyme, a polynucleotide coding for a catalyst, promoter, dye, fluorescent molecule, chemiluminescer, coenzyme, enzyme substrate, radioactive group, a small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like.
  • a catalyst such as an enzyme, a polynucleotide coding for a catalyst, promoter, dye, fluorescent molecule, chemiluminescer, coenzyme, enzyme substrate, radioactive group, a small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may
  • the reporter group can be a fluorescent group such as fluorescein, a chemiluminescent group such as luminol, a terbium chelator such as N-(hydroxyethyl) ethylenediaminetriacetic acid that is capable of detection by delayed fluorescence, and the like.
  • the label is a member of a signal producing system and can generate a detectable signal either alone or together with other members of the signal producing system.
  • a reporter molecule can be bound directly to a -25-
  • nucleotide sequence or can become bound thereto by being bound to an sbp member complementary to an sbp member that is bound to a nucleotide sequence.
  • labels or reporter molecules and their detection can be found in U.S. Patent No. 5,508,178, the relevant disclosure of which is incorporated herein by reference.
  • the signal producing system may have one or more components, at least one component being a label.
  • the signal producing system generates a signal that relates to the presence or amount of target polynucleotide in a sample.
  • the signal producing system includes all of the reagents required to produce a measurable signal.
  • the labels and other reagents of the signal producing system must be stable at the temperatures used in an amplification of a target polynucleotide. Detection of the signal will depend upon the nature of the signal producing system utilized.
  • the signal producing system is characterized in that the nembers of the system are chosen such that the binding of the second and third oligonucleotide probes to their respective duplexes to form termolecular complexes alters the signal generated by the signal producing system of the respective termolecular complexes.
  • the second oligonucleotide probe and the third oligonucleotide probe comprise a member of a different signal producing system and the signals are measured from the ternary complexes and a ratio of signals is determined.
  • the first oligonucleotide probe may comprise a member of both signal producing systems.
  • first oligonucleotide probe in combination with each of the second oligonucleotide probe and the third oligonucleotide probe comprise members of different signal producing systems and the signals measured from the ternary complexes are used to determine a ratio of signals.
  • first oligonucleotide probe comprises a first member of a each of two signal producing systems and the second oligonucleotide probe and the third oligonucleotide probe each respectively comprise a second member of each of the signal producing systems.
  • a signal is produced.
  • the first member is a catalyst such as an enzyme and the second members are catalysts such as enzymes that are different from the first enzyme and from each other and the products of the reaction of the enzyme comprising the first member are the substrates for the other of the enzymes.
  • the second members are catalysts such as enzymes that are different from the first enzyme and from each other and the products of the reaction of the enzyme comprising the first member are the substrates for the other of the enzymes.
  • a list of enzymes is found in U.S. Patent No. 4,299,916 at column 30 to column 33.
  • coupled catalysts usually two or more enzymes, where the product of one enzyme serves as the substrate of the other enzyme.
  • One of the enzymes is used as the label in the first oligonucleotide probe.
  • Different second enzymes are used in the second and third oligonucleotide probes.
  • the solute will be the substrate of any one of the enzymes, but preferably of an enzyme bound to the first oligonucleotide probe.
  • the enzymatic reaction may involve modifying the solute to a product which is the substrate of another enzyme or production of compound which does not include a substantial portion of the solute, which serves as an enzyme substrate.
  • the first situation may be illustrated by glucose-6-phosphate being catalytically hydrolyzed by alkaline phosphatase to glucose, wherein glucose is a substrate for glucose oxidase.
  • the second situation may be illustrated by glucose being oxidized by glucose oxidase to provide hydrogen peroxide which would enzymatically react with the signal generator precursor to produce a signal generator.
  • Coupled catalysts can also include an enzyme with a non-enzymatic catalyst.
  • the enzyme can produce a reactant that undergoes a reaction catalyzed by the non-enzymatic catalyst or the non-enzymatic catalyst may produce a substrate (includes coenzymes) for the -27-
  • Medola blue can catalyze the conversion of NAD and hydroquinones to NADH which reacts with FMN oxidoreductase and bacterial luciferase in the presence of long chain aldehydes to produce light.
  • Examples of particular catalytic systems that may be utilized in the present invention are found in U.S. Patent No. 4,299,916 at column 33, line 34, to column 38, line 32, the disclosure of which is incorporated herein by reference.
  • additional members of the signal producing system include enzyme substrates and so forth.
  • the product of the enzyme reaction is preferably a luminescent product, or a fluorescent or non-fluorescent dye, any of which can be detected spectrophotometrically, or a product that can be detected by other spectrometric or electrometric means.
  • the first member of the signal producing system is a quencher and the second members are fluorescent compounds that emit at different wavelengths or with different decay rates.
  • Fluorescers of interest generally emit light at a wavelength above 350 nm, usually above 400 nm and preferably above 450 nm. Desirably, the fluorescers have a high quantum efficiency, a large Stokes shift and are chemically stable under the conditions of their conjugation and use.
  • the term fluorescer is intended to include substances that emit light upon activation and include fluorescent and phosphorescent substances, scintillators and chemiluminescent substances. In this approach the medium is irradiated with light and the fluorescence is determined.
  • Fluorescers of interest fall into a variety of categories having certain primary functionalities.
  • These primary functionalities include 1- and 2-aminonaphthalene, p,p-diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, imines, anthracenes, oxacarboxyamine, merocyanine, 3-aminoequilenin, perylene, bis-benzoxazole, bis-p-oxazolyl benzene, 1 ,2-benzophenazine, retinal, bis-3- -28-
  • the quencher must be able to quench the fluorescence of the fluorescer when brought into proximity with the fluorescer by virtue of the binding of the probes.
  • Quenchers are chromophores having substantial absorption higher than 310 nm, normally higher than 350 nm, and preferably higher than about 400 nm.
  • the quencher is a fluorescent compound or fluorescer but energy acceptors that have weak or no fluorescence are also useful.
  • quenchers are the xanthene dyes, which include the fluoresceins derived from 3,6-dihydroxy-o- phenyl-xanthhydrol and rhodamines, derived form 3,6-diamino-9-phenylxanthhydrol.
  • Another group of compounds are the naphthylamines such as, e.g., 1-anilino-8- naphthalene sulfonate, 1-dimethylaminonaphthyl-5-sulfonate and the like.
  • quenchers that may be employed are those fluorescers of interest mentioned above wherein one fluorescer can absorb the energy of another fluorescer and quench its fluorescence.
  • Energy acceptors that are non-fluorescent can include any of a wide variety of azo dyes, cyanine dyes, 4,5-dimethoxyfluorescein, formazans, indophenols and the like.
  • quenchers are energy absorbent or quenching particles.
  • examples of such particles are carbon particles, such as charcoal, lamp black, graphite, colloidal carbon and the like.
  • carbon particles metal sols may also find use, particularly of the noble metals, gold, silver, and platinum.
  • Other metal derived particles may include metal sulfides, such as lead, silver or copper sulfides or metal oxides, such as iron or copper oxide.
  • Heller U.S. Patent No. 5,565,322 discloses donor and acceptor chromophores at column 9, line 37, to column 14, line 7, the disclosure of which is incorporated herein by reference.
  • fluorescers and quenchers may also be found in U.S. Patent Nos. 4,261 ,968, 4,174,384, 4,199,983 and 3,996,345, the relevant disclosures of which are incorporated herein by reference.
  • the first member of the signal producing system is a sensitizer and the second members are chemiluminescent compounds that emit at different wavelengths or with different decay rates.
  • the first member is a chemiluminescent compound and the second members are sensitizers that can be independently excited by different wavelengths of light. Examples of chemiluminescent compounds and sensitizers are set forth in U.S. Serial No. 07/923,069 filed July 31 , 1992, the disclosure of which is incorporated herein by reference. Particularly preferred are photosensitizers and photoactivatable chemiluminescent compounds such as described in U.S. Patent No.
  • the sensitizers are those compounds that generate singlet oxygen usually by excitation with light.
  • the sensitizer can be photoactivatable (e.g., dyes and aromatic compounds) or chemi- activated (e.g., enzymes and metal salts).
  • Typical photosensitizers include acetone, benzophenone, 9-thioxanthone, eosin, 9,10-dibromoanthracene, methylene blue, metallo-porphyrins, such as hematoporphyrin, phthalocyanines, chlorophylls, rose bengal, buckminsterfullerene, etc., and derivatives thereof.
  • Photoactivatable chemiluminescent compounds are substances that undergo a chemical reaction upon direct of sensitized excitation by light of upon reaction with singlet oxygen to form a metastable reaction product that is capable of decomposition with the simultaneous or subsequent emission of light, usually with the wavelength range of 250 to 1200 nm.
  • these compounds react with singlet oxygen to form dioxetanes or dioxetanones.
  • the latter are usually electron rich olefins.
  • olefins examples include enol ethers, enamines, 9-alkylidene-N-alkylacridans, arylvinylethers, dioxenes, arylimidazoles, 9-alkylidene-xanthanes and lucigenin.
  • Other compounds include luminol and other phthalhydrazides and chemiluminescent compounds that are protected form undergoing a reaction such as firefly luciferin, aquaphorin, luminol, etc.
  • Other components of the signal producing system may include substrates, enhancers, activators, chemiluminescent compounds, cofactors, inhibitors, scavengers, metal ions, specific binding substances required for binding of signal generating substances, and the like.
  • Other components of the signal producing system may be coenzymes, substances that react with enzymic products, other enzymes and catalysts, and the like.
  • Termolecular complex a complex formed in accordance with the present methods upon the binding of the first oligonucleotide probe and the target polynucleotide to the second oligonucleotide probe.
  • Such complex is termolecular in that it involves three molecules, namely, the two oligonucleotide probes and the single strand of such target polynucleotide.
  • the termolecular complex is a transition complex formed as the target polynucleotide if present displaces the second oligonucleotide probe from a complex of the second oligonucleotide probe with the first oligonucleotide probe.
  • buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components.
  • proteins may be included, such as albumins, organic solvents such as formamide, quaternary ammonium salts, polycations such as dextran sulfate, surfactants, particularly non-ionic surfactants, binding enhancers, e.g., polyalkylene glycols, or the like.
  • one aspect of the present invention provides for detection of a target polynucleotide and its products produced in a nucleic acid -31-
  • the present invention has particular application to amplification reactions conducted at isothermal temperature.
  • all of the necessary reagents for amplification and detection are included in the reaction mixture prior to amplification and it is not necessary to open the reaction vessel after amplification and prior to detection. Thus, contamination is avoided.
  • complexes containing labels can be formed after amplification and without a separation step or opening of the reaction container, and then remaining members of the signal producing may be added, if necessary.
  • a combination for detecting a single stranded target polynucleotide, which comprises (i) a medium suspected of containing the single stranded target polynucleotide, (ii) a first oligonucleotide probe and (iii) a second oligonucleotide probe.
  • the combination of reagents in a single reaction container if desired, is subjected to conditions for amplifying the target polynucleotide sequence to form copies or complements thereof.
  • the first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of the second probe.
  • the first probe is incapable of hybridizing to sequence PP2 of the second probe or to the single stranded target polynucleotide.
  • the single stranded target polynucleotide has a sequence S1 that is capable of hybridizing to PP1 and a sequence S2 that is capable of hybridizing to a sequence PP2 of the second oligonucleotide probe.
  • S1 and S2 are independently about 8 to 100 or more nucleotides in length, preferably, 15 to 40 nucleotides in length.
  • the lengths are such that, under the conditions of the reactions involved, P1 hybridizes with PP1 in the absence of target polynucleotide and S2 hybridizes with PP2 and S1 hybridizes with PP1 sufficiently to displace P1.
  • P1 is about 8 to 100 nucleotides in length, preferably, 15 to 40 nucleotides in length.
  • PP1 and PP2 are each independently about 8 to 100 nucleotides in length, preferably, 15 to 40 nucleotides in length.
  • the degree of complementarity of S1 with PP1 should be sufficiently low to achieve one of the objects of the present invention, namely, the displacement of P1 by S1 upon the binding of S2 to PP2 and sufficiently high to assure binding of PP1 to P1 in the absence of target polynucleotide. Consequently, it is preferred that PP1 be 70-100% complementary, preferably, 90-100% complementary, to P1.
  • the degree of complementary of P 1 to PP1 depends on the relative lengths and nucleotide composition of P1 , PP1 , S2, PP2 and S1.
  • S2 be greater than about 90% complementary, preferably, greater than about 95% complementary, most preferably, fully complementary, to PP2.
  • S1 be greater than about 90% complementary, preferably, greater than about 95% complementary, most preferably, fully complementary, to PP1.
  • a sequence that contains a greater number of G, C nucleotides has a stronger degree of binding to its complementary sequence.
  • S1 and S2 or PP1 and PP2 are noncontiguous.
  • S1 is generally within a relatively few unhybridized (with respect to a single polynucleotide strand to which S1 and S2 or PP1 and PP2 are hybridized) nucleotides, preferably, less than 30 unhybridized nucleotides, more preferably, 1 to 10 unhybridized nucleotides, of S2. It is within the purview of the present invention that noncontiguous sequences can be separated by greater than 30 unhybridized nucleotides, perhaps greater than 100 unhybridized nucleotides. In this event such sequence of nucleotides preferably form a hairpin loop, that is, internally hybridize.
  • an aqueous medium is employed.
  • an aqueous medium is employed for the entire method in accordance with the present invention.
  • Other polar cosolvents may also -33-
  • the pH for the medium is usually in the range of about 4.5 to 9.5, more usually in the range of about 5.5 to 8.5, and preferably in the range of about 6 to 8.
  • Various buffers may be used to achieve the desired pH and maintain the pH during the determination.
  • Illustrative buffers include borate, phosphate, carbonate, Tris, barbital and the like.
  • the particular buffer employed is not critical to this invention but in individual methods one buffer may be preferred over another.
  • the pH and temperature are chosen based on the particular method of amplification employed.
  • the pH and the temperature are selected so as to cause, either simultaneously or sequentially, dissociation of any internally hybridized sequences, hybridization of the oligonucleotide primer with the target polynucleotide sequence, extension of the primer, and dissociation of the extended primer.
  • This usually involves cycling the reaction medium among two or more temperatures.
  • the medium is cycled between two to three temperatures.
  • the temperatures for PCR amplification generally range from about 50°C to 100°C, more usually, from about 60°C to 95°C.
  • Relatively low temperatures of from about 50°C to 80°C are employed for the hybridization steps, while denaturation is carried out at a temperature of from about 80°C to 100°C and extension is carried out at a temperature of from about 70°C to 80°C, usually about 72°C to 74°C.
  • the present method is preferably employed at the conclusion of the temperature cycling.
  • the reaction is conducted at isothermal temperature, which is usually about 38 to 44°C, preferably about 41 °C.
  • isothermal temperature which is usually about 38 to 44°C, preferably about 41 °C.
  • the present invention may be carried out both during or after the amplification reaction.
  • the amplification is conducted for a time sufficient to produce the desired number of complements or copies of the target polynucleotide. This, in turn, depends on the type of amplification reaction and the purpose for which the amplification is conducted, such as, for example, an assay for a polynucleotide analyte. Generally, the time period for conducting the entire method will be from about 10 to 200 minutes. As a matter of convenience, it will usually be desirable to minimize the time period. For amplification involving temperature cycling, the time is about 10 to 200 seconds per cycle and any number of cycles can be used from 1 to as high as 200 or more, usually 5 to 80, frequently 10-60. The time period for amplification involving isothermal temperatures is usually about 10 to 40 minutes.
  • the concentration of the nucleotide polymerase is usually determined empirically. Preferably, a concentration is used that is sufficient such that further increase in the concentration does not decrease the time for the amplification by over 5-fold, preferably 2-fold.
  • the primary limiting factor generally is the cost of the reagent.
  • the concentrations of the first and second oligonucleotide probes will usually be similar, preferably identical, and may be as low as the smallest amounts detectable up to micro ⁇ molar or more, usually 10 "12 to 10 "7 M, more usually 10 "10 to 10 "7 M.
  • the amount of the target polynucleotide to be amplified can be as low as one or two molecules in a sample but generally may vary from about 10 to 10 10 , more usually from about 10 3 to 10 8 molecules in a sample preferably at least 10 "21 M in the sample and may be 10 "10 to 10 "19 M, more usually 10 "14 to 10 "19 M.
  • the amount of the oligonucleotide primer(s) will be at least as great as the number of copies desired and will usually be 10 "13 to 10 "8 moles per sample, where the sample is 1-1 ,000 ⁇ L.
  • the primer(s) are present in at -35-
  • the concentration of the oligonucleotide primer(s) is substantially in excess over, preferably at least 100 times greater than, more preferably, at least 1000 times greater than, the concentration of the target polynucleotide sequence.
  • concentration of the nucleoside triphosphates in the medium can vary widely; preferably, these reagents are present in an excess amount.
  • the nucleoside triphosphates are usually present in 10 '6 to 10 "2 M, preferably 10 "5 to 10 "3 M.
  • the combination is subjected to conditions under which the single stranded target polynucleotide, if present, hybridizes to the second probe and displaces the first probe in a strand displacement.
  • the conditions for the strand displacement normally involve consideration of temperature, ionic strength, pH and time.
  • the combination is incubated at a temperature of 30°C to 75°C, preferably 60°C to 70°C, for at least one minute, preferably, 20 to 40 minutes.
  • the pH for the medium is usually in the range of about 4.5 to 9.5, more usually in the range of about 5.5 to 8.5, and preferably in the range of about 6 to 8.
  • the ionic strength of the medium is usually about 0.1 to 500 mM.
  • first probe and the second probe can comprise members of a signal producing system.
  • the signal is detected and related to the presence of displaced first probe and/or the amount of a duplex comprising the first and second probes.
  • the signal is ultimately related to the presence and/or amount of target polynucleotide present in a sample.
  • Detection of the signal depend upon the nature of the signal producing system utilized. Such conditions are well-known in the art. If the reporter molecule is an enzyme, additional members of the signal producing system would include enzyme substrates and so forth. The product of the enzyme -36-
  • reaction is preferably a luminescent product, or a fluorescent or non-fluorescent dye, any of which can be detected spectrophotometrically, or a product that can be detected by other spectrometric or electrometric means.
  • the reporter molecule is a fluorescent molecule
  • the medium can be irradiated and the fluorescence determined.
  • the label is a radioactive group
  • the first probe can be separated from the duplex of the first and second probes and one of the fractions can be counted to determine the radioactive count.
  • the association of the labels within the termolecular complex may also be determined by using labels that provide a signal only if the labels become part of, or dissociate from, the complex.
  • the binding of the single stranded target polynucleotide, if present, to the second probe causes displacement of the first probe from the second probe and thereby alters a signal generated by the signal producing system. This approach is particularly attractive when it is desired to conduct the present invention in a homogeneous manner.
  • Such systems include en- zyme channeling immunoassay, fluorescence energy transfer immunoassay, electrochemiluminescence assay, induced luminescence assay, latex agglutination and the like.
  • detection of the complex is accomplished by employing at least one suspendable particle as a support, which may be bound directly to a nucleic acid strand or may be bound to an sbp member that is complementary to an sbp member attached to a nucleic acid strand, either first or second oligonucleotide probe.
  • a suspendable particle serves as a means of segregating the bound target polynucleotide sequence from the bulk solution.
  • a second label, attached to the other of the first or second oligonucleotide probes becomes part of the termolecular complex.
  • Typical labels that may be used in this particular embodiment are fluorescent labels, particles containing a sensitizer and a chemiluminescent olefin (see U.S. Serial No. 07/923,069 filed July 31 , 1992, the disclosure of which is incorporated herein by reference), chemiluminescent and electroluminescent labels. -37-
  • the particle itself can serve as part of a signal producing system that can function without separation or segregation.
  • the second label is also part of the signal producing system and can produce a signal in concert with the particle to provide a homogeneous assay detection method.
  • a variety of combinations of labels can be used for this purpose. When all the reagents are added at the beginning of the reaction, the labels are limited to those that are stable to the temperatures used for amplification, chain extension, and branch migration.
  • the particles may be simple latex particles or may be particles comprising a sensitizer, chemiluminescer, fluorescer, dye, and the like.
  • Typical parti- cle/reporter molecule pairs include a dye crystallite and a fluorescent label where binding causes fluorescence quenching or a tritiated reporter molecule and a particle containing a scintillator.
  • Typical reporter molecule pairs include a fluorescent energy donor and a fluorescent acceptor dye.
  • Typical particle pairs include (1) two latex particles, the association of which is detected by light scattering or turbidimetry, (2) one particle capable of absorbing light and a second label particle which fluoresces upon accepting energy from the first, and (3) one particle incorporating a sensitizer and a second particle incorporating a chemiluminescer as described for the induced luminescence immunoassay referred to in U.S. Serial No. 07/704,569, filed May 22, 1991 , entitled “Assay Method Utilizing Induced Luminescence", which disclosure is incorporated herein by reference.
  • detection of the termolecular complex using the induced luminescence assay as applied in the present invention involves employing a photosensitizer as part of one label and a chemiluminescent compound as part of the other label. If the complex is present the photosensitizer and the chemiluminescent compound come into close proximity. The photosensitizer generates singlet oxygen and activates the chemiluminescent compound when the two labels are in close proximity. The activated chemiluminescent compound subsequently produces light. The amount of light produced is related to the amount of the complex formed. -38-
  • both of the oligonucleotide probes are blocked at the 3'-end to avoid any potential interference with and during amplification.
  • the 3'-end of the recognition sequences can be blocked by a group that cannot undergo chain extension, such as, for example, an unnatural group such as a 3'-phosphate, a 3'-terminal dideoxy, an abasic ribophosphate, a polymer or surface, or other means for inhibiting chain extension.
  • a polynucleotide that does not hybridize to the amplicon is attached to the 3'-end.
  • Such an end group can be introduced at the 3' end during solid phase synthesis or a group can be introduced that can subsequently be modified.
  • a ribonucleotide can be introduced at the 3'-end and then oxidized with periodate followed by reductive amination of the resulting dialdehyde with borohydride and aminodextran.
  • the details for carrying out the above modifications are well-known in the art and will not be repeated here.
  • An embodiment of the above is depicted in Fig. 1 by way of illustration and not limitation.
  • Target polynucleotide S comprises a sequence S1 and a sequence S2E, which is 5' of S1 and noncontiguous therewith in the embodiment depicted.
  • First oligonucleotide probe P comprises sequence P1 that may, but need not be, part of a longer sequence and second oligonucleotide probe PP comprises PP1 and PP2, which are contiguous to one another and may, but need not be, part of a longer sequence.
  • P and PP are combined and incubated so that a duplex, P-PP, forms by virtue of the hybridization of P1 to PP1.
  • target polynucleotide S is added and the mixture is incubated under conditions such that a complex capable of strand displacement, P-PP-S, is formed by virtue of S2 of S hybridizing with PP2 of PP.
  • Strand displacement then occurs wherein S1 displaces P1 from PP1 to give free P and a duplex, PP-S, in which S2 and S1 are bound to PP2 and PP1 , respectively.
  • the strand displacement product PP-S has a sequence of nucleotides, S3, in S that lie between S1 and S2 and are not hybridized to PP. Accordingly, S1 and S2 are noncontiguous.
  • P, PP and S may all comprise sequences of nucleotides that lie both 3' and 5' of the respective sequences identified therein, namely, P1 in P, PP1 and PP2 in PP and S1 and S2 in S.
  • the target nucleotide S is present in the reaction medium.
  • the number of target molecules increase during amplification, the number of molecules of the first oligonucleotide probe P displaced by target polynucleotide S from the complex with second oligonucleotide probe PP increases.
  • the progress of the amplification may be monitored by the increase in the amount of P or the decrease in the amount of the P-PP complex.
  • Target polynucleotide S' comprises sequences S'1 and S'2, wherein S'2 is 5' of S'1 and contiguous therewith in the embodiment depicted.
  • First oligonucleotide probe P' comprises sequence P'1
  • second oligonucleotide probe PP' comprises PP'1 and PP'2, which are noncontiguous to one another and connected by sequence PP'3.
  • P' and PP' are combined and incubated so that a duplex, P'-PP', forms by virtue of the hybridization of P'1 to PP'1.
  • target polynucleotide S' is added and the mixture is incubated under conditions such that a complex capable of strand displacement, P'-PP'-S', is formed by virtue of S'2 of S' hybridizing with PP'2 of PP'.
  • Strand displacement then occurs wherein S'1 displaces P'1 from PP'1 to give free P' and a duplex, PP'-S', in which S'2 and S'1 are bound to PP'2 and PP'1 , respectively.
  • the strand displacement product PP'-S' has a sequence of nucleotides, PP'3, in PP' that lies between PP'1 and PP'2 and is not hybridized to S'.
  • PP'1 and PP'2 are noncontiguous.
  • a determination can be made as to the presence of PP'-S' or free P'. -40-
  • FIG. 3 Another embodiment in accordance with the present invention is depicted in Fig. 3 by way of illustration and not limitation.
  • Target polynucleotide S comprises sequences S"1 and S"2, wherein S"2 is 5' of S"1 and contiguous therewith in the embodiment depicted.
  • the amplification reagents are combined with S" and first and second oligonucleotide probes.
  • First oligonucleotide probe P" comprises sequence P"1 and has a sensitizer SN attached on the terminal nucleotide of P".
  • Second oligonucleotide probe PP" comprises PP"1 and PP"2, which are noncontiguous to one another by sequence PP"3.
  • several molecules of PP" are attached at a terminal nucleotide to a particle that is associated with a chemiluminescent compound CL.
  • P" and PP" hybridize so that a duplex, P"-PP", forms by virtue of the hybridization of P"1 to PP"1.
  • Molecules of target S" hybridize with PP" by virtue of the hybridization of S"2 with PP"2 to form a complex capable of strand displacement, i.e., P"-PP"-S".
  • Strand displacement then occurs wherein S"1 displaces P"1 from PP"1 to give free P" and a duplex, PP"-S", in which S"2 and S"1 are bound to PP"2 and PP"1 , respectively.
  • a signal is measured at the outset of the amplification.
  • the signal is produced as a result of the induced luminescence resulting from the interaction of the sensitizer with oxygen to form singlet oxygen, which reacts with the chemiluminescent compound to produce a product that spontaneously produces luminescence.
  • the level of signal is measured periodically during the amplification.
  • the signal measured initially is changed as a result of the increase in concentration of target S" and the displacement of P" from the termolecular complex. Since less sensitizer is in close proximity of the chemiluminescent compound, signal decreases. This decrease in signal can be monitored as an indication of the progress of the amplification of the target polynucleotide.
  • the strand displacement product PP"-S" has a sequence of nucleotides, PP"3, in PP" that lie between S"1 and S"2 and are not -41-
  • Suitable controls can be used for the quantitative measurement of the target polynucleotide in homogeneous systems, which do not provide for separation of free and bound species.
  • the controls may take the form of reference polynucleotides.
  • the reference polynucleotides are present in a predetermined concentration in the medium containing the target polynucleotide. In general, the amount of the reference polynucleotide is at least the minimum amount that will permit detection following amplification. Often, several reference polynucleotides are used, each at a level of about 10 to 100 fold higher than the next. The reference polynucleotides may therefore be present in as little as about 10 copies up to 10 7 or more copies per sample.
  • each of the respective chemiluminescent particles corresponding to the different reference polynucleotides and the target polynucleotide may emit at different wavelengths of light as a result of the sensitization by the sensitizer.
  • the sequences of the reference oligonucleotides are identical to the target sequence except for the S2 sequence that binds to the PP2 sequence of the second oligonucleotide probe.
  • each reference oligonucleotide may have a similar length arbitrary sequence that permits the reference oligonucleotide to be amplified with similar efficiency to the target oligonucleotide.
  • the reference sequence is identical to the target sequence except for the S1 sequence that binds to the PP1 sequence of the second oligonucleotide probe.
  • both the S1 and S2 sequences of the target are replaced by similar length arbitrary sequences in the reference.
  • a particular advantage of the present invention is that excess target polynucleotide does not interfere with the determination. This results from the fact that the target can react stoichiometrically with the P-PP complex and displace P to form an S-PP complex. Once all of the P-PP complex is consumed, no further signal producing reaction can occur because the P-PP complex reagent is used up.
  • kits in packaged combination can be provided in a kit in packaged combination.
  • the kit comprises in packaged combination one or more reagents for conducting an amplification and detection of the amplified product as well as reference compounds.
  • An example of a kit in accordance with the present invention is a kit comprising reagents for conducting an amplification of the target polynucleotide together with first and second oligonucleotide probes.
  • the first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of the second probe.
  • the amplification product in single stranded form has a sequence S1 that is capable of hybridizing to PP1 and a sequence S2 that is capable of hybridizing to a sequence PP2 of the second probe.
  • the first probe is incapable of hybridizing to the PP2 or the single strand.
  • S1 and S2 or PP1 and PP2 are noncontiguous and one or both of the first probe and the second probe comprise members of a signal producing system. The binding of the single stranded amplification product, if present, to the second probe alters a signal generated by the system.
  • kits in accordance with the present invention are kits for RNA amplification.
  • a kit for RNA amplification comprises reference RNA's, a promoter, an enzyme and a plurality of sets of first and second oligonucleotide probes, one set for each different RNA to be analyzed with the present kit.
  • the first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of the second probe
  • the amplification product in single stranded form has a sequence S1 that is capable of hybridizing to PP1 and a sequence S2 that is capable of hybridizing to a sequence PP2 of the second probe
  • the first probe is incapable -43-
  • S1 and S2 or PP1 and PP2 are noncontiguous and (V) one of the probes is labeled with a sensitizer and the other of the probes is labeled with a chemiluminescer.
  • the combination of the sequence pair S1 and PP1 and the sequence pair S2 and PP2 is unique in each of the sets and the respective labels for each of the sets are differentially detectable.
  • the kit can further include any additional members of a signal producing system and also various buffered media, some of which may contain one or more of the above reagents.
  • the kits above can further include in the packaged combination reagents for conducting an amplification of the target polynucleotide.
  • the kit may include a first DNA primer with a 5' tail comprising a promoter, a second DNA primer, reverse transcriptase, RNAse-H, T7 RNA polymerase, NTP's and dNTP's.
  • the kit may include a DNA polymerase and nucleoside triphosphates such as, e.g., deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP).
  • dATP deoxyadenosine triphosphate
  • dGTP deoxyguanosine triphosphate
  • dCTP deoxycytidine triphosphate
  • dTTP deoxythymidine triphosphate
  • the kit may also include various reference oligonucleotides and their respective signal producing system members as necessary.
  • kits can be varied widely to provide for concentrations of the reagents necessary to achieve the objects of the present invention.
  • one or more of the reagents in the kit can be provided as a dry powder, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present invention.
  • Each reagent can be packaged in separate containers or some reagents can be combined in one container where cross-reactivity and shelf life permit.
  • the kits may also include a written description of a method in accordance with the present invention as described above. -44-
  • Tris HCI - Tris(hydroxymethyl)aminomethane-HCI (a 10X solution) from BioWhittaker, Walkersville, MD.
  • Sulfo-SMCC 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.
  • TCEP tris-carboxyethyl phosphine.
  • the cooled supernatant solution was transferred via cannula to an addition funnel and added dropwise (over 2.5 hr) to a solution of phenylglyoxal (11.7g, 87mmol) in THF (300mL) at -30°C under argon.
  • the reaction mixture was gradually warmed to 0°C over 1 hr and stirred for another 30 min.
  • the resulting mixture was poured into a -46-
  • reaction solution was heated at reflux for 23 hr under argon before being cooled to room temperature. To this was added additional TMSCI (50mL, 394mmol); and the reaction solution was heated at reflux for another 3 hr. The resulting solution was cooled, was made basic with cold 2.5N aqueous NaOH and was extracted with CH 2 CI 2 (3x). The combined organic layers were washed with saturated aqueous NaHCO 3 (2x) and brine, was dried over Na 2 SO and was concentrated vacuo to give a brown oily liquid.
  • the solid was washed with 100mL of water and dried (house vacuum, 60°C, P 2 O 5 ).
  • the solid material was placed in a 1 -liter, round bottom flask and concentrated sulfuric acid (500mL) was added with stirring.
  • the mixture was stirred for 4 hr. at 60°C and was then carefully diluted with crushed ice (2000g).
  • the resulting mixture was filtered and the solid wad washed with 100mL of water and dried.
  • the dark blue solid was transferred to a 1 -liter, round bottom flask, concentrated ammonia (500mL) was added, and the mixture was heated and stirred under reflux for 2 hr., was cooled to room temperature and was filtered.
  • Hydroxypropylaminodextran (1 NH 2 / 7 glucose) was prepared by dissolving Dextran T-500 (Pharmacia, Uppsala, Sweden) (50g) in 150 mL of H 2 O in a 3-neck round-bottom flask equipped with mechanical stirrer and dropping funnel. To the above solution was added 18.8g of Zn (BF 4 ) 2 and the temperature was brought to 87°C with a hot water bath. Epichlorohydrin (350mL) was added dropwise with stirring over about 30 min while the temperature was maintained at 87-88°C. The mixture was stirred for 4 hr while the temperature was maintained between 80°C and 95°C, then the mixture was cooled to room temperature. Chlorodextran product was precipitated by pouring slowly into 3L of methanol with vigorous stirring, recovered by filtration and dried overnight in a vacuum oven.
  • the chlorodextran product was dissolved in 200mL of water and added to 2L of concentrated aqueous ammonia (36%). This solution was stirred for 4 days at room temperature, then concentrated to about 190mL on a rotary evaporator. The concentrate was divided into two equal batches, and each batch was precipitated by pouring slowly into 2L of rapidly stirring methanol. The final product was recovered by filtration and dried under vacuum. Hydroxypropylaminodextran (1 NH 2 / 7 glucose), prepared above, was dissolved in 50mM MOPS, pH 7.2, at 12.5 mg/mL.
  • Chemiluminescer particles (TAR beads): The following dye composition was employed: 20% C-28 thioxene (prepared as described above), 1.6%1-chloro-9,10-bis(phenylethynyl)anthracene (1-CI-BPEA) (from Aldrich Chemical Company) and 2.7% rubrene (from (from Aldrich Chemical Company). The particles were latex particles (Seradyn Particle Technology, Indianapolis IN). The dye composition (240-250 nM C-28 thioxene, 8-16 nM 1 -ClBPEA, and 20-30 nM rubrene) was incorporated into the latex beads in a manner similar to that described in U.S.
  • Patent 5,340,716 issued August 23, 1994 (the 716 patent), at column 48, lines 24-45, which is incorporated herein by reference.
  • the dyeing process involved the addition of the latex beads (10% solids) into a mixture of ethylene glycol (65.4%), 2-ethoxyethanol (32.2%) and 0.1N NaOH (2.3%).
  • the beads were mixed and heated for 40 minutes at 95°C with continuos stirring. While the beads are being heated, the three chemiluminescent dyes were dissolved in 2- ethoxyethanol by heating them to 95°C for 30 minutes with continuous stirring. At the end of both incubations, the dye solution was poured into the bead suspension and the resulting mixture was incubated for an additional 20 minutes with continuous stirring.
  • the beads were removed form the oil bath and are allowed to cool to 40°C ⁇ 10°C.
  • the beads were then passed through a 43-micron mesh polyester filter and washed.
  • the dyed particles were washed using a Microgon (Microgon Inc., Madison Hills, CA).
  • the beads were first washed with a solvent mixture composed of ethylene glycol and 2-ethoxyethanol (70%/30%).
  • the beads were washed with 500 ml of solvent mixture per gram of beads. This is followed by a 10 % aqueous ethanol (pH 10-11) wash.
  • the wash volume was 400 ml per gram of beads.
  • the beads were then collected and tested for % solid, dye content, particle size, signal and background generation. -50-
  • the oligonucleotide was immobilized on the surface of the above particles in the following manner.
  • Aminodextran 500 mg was partially maleimidated by reacting it with sulfo-SMCC (157 mg, 10 mL H 2 O).
  • the sulfo-SMCC was added to a solution of the aminodextran (in 40 mL, 0.05 M Na 2 HPO , pH 7.5) and the resulting mixture was incubated for 1.5 hr.
  • the reaction mixture was then dialyzed against MES/NaCI (2x2L, 10 mM MES, 10 mM NaCI, pH 6.0, 4°C).
  • the maleimidated dextran was centrifuged at 15,000 rpm for 15 minutes and the supernatant collected.
  • the supernatant dextran solution (54 mL) was then treated with imidazole (7 mL of 1.0 M solution) in MES buffer (pH 6.0) and into this stirred solution was added the stained chemiluminescer particles (10 mL of 10mg/mL). After stirring for 10 minutes the suspension was treated with EDAC (7 mmol in 10 mM pH 6.0 MES) and the suspension stirred for 30 minutes. After this time, SurfactAmps® (Pierce) Tween-20 (10%, 0.780 mL) was added to the reaction mixture for a final concentration of 0.1%.
  • the particles were then centrifuged at 15,000 rpm for 45 minutes and the supernatant discarded.
  • the pellet was resuspended in MES/NaCI (pH 6.0, 10 mM, 100 mL) by sonication. Centrifugation at 15,000 rpm for 45 minutes, followed by pellet resuspension after discarding the supernatant, was performed twice.
  • the maleimidated dextran chemiluminescer particles were stored in water as a 10 mg/mL suspension.
  • oligonucleotide (oligonucleotide bearing a 5'-bis(6- hydroxyethyldisulfide) group) (Oligos Etc.) was dissolved in water at a concentration of 0.49 mM. To 116 ⁇ L of this solution was added 8.3 ⁇ L of 3.5 M sodium acetate, pH 5.3 and 8.9 ⁇ L of tris(carboxyethyl)phosphine (20 mM). After 30 minutes incubation at room temperature, 548 ⁇ L of cold ethanol. was added and the mixture was maintained at about 20°C for 1.5 hour. The precipitated oligonucleotide was recovered by centrifugation for 2 min. at 15,000 rpm in an Eppendorf centrifuge, then dissolved in 37.5 ⁇ L of 5mM sodium phosphate, 2 mM EDTA, pH 6. -51-
  • the oil bath temperature was maintained at 105°C.
  • the oil bath temperature was slowly allowed to drop to room temperature over 2 hours.
  • the mixture was diluted with 20 mL of ethanol and centrifuged (12,500 rpm, 30 minutes). Supernatants were discarded and the pellets resuspended in ethanol by sonication. Centrifugation was repeated, and the pellet was resuspended in water; and centrifugation was repeated. The pellet was resuspended in 5 mL of aqueous ethanol to a final volume of 40 mL.
  • oligonucleotide bound sensitizer particles The preparation of oligonucleotide bound sensitizer particles was similar to that described for the chemiluminescer particles.
  • the sequence of the oligonucleotide bound to the sensitizer particles was dA 2 .
  • Example 1 The approach utilized in this example is depicted in Fig. 4.
  • a complex of second oligonucleotide probe PPP composed of a sequence PPc ⁇ _1 at the 5'-end and a sequence PP S N2 at the 3'-end, which sequences are complementary to sequences of oligonucleotide probes immobilized on chemiluminescer particles and sensitizer particles, respectively, and a middle sequence P'P'3 that is complementary to a sequence TS3 in the target polynucleotide TS.
  • the sequence P C L1 of the oligonucleotide of the chemiluminescer particle reagent P C L which corresponds to a first oligonucleotide probe
  • the sequence PSN of the oligonucleotide on the sensitizer particle reagent PSN which corresponds to a third oligonucleotide probe.
  • the second oligonucleotide PPP formed a termolecular complex, PCL-PSN-PPP with the respective oligonucleotides on the above particles. This complex generated a signal.
  • the target oligonucleotide also had a sequence TS1 that was complementary sequence PPCL in PPP and was capable of displacement of PCL from the termolecular complex to form a new termolecular complex TS- PSN-PPP- As a result of the displacement, the signal generated by the chemiluminescent -53-
  • TS' had a sequence TS'1 that was the same as TS1 of the first target polynucleotide and consequently TS' was complementary to P'P'3 of PPP.
  • target polynucleotide TS' also had a sequence TS'2, which was complementary to PPSN2 of PPP and, therefore, homologous with PSN2 of the sensitizer particles.
  • the second oligonucleotide PPP formed a termolecular complex, PCL-PSN-PPP with the respective oligonucleotides on the above particles. This complex generated a signal.
  • the target oligonucleotide also had a sequence TS1 that was complementary sequence PPCLI in PPP and was capable of displacement of PSN from the termolecular complex to form a new termolecular complex TS- P C ⁇ _-PPP.
  • the signal generated by the chemiluminescent compound decreases.
  • the more target polynucleotide present the greater the displacement and the larger the decrease in signal.
  • the second oligonucleotides were as follows: OL-1 : 5'-biotin-(TACT)f,GAATGGGATAGAGTGCATCCAGTG-T, 4 (SEQ ID NO:1) OL-4: 5'-biotin-(TACT) fi CATGAATGGGATAGAGTGCATCCAGTG-T ?4 (SEQ ID NO:2)
  • the underlined sequence (P'P'3) is a sequence complementary to that derived form HIV target (see, e.g., GenBank accession no. AF033819 HIV-1 complete genome). The biotin is not relevant to the system and was incorporated for future applications.
  • the second oligonucleotide OL-4 has the additional nucleotides CAT, which formed a gap between two of the sequences of OL-4.
  • the target sequences were as follows:
  • the underlined sequence (TS3 and TS'3, respectively) was complementary to the underlined sequence P'P'3 of the second oligonucleotide probe.
  • the negative controls are not essential to this experiment. They were included for the purpose of showing that displacement is not efficient where the sequence to be displaced is full double stranded DNA.
  • the chemiluminescer particles prepared as described above had 5'-(ATGA) 6 (SEQ ID NO:7) (PC L 1 ) immobilized through the 3'-end and the sensitizer particles had dA 24 (SEQ ID NO:8) (P S N1 ) immobilized thereon through the 3'-end.
  • the particles were mixed with OL-1 or OL-4 to a final concentration of 1 ⁇ g of each of the particles and 5 pM linker, in a total volume of 39 ⁇ l in NL buffer with 15% DMSO (10mM Tris-HCL, 70 mM KCI, 12mM MgCI 2 , pH8.0 acetylated BSA 0.2 mg/ml).
  • Target polynucleotide PL-2 or PL-3 were added and the mixture was subjected to the following incubation conditions: 2 min. at 65°C, 10 min. at 50°C (annealing), and 20 min. at 37°C. Signals were read manually in a reader designed for this purpose. Reaction tubes were irradiated for 0.1 sec. and read for 1 sec. The results are summarized in the following Table 2.
  • the particles, buffers and oligonucleotides used were those described above in Example 1.
  • the particles were mixed with linker OL-1 or OL-4 to a final concentration of 1 ⁇ g of each of the particles and 5.9 pM for the linker, in a total volume of 45 ⁇ l in NL buffer.
  • Mineral oil (20 ⁇ l) was placed on top to prevent evaporation.
  • the linker particle complex was denatured and annealed using the following conditions: 2 min. at 65°C, 10 min. at 50°C (annealing), and 40 min. at 37°C followed by an overnight incubation at room temperature. On the following day, the reaction tubes were preequilibriated at 41 °C for 2 hours. Signals from the linker were read manually in a reader as described above in Example 1 and then 3.38 ⁇ l of the target was added. The displacement of the preformed particle/linker -56-

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Abstract

L'invention concerne un procédé servant à détecter un polynucléotide monocaténaire ciblé. Elle est basée sur une combinaison comprenant (i) un milieu susceptible de contenir ledit polynucléotide monocaténaire ciblé, (ii) une première sonde d'oligonucléotide et (iii) une deuxième sonde d'oligonucléotide. La première sonde possède une séquence P1 capable de s'hybrider à une séquence PP1 de la deuxième sonde. Le polynucléotide monocaténaire ciblé possède une séquence S1 capable de s'hybrider à PP1 et une séquence S2 capable de s'hybrider à une séquence PP2 de la deuxième sonde. Cette première sonde est incapable de s'hybrider à PP2 ou au polynucléotide monocaténaire ciblé. S1 et S2 ou PP1 et PP2 sont non contiguës et une ou les deux de la première sonde et de la deuxième sonde comprennent des éléments d'un système produisant un signal. La fixation du polynucléotide monocaténaire ciblé, s'il est présent, à la deuxième sonde modifie un signal généré par le système produisant un signal. On soumet la combinaison à des conditions dans lesquelles le polynucléotide monocaténaire ciblé, s'il est présent, s'hybride à la deuxième sonde et déplace la première sonde. On détecte alors le signal. On peut mettre ce procédé en application en association avec un procédé servant à amplifier un polynucléotide ciblé ou une pluralité de différents nucléotides. Elle concerne également des trousses servant à mettre en oeuvre ces procédés. Ce procédé peut être appliqué, en particulier, à l'amplification et à la détection de l'ARN.
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